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Giving up hope of ever being exonerated, David Milgaard escaped from prison
For 23 years, Joyce Milgaard battled the judicial system until finally, in April, 1992, she won. That is when the Supreme Court of Canada overturned her son David's conviction for the 1969 murder of Saskatoon nursing assistant Gail Miller and returned to him the freedom he had lost at the age of 16. It is one of Canada's tragic tales of justice gone awry — and of a mother's dogged devotion. In the newly published A Mother's Story: The Fight to Free My Son David, Milgaard recounts her struggles to convince authorities that her son, a restless school dropout, was innocent of the crime. David was finally exonerated by DNA evidence that implicated another man, but not before giving up hope in the sometimesbrutal prison environment. In her book, Joyce Milgaard tells how, after 11 years behind bars, David escaped from custody, bleached his hair and enjoyed the big-city pleasures of Toronto for 2V-¿ months, until police shot him in the back and returned him to prison.
On Aug. 20, 1980, two days before David was to get out on a pass to attend a barbecue party for [his brother] Chris's 27th birthday, he and I met with prison authorities. Once again, they told us that David could not be considered rehabilitated in their eyes until he showed remorse. In addition, the prison officials said they didn't think David could function on the streets, since he had been in custody for so long. It was so ironic and so heartbreaking. They told us about a longrange plan for David to get into a halfway house, but it would mean that he wouldn't get a taste of freedom until four years or so down the road. He had spent 11 birthdays as a prisoner, and the prospect of at least another four was too much to imagine. David left that parole meeting quietly planning to escape.
We had been planning the family barbecue for weeks. We were living then at Quail Ridge, a townhome property in St. James in the west end of Winnipeg that I managed, and it had a lovely country club and tennis courts. [David's sister] Susan was playing racquetball with David's guard when David and [his other sister] Maureen disappeared together. [David's father] Lome and I were
Reprinted with permission from A Mother's Story, copyright Joyce Milgaard with Toronto journalist Peter Edwards, published by Doubleday Canada Ltd., Toronto.
busy preparing the food and didn't think much about it until the guard came back after finishing his game to ask us where David was. The search began in earnest, and we discovered Maureen's car was gone. The guard and Susan drove around the area, thinking perhaps Maureen and David had gone off joyriding. It seemed an eternity, and finally the guard reported David missing. The phone rang, and it was Maureen, and she was sobbing. She told us that David had forced her out of the car, took whatever money she had, and left her.
It wouldn't be until much later that we learned the truth: Maureen helped David escape, then guarded this secret to protect us. We then learned that after he and Maureen left Quail Ridge, she drove to a drugstore to get bleach for his hair and dyed it for him. As he left, David said to Maureen, "Just tell Mom and Dad I have to go. I can't go back."
Wednesday nights are my Christian Science church nights, which David knows. One Wednesday night shortly after this, I returned to my car after church and found a red rose on the seat. I knew it must be from David. The next Sunday, I left an envelope with money inside on the front seat when I went in to church. I could hardly wait for the service to end, and when it was finally over, I rushed to the car, and sure enough, the envelope was gone. Shortly afterwards, I got a letter from Toronto with no return address on the envelope:
Dear Mom,
Where to start and what to say... I am happy... I have a job starting Wednesday, it is part-time 4 hrs, 6 days a week but a start.
I have met a few people and somehow realize I must practice a better regimen of self-discipline in the sense that so far I've been doing or living in a very hedonistic sort of fashion. Freedom is beautiful and Toronto a place that has a strong pulse, if you like; compared to Winnipeg's nite life.
I keep asking myself what is my direction, what do I want from life now and I only come up with to enjoy it. Maybe that will refine itself somehow, I hope so.
Tell father for me that I hope he understands my leaving and that I care for him and hope he knows that. . .
I wish I could come home. Eleven years wanting only that and then putting myself in a position where I can't have what I want most.
I love every one and miss you all.
If I could understand why life has been as it has for me where I am to go in it, where I've come from, I would be content, but it all makes no sense . . . I shall continue to seek good in others and do good as I see it.
I love you, David.
A little while later, my brother phoned from Toronto and said, "The package that you sent has arrived." Of course. I hadn't sent him a package. His code was easy to follow. "Oh, that's great. I'll get back in touch with you," I replied. I called back from a phone booth and talked with David. He had hitchhiked to Toronto. There he changed his name to Ward McAdam, got a job in telephone sales, and was soon making $200 a week selling Grolier encyclopedias. He had fallen in love with a girl named Rhonda whom he met on Yonge Street. He wanted to see me, but on one condition—I couldn't talk to him about turning himself in. I decided to go.
I flew to Toronto under my maiden name, Baxter. I was terrified that I'd meet someone I knew. I was also scared because I knew I was doing something wrong, that I should be turning David in. But I couldn't erase the memory of his voice when he told me on the phone, "If you come here, don't come with the intention of talking me out of it." When I saw David in Toronto, there was a vibrancy about him that I hadn't seen for years. That look had almost died when he was in prison. There was no way I could shut that door on him.
Despite the publicity, David wasn't looking over his shoulder, and would stroll down busy Yonge Street, soaking up the sounds and smells and rhythms of the city's main street. He was determined to savour every moment of freedom. David and I thought that if he could stay out for a while and show that he could function, he would prove the prison authorities wrong. He would show them he could survive outside prison as a productive, peaceful person. It was crazy, but that thought offered us some comfort.
We had a wonderful time in Toronto. We took long walks together. We attended church and had a beautiful Thanksgiving dinner in a restaurant. I went shopping for clothes for him. We found him a furnished apartment in a house, and I bought him a comforter for his bed, and some clothes, including a parka. They were all the mothering things we had both missed so badly.
I David later wrote to me, "My success and I sense of self-pride was diminished only by the I fact that I could not share it [freedom] truly with I those that I love, my family." Meanwhile, in S Toronto, a new man at David's work needed a " place to stay, and David took him in after telling him, "No drugs. I can't stand any heat." The man caught on that David must be hiding something from the police. When the young man left and quickly got into trouble with the police, he told them he knew someone they wanted.
The man David helped told him that he wanted to meet him to thank him for all of his help, and they arranged a get-together near the corner of Queen Street and Roncesvalles Avenue, in the Parkdale district. When David arrived, he saw two big men who looked like police. As he walked by them, one of them said, "David?" "Excuse me," David replied, breaking into a run. Unknown to him, he was running directly into a buildup of police in a parking lot. They yelled for him to stop, and he did, raising his hands above his head. There was a single blast from a double-barrelled shotgun anyway, and David was down on the pavement, bleeding. The pellets had lodged in his spine, and David feared he had lost the use of his legs forever, as well as his taste of freedom, which had now ended after 77 days.
Few questions were asked about the shooting, since David was believed to be a wanton killer and had no public sympathy. Besides our family and a few close friends, who really cared? Police said they thought David was armed when they shot him. Witnesses interviewed by the press said police could have easily reached out and tripped him, but the officer shot him instead. Other bystanders said they never heard the police identify themselves as police officers, and no one said anything about David actually having a weapon.
I flew back to Toronto to comfort him. There was a police officer outside his room at St. Joseph's Hospital, and David was handcuffed to the bed. I sat on his bed, rubbed his feet, and told David that I loved him. He looked so frail, so helpless, so pale. He later explained that he had to escape, despite the risks. "I was dying a little bit... something inside me, every day. I stole my freedom."
That was a turning point for me. I stopped placing hope in appeals or parole or politicians or any part of the system that had already failed us so badly. □


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Making my own spa products, starting with making bath salts, began as a lark. A fun experiment. Then came the Eureka moment.Read More
Add to Favorites By Gayle, Blossom's Herb Farm – We are all inspired to plant a gigantic vegetable garden every year to cut costs at the grocery store. But have you ever …Read More
Add to Favorites If you're just starting your soapmaking journey, learning how to make bath bombs is a good first step. Often ball-shaped, bath bombs are dry, chalky, conglomerations that …Read More

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LG has announced that it will be delaying the launch of its Android-based tablet in order to focus more on smartphones.
According to Bloomberg, an executive within the company said that LG will probably reveal the tablet device in January next year.
The hardware of the unnamed tablet device is ready, the executive said, but the company is still working to configure it with a suitable Android OS.
Industry experts suggest that LG is waiting for Google to launch the next iteration of the Android OS for its tablet device.
The company has gone through a number of senior management changes in the past few weeks after it failed to make an impact on the crowded smartphone market, which is dominated by RIM, Apple and Google.
The LG tablet is said to have a larger screen than Samsung's Galaxy Tab tablet device but no other specifications have yet been released.
Some experts believe that LG might be too late in launching a competitor to the iPad to succeed.


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Co-Hosted by supermodel Tyra Banks, In the Mix tackles smoking prevention in a high energy, in-depth program that dispels common misconceptions among teens. Featured segments include a hard look at the addictive aspects of tobacco as well as the wide range of physical effects; profiles of teens who are kicking the habit; a profile of a young woman suffering from emphysema; and an ex-Lucky Strike cigarette model who has cancer.
11 years - 25 years.
Sue Castle
Castle Works Inc.
Back to Previous screen.


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Funeral services for Beverly Jackson will be held on Wednesday, January 22, 2020 at 1:00 pm at graveside with Bro. Michael Wells officiating. Interment will be in Meridian Memorial Park Cemetery.
Mrs. Jackson, 89, passed away on January 17, 2020, in Philadelphia surrounded by her loved ones. She loved to read. She worked as a clerk in the Lauderdale Circuit Clerks office in her earlier years.
She is survived by her daughter, Merry Irons (Sandy); Son, Dennis Jackson (Christy); Grandson, William Irons and his fiance Ashlyn Pullin; one great granddaughter, Addison Irons; half brother, Donnie Parrent; and numerous nieces and nephews.
She is preceded by her husband, W.H. "Bill" Jackson; Son, Frank Jackson; Two sisters, Mary Liberto and Martha Hickman; and a Brother, Buddy Parrent.
Pallbearers will be Dennis Jackson, Sandy Irons, William Irons, John Robert Hickman, Walt Rudolph, and Jay Bryan.
Family and friends may sign the online memorial, leave a personal note for the family, or order flowers at www.jamesfwebb.com
To send flowers to the family or plant a tree in memory of Beverly Ann (Parrent) Jackson, please visit our floral store.


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Having dental discount plans can save anyone from costly dental work. The right dental plan can help anyone save about 10 to 15 percent per appointment. A dental discount plan can cover all aspects of dentistry. Examples of these services are root canals, teeth cleaning, orthodontia, dentures, crowns and extractions. Discounts on vision and prescriptions are other coverage that dental insurance also include.
Discount plans such as :Dental Plans offer great alternatives to dental insurance. They also help people save loads of cash.
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CIGNAPlus Savings Powered By CIGNA Dental Network Access
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Aetna Vital Dental Savings℠ Plus Rx
Aetna saves an average of 15-50% on most dental services. These discounts include root canals, crowns, implants, braces and more. They also have added savings on prescription medications, vision and hearing. Anyone can save at general dentists and specialist treatments. These are Endodontist, Oral Surgeons, Orthodontist to name a few.
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Anyone can be covered with just $149.95/year for individuals and $199.95/year for families.
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The Careington Care 500 dental plan allows everyone to get discounts on dental work and covers most procedures. They also cover discounts on drugs and vision. The dental discount is available for individuals, families through over 90,000 providers.
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Members of this plan can save 20% to 50% on most dental procedures. They also offer 20% savings on orthodontics including braces and retainers for children and adults. The plan includes Dental, Telemedicine and 8 more savings program to keep everyone healthy and happy.
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Dear Ernest,
When will everyone be told what the Big Building with No Name is all about?
Dear Francesca,
That's a really good question. I have that question myself.
You have to understand that Christmas is a season of secrets. Even at the North Pole. Did you know that most of the time when an elf works on a toy or a gift he doesn't know WHO's gift he is working on? Santa just tells us to make the toy, he never tells us who it is for.
So Christmas is always full of surprises.
Now, usually, I am know most things that happen at the North Pole. I cannot talk about everything I know but my job as an elf is a little different — it is my job to tell you guys stuff.
But I honestly don't know the answer to the Big Building. I can see it right outside my window. I tell you everything I see, everything I know about it. But even I don't know what it is for and why so many elves are in there.
But over the course of the season we're going to figure it out. I'm fairly certain that by the time Christmas comes around we'll know.


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Oxford Analytica Staff
Leaders in Burkina Faso face a serious quandary this week, as unions threaten a general strike on Tuesday and Wednesday to protest against the rising cost of living.
In February, riots erupted in towns across the country, and more than 300 people were arrested, leading the government to lower tariffs on imports of basic foodstuffs. Unions demanded further measures to reduce food prices, as well as a public sector wage increase, at marches in early March.
Soaring commodity prices
Burkina Faso is not alone: many of its neighbours in the Sahelian region of West Africa -- and elsewhere in Africa and beyond -- are facing difficulty over the rising cost of living, due to soaring commodity prices:
Africa: constrained
Africa is not unique in being affected by rising costs of living, but food price rises are hitting the region particularly hard. The IMF last economic outlook suggests that food prices contribute most to headline inflation in Africa at 60%, compared to 18% in the OECD countries. Asia (56%) and the Middle East (52%) are also strongly affected.
African governments are also constrained in their ability to react to price increases, either to cushion the blow to the poor, or to stimulate increased production:
With no end in sight for the commodity price boom, the political backlash in the region can be expected to continue for the foreseeable future.
Oxford Analytica (Reino Unido)


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This year I am walking in the Candlelighters "Virtual" Fun Walk and I would love for you to support me! I am walking in honor of all the families who face childhood cancer. I know that Candlelighters makes a difference in the lives of childhood cancer families each and every day. Get inspired – watch this video
Last year I was blessed to be a counselor at the camp (go Cabin 13!). It was an amazing experience to see the resiliency of the survivors and their families.
Here's what Fun Walk donations will be used toward this year:
1. Hospital Parking Assistance - Candlelighters has still been able to provide hospital parking financial assistance to families digitally. {Families have to pay $13 per day to park at the hospitals.}
2. Emotional Support - families more than ever before are nervous to leave their homes, feeling isolated, being torn apart (thanks to hospital visitor restrictions) and we've been ramping up our peer-to-peer support efforts. Our weekly support group meetings have grown, and Candlelighters Parent Consultants (staff who are also parents of childhood cancer survivors) are working around the clock to touch base and check-in with our families and their needs.
3. Gas Cards & Grocery Cards - these have been Candlelighters top request throughout the COVID-19 crisis as families more than ever are strapped for basic needs due to furloughs and layoffs.
4. Memorial Funds - cancer hasn't quit, unfortunately. Children are still being diagnosed and gaining their angel wings. Candlelighters provides financial assistance for families to hold memorial services for their children.
In addition to Candlelighters daily efforts, we're staying on top of the CDC and American Oncology Camping Association message boards to make the best decision for our weekend family camp, Camp CLIFF. The Fun Walk usually completely funds this weekend getaway for Candlelighters families – a getaway that is MUCH needed from the daily grind of a childhood cancer diagnosis. Should camp be unable to happen this fall, it is Candlelighters goal to be able to provide a virtual camping experience and activities for families instead of a physical weekend getaway.
Thank you for supporting my efforts to help keep Candlelighters afloat so we can continue to serve childhood cancer families. Whatever you can give, thank you for helping all the children with cancer and their families that Candlelighters serves!


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- © 2011 American Society of Plant Biologists. All rights reserved.
Boron (B) is an essential plant micronutrient that is toxic at higher levels. NIP5;1 is a boric acid channel required for B uptake and growth under B deficiency. Accumulation of the NIP5;1 transcript is upregulated under B deficiency in Arabidopsis thaliana roots. To elucidate the mechanism of regulation, the 5′ untranslated region (UTR) of NIP5;1 was tested for its ability to confer B-dependent regulation using β-glucuronidase and green fluorescent protein as reporters. This analysis showed that the 5′ UTR was involved in NIP5;1 transcript accumulation in response to B conditions. We also found that high-B conditions trigger NIP5;1 mRNA degradation and that the sequence from +182 to +200 bp in the 5′ UTR is required for this mRNA destabilization. In the nip5;1-1 mutant background, a NIP5;1 complementation construct without the 5′ UTR produced high levels of mRNA accumulation, increased B concentrations in tissues, and reduced growth under high-B conditions. These data suggest that the 5′ UTR controls B-dependent NIP5;1 mRNA degradation and that NIP5;1 mRNA degradation is important for plant acclimation to high-B conditions.
Boron (B) was established as an essential micronutrient for plants in 1923 (Warington, 1923). Since then, B was shown to be essential for diatoms (Smyth and Dugger, 1981) and cyanobacteria (Bonilla et al., 1990) as well as for animals, including zebra fish (Danio rerio) (Rowe and Eckhert, 1999), trout (Oncorhynchus mykiss) (Eckhert, 1998; Rowe et al., 1998), frogs (Xenopus laevis) (Fort et al., 1998), and mice (Mus musculus) (Lanoue et al., 2000). Although the precise physiological role of B has not been established, it is beneficial for human health (Nielsen, 2000).
B in neutral solution is predominantly present as boric acid (H3BO3), an uncharged molecule. Boric acid is a very weak Lewis acid with a pKa of 9.24 (Woods, 1996), and plants take up B from the soil mainly in the form of boric acid. B availability in soils is limited in many high rainfall areas because boric acid is easily leached out from the soil due to its high solubility (Shorrocks, 1997). B, however, is toxic to plants when present in excess, and arid or semiarid areas often have B toxicity problems. Water reaches the topsoil by capillary action and then evaporates, causing B to accumulate to a high concentration (Yau et al., 1995). Because B is relatively phloem immobile in most crop plants, B deficiency symptoms often occur in the growth of apical meristems (both shoots and roots), and toxicity symptoms often appear as necrosis along the margins of old leaves (Marschner, 1995; Dell and Huang, 1997; Shorrocks, 1997). Since both B deficiency and B toxicity reduce crop yield, managing B availability in soils is important to maintain high crop productivity.
The main known function of B in plants is to maintain cell wall structure, and B is a component of rhamnogalacturonan-II, a complex pectic polysaccharide. Cross-linking of rhamnogalacturonan-II by borate is essential for normal expansion of leaves (O'Neill et al., 2001, 2004).
After B is taken up from root surface, it must be transported across the plasma membranes of various cells. Boric acid, the major B form in the plant, is a small, uncharged molecule that can diffuse relatively easily across membranes. Under optimum B conditions, plants can acquire enough B for normal growth mainly by passive diffusion (Dordas et al., 2000; Dordas and Brown, 2001; Stangoulis et al., 2001). By contrast, under low B conditions, two types of B transporters, BORs and the nodulin 26-like intrinsic proteins (NIPs), are necessary for effective B movement from roots to shoots and for effective B uptake from soils to roots.
BOR1, identified as the first B efflux transporter in Arabidopsis thaliana, is localized to the plasma membrane and is involved in xylem loading of B in roots under B limitation (Takano et al., 2002). BOR1 is polarly localized on the inner side of the cells (Takano et al., 2010), and BOR1 accumulation is regulated by posttranscriptional mechanisms in response to B deficiency (Takano et al., 2005). Higher accumulation of BOR1 fused with green fluorescent protein (GFP) was observed in transgenic plants grown under low-B conditions compared with those under high-B conditions. BOR1-GFP was internalized through endocytosis and degraded in vacuoles in response to high-B supply (Takano et al., 2005, 2010). A BOR1 paralog, BOR4, also encodes a functional efflux B transporter in Arabidopsis. BOR4 is stable under high-B conditions and excludes B from the roots under such conditions. Overexpression of BOR4 was found to significantly improve excessive B stress tolerance in Arabidopsis plants (Miwa et al., 2007).
Another type of membrane protein, NIP5;1, was discovered as a boric acid channel (Takano et al., 2006). NIP5;1 is a member of the major intrinsic protein family; major intrinsic protein is known to facilitate the passive flow of water and small uncharged molecules and is widely present in various organisms, such as mammals, amphibians, yeast, bacteria, and plants (Johanson et al., 2001; Zardoya, 2005; reviewed in Tyerman et al., 2002; Maurel, 2007; Tanaka and Fujiwara, 2008).
Under B limitation, NIP5;1 is required for B uptake for normal growth in Arabidopsis. NIP5;1 is localized to the plasma membrane on the outer side of epidermal cells in the roots (Takano et al., 2006, 2010). Accumulation of NIP5;1 mRNA is upregulated by 10-fold in response to B deprivation. NIP6;1, the Arabidopsis protein most similar to NIP5;1, is also a boric acid channel and functions in preferential B distribution to shoot sink tissues under B deficiency (Tanaka et al., 2008). NIP6;1 is localized to the plasma membrane in the vascular bundle, especially in phloem parenchyma cells and companion cells in the nodal region in shoots. NIP6;1 mRNA accumulates significantly even under high B supply and is upregulated by approximately twofold in response to B deprivation.
To adjust to the environmental changes required for growth and development, gene expression can be regulated at multiple levels. Posttranscriptional control of mRNA stability can be beneficial, providing a rapid response to changes in the intracellular and extracellular environments (reviewed in Abler and Green, 1996). In many cases, mRNA stability is regulated by sequences in the 3′ untranslated region (UTR). One A-rich element located in the 3′ UTR is well known as an mRNA stability element for translation-dependent destabilization in eukaryotic cells (Ohme-Takagi et al., 1993; Chen et al., 2001; Sarkar et al., 2003). The downstream element, containing an mRNA instability sequence located in the 3′ UTR, was also characterized in plants (Gil and Green, 1996; Pérez-Amador et al., 2001). In some cases, mRNA stability is controlled by sequences in the 5′ UTR. For example, dark-dependent FERREDOXIN-1 mRNA destabilization in transgenic tobacco (Nicotiana tabacum) is regulated by multiple CAUU sequences located in the 5′ UTR (Bhat et al., 2004). Lhcb1*4 encodes light-harvesting complex II proteins in peas (Pisum sativum), and the Lhcb1*4 transcript is destabilized by blue light through a 5′ UTR sequence (Anderson et al., 1999).
In this study, to obtain insight into the mechanisms regulating NIP5;1 in response to B conditions, we performed deletion analyses of the NIP5;1 promoter and the 5′ UTR and investigated the physiological role of NIP5;1 regulation. We demonstrated that B-responsive NIP5;1 expression is regulated through sequences in the 5′ UTR and that a conserved element located in the 5′ UTR controls mRNA stability in a B-dependent manner in Arabidopsis roots. This regulatory mechanism plays an important role in maintaining B concentration in plants within the range that allows normal growth, even during rapid changes in B availability.
Deletion Analysis of the NIP5;1 Promoter to Identify Cis-Acting Elements for B-Regulated Expression
In a previous report, Takano et al. (2006) demonstrated that β-glucuronidase (GUS) activity driven by the NIP5;1 promoter (2180 bp) and 5′ UTR (312 bp) was higher in Arabidopsis roots grown under low-B (0.3 μM B) conditions than those under high-B (100 μM B) conditions. To further investigate the induction pattern, the same transgenic plants containing the full-length promoter with the 5′ UTR fused to GUS, referred to as P-2,180 UTR312-GUS in this article (see Supplemental Table 1 online for the nomenclature of the constructs), were grown for 14 d on solid medium containing a wide range of B concentrations (0.1 to 3000 μM B), and the GUS activity in the roots was determined (Figures 1A and 1B). The –2180 refers to the nucleotide number from the transcriptional start site (+1) of NIP5;1 (based on The Arabidopsis Information Resource [TAIR] database), and 312 is the length of the 5′ UTR in NIP5;1 (Figure 1A). GUS activity strongly increased under B-deficient conditions at 11.6- and 10.4-fold higher at the 0.1 and 0.3 μM B, respectively, than at the 100 μM B condition and gradually declined as the B concentration in the medium increased (Figure 1B). At 3 mM B, GUS activity was below the detection limit.
To clarify the regulatory mechanism of expression by the NIP5;1 promoter fragment, a 5′-deletion series (–1248, –589, –508, –389, and –289 bp) from the transcriptional start site (+1) containing the 5′ UTR was constructed and fused to the GUS reporter gene (referred to as P-1,248 UTR312-GUS, P-589 UTR312-GUS, P-508 UTR312-GUS, P-389 UTR312-GUS, and P-289 UTR312-GUS, respectively) and introduced into Columbia-0 (Col-0) plants (Figure 1C). Independent T3 transgenic lines homozygous for the T-DNA insertion were grown for 7 d on solid medium containing 0.3 or 100 μM B. For each construct, GUS activity was measured in the roots of two to three independent lines (Figure 1D). The measured GUS activity was lower in Figure 1D than in Figure 1B presumably because plants were grown for 14 and 7 d in Figures 1B and 1D, respectively. GUS activity at 0.3 μM B was around 12-fold higher than at 100 μM B in transgenic plants containing –2180 promoter in Figure 1D, and the ratio (12.0) is almost similar to that in Figure 1B (11.6), suggesting that plants grown for 7 d are sufficient to detect induction of GUS activity under the low-B condition compared with that under the high-B condition. GUS activity in the transgenic lines carrying P-1,248 UTR312-GUS, P-589 UTR312-GUS, P-508 UTR312-GUS, and P-389 UTR312-GUS was elevated under B deficiency similarly to the transgenic lines carrying the full-length P-2,180 UTR312-GUS. The GUS activity was ~2.6- to 15.2-fold higher under the 0.3 μM B condition compared with the 100 μM B condition in these lines. By contrast, GUS activity in the transgenic line carrying P-289 UTR312-GUS was low and close to the detection limit. These results suggest that a cis-acting element that acts in the response to B status is likely to exist downstream of –389.
Role of the 5′ UTR in the Regulation of Gene Expression in Response to B Concentration in Arabidopsis Roots
The above results suggest that regions of NIP5;1 required for regulation of B-dependent expression may be present in the promoter region between −389 and +1 or in the 5′ UTR. It is known that the 5′ UTR can affect regulation of gene expression, including regulation of translation efficiency and mRNA stability. To investigate the contribution of the 5′ UTR to NIP5;1 expression in response to B status, we tested the NIP5;1 promoter fragment without the 5′ UTR by making a construct that fused the promoter to the GUS reporter gene (P-2,180 ΔUTR312-GUS; Figure 2A) and then introducing that construct into Col-0 plants. Independent T3 transgenic lines homozygous for the T-DNA insertion were grown for 11 d on 0.3 or 100 μM B. The GUS activity controlled by P-2,180 UTR312 construct containing the 5′ UTR was higher under low-B conditions (0.3 μM B) than under high-B conditions (100 μM B), but the GUS activity controlled by the P-2,180 ΔUTR312 construct without the 5′ UTR was similar under both conditions (Figure 2B). This result suggests that the 5′ UTR is required for B-dependent expression of NIP5;1.
To further investigate the requirement of 5′UTR for B-dependent expression of NIP5;1 protein, NIP5;1 cDNA was fused to GFP (GFP-NIP5;1) and placed downstream of the NIP5;1 promoter fragment with or without the 5′ UTR (referred to as P-2,180 UTR312-GFP-NIP5;1 and P-2,180 ΔUTR312-GFP-NIP5;1, respectively, in Figure 2C). The constructs were introduced into the NIP5;1 T-DNA insertion line (nip5;1-1). Independent T3 transgenic lines homozygous for the T-DNA were grown for 14 d on solid medium containing 0.3, 1, 100, or 1000 μM B. GFP fluorescence in the root differentiation zone was detected by confocal laser scanning microscopy. Three independent transgenic lines were observed, and representative images are shown in Figures 2D to 2G. The GFP-NIP5;1 fluorescence in the transgenic plants carrying P-2,180 UTR312 is localized in the outer (distal) plasma membrane domain of the epidermal cells under low-B conditions as previously reported (Figure 2D; Takano et al., 2010), and the signal intensity gradually declined as B increased (Figure 2E). The GFP-NIP5;1 fluorescence in the transgenic plants carrying P-2,180 ΔUTR312 was also localized to the outer (distal) plasma membrane domain of epidermal cells (Figure 2F). In contrast with the transgenic plants carrying P-2,180 UTR312, the signal intensity of transgenic plants carrying P-2,180 ΔUTR312 was maintained even under excess B conditions (1000 μM B) (Figure 2G). These results suggested that the 5′ UTR controls B-dependent expression of NIP5;1.
We also determined the accumulation of the GFP-NIP5;1 transcript in the roots of these transgenic plants by quantitative RT-PCR (qRT-PCR) (Figure 2H). Plants were grown for 11 d on solid medium containing 3 or 1000 μM B. The primers used in this study detect both the NIP5;1 and GFP-NIP5;1 transcripts, but in nip5;1-1, the NIP5;1 mRNA is reduced to around 1% of that in Col-0 plants (Takano et al., 2006); thus, the endogenous NIP5;1 mRNA in these transgenic plants is negligible. GFP-NIP5;1 mRNA accumulation in transgenic plants carrying P-2,180 UTR312 decreased under the excess B conditions (1000 μM B) compared with the low-B conditions (3 μM B) as with NIP5;1 mRNA accumulation in Col-0 plants, while GFP-NIP5;1 mRNA accumulation in transgenic plants carrying P-2,180 ΔUTR312 remained high under the excess B conditions compared with the low-B conditions. These findings suggest that the NIP5;1 5′ UTR regulates mRNA accumulation in response to B and that posttranscriptional regulation may be important for this regulation.
Identification of a Region in the 5′ UTR Required for B Response
To search for regulatory element(s) in response to B status in the 5′ UTR, the 5′ UTR of Arabidopsis NIP5;1 was compared with the corresponding sequences in Os NIP3;1, the rice (Oryza sativa) NIP5;1 ortholog, which is also increased by B deficiency (Hanaoka and Fujiwara, 2007). Note that the 5′ UTR regions of these two genes are highly conserved (see Supplemental Figure 1 online). Both sequences contain three upstream open reading frame (uORF). The distances of the uORFs from the putative transcriptional start site (+1) are +1, +80, and +191 bp, and the uORFs consist of one, six, and one codon(s), respectively, in NIP5;1. In Os NIP3;1, the corresponding ORFs are at +1, +85, and +190 bp and consist of one, three, and one codon(s), respectively. The sequence (CAUGUAA) including the first uORF and the sequence (UCAAAUCAUGUAA) including the third uORF were identical between the Arabidopsis and rice genes (Figure 3).
To confirm whether the 5′UTR is sufficient to control B-dependent expression, the NIP5;1 5′ UTR was fused to the cauliflower mosaic virus 35S RNA promoter (P35S) (referred to as P35S-100UTR+312-GUS and P35SUTR+7-GUS in Figure 4A) and introduced into Col-0 plants. In this experiment, we used P35S to eliminate other possible transcriptional regulation in response to B. For each construct, two to three independent T3 transgenic lines homozygous for the T-DNA insertion were established and used for the analysis of B-regulated expression. Plants were grown for 11 d under 0.3 or 1000 μM B conditions, and the GUS activity in roots was determined (Figure 4B). GUS activity in transgenic plants carrying P35S-100UTR312 and P35SUTR+7 was higher under the 0.3 μM B condition compared with the 1000 μM B condition. Ratios of GUS activity in B-deficient roots to that in B-sufficient roots were ~4.2 to 10.4, indicating that NIP5;1 5′ UTR is sufficient to regulate B-dependent expression. These findings suggested that B-dependent expression is regulated by the 5′ UTR through posttranscriptional mechanisms.
To identify the element responsible for B-dependent regulation in the 5′ UTR, a series of truncated 5′ UTRs in NIP5;1 was generated and fused to the GUS reporter gene under the control of P35S (referred to as P35SUTR+101-GUS, P35SUTR+198-GUS, P35SUTR+7 to +79-GUS, and P35SUTR+7 to +183-GUS in Figure 4A) and introduced into Col-0 plants. For each construct, two to three independent T3 transgenic lines homozygous for the T-DNA insertion were established and used for the analysis of B-regulated expression. Plants were grown for 11 d under 0.3 or 1000 μM B conditions, and the GUS activity in roots was determined (Figure 4B). GUS activity in transgenic plants carrying P35SUTR+101 as well as P35S-100UTR312 and P35SUTR+7 was higher under the 0.3 μM B condition compared with the 1000 μM B condition. Ratios of GUS activity in B-deficient roots to that in B-sufficient roots were ~4.9 to 9.5. By contrast, the GUS activity in transgenic plants carrying P35SUTR+198, P35SUTR+7 to +79, and P35SUTR+7 to +183 was not downregulated by external B concentration (0.3 or 1000 μM B). Ratios of the GUS activity in B-deficient roots to that in B-sufficient roots were ~0.6 to 0.8. These results suggest that the 13-bp region from +184 to +197 bp in the 5′ UTR is important in the regulation of NIP5;1 expression in response to low B.
To confirm the above results, transgenic plants carrying P35SUTR+7 harboring an internal deletion between +182 and +200 bp were established (referred to as P35SUTR+7Δ+182 to 200-GUS in Figure 4C) and introduced into Col-0 plants. Two independent T3 transgenic lines homozygous for the T-DNA insertion were grown for 11 d under 0.3 and 1000 μM B conditions, and GUS activity in the roots was determined (Figure 4D). GUS activity in the roots of transgenic plants carrying P35SUTR+7 increased 4.5- to 4.8-fold in 0.3 μM B compared with 1000 μM B, whereas GUS activity in the roots of transgenic plants carrying P35SUTR+7Δ+182 to +200 grown under 0.3 μM B was similar to that under the 1000 μM B condition. This result demonstrated that the sequence from +182 to +200 bp in the 5′ UTR is required for the regulation of NIP5;1 expression in response to B.
To investigate whether NIP5;1 5′UTR can affect the mRNA accumulation driven by a heterogeneous promoter, P35S, GUS mRNA accumulation in roots was measured by qRT-PCR (Figure 4E). GUS mRNA accumulation in transgenic plants carrying P35SUTR+7 increased 1.7- to 2.1-fold under the 0.3 μM B condition compared with the 1000 μM B condition, suggesting that the 5′ UTR affects the mRNA accumulation driven by the P35S. By contrast, GUS mRNA accumulation in transgenic plants carrying P35SUTR+7Δ+182 to +200 grown in 0.3 μM B was similar to that in the 1000 μM B condition, indicating that the sequence from +182 to +200 bp in the 5′ UTR influences the regulation of mRNA accumulation in response to B in Arabidopsis roots.
The 5′ UTR Affects NIP5;1 mRNA Stability in a B-Dependent Manner
A possible mechanism of NIP5;1 transcript regulation in response to B by the 5′ UTR is mRNA decay. To test this possibility, the half-life of NIP5;1 mRNA in intact Col-0 roots was estimated by determining a time course for mRNA decay after exposure to 3′-deoxyadenosine (cordycepin) (Seeley et al., 1992), an RNA synthesis inhibitor, under high- and low-B conditions. Col-0 plants were grown with 0.3 μM B for 23 d and then preincubated for 10 min in hydroponic culture medium containing 0.3 or 100 μM B. After the preincubation, cordycepin was added to a final concentration of 0.6 mM. Roots were harvested at 0, 10, and 30 min after cordycepin treatment, and the NIP5;1 mRNA level was measured by qRT-PCR (Figures 5A and 5B).
After cordycepin treatment, the NIP5;1 mRNA level in Col-0 plants exposed to high-B conditions decreased rapidly compared with that in Col-0 plants exposed to low-B conditions (Figure 5B). The estimated half-lives of NIP5;1 mRNA were 5 and 17 min in Col-0 plants exposed to high-B and low-B conditions, respectively, suggesting that the rate of NIP5;1 transcript degradation is more than 3 times faster under high-B conditions than under low-B conditions.
Requirement of the +182- to +200-bp Region in the 5′ UTR for the B-Dependent Regulation of mRNA Stability
To determine if the sequence from +182 to +200 bp in the 5′ UTR is involved in mRNA degradation in response to B, the half-lives of GUS mRNA in transgenic plants carrying P35SUTR+7 and P35SUTR+7Δ+182 to +200 were measured under high- and low-B conditions (Figures 5C and 5D). Transgenic plants were grown in 0.3 μM B for 14 d, then cordycepin was added and whole plants were harvested at 0, 10, and 20 min after cordycepin treatment. The GUS mRNA level in transgenic plants carrying P35SUTR+7 exposed to high-B condition decreased faster than in transgenic plants carrying P35SUTR+7 exposed to low-B condition (Figure 5C). The estimated half-lives of GUS mRNA were 10 and 55 min in the transgenic plants exposed to high-B and low-B conditions, respectively, suggesting that the rate of GUS transcript degradation is 5 times faster under high-B condition than under low-B condition. In P35SUTR+7Δ+182 to +200-GUS transgenic plants, the rate of GUS mRNA accumulation did not significantly differ under high- and low-B conditions (Figure 5D). The estimated half-lives of the GUS mRNA were 24 and 38 min in transgenic plants exposed to high-B and low-B conditions, respectively. These results indicate that the sequence from +182 to +200 is involved in B-dependent mRNA degradation.
Physiological Roles of the Regulation of NIP5;1 Expression by the 5′ UTR in Roots under Low- and Excess-B Conditions
To investigate the role of the 5′ UTR on growth characteristics, three independent T3 lines carrying P-2,180 ΔUTR312-GFP-NIP5;1 and P-2,180 UTR312-GFP-NIP5;1 were grown on plates containing 0.3, 100, and 3000 μM B. The growth patterns of the three independent transgenic lines were very similar, and images of two of the transgenic lines are shown in Figures 6A to 6C. All transgenic plants showed growth recovery compared with nip5;1-1 mutant plants under low-B conditions (0.3 μM B; Figure 6A), suggesting that GFP-NIP5;1 functions as a B transporter and complements the nip5;1-1 mutation. Under high-B conditions (100 μM B), growth was similar among all plants (Figure 6B). Under excess-B conditions (3000 μM B), growth of the transgenic plants carrying the P-2,180 ΔUTR312 construct was reduced in both the roots and rosette leaves compared with Col-0 and nip5;1-1, whereas growth of the transgenic plants carrying P-2,180 UTR312 was similar to that of Col-0 and nip5;1-1 (Figure 6C). Moreover, leaves of the transgenic plants carrying the P-2,180 ΔUTR312 construct were yellower and smaller compared with other plant lines (Figure 6C). These results established that the construct lacking the 5′ UTR inhibited growth under excess-B condition, whereas the construct with the 5′ UTR did not.
To further characterize the phenotype of transgenic plants carrying P-2,180 ΔUTR312 under excess-B conditions, the root length and B concentration were determined in transgenic plants grown under 3000 μM B for 14 d (Figures 6D and 6E). Root length in transgenic plants carrying P-2,180 ΔUTR312 was reduced compared with Col-0, nip5;1-1, and transgenic plants carrying P-2,180 UTR312 (Figure 6D). B concentrations in shoots (two lines) and roots (one line) were significantly higher in transgenic plants carrying P-2,180 ΔUTR312 than in Col-0 plants (Figure 6E). Because boric acid is an uncharged small molecule that can relatively easily diffuse across membranes, under high-B conditions, B can be absorbed from roots without specific B transporters. It is considered that the additional B uptake by NIP5;1 resulted in higher B accumulation in leaves and caused B toxicity in the transgenic plants. Therefore, these data further confirmed the importance of the 5′ UTR in avoiding the negative effects of NIP5;1 expression under excess B conditions.
In this study, we demonstrated that changes in mRNA stability are involved in B-dependent accumulation of the NIP5;1 transcript and that destabilization in response to high-B conditions is regulated by the sequence in the 5′ UTR from +182 to +200 bp. The B-dependent regulatory mechanism controlled by the 5′UTR plays an important role in growth of Arabidopsis, taking up B from soils under low-B conditions and avoiding excess-B uptake under high-B conditions.
The NIP5;1 Transcript Is Destabilized under High-B Conditions through the 5′ UTR
We demonstrated that the NIP5;1 5′ UTR acts to repress transcript accumulation under high-B conditions but not to induce transcript accumulation under low-B conditions. In addition to the involvement of the 5′ UTR in response to B, measurement of the mRNA half-life revealed that the sequence from +182 to +200 bp in the 5′ UTR is required for B-dependent NIP5;1 mRNA destabilization (Figures 2 and 5).
The region from +182 to +200 bp in the 5′ UTR contained a highly conserved sequence (UCAAAUCAUGUAA) found in both Arabidopsis and rice (Figure 3). From the database, the maize (Zea mays) and grape (Vitis vinifera) orthologs of NIP5;1 also contained the conserved sequence, suggesting that it may also function as a boric acid channel and that its expression is repressed under high-B conditions in the roots (Figure 3). We also investigated whether a highly conserved sequence exists in the 5′ UTR of Arabidopsis NIP6;1, a boric acid channel responsible for B distribution in shoots (Tanaka et al., 2008). No conserved sequence was detected in the 5′ UTR of NIP6;1 compared with that of NIP5;1 (see Supplemental Figure 2 online), suggesting that the identified regulatory mechanism for B via mRNA decay is specific to NIP5;1. Consistently, B-dependent regulation of NIP6;1 mRNA levels is not as drastic as that of NIP5;1 (Tanaka et al., 2008). Orthologs of NIP5;1, which are expressed in the roots, might have retained these elements during plant evolution.
So far, mRNA stability controlled by the 5′ UTR has been characterized for a few genes, including light-regulated genes (Gil and Green, 1996; Pérez-Amador et al., 2001; Bhat et al., 2004) and salt and heat stress–regulated genes (Hua et al., 2001) in plants. Here, we identified an example of mRNA stability control by nutrient conditions through the 5′ UTR in Arabidopsis. Multiple transcriptional and posttranscriptional regulatory mechanisms of gene expression have been reported for acclimation to nutrient conditions. Our study demonstrated that mRNA stability control through the 5′ UTR is another important mechanism for plant acclimation to nutritional conditions.
Possible Mechanisms of mRNA Stability Regulation via the 5′ UTR in NIP5;1
The mechanism by which the NIP5;1 5′ UTR modulates mRNA stability in a B-dependent manner remains to be studied. Several different mechanisms of regulating mRNA stability through the 5′ UTR are reported, including the influence of the uORF, the 5′ UTR–specific RNA binding protein, the riboswitch and ribozyme, and microRNA (miRNA). A brief description of the current understanding of each mechanism is as follows: The uORF-dependent degradation can be classified into nonsense-mediated mRNA decay (NMD)–dependent or –independent degradation in yeast (Linz et al., 1997; Vilela et al., 1999). NMD is a well-studied mRNA surveillance system that degrades aberrant mRNA with premature termination codons. The NMD-independent pathway was shown in yeast, but the details are not well understood. In yeast, RNA binding proteins interfere with ribosome–mRNA interaction, which results in translational inhibition. This inhibition then leads to mRNA destabilization (Linz et al., 1997). Binding of metabolites and/or metals (Mg+) to riboswitches induces conformational changes in mRNA. The altered structures could affect transcriptional control, translational control, alternative splicing, and mRNA processing or stability in bacteria (Sudarsan et al., 2003; Cromie et al., 2006; Henkin, 2008). Ribozymes efficiently cleave specific sites in mRNA, resulting in the inhibition of gene expression in Gram-positive bacteria (Winkler et al., 2004). miRNAs can interact with a target region, mainly in the 3′ UTR, that influences mRNA degradation in melanoma cells (Goswami et al., 2010).
Each of these mechanisms could play a role in NIP5;1 regulation. Regarding the possibility of the involvement of a uORF in regulation, the NIP5;1 5′ UTR contains three short uORFs located at positions +1 (one codon), +80 (six codons), and +191 bp (one codon). The identified sequence (182 to 200 bp in the 5′ UTR) includes the third uORF position at +191 bp (Figure 3). According to Nyikó et al. (2009), longer uORFs (>50 amino acids) can trigger NMD, but shorter uORFs cannot effectively activate NMD. In NIP5;1, all uORFs are short and, per this mechanism, mRNA decay is to be controlled by the third uORF encoding 1 amino acid. However, we think that this does not necessarily exclude the possibility of involvement of the NMD-mediated pathway because if the stop codon of the third uORF was read through, a peptide with 51 amino acids overlapping the main ORF could be produced. Considering that boric acid can bind with cis-diols (Springsteen and Wang, 2002), it may be possible that boric acid binding causes a stop codon read-through. It could then inhibit translation from the main ORF and induce NIP5;1 mRNA destabilization by the NMD-dependent pathway. Alternatively, NMD-independent mechanisms may be involved in the regulation of mRNA stability through the NIP5;1 5′ UTR. The excess B may induce and/or activate an RNA binding protein(s) that recognizes the identified sequence in the NIP5;1 5′ UTR, which in turn leads to destabilization. A third possibility is that high-B conditions may trigger induction of miRNAs that interact with the identified sequence in the NIP5;1 5′ UTR, causing mRNA degradation. However, no miRNA that targets the NIP5;1 5′ UTR was found by massively parallel signature sequencing (Brenner et al., 2000) and none are in the plant miRNA database (Zhang et al., 2010).
Possible Involvement of Translational Regulation
In addition to controlled mRNA degradation, modulation of translation efficiency may also be involved in the regulation of B-dependent expression through NIP5;1 5′ UTR. In Figures 4D and 4E, ratios of GUS activity in two independent transgenic plants carrying the NIP5;1 5′ UTR were 4.8- and 4.5- fold higher under low-B than high-B conditions; by contrast, ratios of GUS mRNA accumulation in these transgenic plants were only 2.1- and 1.7-fold higher under low-B than high-B conditions.
To further support this hypothesis, immunoblot analysis of microsomal protein from transgenic plants carrying P-2,180 UTR312-GFP-NIP5;1 was also performed, and the NIP5;1-GFP protein accumulation in roots was at least 16-fold higher under low-B than high-B conditions, but ratios of GFP-NIP5;1 mRNA accumulation in roots were 6.7-fold higher under low-B than high-B conditions (see Supplemental Figure 3 online). These findings imply that translational regulation reduces protein accumulation under high-B conditions. Such a difference was not observed in the constructs carrying the deletion between +182 to +200 bp in the 5′ UTR (Figures 4C and 4D), suggesting that the deleted sequence may be required for the regulation of translation efficiency as well as mRNA stability in response to B in Arabidopsis roots. Further experiments are needed to confirm this hypothesis.
The Control of mRNA Degradation Plays an Important Role in Plant Growth in Acclimating to B Conditions
Growth defects in nip5;1-1 plants under low-B conditions are complemented by introduction of not only P-2,180 UTR312 GFP-NIP5;1 but also P-2,180 ΔUTR312-GFP-NIP5;1, suggesting that NIP5;1 produced from the construct without the 5′ UTR functions similarly to that from the construct with the 5′ UTR (Figure 6A). The nip5;1-1 plants carrying P-2,180 ΔUTR312 GFP-NIP5;1 showed higher sensitivity to toxic B conditions than the nip5;1-1 plants carrying P-2,180 UTR312 GFP-NIP5;1 (Figures 6C to 6E). This suggests that the 5′ UTR is essential in circumventing B toxicity.
In general, nutrients, including B, are nonuniformly distributed in soils. Rainfall often reduces the B content in soil, while B accumulates on the soil surface under dry conditions. Furthermore, the optimum B range for plant growth is reportedly very narrow (Marschner, 1995). To maintain optimum B concentrations for normal growth, roots must recognize the B concentration in the soil and control B uptake through NIP5;1 expression. mRNA degradation may be advantageous for the rapid change in gene expression to acclimate to changing B conditions.
In conclusion, our study demonstrated the importance of NIP5;1 transcript stability, controlled by a conserved sequence in the 5′ UTR, in a B-dependent manner. Our study provides further understanding of B transport wherein NIP5;1 is downregulated through controlled mRNA decay mediated by 5′ UTR sequences under high-B conditions to avoid excessive B transport.
Plant Materials
The Col-0 ecotype of Arabidopsis thaliana was derived from our laboratory stocks. Information on the T-DNA insertion lines (nip5;1-1) was obtained from the Salk Institute Genomic Analysis Laboratory (SIGnAL) database (Alonso et al., 2003), and the seeds were obtained from the ABRC. The genotype of plants was determined by PCR using left border T-DNA–specific primers and gene-specific primers (Takano et al., 2006).
Plant Growth Conditions
For phenotypic analysis, GUS activity, confocal imaging experiments, mRNA analysis, immunoblot analysis, and B concentration measurement, plants were grown on solid medium (Fujiwara et al., 1992) containing 1% (w/v) Suc and 1.5% (w/v) Gellan Gum (Wako Pure Chemical) with different concentrations of boric acid (Wako Pure Chemicals) and with identical pH. Surface-sterilized seeds were sown on the plates and incubated for 1 to 2 d at 4°C, and the plates were placed vertically at 22°C in a growth chamber under long-day conditions (16-h/8-h light/dark cycle). For the mRNA half-life experiment in Col-0, plants were grown on solid medium containing 1% (w/v) Suc and 0.15% (w/v) Gellan Gum containing 0.3 μM B and then transferred to hydroponic culture containing 0.3 μM B for 2 d at 22°C in a growth chamber under short-day conditions (10-h/14-h light/dark cycle). For the mRNA half-life experiment in transgenic plants, plants were grown on solid medium containing 1% (w/v) Suc and 1.5% (w/v) Gellan Gum containing 0.3 μM B at 22°C in a growth chamber under long-day conditions.
Quantification of Transcript Accumulation by qRT-PCR
Total RNA was extracted using an RNeasy plant mini kit (Qiagen) as recommended by the manufacturer. Total RNA (500 ng) was reverse transcribed into cDNA in a 20-μL reaction using an ExScript RT reagent kit (Takara Bio) with oligo-d(T)16 primers. The cDNA was amplified by PCR in a Thermal Cycler Dice Real-Time System TP800 (Takara Bio) with a SYBR Premix Ex Taq kit (Takara Bio). The eukaryotic elongation factor 1-α (eEF1-α) gene was used as a control for quantification. The primers used in qRT-PCR are shown in Supplemental Table 2 online. Specific amplification of target genes was confirmed by electrophoresis of PCR reactions and by melting curve analysis of PCR products using the Thermal Cycler Dice instrument.
Plasmid Construction and Plant Transformation
The NIP5;1 promoter was serially deleted and fused to the GUS reporter gene in the binary vector pTkan+ (kindly provided by K. Schumacher, University of Heidelberg). The promoter and 5′ UTR (from –2180 to +312 bp) of the NIP5;1 gene was previously isolated (Takano et al., 2006). Briefly, the region from –2180 to + 312 bp of the NIP5;1 gene fused to the GUS reporter gene was subcloned into the pUC18 vector (named pMW1). The nucleotide number corresponding to that from the transcription start site and the 5′ end of the full-length cDNA (TAIR database) was used as the predicted transcript start site (+1). Using the pMW1 construct as a template, the 5′-upstream regions of the NIP5;1 gene, starting from the –1248, –589,–508, –389, and –289 bp to +312 bp and –2180 to +1 bp, were amplified by PCR. The primers used in plasmid construction are shown in Supplemental Table 2 online. The amplified fragments were digested using a BamHI site at the 5′ end of the primers (underlined) and a NotI site at the 3′ end of the GUS gene (underlined). The BamHI-NotI fragment of the resulting plasmids contains deleted promoter fragments fused to the GUS gene. The fragments were then subcloned into the BamHI-Bsp120-I vector fragment from the binary vector pTkan+.
To construct P-2,180 ΔUTR312-GFP-NIP5;1, the NIP5;1 promoter region from –2180 to –1 bp was amplified by PCR from pMW1. The amplified fragments were digested using a BamHI site at the 5′ end of the primers and an NcoI site at the 3′ end of the primers. Using BamHI and NcoI, the amplified fragment was subcloned into the BamHI-NcoI vector fragment from the binary vector P-2,180 UTR312-GFP-NIP5;1 (Takano et al., 2010).
A plasmid carrying a truncated 5′ UTR fused to the GUS reporter gene under the control of P35S was constructed as described below. The truncated 5′ UTR, the 5′-upstream regions of the 5′ UTR, starting from –100, +7, +101, and +198 to +312 and +7 to +79, +7 to +183, and +7 Δ+182 to +200 bp was amplified from pMW1 by PCR using primers (see Supplemental Table 2 online). Accuracy of the cloned PCR product was confirmed by DNA sequencing. The amplified fragment was subcloned into the pENTR/D-TOPO vector via the TOPO cloning reaction (Gateway technology; Invitrogen). The cloned promoter fragment was subsequently subcloned into pMDC140 (Curtis and Grossniklaus, 2003), which contains the dual P35S, gusA, and terminator of the gene for nopaline synthase (Nos T) using the Gateway system according to the manufacturer's instructions (Invitrogen).
Arabidopsis Col-0 and nip5;1-1 plants were used for plant transformation using the floral dip method (Clough and Bent, 1998). Homozygous T3 plants were established for each transgenic line and used for analysis.
Fluorometric Assays of GUS Activity
T3 (homozygous) transgenic lines carrying the P-2,180 UTR312-GUS construct were grown with solid medium containing 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, and 3000 μM B for 14 d to observe the NIP5;1 expression pattern by GUS assay. T3 (homozygous) transgenic lines carrying the promoter deletion series constructs were grown with solid medium containing 0.3 or 100 μM B for 7 or 11 d to observe the NIP5;1 expression pattern by GUS assay. T3 (homozygous) transgenic lines carrying the 5′ UTR deletion series constructs were grown on solid medium containing 0.3 or 1000 μM B for 11 d to observe the NIP5;1 expression pattern by GUS assay. Quantitative GUS assays were conducted as described by Jefferson et al. (1987). Five to ten transgenic seedlings were collected as one sample and used for the GUS assay. The seedlings were homogenized in 300 μL of extraction buffer (50 mM sodium-phosphate, pH 7.0, 10 mM Na2EDTA, 0.1% Na-sarcosyl, and 0.1% Triton X-100). The samples were centrifuged for 10 min at 4°C, and the supernatant was used for subsequent analysis. The enzyme activity was measured fluorometrically using 4-methylumbelliferyl-β-d-glucuronide as the substrate, and the reaction product 4-methylumbelliferone was fluorometrically detected. The protein concentration of extracts was measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories). GUS activity was shown in units of micromole 4-methylumbelliferone produced per hour per microgram of protein.
Measurement of B Concentration
Plants were grown on solid medium containing 3000 μM B for 14 d. Roots and rosette leaves were harvested, and B concentrations of these samples were determined using inductively coupled plasma–mass spectrometry (SPQ-9000; Seiko Instruments) as previously described (Takano et al., 2006).
Half-Life Measurement of mRNA
mRNA half-lives were determined according to Seeley et al. (1992) with a few modifications. The growth conditions for hydroponic culture in Col-0 plants were described previously (Takano et al., 2005). Briefly, seeds were sown on nylon mesh (10 × 10 cm2 and 300-μm pore size) on solid medium (Fujiwara et al., 1992) containing 1% (w/v) Suc and 0.15% (w/v) Gellan Gum for 21 d under short-day conditions. Plants were then transferred to liquid medium supplied with 0.3 μM B and grown for 2 d. For transgenic plants, seeds were sown on solid medium containing 1% (w/v) Suc and 1.5% (w/v) Gellan Gum for 14 d under long-day conditions. Col-0 and transgenic plants were preincubated with hydroponic culture solution (Fujiwara et al., 1992) containing 0.3 or 100 μM B for 10 min. After preincubation, cordycepin (Funakoshi) was added to a final concentration of 0.6 mM (0 min) into the hydroponic medium, and vacuum infiltration was immediately applied for 45 s. Root samples in Col-0 and whole-plant samples in transgenic plants were harvested at 0, 10, or 30 and 20 min, respectively, and frozen in liquid nitrogen. In this study, cordycepin was directly added to the hydroponic medium supplemented with 0.3 or 100 μM B instead of using incubation buffer (Seeley et al., 1992), and total RNA was isolated and analyzed by qRT-PCR. The RNA remaining at each time point was determined relative to the amount at the zero time point and plotted on a log scale. To test whether cordycepin conferred a strong block to transcription in plants grown under hydroponic culture, NIA2 (At1g37130) encoding nitrate reductase identified as an unstable transcript was used as a control for cordycepin activity and measured mRNA accumulation (Gutierrez et al., 2002) (see Supplemental Figure 4 online). Similar results were observed and the mRNA levels rapidly dropped in hydroponic culture supplied with 0.3 μM B. We thus used this condition in further experiments.
Expression of GFP-Tagged NIP5;1 in Arabidopsis Roots
The transgenic plants carrying P-2,180 UTR312-GFP-NIP5;1 and P-2,180 ΔUTR312-GFP-NIP5;1 were grown on vertically placed solid medium containing 0.3, 1, 100, or 1000 μM B for 14 d. Confocal laser scanning microscopy was performed using an LSM 510 (Carl Zeiss) with an excitation wavelength of 488 nm and a detection wavelength of 515 to 545 nm for GFP imaging in the root differentiation zone.
The relative expression of NIP5;1 in transgenic plants carrying P-2,180 UTR312-GFP-NIP5;1 was assayed by immunoblot analyses of microsomal proteins with an anti-GFP antibody as described previously (Takano et al., 2005). The membrane was then stripped and reprobed with plasma membrane marker and plasma membrane aquaporin antibody (Ohshima et al., 2001) as loading control.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative or GeneBank/EMBL data libraries under the following accession numbers: At NIP5;1, At4g10380; At NIP6;1, At1g80760; NIA2, At1g37130; Os NIP3;1, Os10g36924; Zm NIP3;1, AF326486; and Vv 00000446001, CAO62847.1.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Sequence Comparison of the 5′ UTR of Arabidopsis NIP5;1 and Rice NIP3;1.
Supplemental Figure 2. Sequence of the NIP6;1 5′ UTR.
Supplemental Figure 3. NIP5;1-GFP Protein and NIP5;1-GFP mRNA Accumulation under Low- and High-B Conditions.
Supplemental Figure 4. Time-Course Analysis of mRNA Degradation of NIA2 in Col-0 Plants.
Supplemental Table 1. List of Construct Names.
Supplemental Table 2. List of Primers.
The T-DNA insertion lines were kindly provided by the ABRC at Ohio State University. Plasma membrane aquaporin antibody was kindly provided by M. Maeshima (Nagoya University). We thank Y. Kawara and K. Aizawa for excellent technical assistance and K. Miwa (Hokkaido University) for inspiring discussion. This work is supported in part by a Grant-in-Aid for Scientific Research S (to T.F.) and a Grant-in-Aid for Scientific Research on Innovative Areas (to T.F.) from the Ministry of Education, Culture, Sports, Science and Technology and from the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation Grant IPG-0005 (to T.F.).
M.T., J.T., Y.C., H.O., S.N., and T.F. designed the research. M.T. and Y.O. performed the research. M.T., J.T., Y.C., F.L., H.O., S.N., and T.F. analyzed data. M.T., J.T., S.N., and T.F. wrote the article.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Toru Fujiwara (atorufu{at}mail.ecc.u-tokyo.ac.jp).
↵[W] Online version contains Web-only data.
- Received June 28, 2011.
- Revised August 5, 2011.
- Accepted August 24, 2011.
- Published September 9, 2011.


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Asked by:
Vote as Helpful: Good Content vs. Kudos
General discussion
I was thinking of something the other day and thought i'd toss the idea here. What do you think?
There are two reasons to vote up posts:
- To help identify good content.
- To gives kudos to a poster of a helpful reply.
These are not the same thing, nor do they apply the same way.
- Good content merely needs identification, so one point is usually enough; Kudos have no limit.
- Good content may be proved incorrect, and thus should be downvoted as well; Kudos are for the effort, not the content.
- Good content recognition is obviated when it becomes an answer (which has its own recognition); kudos are separate from being the answer.
- Good content recognition does not thank the poster's attitude; kudos do.
I have been assuming the "vote as helpful" means the reply is helpful (as opposed to the poster), which leaves me wanting a way to thank a poster for good effort. A few times i have voted a post as helpful and also posted a reply expressing gratitude.
This may be a bit much, but i would almost want to see two separate vote boxes. One for the post and another for the poster. Having two votes boxes next to each other, however, would be confusing, intrusive, tiring, and probably abused. I think i just want a way to identify good posters.
The obvious answer is that's what points are for. To me, points identify activity. Well, the new point system does address this, but it rewards frequent posters of answers, as opposed to those otherwise helpful posters.
Just some thoughts which i am not quite clear on myself. But i thought i'd share. :)
- Edited by Brian Tkatch Friday, February 10, 2012 1:56 PM
All replies
And who decide if it is good?
The reply which is wrong, but understood by the original poster because it is in line what the beginner is doing and he/she wants to hear that?
The reply which is very correct and in fact how something should be done, but the answer is completely not understood by a beginner who asks the question?
Helpfull votes are in my not related to this decission, any post can be helpful which has in fact nothing to do with the answer itself for others.
Be aware there is nothing against writting a thank you message in addition to a vote as helpfull, that stimulates many persons more than to get points.
I do agree that kudos and identifying good content is not the same in general, but in this case it is:
- I wouldn't give kudos to a post which hasn't good content.
- I wouldn't identify a post as "has good content" and at the same time denying the poster kudos. :)
In fact the only time these two are not entwined, would be a post with good content, but the poster has a bad attitude. This happens (at least in my experience) very rarely and i wouldn't think we need an extra button for that.
And if you feel the need to express your special gratitude or appreciation for someone, i think its not to much to ask to put that in words. Its much more rewarding for the target of your appreciation anyway. :)
Blog: http://bytecookie.wordpress.com
Hilf mit und markiere hilfreiche Beiträge als "Hilfreich" und Beiträge die deine Frage ganz oder teilweise beantwortet haben als "Antwort".


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Jacket Ally Jacqueline Jacquets
Personalised Hand-Painted Groom Jacket with Skulls
A Halloween-inspired groom jacket that shows you're committed til death. Perfect for an intimate elopement or an eerily beautiful wedding! Get a matching set or rock it solo. You may choose to change the colors, text, and font used if you prefer! Price includes 'The Leather Bomber Jacket' from Everlane ($300 USD) - it's made of ulta-soft, premium leather that's fully traceable and will stand the test of time. If you prefer vegan leather or want to use a different style jacket, please just contact me!
- Condition: New - with tags
- Material: Leather
- Colour: Orange
- Brand: Ally Jacqueline Jacquets
Size Details
Please see sizing info on Everlane's website: https://www.everlane.com/products/mens-leather-bomber-jacket
Garment Care
Please understand this item is hand-painted and hand-sewn so care should be taken when wearing and looking after your jacquet. Spot clean only.
Returns & Exchanges
You may cancel an order from your account before it is shipped. Please note that if the item is made-to-measure you may only be able to cancel the order before it is shipped provided that the garment hasn't been altered or made yet.
If you receive an item and wish to return it for any reason you must inform the Seller in writing within 14 days of receiving the item.
You may also return any item to the Seller within 28 days if the item becomes faulty.
The Seller will then provide you instructions on how to return your item and once your item is received back, they will process your refund through Paypal.
Return postage is your responsibility unless the item is incorrect or faulty, in which case the Seller will refund the return postage. If you cancel your contract under the Consumer Contracts (Information, Cancellation and Additional Charges) Regulations 2013 any standard shipping charges that you have paid to receive your purchase will be included as part of your refund.
Please note that the Seller may refuse a refund if the garment has been made according to your precise measurements ie) made-to-measure. The items you return must be unworn and in their original condition save for your right to inspect the goods and try on the garments. It is your responsibility to ensure that you package the returned goods adequately so that it arrives back to the Seller undamaged.
If you have any further questions on returns, refunds or cancellations please contact the Seller.
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The availability of key skills and expertise is the foremost business threat for ASEAN CEOs.
Findings from PricewaterhouseCoopers' (PwC) 17th Annual Global CEO survey revealed 90% of ASEAN CEOs are concerned about the amount of fundamental talent available to them.
According to the report, which surveyed 1,344 respondents in 68 countries in Q3 2013, this figure is the highest in the world.
"While interest in Southeast Asia continues to develop, it must be matched by available key talent, lest the growth might not reach its full potential," Alywin Teh, Singapore consulting leader at PwC Southeast Asia Consulting, said.
"Moreover, with 65% of ASEAN CEOs indicating that they plan to boost headcount, the attraction, development and retention of talent in the region is essential to successful growth in the region."
Other top concerns included rising labour costs in high-growth markets (80%), as well as high and volatile raw materials prices, and bribery and corruption – both coming in at 68%.
When it came to businesses' top potential economic and policy threats, ASEAN leaders cited exchange rate volatility (79%), continued slow or negative growth in developed economies (77%), over-regulation (77%), slowdown in high-growth markets (75%), and government response to fiscal deficit and debt burden (75%) among others.
However, despite these pressing concerns, regional viewpoints on the economy continued to slant upward, with only 6% of ASEAN CEOs believing the global economy will decline in the coming year.
Slightly less than half (48%) of regional bosses anticipated it will stay the same, while 45% expected an improvement in the economy.
"For the future, ASEAN CEOs tell us they expect three major global trends – technological advances, demographic shifts and resource scarcity and climate change – will have a major impact on the future of their businesses," Yeoh Oon Jin, executive chairman of PwC Singapore, said.
"Finding ways of turning these global trends to their advantage will be the key to future success."
ASEAN CEOs also named China (41%), Indonesia (24%) and India (21%) as the countries most important to them for their growth in the next 12 months.
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New Releases for March 6, 2020
Honey Harper – Starmaker CD/LP (ATO)
Honey Harper's debut album, Starmaker, comes as a collection of contradictions in both subject matter and style. Written with his wife and writing partner Alana Pagnutti over the past three years, the album itself is primarily about the journey to create it. It's an attempt at honesty through the veil of a country singer lost in the stars. On Starmaker, Harper seeks to revitalize country music for people who don't like country music.
Lauv – How I'm Feeling CD/2xLP (Lauv)
Most pop genre albums can be categorized by their peppy messages along the lines of "Believe in yourself no matter what!" or "Life is always amazing!" That's what Lauv's second album – How I'm Feeling – subverts. Instead of following industry standards, the record acts as a refreshing take on pop music by taking an honest introspective look on himself.
Mandy Moore – Silver Linings CD/LP (Verve)
Emmy, Grammy & Golden Globe-nominated actress/singer/songwriter Mandy Moore returns to music with her first album in a decade, Silver Landings. In a purposeful departure from the pop sound of her previous material, the album deftly moves from '70s inspired soft-rock to contemporary pop.
Esmé Patterson – There Will Come Soft Rains CD/LP (BMG)
Esmé Patterson is folk-rock singer/songwriter who began as a member of the Denver folk pop septet Paper Bird and has written three records as a solo act including All Princes, I (2012), Woman To Woman (2015) and We Were Wild (2016). The Guardian called her "defiant and witty," the New York Times found her voice "wiry and candid" with songs that "hint at mystery and mortality." Now she returns with her fourth solo endeavor, There Will Come Soft Rains, produced by Tennis aka Patrick Riley and Alaina Moore. Named after a story from Ray Bradbury's The Martian Chronicles, it finds Patterson adding synthesizers and dream-pop arrangements to the mix with stunning results.
Silverstein – A Beautiful Place To Drown CD/LP (UNFD)
Ontario-based post-hardcore band Silverstein celebrate their 20th anniversary with the release of a new album. They may be proud torchbearers of the emo and post-hardcore scenes but appearances from Underoath's Aaron Gillespie, Beartooth's Caleb Shomo, Simple Plan's Pierre Bouvier, and perhaps most surprisingly, hip-hop firebrand Princess Nokia, are indicative of the band's wide-reaching impact and their ability to reach listeners in all worlds of music.
Sadler Vaden – Anybody Out There? CD/LP (Dirty Mag)
Acclaimed solo artist. Songwriter. Grammy-winning member of Jason Isbell and the 400 Unit. Producer. Studio musician. Modern-day guitar hero. It's a modern record that doesn't ignore the past, balancing frequent nods to Vaden's 20th century influences – including Neil Young, The Who, Tom Petty, Joe Walsh, and Oasis – with a contemporary sound that mixes raw, live performances with digital recording technology.
Atmosphere – Whenever CD/2xLP (Rhymesayers)
For over 20 years, Atmosphere have pursued the underbelly of what it means to be human with a rabid curiosity. Ant's dusky production has provided the pulse for Slug's evolving and matter-of-fact pen. As Slug transitioned from throwing-up-in-the-backseat-raps to holding-hands-when-you-cross-the-street-raps, Ant has grown to be a master of crate digging and unearthing the humanity of a drum loop. On Whenever, the duo continue to move in lockstep. Within the minutiae lie clues they're still struggling with mortality and the need to protect emotional energy, as on previous releases, but there's a quiet romanticism now, with Slug sounding – reluctantly – in love with life.
Body Count – Carnivore CD/LP (Century Media)
Seventh album from the metal band fronted by rapper/actor Ice T.
Brandy Clark – Your Life Is A Record CD (WB)
Few artists understand the human condition like six-time Grammy nominee Brandy Clark. Working again with acclaimed producer Jay Joyce, Your Life Is A Record takes her unerring eye for detail and a genre-melting sense of melody to examine personal truths, revelations and emancipations with wit, grace and compassion. Working with just four acoustic-based musicians, intimacy was the goal. Memphis strings and horns, guest appearances from Randy Newman (Bigger Boat) and guitarist John Osborne color in the sketches – creating a depth to what is easily Clark's most revelatory record yet.
Crematory – Unbroken CD/2xLP (Napalm)
15 energetic tracks within brutal riffs, deep growls and tight drum grooves from the German pioneers of gothic metal.
Disq – Collector CD/LP/Cassette (Saddle Creek)
Disq have assembled a razor-sharp, teetering-on-the-edge-of-chaos melange of sounds, experiences, memories, and influences: gnarled power pop, anxious post-punk, warm psych-folk, and hectic, formless, tongue-in-cheek indie rock. [Limited colored vinyl pressing also available.]
Luke Haines/Peter Buck – Beat Poetry For Survivalists CD/LP (Omnivore)
Beat Poetry For Survivalists is the collaboration between Peter Buck and Luke Haines. Peter Buck was the guitarist for one of the biggest bands in the world, R.E.M. Luke Haines was the guitarist for The Auteurs.
The James Hunter Six – Nick Of Time CD/LP+MP3 (Daptone)
The James Hunter Six return with another sublime offering of no nonsense rhythm and blues.
Jose James – No Beginning No End 2 CD/2xLP (Rainbow Blonde)
The satin-voiced songwriter's latest is No Beginning No End 2 – a sequel to his 2013 album that resurrects the bold eclecticism we first fell in love with, while taking us on a journey through both celebration and introspection. The album is chock-full of collaborators including; Laura Mvula, Aloe Blacc, Ledisi, Erik Truffaz, and Hindi Zahra – appearing in unexpected sonic contexts.
Stephen Malkmus – Traditional Techniques CD/LP+MP3 (Matador)
Malkmus breaks out his 12-string acoustic guitar and touches on half a century of folk-rock reference points on his new album. Of course, this is Stephen Malkmus of Pavement and those great records with the Jicks, so this might be rootsy but it's also fresh, unpredictable, infectious and imaginative: ACC Kirtan, Xian Man, The Greatest Own in Legal History, Cash Up and more tracks made with Matt Sweeney, Spooner Oldham and the Decembrists' Chris Funk.
The Mastersons – No Time For Love Songs CD/LP (Red House)
The Mastersons are singer-songwriters/multi-instrumentalists Chris Masterson and Eleanor Whitmore. Longtime members of Steve Earle's band the Dukes, the musical and marital twosome make inspired albums of their own vivid, deeply humanistic songs. Their fourth album, No Time For Love Songs, marks the 10th anniversary of their partnership and creative journey with a collection of songs that could easily serve as the soundtrack to our lives-or at least one version of it.
Mac Miller – Circles CD (WB)
Conceived as a sister album to 2018's full length Swimming, Circles was completed with the assistance of Jon Brion, with whom Miller worked on Swimming and had been working together on Circles at the time of his passing. While sonically distinctly different than its predecessor, Circles features many of the hallmarks for which Swimming was critically-acclaimed upon its release – Miller further realizing his singing voice in addition to rapping, live instrumentation and earnest, confessional lyrical content.
My Dying Bride – The Ghost Of Orion CD/2xLP (Nuclear Blast)
Formed in 1990 My Dying Bride became forerunners and trend makers in the gothic, doom, death metal genre.
Phantogram – Ceremony CD/LP (Republic)
Genre-defying alt-rock duo Phantogram – comprised of lifelong friends Josh Carter and Sarah Barthel – have continued to change the zeitgeist for almost a decade now by consistently challenging it with their signature blend of hard-hitting beats, guitar-driven dark psychedelia and electronic pop. Phantogram's fourth studio album, Ceremony, marks a rebirth and embodies a dark, but hopeful, chapter for the band.
Caroline Rose – Superstar CD/LP (New West)
Here comes Superstar the bigger, badder, glitter-driven record by Caroline Rose. Written as a sequel to 2018's Loner, Superstar "plays out like an epic movie about the pursuit of fame and fortune,' Rose states. 'I've always been fascinated by this pursuit, but what's even more fascinating is what happens when it fails." Indeed, gone are the successful Hollywood hunks and starlets of old. Superstar chronicles a quirky anti-hero, who after receiving a wrong call from the elite hotel Chateau Marmont, decides to leave their old life behind in order to become a big Hollywood star.
U.S. Girls – Heavy Light CD/LP (4AD)
Recorded live at Montreal's acclaimed studio Hotel 2 Tango and featuring 20 odd session musicians, Heavy Light is an architectural achievement for an artist whose career began with a 4 track tape recorder and a Greyhound bus pass. Following the subversive sheen of 2018's In A Poem Unlimited, Remy has taken a deeply introspective about-face to produce a set of mindful compositions on hindsight, the passage of time, the delicate balance of the conscious and the unconscious, and the pursuit of a radical empathy. Remy worked with co-writers Basia Bulat and Rich Morel to develop the core of Heavy Light, a set of songs conceived as a richly arranged balance between orchestral percussion (as arranged by percussionist Ed Squires) and the human voice.
Jason Boland & The Stragglers – Pearl Snaps [Reissue/1999] LP (Proud Souls Ent.)
Vinyl reissue of the debut album by red dirt/Texas country band.
Noel Gallagher's High Flying Birds – Blue Moon Rising 12" (Caroline)
New EP from the former Oasis guitarist and songwriter. Blue Moon Rising features new songs plus a remix of the title track.
The Who – The Kids Are Alright [Reissue/1979] LP (MCA)
The Who – Quadrophenia [Reissue/1973] 2xLP (Geffen)
The Kids Are Alright is the legendary companion album to The Who's classic documentary film of the same name from 1979, featuring the extraordinary 1967 Smothers Brothers TV show version of "My Generation" – including exploding drum-kit finale. Quadrophenia is the soundtrack from the classic and influential 1979 film, adapted by director Franc Roddam from the Who's 1973 rock opera, set against a backdrop of the mods vs rockers battles of the period.


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Snake found in Phenix City bank's restroom
A Phenix City bank employee got quite the scare Monday when taking a trip to the restroom.Continue Reading »
A Phenix City bank employee got quite the scare Monday when taking a trip to the restroom.Continue Reading »
A second woman is now coming forward claiming she has also been sexually abused by a long-time local pastor.Continue Reading »
Jury selection is underway for the trial of a Columbus man accused of murder.Continue Reading »
Football season is right around the corner, but before teams take the field, they'll approach the stage for SEC Media Days.Continue Reading »
Columbus Police Department's Financial Crimes Unit arrested a man on 43 counts of financial transaction card fraud.Continue Reading »
A group of fishermen, some of them fathers and sons from Eufaula and Phenix City, have been arrested for an illegal head start on the lobster season. That's according to officers with the Florida Fish and WildlifeContinue Reading »
A Columbus teen was arrested for allegedly stabbing his brother with a knife, according to a police report.Continue Reading »
St. Francis has announced that the hospital will eliminate 50 position due to the restructuring of many departments. According to St. Francis' Marketing Director Kendra Wright, these positions will not impact bedside care.Continue Reading »
Auburn Police Division arrested 19-year-old Eric Nelms Jr. on felony warrants Monday. July 10. Nelms, an Auburn resident was charged with criminal mischief in the first-degree and two counts of theft of property in the first-degree.Continue Reading »
Columbus makes the Top 50 in a study that measured the best and worst cities in the United States. Out of 150 cities, Columbus landed at 42.Continue Reading »
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When I began learning to shoot in manual mode all the time it took a lot of practice. The information provided in posts What is Aperture?, What is ISO?, and Figuring out Shutter Speed will help you to move on the full manual mode. It is a scary thought I know but you can do it! I learn best by doing and looking at examples so I hope this helps you as well.
I gathered some photographs taken with different lenses and in different lighting situations all with a special purpose. The first image I took while my family went on a walk. We had just adopted our dog from the Bella Vista Animal Shelter and was enjoying a warm fall day. I wanted to capture the dog as the focal point of the image and show what was going on in the scene. I needed an ISO of 100 because it was bright outside, an f-stop of 4.5 because I wanted the background blurred but still visible and the shutter worked at 1/100 sec to balance the lighting. If I needed a slower shutter due to strong shade cover I would have bumped my ISO up.
In this next photograph, I wanted to blur the background completely. I used aperture as my priority to get right for what I needed to accomplish. I set the camera to f/2.8 and focused on her right eye and used ISO 400 since the sun was bright but beginning to go down. Lastly, my shutter was 1/640 of a second much faster than it needed to be for a still image but fast because I was letting in so much light using f/2.8. I ended up changing my white balance on this one because the setting sun made the image too warm for a summer day.
This next image is framed as a square metal print in my bedroom. I love this picture because it represents new beginnings. This was the first bloom of spring from the magnolia tree in our yard. I don't have a true macro lens so I used my 85 mm prime lens. My settings are very similar to what they were in the previous image. I prioritized my f-stop to 2.8 so I could get a creamy background blur so that the flower would pop. ISO was 400 because it was somewhat overcast and I had a fast shutter mostly due again to the aperture I prioritized but also I needed a super sharp image.
For this photograph I had to use a high ISO due to the darkness and balance the shutter speed so that I could get the lights coming from the spot light and the building in the background. My f-stop needed to be 8.0 so I could get as much visible as I could. I wanted to get the design of the Crystal Bridges building across the water. At concerts I use 1600 ISO and f/2.8 so I can use a faster shutter. In this situation a fast shutter wasn't necessary since the subject was relatively still.
I have to throw in a picture of my new nephew who is two days old in this photograph. I placed him in front of a window and unfortunately it was severely overcast and very little light was coming in so I had to bump my ISO much higher than I wanted to at 4000. Babies should be photographed softly so I used 3.2 as my aperture and 1/160 sec shutter. Even at this setting I had to brighten the photo through exposure and because the photo was taken in a house my white balance had to be adjusted for the tungsten lights.
I hope this helps you get started playing with your manual setting! Have fun and share your photographs with me!
If you want to schedule a photo session where you can relax and have someone else take your family portraits, give me a call.


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Welcome to Labh Group!
Labh Group of Companies is a fast growing, well- recognized and an ISO 9001:2008 certified, Indian business group of global repute having presence in more than 100 countries worldwide with strong base of more than 10000 satisfied customers. We have prestigious STAR EXPORT HOUSE STATUS from Government of India. The group has a very modest beginning in the year 1991 with a business of manufacturing of packaging machineries. In span of over 20 years, group has spread its wings and today, under the Group umbrella there are vividly diversified business segments ranging from execution of turn-key projects for various industries, manufacturing and exporting of wide range of plant and machineries for different industries and applications, export and import of wide range of technical and not technical products like packaging materials, raw materials, consumer goods, engineering items etc. to service business like global placement, global health tourism, management and technical consultancy, knowledge process outsourcing (KPO), software development etc. The group has many ambitious plans to foray into many new promising segments in near future.
The group is having strong hold in the fast developing domestic market of India as well international market through efficient network of branches, agents, distributors and partners. The group is having its presence all around the world in countries like U.K., U.S.A., Canada, Brazil, Mexico, Argentina, Panama, Australia, Germany, France, Italy, China, Mauritius, Dubai, Saudi Arabia, Iran, Yemen, Russia, Uzbekistan, Indonesia, Vietnam, South Africa, Nigeria, Liberia, Uganda, Kenya, Ethiopia, Sudan, Bangladesh, Nepal and many more. We have many reputed names in our clientele, both from the domestic as well as international market.
The group name Labh is a part of very auspicious and respected words Shubh- Labh featuring together in Indian scriptures. Shubh means auspicious and Labh means benefit and you will find Shubh- Labh words used in one or the other manner in most of the Indian households and even business set ups in India. Taking a cue from there, we aim at making it auspiciously beneficial for everyone who associates with us. Always give people more than they expect remains our guiding principle and philosophy. An amalgamation of the two has made our motto as Benefits Unlimited which we consistently Pursue- Benefits to all the people we interact with whether they are our customers, our suppliers, our business associates, our employees or our shareholders. We want to imprint Labh i.e. Benefit, in their heart and mind as the end result of their association with us.
Address:4th Floor, Sarthik Square, Besides Info Tower, S. G. Road
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