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[[Side Note: Dear Body
Dear Body,
Is it too late for me to apologize to you? For not treating your like you should have been treated for so many years?
I'm sorry for all those years that I bullied you, telling you you weren't enough, that you weren't strong enough, pretty enough, or worth it.
I'm sorry for all the food I deprived you of that would help you grow and develop correctly. I'm sorry you weren't able to enjoy so many of your favorite foods for so long and I made you eat things with no flavor and so very littler nutrients.
I'm sorry for all the times I told you you weren't good enough to hang out with your friends, go out to a restaurant, have a drink with your friends and do stupid things that would make the most awesome memories.
I'm sorry for all the miles that I made you run, and not for fun. I'm sorry all those miles were solely to burn off calories for even the little food you ate at the time. I'm sorry you didn't enjoy so many of those and took precedence over the things you could have been doing otherwise and actually enjoying.
I'm sorry for all the things I have put you through you should not have gone through in the past 7 years. I'm sorry it has taken me so long to realize that what I was doing was wrong and destructive to you.
But now, I hope that I can treat you better. I hope that I have learned how to fuel you properly and provide you with the vitamins and minerals you need to perform and repair yourself correctly.
I hope that I won't beat you up and you will become stronger and more confident as I embark on a mission to run another 1/2 marathon and train for my first sprint triathlon by the end of the year.
I hope that you realize how beautiful you are, how you have the ability to do so many things and accomplish so many dreams. I hope you know you can travel to far away countries and try foods you've never even heard of, hike mountains that poke into the clouds, play in rivers with all sorts of crazy things, and have fun all the while.
I hope you know how much I love you, how much I appreciate you, how grateful I am that you continue to put one foot in front of the other and help me live the life I want to live doing the jobs I love to do outdoors saving our planet and mother nature.
What would you tell your body today? ]]
Whew! Another long day in the field work front! I got trained today to do anther aspect of my job which was cool, but man, the weather conditions were totally not in my favor! Arg!
I woke up this morning freezing my toosh off and knew that being outside was going to be even worse. So what better than to start off with a warm and comforting breakfast?
I mixed a packet of plain instant oatmeal together with a jazz apple and nuked it before adding in the pb blob and kashi cinnamon h2h. I had yet to take my cinnamon out of my food box in the car so I needed the cereal for some flavor to the oats! You can bet I rummaged around for that cinnamon this afternoon after work!
I headed out to the car to leave and holy smokes: THERE'S ICE ON MY CAR! AND SNOW ON THE SIDES OF THE HILLS AND MOUNTAINS?!!?! WHAT?!?!!?! IN MAY!?!!?
Madness I tell you. And the winds were gusting at over 35mph. Oh yeah, winter is still here in the highland desert of California! Boo! Go away!
I headed out to do my surveys and such below the big 3+ megawatt wind turbines…
Did you know that at the tips these things can go up to 200mph?
And if you put two of the blades directly across from each other they're the length of a football field?
But that's only for the really big ones, obviously many of them vary in shape and size.
It was a bitter cold morning (temps in the 30s, and that doesn't include the wind chill!) and I was definitely thankful for my wind breaker jacket and pants. Otherwise I would have died. No joke, I would have frozen and been left to the turkey vultures.
When I get cold I have absolutely no appetite…until I warm up in the car. Then I become ravenous! I packed up this nice tupperware to snack on between survey areas: strawberries and a small jazz apple with natural pb.
And 6 hours later I was done. And ate a banana on the way home. I think the sucker was as long as my arm. Ok, my forearm. Ok not really; it was huge though!
I had packed a lunch to eat out in the field but because we started early enough I got home in time to eat my sandwich at leisure at home! A simple hummus/lettuce/tomato sandwich with a side of two chopped carrots…and extra unpictured hummus.
I worked on some work stuff throughout the afternoon and finally started to catch up on some blogs. I had a sweet snack to keep my tired self going throughout the afternoon: cinnamon kashi and guittard chocolate? take this times two, and extra chocolate chips.
In spite of my efforts to stay awake I passed out watching tv and then finally got around to working out. I didn't do very much walking or anything today so I thought it a good opportunity to get a run and ST session in.
I did a new set of self cards, and two bikini workouts I found in two different Fitness Magazines. I'm gonna have a sore booty and arms/shoulders tomorrow. No hate, it'll be good soar.
After the ST session I set out for my run on the DREADMILL. I know you've heard me complain about that damned thing, that it makes me feel like I'm drunk when I get off, but today, and probably for some time, I have no choice.
It is simply too cold for me to run outside without the proper cold weather running gear, as I own…none. I mean, I just finished living in Miami for goodness sakes! Haha…so yes, this weekend I'm on a mission to find some good running tights and tops…thanks for all the suggestions too! I'll be looking into all those different brands!
I ended up running 4 miles today, starting at 6.5 in the beginning and making my way up to 7.5 for at least the last 2.5 miles. The last .25 was done at 8.5 though…that was killer, but freak'n awesome all at the same time. I brought along a magazine to read afterwards during my mile walk cooldown at a 5% incline…I think that's not going to help with all the walking I'm going to be doing tomorrow. Oops.
You can bet I was ravenous after all of that so I cleaned up quick and made a lazy dinner. But what can be better than starting it off with one of my favorite types of bread: SHEPHERDERS BREAD!!!
I went over to the local German Bakery in town and when I saw they had some loaves, I couldn't pass it up. It is one of my favorite breads that I used to demolish by the loaves when we took family vacations up to Mammoth Lakes, CA during the summer.
This bread tasted great, but not as good as the Sheepherder's bread we get at Eric Schat's Bakery in Bishop, CA and Mammoth, CA. I need to get my hands on some of it asap. This will suffice for now. I started off with two pieces covered in a thin layer of Sunbutter's Omega-3 Nutbutter and a bit of Crofter's America Spread…along with another small piece that was plain and somehow avoided a picture…
I've had my heart set on the mangoes I purchased the other day but because my mini fridge is stupid it froze half of it…making it basically inedible. Boo. I salvaged some of it…
And rounded out the meal with my fair share of vegetables…until I found a ladybug crawling on my fork. Ok…and I had already consumed just over 1/2 of it. Chucked the rest.
Now I'm off to chill with my book and get some shut eye! I have a long day of walking surveys starting bright and early tomorrow…and all by myself! Thank goodness for Ipods!


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Running Back
Height: 6-foot-0
Weight: 245 pounds
Class Year: 3V
Hometown: New Ellenton, SC
High School: Silver Bluff
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NASHVILLE, Tenn.�Fans and media spent much of this week discussing whether six wins could get Vanderbilt to its first bowl since 1982. But the Commodores' play on the field Saturday afternoon at Vanderbilt Stadium quickly rendered that discussion moot. Vanderbilt rallied late behind the arm of...[details]
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How Microsoft IT Approaches Organizational Change Management
About This Video
The following content may no longer reflect Microsoft's current position or infrastructure. This content should be viewed as reference documentation only, to inform IT business decisions within your own company or organization.
Understand how Microsoft IT created an organizational change management framework to drive an organization wide change management competency around planning, managing and implementing organizational change. By utilizing a structured process and specific set of resources for addressing the impact of change on people, Microsoft IT is able to realize faster change adoption, maintain momentum and its commitments while mitigating the natural resistance to change.
Published Date: June 25, 2010
Tags: TechNet Edge , Microsoft IT Showcase , Zune , iPod , MSIT , IT Showcase , Microsoft IT , Cost Savings , Microsoft IT Showcase , ROI , Videos , English


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Flicks in Five: Dances with Wolves
'Dances with Wolves' changed both Kevin Costner's career as an actor and director as well as the place of Westerns in American film history. On Flicks in Five, we hear John Barry's Oscar-winning score.
Lynne Warfel returns to a 24-year radio career after a four-year hiatus. In this "Cavorts with Collies" period, she followed her lifelong passion for working with dogs as a trainer and behavior consultant. She started "Good Dog Productions," her in-home dog training business in 2006. Previous to The Dog Mutterer phase, she was one of the first national hosts back in 1995. She was also heard locally on Minnesota Public Radio from 1993-2005. While at MPR in her former incarnation, she hosted the Minnesota High School Music Listening Contest, Echoes of Christmas with the Dale Warland Singers, Sommerfest broadcasts, was a guest narrator with The SPCO and Minnesota Orchestra, and was a frequent guest host on The Morning Show with Dale Connelly and Jim Ed Poole.
'Dances with Wolves' changed both Kevin Costner's career as an actor and director as well as the place of Westerns in American film history. On Flicks in Five, we hear John Barry's Oscar-winning score.
On this week's Flicks in Five, Lynne Warfel takes a look at the career of one of film history's great leading men -- Clark Gable. We'll hear music from the score to Gable's final film, 'The Misfits'.
On this week's Flicks in Five, Lynne Warfel discusses the 1955 musical film version of 'Kismet'. We'll hear some of Borodin's melodies, and a song featuring actors Howard Keel, Ann Blyth, and Vic Damone.
One of Hollywood's most lauded director-composer collaborations was that between David Lean and Maurice Jarre for 1984's 'A Passage to India'. This week on Flicks in Five, we'll hear Jarre conducting his own music for the film.
On this week's Flicks in Five, Lynne Warfel looks at the career of Ron Howard, and shares music from his 1995 film, 'Apollo 13'.
One of Robin Williams's most memorable roles was that of unconventional teacher John Keating in 1989's 'Dead Poets Society'. In this week's Flicks in Five, Lynne Warfel will share some of Maurice Jarre's music from that award-winning film.
He considered film music to be 'operas without words' and his movie scores earned three Oscar nominations -- including one win for Best Original Score. This week on Flicks in Five, Lynne Warfel discusses Erich Wolfgang Korngold, and shares music from his soundtrack to 'The Private Lives of Elizabeth and Essex'.
Stanley Kramer became known as a director of 'message movies', taking on tough subjects of the day, but it was his big budget, all-star shot at comedy that endeared him to many. This week on Flicks in Five, Lynne Warfel looks at Kramer's 'It's a Mad, Mad, Mad, Mad World'.
Austrian-born American director Billy Wilder brought us many films in his trademark black and white, but it was his 1958 comedy starring Jack Lemmon, Tony Curtis and Marilyn Monroe that earned him a place in the annals of film history. On this week's Flicks in Five, listen to music from 'Some Like It Hot'.
Alfred Hitchcock arrived in Hollywood in the 1930s, and by the late 1950s, his nine-stroke pencil caricature had become internationally famous. In 1960, Hitch released of one of his best known films, 'Psycho'. On this week's Flicks in Five, Lynne Warfel will share some of Bernard Herrmann's unforgettable music from that soundtrack.
August 1st marks the birthday of film composer Jerome Moross -- best known for his soundtrack music composed for 1958's "The Big Country." On this week's Flicks in Five, Lynne Warfel discusses Moross and that Academy Award-nominated score.
Film composer Randy Newman didn't have Hollywood success handed to him on a silver platter. 'Ragtime' gave Newman a chance to reach new heights.
On this week's Flicks in Five, Lynne Warfel looks at the Michael Giacchino score to 2009's 'Star Trek', which cleverly interweaves elements of Jerry Goldsmith's melodies from the original TV series score from the 1960s.
Meryl Streep is widely considered to be the greatest living actress. She has garnered more Academy Award nominations than any other, although Katherine Hepburn has won more Oscars. Lynne Warfel looks at 'Out of Africa', for which Streep earned an Oscar nomination for Best Actress in a Leading Role.
As the Fourth of July approaches, Flicks in Five features one of James Cagney's most enduring roles, and one of his favorites. "Yankee Doodle Dandy" gave Cagney the chance to shine as a song and dance man.
Inconceivable! On this week's Flicks in Five, Lynne Warfel takes a look at 'The Princess Bride', and its Academy Award-nominated score by Mark Knopfler.
Overdubbing is when an actor portraying a part in a musical doesn't do his or her own singing. That doesn't mean the actor can't sing; often there are other factors at work. Lynne Warfel has some stories about overdubbing on this week's Flicks in Five.
In the latter part of the 20th century, Kenneth Branagh reigned supreme as the actor putting Shakespeare plays on film. In the cinematic version of Shakepeare's best comedy, Patrick Doyle provided the music.
With May and June being the Triple Crown months in thoroughbred horse racing -- the Kentucky Derby and Preakness Stakes in May and the Belmont Stakes in June -- this week's Flicks in Five focuses on 'Seabiscuit' and its score by Randy Newman.
100 years ago this month, Orson Welles was born in Kenosha, Wisc. In this week's Flicks in Five, Lynne Warfel examines his 'Citizen Kane', and its Academy Award-nominated score by Bernard Herrmann.


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The home at 4923 Kessler Blvd. E. Dr., is on the market for $1,295,000.
See the full listing here: http://bit.ly/1cyrpQe
More real estate galleries:
Got $5M to spend? Check out this house -- http://bit.ly/17s0znn
Las Vegas underground home on sale for $1.7M -- http://bit.ly/158UQSP
PICS: 10 churches converted to homes -- http://bit.ly/17rXy6B


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Generac Power Systems Jobs
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How do you run an open source project?
Managing Your Open Source Project
Many want to know about getting people to join and how to manage their project.
This is asking quite a hard question :o) Attracting and vetting people in open source is something that can be done in a lot of ways. You also will find an issue with people that want to join as a developer or even as an owner, but they are asking for something that they may never act upon. So, it is really three things:
1) How do you get people to join?
2 ) How do you manage those on the team?
3) What to do with those that are asking for more than they need or want?
Getting Volunteers to Join
For 1, getting people to join, the keys are organization, information and promotion. Be clear about your goals. Be specific about the tasks and what needs to be done and in what order. Lay out the phases. Make sure people understand why this project is important. Also talk about why participating in the project is good for them too from the types of technology to what they may be able to put on their resume.
The primary place to do this is with your project pages, but you can also use the wiki, forums, documents and files, the issue tracker, the help wanted wiki, and finally blogs. The more that you talk about your project in both volume, location, and frequency, the greater the probalility that you will get someone that will help.
Beyond that, you have one key volunteer to do the important work and that is you.
How Do You Manage Your Project?
For 2, this depends on how you want to manage. Open source should not be about code changes happening at the will of every member. Usually there is an owner (you) that acts as architect. This person often creates the first prototype. It does not need to be big. Either it lays out the features, and does not implement them, or is a pilot, or just the first couple features. Work from there still rests with the architect and getting people to help design or to shepherd others on tasks.
Once a project has actively participating members it is a matter of passing out assignments. Very often it is volunteers that pick assignments. Rarely can you assign someone with something unless the task is related to a request,bug, or area that the participant is very familiar with. Remember that this is about voluntary involvement and not a boss/worker relationship.
If tasks are laid out in a way that people can see something interesting to do or something that they want so bad that it must be done by them, then they will do it. People must be drawn to their tasks. Your tools are coolness, interesting problems, learning opportunities, resume filler, irritating bugs, needs from other tasks, and even self promotion.
What About People That Join To Just Join?
For 3, the problem is in part about 1 and 2 and also not reading up on 1 and 2. The more you make it clear what the responsibilities are, the greater the chance that they will select the right option. Many request that the joiner email prior to joining other than observer. Some require observer before any other request. Some require joining the email lists and the forums. The key is to require something for being other than observer. You need to be careful about anything beyond observer because there are people that can really mess up your site. Only grant higher rights to people that are properly vetted to your satisfaction.
In the jxta.org platform project (one I think has one of the better models), you need to be an observer and submit a fix or enhancement via the issue system. If the proposed patch is good or that there are a series of good patches, the user is upgraded to developer. All code changes are submitted as patches which are attached to feature/bug issues. Each patch must be approved by at least one other member of the development team before the code is allowed to be physically changed in the cvs. The cvs commit must contain a reference to the issue and the person that approved the patch. Of course this is with an established project that has had many versions, but it is a good model that can be expanded to larger features.
Remember, open source is not anarchy. Linux is run by Linus and his closely held team - not by the millions of Linux users and contributers. It is a commons with agreement from a gentile monarch or a tight team. Information is power both to keep things on track and to cause people to take action. Active forums, email lists, issues, project pages, documents, demos, releases, and blogging can help cause people to take action.
Note that this has been added to the wiki. See ManagingYourOpenSourceProject
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RCX 1.0 Case Replacement
Member-Contributed Guide
An awesome member of our community made this guide. It is not managed by iFixit staff.
How to access the RCX's circuit board.
- Author: notexactly
- Time estimate: 1 - 10 minutes
- Difficulty: Moderate
How to take the top (yellow) and bottom (green) case halves off the RCX 1.0. Once you've taken off the case, you can clean the electrical contacts and replace any fried electronic components.
This guide is based on Kekoa Proudfoot's instructions on his RCX page, which includes two sentences of disassembly instructions and some photos.
Embed this guide
With the HTML code below, you can embed this guide as a small widget on your website.
- Phillips #1 Screwdriver The screws have Pozidriv heads, so you can use a Pozidriv #1 screwdriver if you have one.
Remove the gray battery cover by lifting from the front (near the IR window). Raise it at an angle and remove it to reveal the green battery compartment.
You can now remove the batteries and insert new ones.
Remove the four screws in the battery compartment. These hold the yellow top on.
Turn the RCX so the IR window points up.
Pull the right side of the battery compartment away from the top case. The left side will be held in place by the battery clip.
Lift the bottom of the black IR window. Pull it out.
Hold the exposed edge of the circuit board with your fingers and pull the top case off, starting at the front. This takes considerable force.
Make sure the case doesn't get stuck on the power connector on the back of the RCX.
Hold the battery compartment and twist the circuit board counterclockwise.
There is a little nub of plastic at each end of the battery compartment that you must lift the board over as you turn it.
Comments Comments are onturn off
As you can see, I was rushing when I did this guide. I'm planning to redo the disassembly next weekend and take landscape-oriented photos with better lighting and a white background.
notexactly, · Reply


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nii holdings inc
(NIHD:Consolidated Issue Listed on NASDAQ Global Select )
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Hat tip to Father Bob for this cartoon.
« August 2006 | Main | October 2006 »
Hat tip to Father Bob for this cartoon.
Curious Christian in Ethics | Permalink | Comments (0) | TrackBack (0)
What is emerging church? Is it always missional? Or is it something else? Is involvement in alternative worship a defining mark? Or not? Or is it all of the above?
What does emerging church encompas? What does it mean?
Fernando Gros has helpfully summarised some of the recent buzz and I recommend you also check out (e)mergent Voyageur's reflections on Redemptive Taxonomy In The Emerging/Missional Divide.
Here are some comments I made in an email conversation the other day to someone who discribes himself as an interested observer of the Emerging Church. This was all off the cuff but is fairly representative of my thoughts on the taxonomy issues:
To tell you the truth I see the 'emerging church' label as more of a conversation starter than a last word. I'm yet to hear a definition everyone agrees on. At times it means little more than, hey we're trying to follow Christ in ways that don't fit the conventional definitions. But all that does is tell people what EC isn't, not what EC is. It's an apophatic ecclesiology in a way.
See, why I say this is that most of what I do is outside the church too, at least in terms of how church is conventionally understood. I have a loose network of mission-minded companions who I share the journey with as well as a loose relationship to a more conventional, if somewhat informal, church. We don't worship together often at all but they're a lifeline in terms of companionship and support. Most of all I see us somewhat in the mode of William Carey's to the west, mission-minded Christians incarnating into the new globalised subcultures so that the people within them might know the love of Christ in their own terms. Being a companion on a quest doesn't require us to live in each others pockets, it just requires us to share a commitment to a cause and one another. Think 'Fellowship of the Ring' with how the companions scattered and came together at different stages in the quest.
Sure, some ECer's get really into close knit alternate worship services, but to be honest I don't think they are not always the most missional experiments around as I see them attracting burnt out Christans a lot more than non-Christians. So don't think that just because you're not connected with an alt. worship group that you can't be involved. Not all of us are either! Then again, not all would call themselves ECers either. Some would prefer to be called church exiles, some just missional Christians. We don't care.
Curious Christian | Permalink | Comments (3) | TrackBack (0)
Oh, I should just add that I'm off to Black Stump tomorrow, so if any of you are going and are interested in catching up at any stage please leave an email tonight and we can swap mobile numbers.
For the uninitiated, Black Stump is Christian art and music festival held south of Sydney every October long weekend. For the Brits I imagine it's a bit like your Greenbelt festival.
The theme for Black Stump 2006 is "iGod: portable faith in a portable culture."
I see Darren Wright of Planet Telex is running an elective and hanging out in Sacred Space so I expect I'll be dropping in there at some stage. Hmmm, lets' see if I can remember the camera for a spot of video blogging!
Curious Christian in Community | Permalink | Comments (0) | TrackBack (0)
Avast, me hearties! In honour of International Talk Like A Pirate Day I direct ye to heed the words of these hear scalleywags on pirates and spirituality.
God Considers Smiting Bible Pirates
When he prophesies, it's in pirate
Curious Christian in Laughs | Permalink | Comments (3) | TrackBack (1)
I have been venturing into www.youtube.com territory.
My pocket digital camera has short span video capability so if I can master the medium this could open up some interesting possibilities. Video blogging is an idea I have been toying with a little but even more interesting is the possibility of publishing footage from alternate spirituality festivals and community events.
A couple of years back I took some footage of pagan drummers at the winter magic festival and I am thinking it may also be interesting to film some interfaith conversations or a tarot gospel presentation.
I am not expecting short segments will allow for great depth but I take to heart Hugh Mackey's comments in 'Why Don't People Listen' that a new message in combination with a new experience is more powerful than a new message alone. I would like to give people a more multi-media taste of what we get ourselves into.
As a test though, this is just some footage of my younger son, Aaron.
Curious Christian in Geek | Permalink | Comments (1) | TrackBack (0)


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7 killed, 24 injured in Chicago shootings in 48 hours
On Thursday, nineteen people were shot in seven attacks in the city. Thirteen of the victims were shot within a half-hour period.Chicago officials have been battling a sharp increase in shootings and homicide. The city's murder rate has increased 29 percent year-over-year as of this month. MN/MR/MA/HJL


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About Ohr Pharmaceutical, Inc.
Ohr Pharmaceutical, Inc. (NasdaqCM:OHRP) is a pharmaceutical company dedicated to the clinical development of new drugs for underserved therapeutic needs in large and growing markets. The Company is focused on advancing its pipeline products currently in phase II clinical development: Squalamine Eye Drops for the treatment of the wet form of age-related macular degeneration, and OHR/AVR118 for the treatment of cancer cachexia. Additional information on the Company can be found at www.ohrpharmaceutical.com.
Safe Harbor Statement under the Private Securities Litigation Reform Act of 1995:
This news release contains forward-looking statements within the meaning of the "safe harbor" provisions of the Private Securities Litigation Reform Act of 1995. These forward-looking statements are made only as the date thereof, and Ohr Pharmaceutical undertakes no obligation to update or revise the forward-looking statement whether as a result of new information, future events or otherwise. Our actual results may differ materially and adversely from those expressed in any forward-looking statements as a result of various factors and uncertainties, including the future success of our scientific studies, our ability to successfully develop products, rapid technological change in our markets, changes in demand for our future products, legislative, regulatory and competitive developments, the financial resources available to us, and general economic conditions. Stockholders and prospective investors are cautioned that no assurance of the efficacy of pharmaceutical products can
|SOURCE Ohr Pharmaceutical, Inc.
Copyright©2012 PR Newswire.
All rights reserved


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Accenture – Montreal
Accenture is the world's largest consulting firm, specializing in technology services. They have over 150,000 employees in 150 offices. That's almost as many people as Office Snapshots employs (almost). These pictures are from their attractive Montreal offices.
Accenture sponsors Tiger Woods, we wrote about Accenture, so that basically means that Tiger Woods is our best friend. You might need to brush up on your logic if that doesn't make sense.
Find out if Accenture is hiring and see what employees are saying:
Accenture Reviews, Accenture Salary, Accenture Interview Questions, Accenture Jobs


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Carol Cloud Bailey: Pests are eating night-blooming jasmine
Q. I love my night-blooming jasmine, but for the past year something is eating it. All the new blossoms are gone as soon as they appear. I've gone over this plant with a fine-toothed comb and I've never seen a bug, a spot, anything. I've sprayed it with Seven.
Someone suggested I treat the root system with a pesticide but I live too close to the lake and prefer not to do that.
Linda, Jensen Beach
A. There are several things that could be causing the loss of flowers from Linda's night-booming jasmine (Cestrum nocturnum). This plant's flowers open at night and sometimes it appears they are falling unopened.
Though this is a very drought-tolerant species, very dry weather such as we have experienced along the Treasure Coast could cause any plant to drop leaves or flowers. Check the ground around the plant for unopened buds or spent flowers to diagnose either of these problems.
If the flower buds seem to be disappearing, then something may be eating the buds; the most likely candidate is a caterpillar. Caterpillars or worms are the larva or immature form of insects in the Lepidopterous group — moths and butterflies.
Worms can and do eat just about any portion of a plant depending on species. Some are fond of leaves or bark and some feast on flowers. Sometimes the caterpillars are tough to find; they may be small, and feed underneath the leaves or at night. Nocturnal caterpillars often spend the day in the soil or mulch under the host plant.
It is never a good idea to apply a pesticide if the pest has not been identified. Control options for caterpillars include hand picking, spraying or waiting for natural enemies to do the job. Low-toxicity sprays are available. Products containing the bacteria Bacillus thuringiensis or BT are applied like traditional pesticides but do little damage to non-target pests.
Natural enemies of caterpillars do exist; birds, wasps and fungi all use worms for food or incubators. Once natural enemies have been observed, leave them to their work and check back in a day or so to evaluate the need for other control tactics.
Carol Cloud Bailey is a landscape counselor and horticulturist. She can be reached at firstname.lastname@example.com.
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Mom at Sixteen is a 2005 Lifetime Television film about a sixteen-year-old mother, dealing with problems at school and her decaying relationship with her mother, because of her seeing her boyfriend for the first time since before Charley was born.
List of links to Mom at Sixteen fansites:
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hand protection Companies in Spain
Hand Protection Int
based in Sialkot, PAKISTAN
Hand Protection Int, A Brand Of Quality Leather Working Gloves, Chainsaw Gloves, Gardening Gloves and Mechanic Gloves. Hand Protection Int ISO, CE, EN:388, EN:420 Certified Company, Registered with D & B`s global under RMS D-U-N-S No645749511. Hand Protection Int stands apart as the first choice for global sourcing in the highly competitive arena of Gloves manufacturing. ...
TST Sweden AB
based in Kinna, SWEDEN
TST Sweden AB develops and supplies personal a complete assortment of personnel protection equipment for the UHP waterjetting, waterblasting, heat protection and chemical protection markets. Ever since we developed our first products for personnel protection in 1990, we have consulted end users across the world. Today we offer a broad range of protective equipment with ...
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based in Virginia Beach, VIRGINIA (USA)
Coastal Training Technologies is now part of DuPont Sustainable Solutions. This new entity integrates a unique combination of collaborative consulting services, technologies and blended learning solutions which reflect market needs in the areas of workplace safety, sustainable operations, employee training and clean technologies.
Protective Packaging Corporation
based in Carrollton, TEXAS (USA)
Veteran-owned ISO 9001:2000 certified manufacturer of custom & standard barrier packaging products. Packaging products include anti-static & static shielding flexible bags, moisture barrier bags, U.S. government specified bags, corrosion inhibiting bags, desiccant bags & Military Spec. bags. Crating supplies as well as heat & vacuum sealing equipment are also available. ...
NOAQ Flood Protection AB
based in Näsviken, SWEDEN
NOAQ Flood Protection AB is a Swedish company, established with the aim of developing and selling flood protection on the global market. There is a big dammed up (!) need for more efficient and faster protection than sandbags and earthen levees and with the foreseen changes in climate the need will increase even further in the years to come. NOAQ Tube Wall, is built upon ...
Dupont Protective Apparel
based in Wilmington, DELAWARE (USA)
DuPont is committed to protecting people. From first responders to industrial workers, DuPont provides a solution to meet your safety needs. DuPont Personal Protection offers a wide range of proven, science-based solutions including some of the most trusted brands in the industry: Tyvek® for dry particulate hazards, Tychem® for liquid and gas chemical hazards, NOMEX® ...
asecos GmbH - Safety and Environmental protection
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asecos - Europe's leading manufacturer of fire resistant safety storage cabinets.Since its establishment in 1994, asecos has dedicated itself to the development and production of innovative products for the storage, handling, transportation and ventilation of hazardous materials:The quality and safety of our products are recognized and certified by the relevant ...
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In 1947 Ameron introduced T-Lock, a T-ribbed poly-vinyl chloride sheet lining material designed specifically to protect new concrete sewer pipe, structures and monolithic tunnels against the ravages of hydrogen sulfide gas/ sulfuric acid attack and other forms of corrosion. Now, more than fifty years later with millions of square feet of the material installed, we are ...
Environmental Sensors Company
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Environmental Sensors Co. offers a complete line of analytical instruments for toxic chemical vapor detection and monitoring. ESC gas monitors are designed to monitor indoor air quality where varying amounts of the targeted gas are expected to be present. ESC gas monitors are often used for personal or area monitoring to protect employees` safety. All of ESC gas monitors ...
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Globus, the UK partner for Showa Best gloves, provide high-performance solutions to the problems of hand protection in the workplace. Globus' partnership in the UK and Ireland with Showa Best has proved invaluable to companies in many diverse industry sectors. Why choose hand protection from Showa Best: For over fifty years, Showa Best has offered high-performance ...


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Lisa Orrell, CPC, is globally recognized as "The Generations Relations & Leadership Expert", and she's the award-winning author of FOUR business books currently on Amazon: Millennials Incorporated; Millennials into Leadership; and Boomers into Business. Her 5-star rated fourth book, Your Employee Brand is in Your Hands, was recently named a finalist in ForeWord Magazine's 17th Annual Book Awards competition (in both the Careers AND Leadership book categories).
As a respected expert, Lisa is an in-demand Keynote Speaker, Consultant, and Thought Leader hired by many well-known organizations and professional associations. A sample of her client list includes: Wells Fargo, Johnson & Johnson, Intuit, Chico's, Pepsi, Applied Materials, Paul Mitchell The School (100+ locations nationally), eBay, PayPal, Cisco, USC's Marshall School of Business, Blue Cross/BlueShield, PBWC, NCHRA, SBODN, and Monster.com.
She was recently even hired by the Brunei Government to be a featured speaker in their country at a Millennial Leadership Conference!
Aside from Lisa's fun, candid, motivating, and engaging presentation style, audiences enjoy her interesting content and perspectives on leadership, communication, employee engagement, and Personal Branding…with the unique "twist" of how generational dynamics greatly impact relationships in today's workforce.
She offers a variety of popular keynotes and workshops that: Improve generations relations at work; educate Leadership Teams on workforce trends; improve the recruitment, management and retention of Millennial (aka: Gen Y) Talent; and that educate Millennial employees and college students on how to be effective young leaders in the workforce. She also conducts Personal Branding workshops for employees to support their career growth and leadership development (with audiences often ranging in age from 21 – 60+ years old).
In addition to speaking, consulting, and being an author, Lisa is a Certified Leadership Coach who primarily works with Millennial and Gen X employees, entering or currently in, a leadership role at work. She received her Leadership Coach Certification through an intensive training program accredited by the International Coach Federation.
Prior to her current career, Lisa owned a full-service Marketing Agency in the San Francisco Bay Area for 20-years, and her company received over 75 national and international awards for Marketing & Branding excellence. Lisa remains a Marketing & Branding Consultant today for select clients, and in 2013 AND 2014 was voted one of the "Top 30 Most Influential Branding Gurus in the World" by over 22,000 industry professionals. She holds a Bachelor's Degree in Advertising with a Minor in Marketing from San Jose State University.
Based on her expertise and notoriety, Lisa has been interviewed by, or written articles for, countless media and research firms, including (partial list): ABC, NPR, MSNBC, CBS, The NY Times, Wall Street Journal, TIME, Cosmo, U.S. News & World Report, Human Resource Executive, The Business Journals (throughout the U.S.), Recruitment & Retention, Universum's Trainee Guides for Norway, Sweden and Denmark, China's HerWorld Magazine, Diversity Business, HR.com, FoxBusiness.com, BNET.com, Monster.com, and CareerBuilder.com.
"Lisa Orrell is an expert in this breaking new field of 'generation relations' at work.…her seminars and books are resources that every innovative company needs. And I've referred her to several of my largest corporate clients who were thrilled with her presentations and expertise!"
– Rayona Sharpnack, Founder & CEO of the Institute for Women's Leadership, Speaker, Author and 25-Year Executive Coach to Fortune 500 Execs


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By British Gas
Open iTunes to buy and download apps.
This app is only for British Gas customers who are using the Remote Heating Control product.
Say hello to a smarter home. Control your heating virtually anywhere, anytime, using the myHome app from British Gas.
Remote Heating Control customers can
Turn your home's heating on and off
Control and set your central heating schedule
View your home's current temperature
Set alerts to let you know if the temperature in your home goes above or below a certain level
Add other family members so they can also control your heating remotely
Please Note:
If you are experiencing problems please logout and try to log back in again. If the issue continues email us at firstname.lastname@example.com.
What's New in Version 4.3
Minor changes to contact details and help section.
iPhone Screenshot
Customer Reviews
Overall pretty good
For me, this app does what it says on the tin, bar a few issues with RHC in general:
1. Current problem with thermostat time running ten minutes slow, which is, to be fair, inconvenient rather than catastrophic. Seemingly BG still working on a fix, but given others have reported this bug some months ago, I suspect we'll not see it till they integrate RHC with Hive, which is supposedly some months off.
2. I have a long standing issue with their web interface being unable to display the temperature graphs for today and yesterday, but only on a Sunday, across my iPad, phone and laptop. It appears I'm the only one they know of with this weird issue!
3. Inability to program more than four on/off points even although thermostat supports six. I've got round this by using a freebie SMS scheduler app on my phone which sends on/off texts mid-evening, as well as other times in the week when I know I'll be out. Shame they couldn't incorporate that into the app and overcome any limitations the hub/ thermostat might have now or in the future.
Great device but time needs sorting.
Great device and control it loads when I'm out and about, plus with new born baby my partner can turn heating on and off when she's out. the time issue needs sorting for us early adopters, I don't need hive branding as this doès what I need for my combi boiler, please sort the time issue out British Gas. Noted numerous times on the reviews.
My Home - British Gas
App needs tweaking further thus:
Retain "Log In" details especially when WiFi signal is lost!
- Free
- Category: Utilities
- Updated: 05 February 2014
- Version: 4.3
- Size: 5.9 MB
- Languages: English, Dutch, French, Italian, Traditional Chinese
- Developer: British Gas Trading
- © 2011 British Gas Trading Limited (British Gas)
Compatibility: Requires iOS 5.0 or later. Compatible with iPhone, iPad, and iPod touch.


text:
White Oak man jailed for exposure, intimidation issues
A White Oak man is in Allegheny County Jail in lieu of $3,000 bond pending a hearing on charges of ethnic intimidation and exposure of his private parts to a group of youngsters playing basketball at a borough playground.
Police said William Elyes, 54, threatened the youngsters, ages 9-18, on Tuesday and twice on Wednesday at the Larry Bishop Playground along Vermont Avenue.
"He was yelling that he would get a gun unless the basketball dribbling stopped," police Chief Lou Bender said.
Bender said the incidents happened around 5:15 p.m. He said the group included white and black youngsters.
When Elyes confronted the group on Wednesday, Bender said, "he began yelling obscenities and racial slurs."
Elyes left the playground, but returned and, according Bender, told the group that he had a weapon that was "locked and loaded."
"At that point he proceeded to drop down his pants and exposed his genitals to the children," Bender said. "Prior to his leaving he made one more threat and he walked toward his house."
Bender said the children were panicked.
"All of them fled to nearby houses and the police were called," the chief said. "Officer Ken Wehrli and Sgt. Bob Loera confronted Elyes at his house and found drug paraphernalia there."
Elyes was arraigned early on Thursday in Pittsburgh before Allegheny County Common Pleas President Judge Donna Jo McDaniel.
Bender said Elyes is charged with a felony count of ethnic intimidation and misdemeanor counts of harassment, disorderly conduct, terroristic threats, indecent exposure and possession of drug paraphernalia.
According to court records the last count is listed as "prohibited acts."
A preliminary hearing for Elyes is scheduled on Dec. 16 at 1:30 p.m. before Magisterial District Judge Thomas Miller Jr. in White Oak.
Patrick Cloonan is a staff writer for Trib Total Media. He can be reached at 412-664-9161 ext. 1967, or email@example.com.
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Are YOU In The Right Job? Check Your Mirror!
by Sheryl Stewart
posted Jan 26 2010 1:48PM
According to a professional face reader (btw...doesn't that sound like a fun job?) YOU might not be on the career path that best suits you.....and all you need to do to find out, is look in the mirror. Check out this article below:
SAN DIEGO (Wireless Flash - FlashNews) – In today's tough job market, people can find their true calling by examining their own face.
Face reader Barbara Roberts, author of Face Reading: How To Read Anyone At A Glance (Roberts), says certain facial features make people adept at certain jobs.
Someone with close-set eyes, a narrow nose bridge, or a tall, wide forehead would do well in a computer/tech career.
Those with a widow's peak or large ears would be highly successful in a music career.
Roberts says people meant to be interior designers or artists often have long, thin ears or fine hair.
Teachers, counselors, or anyone in communications usually have a rounded hairline, meaning they're great with people.
A ball on the tip of their nose means they're the "center of all news" and people often come to them with gossip.
Finally, those with thick hair and thick eyebrows – representing "physical endurance" – are destined for outdoor jobs.
Back To Blog
Are YOU In The Right Job? Check Your Mirror!
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DNA methylation has been linked to many important biological phenomena. Researchers have recently begun to sequence bisulfite treated DNA to determine its pattern of methylation. However, sequencing reads from bisulfite-converted DNA can vary significantly from the reference genome because of incomplete bisulfite conversion, genome variation, sequencing errors, and poor quality bases. Therefore, it is often difficult to align reads to the correct locations in the reference genome. Furthermore, bisulfite sequencing experiments have the additional complexity of having to estimate the DNA methylation levels within the sample.
Here, we present a highly accurate probabilistic algorithm, which is an extension of the Genomic Next-generation Universal MAPper to accommodate bisulfite sequencing data (GNUMAP-bs), that addresses the computational problems associated with aligning bisulfite sequencing data to a reference genome. GNUMAP-bs integrates uncertainty from read and mapping qualities to help resolve the difference between poor quality bases and the ambiguity inherent in bisulfite conversion. We tested GNUMAP-bs and other commonly-used bisulfite alignment methods using both simulated and real bisulfite reads and found that GNUMAP-bs and other dynamic programming methods were more accurate than the more heuristic methods.
The GNUMAP-bs aligner is a highly accurate alignment approach for processing the data from bisulfite sequencing experiments. The GNUMAP-bs algorithm is freely available for download at: http://dna.cs.byu.edu/gnumap webcite. The software runs on multiple threads and multiple processors to increase the alignment speed.
Keywords:DNA methylation; Bisulfite sequencing; Probabilistic alignment; Parallel processing
DNA methylation is a chemical process in which a methyl group is added to the carbon-5 position of a DNA cytosine. In most vertebrates, DNA methylation typically occurs on the cytosine of a CpG dinucleotide [1,2], although some specific examples of other types of methylation have been shown to play roles in specific tissues [3-6]. Since its discovery over 60 years ago, DNA methylation has been linked to many important biological phenomena such as the suppression of gene expression [7,8], imprinting [9], X chromosome inactivation [10], epigenetic reprogramming during mammalian development [11], and cancer development [12]. Therefore, the study of genome-wide methylation patterns is currently of great interest to researchers, particularly in areas related to the molecular mechanisms of development, cancer, and chromatin dynamics.
When DNA is treated with sodium bisulfite, unmethylated cytosine residues are converted to uracil, while 5-methylcytosine residues are unaffected. Later in bisulfite sequencing (BS-seq) experimental protocols, PCR amplification or sequencing converts the uracil residues to thymines. The next step of finding the correct genomic location for a bisulfite-treated read (BSR) is a complicated and difficult process. Although cytosine to thymine changes are allowed for when mapping the BSRs to the genomic sequence, methylated and unmethylated CpG locations are often identified and, in some cases, it is impossible to distinguish between a bisulfite (BS)-treated thymine that originated from an unmethylated cytosine, and a true thymine from a different genomic location or an individual genomic variation at that location [13,14]. This ambiguity is often magnified by the presence of sequencing errors or low-quality bases. As a result, sophisticated computational strategies are required for aligning reads from BS-seq experiments.
The first whole-genome methylation profiles were performed on Arabidopsis thaliana. To map the resultant BSRs, alignment algorithms based on a probabilistic formulation and a suffix tree [15] as well as reference genome conversion [16] were used. However, it is not computationally feasible to apply this approach to larger genomes such as the human genome, or to experiments with the current deeper sequencing depths. Later algorithms, such as BSMAP[14], constructed seed tables of locations from both the original reference sequence and the BS variants, and then extends the seeds to form a possible mapping location. This seed extension process can be somewhat unreliable because the seeds must be exact and do not take into account BS-treated variations. BS alignment methods such as BS Seeker[17], Bismark[18], and BRAT-BW[19] have been used to map BSRs. These methods employ a Burrows-Wheeler transformation [20] for fast (in)exact string matching and then combine the results with either a pre-processing or post-processing script to handle BSRs with three letters after converting all Cs to T. The strength of these methods lie with reads with fewer mismatches, but they offer very limited support for aligning reads with insertions or deletions (indels). These methods also have difficulty in aligning Ts in the reads to Cs in the genome without also (incorrectly) aligning for cysteines in the reads to thymidines in the genome. More recently, the LAST alignment program has been adapted to align BSRs [21]. LAST uses a seed extension approach similar to the one used by NCBI BLAST [22], but the speed and mapping accuracy are increased by using variable-length seeds and base quality information [23].
In this study, we present a highly accurate probabilistic mapping algorithm, Genomic Next-generation Universal MAPper for Bisulfite Sequencing (GNUMAP-bs), for BSR alignment. GNUMAP-bs is an extension of the Genomic Next-Generation Universal MAPper (GNUMAP) [24] that can accommodate the alignment of BSRs to a reference genome. GNUMAP-bs was developed to achieve higher accuracy than other BS-seq approaches by including base quality scores in the alignment process. In addition, the GNUMAP-bs probabilistic mapping approach allows for the unbiased estimation of DNA methylation, especially when reads are aligned to multiple genomic locations.
Results and discussion
We compared GNUMAP-bs with several commonly-used BS alignment methods, namely BSMAP (v2.3) [14], Bismark(v0.7.9) [18], LAST[21], and BRAT-BW[19]. We also compared GNUMAP-bs with the unpublished proprietary probabilistic aligner Novoalign (v2.07.09, http://www.novocraft.com webcite) because it uses a probabilistic approach that is similar to the approach used in GNUMAP-bs. For a fair comparison, we considered only common options that were shared by all the alignment methods; otherwise, we used the default values or slight modifications of the defaults to achieve the best performance for each method. For each aligner, we allowed up to three or four base pair differences within 100-bp BSRs. For GNUMAP-bs and Novoalign, this resulted in cutoff values of -a 0.90 and -t 75, respectively. Considering that repeated genomic sequences often appear in mammalian genomes, we allowed up to 20 multiple mapping locations for each read. This approach to handling multiple alignments has only a small impact on the sensitivity of the more heuristic algorithms, but has moderate sensitivity gains in more traditional approaches like GNUMAP-bs. The Bismark software only reports up to two valid alignments. The parameter values that were used for each alignment method are given in Table 1.
Table 1. Parameters used in the two experiments for each of the aligners tested
We used two datasets to evaluate the performance of each of the alignment methods. The first was a simulated BSR dataset, which was carefully designed to mimic a typical human methylation experiment. The second was a real human BS-seq dataset, which was compared with an experimentally-derived human methylome profile.
Simulated bisulfite sequencing experiment
For the simulation study, we randomly assigned 20% of the CGs in the whole human genome (NCBI build37/HG19) to represent unmethylated cytosines (Cs) by changing them to thymidines (Ts), thereby simulating complete BS conversion. For the remaining 80% of the CGs, 75% was randomly assigned to be fully methylated and, therefore the Cs remained as Cs. The remaining 5% was assigned to be methylated in proportions between 0.1 and 0.9. In this dataset, we assumed that all non-CG sites remained unmethylated, so these Cs were all changed to Ts for read generation. We used the dwgsim ( http://sourceforge.net/projects/dnaa/files/dwgsim webcite) simulation tool with parameters -e 0.001-0.008 -1 100 -y 0.05 -r 0.002 -R 0.2 -C 10, to generate 100-bp BSRs with a 10? read depth across the genome. This simulation produced a BS-seq dataset that contained approximately 180 million (M) reads with a sequencing error rate that ranged from 0.001 to 0.008 and increased from the 5' to 3' ends, plus 5% randomly generated sequence, and a mutation rate of 0.002 (in which 0.2% were indels).
We found that the mapping sensitivities of the probabilistic aligners GNUMAP-bs (97.0%), Novoalign (96.3%), and LAST (96.9%), were higher than the sensitivities of BSMAP (93.9%) and Bismark (93.2%), as shown in Table 2. The Bismark-Bowtie2 algorithm had the lowest false positive rate (0.1%), but at the cost of an approximately 7% decrease in its sensitivity. Overall, the Bismark-Bowtie2 algorithm had 1.2 M fewer false positives than GNUMAP-bs, but approximately 10 M fewer BSRs were aligned correctly to the genome. The Bismark-Bowtie algorithm performed better than Bismark-Bowtie2 in that it aligned 5 M more reads with only a slight increase in the number of incorrectly aligned reads. The LAST algorithm aligned nearly all the BSRs (99.2%) to the genome, but although the proportion of correctly mapped reads was similar to that of GNUMAP-bs, the error rate was much higher. BRAT-BW was not included in this comparison because the application software removed the original sequence header/name which contained information pointing to the correct alignment location of the BSRs.
Table 2. Simulated bisulfite read experiment
Differences in sensitivities of these methods were much more pronounced for BSRs that contained at least one sequencing error or genome variant, and the GNUMAP-bs sensitivity was clearly better than the sensitivities of the other approaches (Table 2). We also evaluated how accurately each aligner predicted the CG methylation levels when the true methylation level ranged from 10% to 90%. All the alignment methods tested performed well with mean absolute errors less than 0.01 and with standard deviations less than 0.07.
When the computational performances of each of the methods were compared (Table 2), we observed that GNUMAP-bs required the most RAM (44.8 GB) and Bismark required the least (5.9 GB). Because some of the software applications presented here support computation on multiple threads and some do not, we presented two different measures of computational speed: 1) the total run time on a 16 CPU linux server and 2) the number of reads processed per CPU per second. GNUMAP-bs required approximately 40 hours of total compute time to process the 180 M BSRs, while BSMAP, the fastest algorithm, was nearly nine times faster than GNUMAP-bs. The LAST application does not support parallel computing, so LAST had the longest total alignment time. However, the LAST algorithm aligned the most reads per second per CPU (753), while Bismark-Bowtie2 aligned the least (26).
Human BS-seq dataset
We also evaluated the performances of the alignment methods using a BS-seq dataset generated from samples from a healthy human donor collected at the Andrology Laboratory at the University of Utah (Salt Lake City, UT, USA). The BS-seq data were generated by coupling BS conversion and the Illumina HiSeq2000 platform, which generated 101-bp BSRs for analysis. We aligned 283.6 M reads from three lanes of BS sequencing data containing 85 M to 100 M sequencing reads each, to the recent build of the human genome (NCBI build37/HG19). The human BSRs were processed for quality control as suggested previously [25]. Briefly, the quality control involved masking low quality bases or trimming consecutive lowest quality bases at the 3' ends of the reads.
We used the same parameters on these data as were used for the simulation experiment (Table 1) with two exceptions: -a 0.92 for GNUMAP-bs and -t 90 for Novoalign. The LAST algorithm again aligned the highest proportion (93.7%) of BSRs to the genome, followed by 70.0% for GNUMAP-bs, 68.2% for Novoalign, 67.1% for BSMAP, 67.0% for Bismark, 62.9% for Bismark-Bowtie2, and only 50.3% for BRAT-BW.
We compared these mapping results with the Sanger-based BS sequencing control data available from the Human Epigenome Project (HEP) [26] by selecting the data that were obtained using the same type of tissue as was used in our BS-seq dataset. The HEP data provides a natural gold standard for algorithmic evaluation. For this comparison, we focused on the 13,563 HEP CG sites on chromosome 22 (chr22), because these data provide the most comprehensive chromosomal CG coverage that is available in the HEP database. The data for the other chromosomes in the HEP showed similar profiles as chr22, but the coverage was much sparser.
Overall, the mapping results produced by the different alignment methods for the human BS-seq dataset were consistent with the results for the simulation dataset. Compared with GNUMAP-bs, LAST aligned almost twice as many reads to chr22 (3.02 M compare with 1.57 M); however, the CG read coverage for LAST (7.6 reads/CG) was only about 20% higher than for GNUMAP-bs (6.3 reads/CG) (Table 3). While Novoalign aligned more reads to and covered more CG sites on chr22 than GNUMAP-bs, GNUMAP-bs had better CG read coverage (6.3 reads/CG) than Novoalign (6.1 reads/CG) and the other aligners, BSMAP (5.8 reads/CG), Bismark (5.8 reads/CG), Bismark-Bowtie2 (5.7 reads/CG), and BRAT-BW (5.0 reads/CG) (Table 3).
Table 3. Human bisulfite read experiment
The differences between GNUMAP-bs and LAST become even less pronounced when the overlap between the mapped BSRs and the 13,563 CGs with HEP coverage on chr22 was considered. Last aligned the BSRs to 63.5% (8,606) of the CGs HEP sites compared with 58.3% (7,902) for GNUMAP-bs, and 57.5% (7,802) for Novoalign, 57.1% (7,747) for BSMAP, 55.7% (7,561) for Bismark, 54.1% (7,331) for Bismark-Bowtie2, and 49.3% (6,690) for BRAT-BW (Table 3).
To determine if increased coverage resulted in decreased alignment quality, we computed the correlation coefficient between the estimated HEP methylation levels and the methylation levels estimated from each alignment method. The correlation coefficients were 0.887 for GNUMAP-bs and ranged from 0.895 for Bismark-Bowtie2 to 0.882 for LAST (Table 3). We also computed a concordance statistic for each of the methods as defined previously [27]. Briefly, the concordance is the fraction of sites for which the methylation levels (aligner vs. HEP) differ by less than a predefined cutoff (we used 0.25). Based on this statistic, we found that the performances of most of the aligners were similar, with concordance values of 0.869 for GNUMAP-bs and ranged from 0.872 for Bismark-Bowtie2 0.872 and BRAT-BW to 0.858 for LAST (Table 3). These results showed that the increased coverage levels produced by LAST result in lower consistency with the HEP data in terms of both correlation and concordance. Pairwise comparisons of the concordance for the CGs covered/not covered between GNUMAP-bs and the other aligners are shown in Figure 1. For example, Figure 1 shows 577 CGs that were covered by GNUMAP-bs but not covered by Bismark; the concordance of the GNUMAP-bs methylation estimates for these sites was 0.78. In contrast, only six CGs covered by Bismark were not covered by GNUMAP-bs, and the concordance for these sites was extremely low (0.33). These results clearly showed that the increased mapping coverage (for both numbers of CGs covered and reads per CG) obtained using GNUMAP-bs did not result in decreased alignment quality or methylation estimates.
Figure 1. GNUMAP-bs workflow for mapping high-throughput bisulfite reads. A flow-chart of the GNUMAP-bs algorithm is shown. For the reference genome, all Cs are converted to Ts and then each k-mer in the genome is hashed, producing a list of positions in the genome to which the k-mer sequence is mapped. Given a completely BS-converted read (e.g. TGGTGTT) where the corresponding original bisulfite read (BSR) is known (e.g. CGGTGTC), a query k-mer (e.g. TGGTG) is searched against the BS-converted hash table. Locations with a k-mer match to both the genome and the read are aligned using the original read and original genome and the GNUMAP-bs probabilistic Needleman–Wunsch (N-W) algorithm. If the alignment score passes a quality threshold, the location is considered a match and recorded on the genome for future output. The posterior probability is computed for each mapped location using Equation (1). Finally, all the mapped BSRs are used to quantify the fraction of methylation at each CG location across the reference genome.
BS sequencing presents difficult challenges to researchers attempting to process the sequencing reads from BS-seq experiments. In this work, we present GNUMAP-bs, a highly accurate and effective alignment algorithm that is specifically designed to estimate DNA methylation levels with base-level resolution in BS-seq data. GNUMAP-bs uses a probabilistic approach to align BSRs to a reference genome. GNUMAP-bs was developed to achieve higher coverage and accuracy than other published BS-seq approaches by integrating base quality and alignment quality information in the mapping process. We have shown that the GNUMAP-bs probabilistic mapping approach results in an improved unbiased estimation of DNA methylation across the human genome. In simulated and real datasets, we showed that GNUMAP-bs outperforms other BS-seq alignment methods when both coverage and consistency were balanced with Sanger based BS sequencing controls.
In addition, GNUMAP-bs provides many high-demand features needed for constructing a high quality methylome from BS-seq data. First, GNUMAP-bs incorporates quality sequencing data into a dynamic programming framework. This feature gives GNUMAP-bs the best balance between sensitivity and specificity of the tested BSR aligners, especially for reads that contain short polymorphisms. Second, GNUMAP-bs adaptively assigns an optimal mapping stringency based on an effective read length after the original read is trimmed. Third, GNUMAP-bs not only relies on the maximal score alignment but also probabilistically considers suboptimal alignments; that is, the alignment score is converted to a posterior probability and the probabilistic scores quantify the likelihood of the true source location for each read across the reference genome. As a result, for both the simulated and real datasets, we showed that GNUMAP-bs was more effective that the other methods in detecting read locations in the presence of sequencing errors. GNUMAP-bs also displayed the highest consistency with a known HEP methylation database. Because GNUMAP-bs supports Message Passing Interface (MPI) processing, the computational burden of the dynamic programming can be alleviated. Moreover, with computer resources becoming cheaper, computing clusters with large numbers of nodes and cores, and more computing clouds are becoming available. Therefore, memory and CPU running times are less of a bottleneck, which is especially useful for GNUMAP-bs alignments. For this reason, accuracy should currently be a more important concern in BS-seq data analysis.
The GNUMAP-bs alignment algorithm is a modification of the GNUMAP algorithm, which consists of three main steps, all of which needed to be modified to align BSRs to a reference genome. A flow chart of the GNUMAP-bs algorithm is displayed in Figure 2. The first step is the construction of a hash table using all genomic subsequences, where the k nucleotide (nt) long (k-mers) are the keys and the hash table values store the genomic locations of the k-mer. In addition, k-mers from the BSRs are incrementally referenced in the reads in the genomic hash table. In GNUMAP-bs, the genome and the reads are artificially 'BS-converted' by changing all Cs to Ts before the hashing step. This process ensures that all BSRs can be referenced into the hash table regardless of whether or not they contain methylated bases.
Figure 2. Relative complement mapping consistency of GNUMAP-bs with HEP methylation profiles of human chromosome 22. Venn diagrams between GNUMAP-bs and (a) Novoalign, (b) BSMAP, and (c) Bismark showing both the number of covered/uncovered CG sites and the concordance (in parenthesis) of these sites with the HEP methylation profiles. The estimated levels of methylation in the additional CG sites covered by the probabilistic aligners but not by the other aligners are highly concordant with the HEP results.
In the second step of the GNUMAP algorithm, all the BSRs are aligned to the genome at the hashed locations. The alignment is performed using a novel probabilistic alignment algorithm that uses base quality information. All the regions that meet a predefined alignment score threshold are retained for the final step. In GNUMAP-bs, the probabilistic alignment algorithm matches the unconverted (original) reads to the unconverted genome while allowing matches between Ts in the reads and Cs in the genome. Using dynamic programming in probability space for mapping BSRs to a genome has several benefits: 1) the Needleman–Wunsch algorithm is guaranteed to find the optimal alignment for a BSR; 2) by incorporating base qualities into the probabilistic algorithm, true DNA methylation can be more accurately identified in the alignment, especially in areas where the reads have low base quality; and 3) by making only a small change in the alignment scoring matrix, namely by removing the T (read) to C (genome) 'mismatch' penalty and scoring these alignments as a 'match', the probabilistic Needleman–Wunsch algorithm can accurately account for the BS changes in BSRs without losing reads that are partially converted, or that have genome variations or sequencing errors.
During PCR amplification after BS conversion, the C to T conversions in the BSRs lead to G to A conversions on the PCR-synthesized strand as described previously [14]. These G to A changes provide evidence of non-methylation on the original strand. The phenomenon also occurs for C to T changes on the PCR-synthesized strand. Therefore, the GNUMAP-bs algorithm maps each BSR twice, once looking for only C to T changes and then looking for only G to A changes. When a G to A change is observed, the read is considered to be a PCR-synthesized strand, and the non-methylation event is attributed to the original strand.
In the final step, the GNUMAP-bs algorithm quantifies the significance of an alignment (e.g. a posterior probability) by assigning a mapping score that is proportional to the relative quality of alignments and inversely proportional to the number of matching sites in the reference genome. Given a BSR, r, assume that there are J "best match" locations (potential mapping locations) for which the alignment score is q[ d] for alignment to genomic position d, which is also aligned to the first base pair location of r, matches some user-defined threshold. Then, the posterior probability at each location P(rd) can be computed as:
It is worth noting that the calibrated posterior probability is a special case of the LAST[21] mapping probabilities equation where a scaling factor for a bit score in the simple E-value statistics is not explicitly computed.
This probability provides an intuitive value between 0 and 1 representing the methylation signal for each read at each CG site. Other BS mapping programs use instead of Equation (1), which may not accurately capture the true posterior probability. The posterior probability indicates the relative significance of mapping a read to a particular position. The probabilistic Needleman–Wunsch computes a log-likelihood score; therefore, if a particular BSR has multiple possible mapping locations in the reference genome, the significance of each mapping decreases. After GNUMAP-bs computes P(rd) for each read, the algorithm combines the probabilities into one methylation profile vector and infers a true methylation ratio at each CG site.
For example, consider a nucleotide, ni at genomic location i, and assume that ni is a C residue in a reference genome. Let C(m) be a methylated C residue and L(r) be the read length. For a set of aligned reads covering i, the methylation ratio c[i] can be computed as:
Intuitively, the reads that cover each C location across the reference genome provide evidence of true mapping so that a reliable methylation percentage can be obtained.
Software implementation and availability
The GNUMAP-bs algorithm is integrated into the is GNUMAP software suite, and is freely available for download at http://dna.cs.byu.edu/gnumap webcite. The GNUMAP-bs pipeline can accommodate single-end or pair-end reads in either FASTA or FASTQ file formats. A reference genome file (or multiple reference files) in FASTA format is also required. However, to increase the efficiency of the workflow, users can opt to write the reference genome hash table to a file, which can be used in future runs. GNUMAP outputs read alignments in standard SAM file format. The GNUMAP-bs software suite contains an addition application function (sam2gmp) that summarizes a SAM file and writes it into a text file that contains one line for each cytosine in the genome. Each line in this file contains the chromosome number, the location of the cytosine on the chromosome, the number of reads, and the numbers of As, Cs, Gs, Ts, and Ns covering the location. In addition, the text file gives a likelihood ratio p-value that indicates whether there is a significant number of Cs at the location (i.e. methylation significance).
In addition to the adaptations for BSRs, the GNUMAP-bs software also contains several modifications to reduce the computational time and memory needed for the alignments. For example, the initial read and genome conversion step reduces the genome alphabet to three bases, leading to an increase in the number of genome locations identified in the hash table. To reduce this effect, multiple seeds from each read are referenced by GNUMAP-bs, and the alignment is only conducted on the locations with the top two most k-mer hash references. Furthermore, although the original GNUMAP software supports multi-threaded computing within the same node using pthread, GNUMAP-bs is fully enabled to support MPI processing. This feature allows a large-scale alignment to be spread across multiple nodes in a cluster or supercomputing facility [28]. In this implementation, the genome is divided into equal parts across nodes, and then the same batches of reads are aligned by each node to their individual portion of the genome. Once the batch is completed, the nodes communicate via MPI to calculate the posterior probability scores. Because most of the CPU time is spent on the alignments, the communication overhead is relatively small, resulting in a highly efficient parallel algorithm.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
QS, MJC, and WEJ conceived the study and developed the algorithm. CH and NLC implemented the algorithm and developed the software. CH and SC generated the simulated read dataset and analyzed both datasets. SSH, DTC, and BRC generated the human BS-seq datasets. CH and WEJ wrote the paper. All authors read and approved the final manuscript.
This research was conducted using the Fulton Supercomputing Lab at Brigham Young University (Provo, UT, USA) and the Linux Clusters for Genetic Analysis (LinGA) computing resources at Boston University Medical Campus (Boston, MA, USA). The study was supported financially by a grant from the National Institutes of Health (NIH) (R01 HG005692).
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What Jung Shin Lee, Ph.D., a postdoctoral researcher and the study's first author, and Alexander Garrett, a bioinformatics specialist in the Shilatifard lab and co-author, found was unexpected: although yeast carrying mutant Chd1 showed a loss of H2B ubiquitination, H3 methylation was fairly unaffected. "Surprisingly, when we looked genome-wide, we observed that methylation patterns of other histones within nucleosomal complexes were fairly normal in Chd1 mutants," says Lee. "That told us that there are other functions associated with ubiquitination of H2B besides methylation of H3."
To reveal what those functions were, the investigators applied a technique that has been mastered by Penn State biochemist Frank Pugh, Ph.D. The methodology, known as nucleosomal occupancy mapping, can nail down to a single DNA base the very spot on a DNA strand where a complex of histone proteins is sittingnot in just one gene, but in all 5,700 or so yeast genes simultaneously.
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