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http://www.goal.com/en-us/teams/united-states/224/new-york-red-bullsGoal.com News - New York Red Bulls2015-10-05T00:12:55+00:00http://i1.goal.com/web/goal/2015.10.01-v1/images/default/logo500x500.png Copyright 2015 Goal.comhttp://www.goal.com/en-us/news/1110/major-league-soccer/2015/10/04/15993562/mls-review-revs-fail-to-clinch-playoff-berth-toronto-movesMLS Review: Revs fail to clinch playoff berth, Toronto moves closer2015-10-04T06:15:17+00:00By Sacha Pisani
The Revs' playoff spot was put on hold after losing at Chicago, while Toronto FC closed in on a post-season berth. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/09/24/15705372/mls-week-30-preview-tv-scheduleMLS Week 30 preview, TV schedule2015-09-25T02:28:39+00:00
The playoff race in the Western Conference could clear up some as several contenders go head-to-head. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/09/20/15571042/mls-review-red-bulls-secure-playoff-spotMLS Review: Red Bulls secure playoff spot2015-09-21T01:42:58+00:00By Michael Huguenin
New York Red Bulls became the first club to secure a MLS play-off spot this season after a 2-0 win over Portland Timbers. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/09/20/15570022/nat-borchers-timbers-owe-fans-an-apology-after-lossNat Borchers: Timbers owe fans an apology after loss2015-09-21T00:31:40+00:00By Jon Arnold
The center back said the home team lacked energy in Sunday's defeat to the New York Red Bulls. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/09/17/15449212/mls-review-revolution-move-atop-eastern-conferenceMLS Review: Revolution move atop Eastern Conference2015-09-17T05:02:17+00:00By Sacha Pisani
The New England Revolution jumped to first place in the Eastern Conference with a 2-1 win over the New York Red Bulls. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/09/12/15280672/marsch-red-bulls-comeback-was-good-prep-for-playoffsMarsch: Red Bulls comeback was good prep for playoffs2015-09-12T14:26:04+00:00By Thomas Floyd
The New York Red Bulls coach was pleased how his team overcome a two-goal deficit en route to a 3-2 win over the Chicago Fire on Friday. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/09/11/15272632/new-york-red-bulls-3-2-chicago-fire-red-bulls-go-topNew York Red Bulls 3-2 Chicago Fire: Red Bulls go top2015-09-12T03:26:40+00:00By Dejan Kalinic
Sacha Kljestan netted a late penalty as the New York Red Bulls claimed a comeback win against the Chicago Fire. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/08/30/14905892/mls-review-red-bulls-keep-dc-down-sounders-trump-timbersMLS Review: Red Bulls keep D.C. down, Sounders trump Timbers2015-08-31T01:35:04+00:00By Michael Huguenin
Bradley Wright-Phillips led New York Red Bulls to within two points of D.C. United in MLS with a brace against their rival. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/08/26/14789792/mls-review-fire-stun-red-bullsMLS Review: Fire stun Red Bulls2015-08-27T03:46:28+00:00By Michael Huguenin
Chicago Fire finally had a reason to celebrate with a shock victory, while Kevin Doyle claimed a win for the Colorado Rapids. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/08/16/14475342/mls-review-galaxy-surge-past-whitecaps-after-sportingsMLS Review: Galaxy surge past Whitecaps after Sporting's stunning win2015-08-16T05:33:16+00:00By Michael Huguenin
The LA Galaxy's 2-1 win, combined with the Vancouver Whitecaps, 4-3 loss, sent the Galaxy to the top spot in the Western Conference. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/08/16/14475112/red-bulls-midfield-ascendant-as-new-york-cruises-pastRed Bulls midfield ascendant as New York cruises past Toronto FC2015-08-16T04:08:07+00:00By Ben Valentine
For the second week in a row, the Red Bulls faced off against a star-studded MLS side and came through with a dominant performance, with their midfield controlling the game. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/08/05/14198392/mls-review-giovinco-hat-trick-downs-orlando-montreal-drawsMLS Review: Giovinco hat trick downs Orlando, Montreal draws Red Bulls2015-08-06T02:20:05+00:00By Michael Huguenin
Toronto FC returned to the win column list thanks to Sebastian Giovinco, while the Montreal Impact earned a draw. http://www.goal.com/en-us/news/1110/major-league-soccer/2015/08/02/14087112/mls-review-goals-galore-as-dc-united-win-10-goal-thrillerMLS Review: Goals galore as D.C. United win 10-goal thriller2015-08-02T04:40:34+00:00By Sacha Pisani
D.C. United came from behind, scored six goals and conceded four in a remarkable match against Real Salt Lake in MLS action on Saturday. http://www.goal.com/en-us/news/69/transfer-zone/2015/07/27/13931002/shaun-wright-phillips-joins-brother-bradley-at-red-bullsShaun Wright-Phillips joins brother Bradley at Red Bulls2015-07-27T14:39:13+00:00By Jack Davies
Shaun Wright-Phillips has joined New York Red Bulls following his release by QPR. http://www.goal.com/en-us/news/85/england/2015/07/23/13828172/mourinho-happy-with-chelsea-testMourinho happy with Chelsea test2015-07-23T13:44:46+00:00By Sacha Pisani
Chelsea was stunned 4-2 by a youthful New York Red Bulls outfit in the International Champions Cup on Wednesday.


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1 Large Bedroom for rent. All inclusive
Spacious 2 bed ground floor flat currently undergoing refurbishment. Will be available at the beginning of November. Property located on Okebourne Road. Unfurnished main bedroom available 4.49 x 3.12 (14'9 x 10'3). Has entrance hall, conservatory,
- Date available: 10 Nov 2015
- Flat

subset: commoncrawl


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2011 Peterbilt 387
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2016 Freightliner BUSINESS CLASS M2 106
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2012 Dodge RAM 5500SLT
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2007 International 9400i
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2012 Volvo VNL64T300
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Ken Loach(1936-), Film director
Kenneth Charles ('Ken') Loach
Sitter in 7 portraits
Film and television director, best known for gritty realist dramas motivated by social concern. Loach made his name with Cathy Come Home (1966), and subsequent works include Poor Cow (1967), Kes (1970), Ladybird, Ladybird (1994), My Name is Joe (1998) and Sweet Sixteeen (2003).
by Jorge ('J.S.') Lewinski
bromide print, May 1972
by Nick Cudworth
pastel, 1998
- Larger Image
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- Recent acquisition
by Mayotte Magnus
modern bromide print from original negative, 1973
NPG x138820
by Neil Libbert
bromide print, 1987
NPG x136290
by Steve Pyke
colour print, 1993
NPG x45588
British film directors (Stephen Frears; Sir Richard Eyre; Ken Loach; Mike Leigh; Michael Winterbottom; Stephen Daldry)
by Mary McCartney
C-type colour print, December 2002
NPG x126206
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- Recent acquisition
by Eamonn McCabe
archival inkjet print, 2006
NPG x199150
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Eser Facilitators and Committee
Rabbi Jeff Amshalem
Rabbi Jeff Amshalem teaches Jewish Studies at Gann Academy and recently began working with the Moishe House. Before moving to Boston, Amshalem studied in Israel at the Pardes Institute in Jerusalem and at Ben-Gurion University. He received smicha from Rabbi Zalman Nechemia Goldberg and an MA in Jewish Thought with a focus on Hasidism and Kabbalah. Amshalem is currently preparing a new translation of Shivhei haBesht, the earliest collection of stories about the Baal Shem Tov.
Benjamin Barer
Benjamin is a rabbinical school student at Hebrew College, as well as the Editorial Director of State of Formation, an international blog for emerging religious and ethical leaders. Benjamin is passionate about interfaith dialogue and Jewish text, and blogs at whatibelievein.wordpress.com.
Rabbi Getzel Davis
Getzel Davis is associate rabbi and Jewish educator at Harvard Hillel. Before joining Harvard, he was a rabbinic intern at The Boston Synagogue and spiritual director at Eden Village Camp. He has also worked as an educator for American Jewish World Service, Bend the Arc, the Teva Learning Alliance and Larchmont Temple. A graduate of Brandeis University, Davis received his rabbinic ordination and Master of Jewish Education from Hebrew College. He loves to get lost in deep forests and rock out to brass-band music.
Elisha Gechter
Elisha Gechter, former associate director of community engagement for adult learning at Hebrew College, is program manager of the Wexner Israel Fellowship Program at Harvard University's John F. Kennedy School of Government. A graduate of the Heller Hornstein master's program at Brandeis University, she lives in Cambridge with her husband and daughter. Gechter was named a 2015 Chai in the Hub honoree by Combined Jewish Philanthropies.
As a Prozdor alum and inaugural Eser member, Debbie is excited to be back at Hebrew College to teach in Eser's new venture, Expressing Our Book: Creative Approaches to Jewish Text Study. Debbie received her Master's in Social Work from Simmons College in 2002 and currently specializes in in-home family therapy. Debbie is also a student at the Grub Street writing center in Boston, focusing on creative non-fiction, and serves on the Executive Council for the New Center Now, as chair of the film circle.
Rabbi Hillel Greene, Rab'14
Hillel Greene, a 2014 graduate of the Rabbinical School of Hebrew College, is a Jewish educator at Gann Academy in Waltham, Mass. He previously served as rabbinic adviser and Jewish chaplain at Boston College and Northeastern University. An avid reader, Greene is slowly working his way through the complete works of William Shakespeare.
Rabbi Neil Hirsch
Neil Hirsch joined the staff of Temple Shalom in Newton, Mass., in 2010. Born and raised in Houston, he attended Tufts University and received rabbinical ordination from Hebrew Union College. Hirsch is an avid cyclist and runner, having completed the New York City Marathon in 2009.
Rabbi Avi Poupko
Rabbi Avi Poupko served as the Hillel Campus Rabbi at Harvard University from 2005-2007 and then spent four years teaching in Israel, first at the Pardes Institute in Jerusalem and then at Midreshet Ein Prat-The Israeli Academy for Leadership. Poupko is currently the Rabbi of Congregation Ahavas Achim in Newburyport in addition to serving as a chaplain at the Essex County Prison in Middleton, MA.
Becky graduated from Emerson College in 2009 with a BA in Theatre Education, and has since been teaching drama in Newton Public Schools as well as in Jewish educational programs including Prozdor Makor, Kesher, and Congregation Shir Tikva. Becky recently completed her Masters coursework toward an MA in Directing at Roosevelt University and looks forward to completing her thesis production this fall. Having served as an Eser committee chair for 3 years, she is excited to venture into her new role as group facilitator for Eser's new Expressing Our Book series.
Rabbi Rachel Silverman
Rachel Silverman is director of congregational learning at Congregation Kehillath Israel in Brookline, Mass. A graduate of Brandeis University, she received rabbinic ordination from the Jewish Theological Seminary. Silverman created a line of T-shirts, Real Men Marry Rabbis, and launched Homegrown Judaica, an online art marketplace.
Karen Nahary
Lisa Jacobs
Dimitry Neyshtadt
Pete Ziegler
Andrea Bullock
Emily Kates
Ruth Ginsburg
Amanda Dropkin
Debbie Gerofsky
Ben Ginsburg
Becky Price
Andrew Oberstein
Hillary Kern
Robin Cohen
Hannah Delucia
Matt Bubley
"The program made me realize just how important Judaism is to my identity and how I long to be around others who are strongly connected to our heritage."
"The Shabbat dinner was the best Friday night of my life. It made me realize how much I enjoy being a part of the Jewish community."


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SpinKit just got three new additions: a folding cube spinner (above), a classic circle spinner, and a scaling grid spinner. Just like the other spinners, they are animated using only the
transform and the
opacity properties, making them perform well, and easy to customize: just change the background color to match your site.
If you want to use SpinKit in your project and stay up to date with new changes and additions, the project is now also available on npm and bower. Just run:
npm install spinkit
bower install spinkit
With the recent release of SpinKit 1.2, we refactored a lot of the SCSS. The spinners are now easier to customize, and we fixed all known bugs. I highly recommend updating.
Do you have a spinner in mind that you're missing from SpinKit? Let me know, and it may just join the SpinKit family in the next update.
Happy spinning!
Bakken & Bæck, announcing Wake:
We couldn't find anything that would allow us to quickly share visual work as a team, and we needed a better tool to keep us all in sync. Our design process was fragmented across different tools and channels. We were storing files in Dropbox, and giving feedback on email, Skype or Slack.
These tools are great for text-based communication, but they were not designed for the type of visual work we do. We needed a solution to keep everyone in the loop built specifically for designers.
Together with Chris we started building a prototype for a tool that would improve our own design workflow.
Wake is a new tool by Bakken & Bæck for collaborating around design, that let's you "explain your thinking and get feedback". It looks gorgeous.
Alan Kay did a talk at OOPSLA in 1997 titled "The computer revolution hasn't happened yet," in which he argued that we haven't realized the potential that computers can provide for us. Eighteen years later, I still agree with him - it hasn't happened yet. And teaching people how to loop over a list won't make it happen either. To realize the potential of computers, we have to focus on the fundamental skills that allow us to harness external computation. We have to create a new generation of tools that allow us to express our models without switching professions and a new generation of modelers who wield them.
— Chris Granger in Coding is not the new literacy
Brandon Walkin, on Facebook's engineering blog:
Today, we're excited to release Origami Live for iOS, along with a major new version of Origami for Mac. Origami Live is a new app that lets you use your Origami prototypes on your iPhone or iPad. Alongside it, we're releasing Origami 2.0, which has a lot of new features, including code exporting, powerful gesture support, Sketch integration, presentation mode, and more.
They also launched a new site with tutorials and documentation to get started with Origami.
I recently added a simple visual effect to this blog that I quickly fell in love with: when you hover blog headers, the link's underline is revealed by animating it out from the center. You can try it in the banner above.
Creating this effect is surprisingly easy, doesn't require any additional DOM elements to be added through HTML, and falls back nicely for browsers that don't support CSS animations (it will just show up as a regular underline).
The first thing we need to do is turn off
text-decoration, and set the link's
position to
relative. For simplicitiy's sake, we'll also make sure the link doesn't change color on hover. Here we're applying the effect to all link elements inside
h2 > a {
position: relative;
color: #000;
text-decoration: none;
h2 > a:hover {
color: #000;
Next, we want to add the border, and hide it through a transformation. We do this by inserting it with
:before, and setting its X scale to
0. As a fallback, we hide it with
visibility: hidden for browsers that don't support CSS animations.
h2 > a:before {
content: "";
position: absolute;
width: 100%;
height: 2px;
bottom: 0;
left: 0;
background-color: #000;
visibility: hidden;
-webkit-transform: scaleX(0);
transform: scaleX(0);
-webkit-transition: all 0.3s ease-in-out 0s;
transition: all 0.3s ease-in-out 0s;
At the very bottom we tell the element to animate all changes applied to it, with a duration of
0.3 seconds. For the animation to appear, now we just need to make the element visible again on
hover, and set its X scale back to
h2 > a:hover:before {
visibility: visible;
-webkit-transform: scaleX(1);
transform: scaleX(1);
And that's it! Firefox has had support for
animation and
transform without the
moz prefix since version 16.0 (for over a year), so I've left out the prefix in the code. If you want to be safe, you should add
-o and
-moz to all animations and transforms.
Caught up in a race for money and fame, we lost our focus on the important. We talk of venture capital, recruiting tactics, dreams of disrupting industries, stock options, growth hacks, and the superiority of our tools. We do not talk of the bugs, the quirks, the difficulties of using our creations, the exploitation of the public, or the worst secret of all: software is broken, we are responsible, and we're making a lot of money off it.
We've become obsessed with process and tools. We've stopped caring about the product.
Poole is the Jekyll butler. It's a fully furnished Jekyll setup, designed to be a consistent and flexible starting point for Jekyll projects.
I wish I would've had Poole when I made the transition to GitHub Pages and Jekyll. Out of the box, it offers layouts, a custom 404 page, RSS feeds, posts, and an example page.
Along with Poole, Mark released two excellent Jekyll themes today: Hyde 2 and Lanyon.
Typography is the craft of arranging type with the goal to make language visible. We're arranging type multiple times throughout the day; whether it be writing essays, summarizing meeting minutes or creating slides for a presentation. What usually happens is, we end up thinking more about what we write than how we write it.
The guide is beautifully illustrated. If you want to contribute, it's available on GitHub.
It's easy to design something attractive that's not very usable, and it's easy to design something usable that's unattractive. The challenge is striking a balance, and iOS 7 made too many usability sacrifices to achieve attractiveness.


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Our new "How to Study the Bible" series starts soon! Sign up today.
marsh. (1.) A town in the low country or shephelah of Judah, near Zorah (Josh. 15:34). It was re-occupied after the return from the Captivity (Neh. 11:30). Zanu'ah in Wady Ismail, 10 miles west of Jerusalem, occupies probably the same site.
(2.) A town in the hill country of Judah, some 10 miles to the south-west of Hebron (Josh. 15:56).


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"Iron Man 3" Review and Discussion Thread (spoilers)
View Single Post
May 4 2013, 04:42 PM
Vice Admiral
Location: Sac, Ca
Re: "Iron Man 3" Review and Discussion Thread (spoilers)
Thought it was pretty good, but unfortunately I thought the second half still suffered from the same kind of aimless and unfocused storytelling the second movie did.
And for whatever reason the whole Extremis thing didn't grab me either. The villains made of lava or fire who could heal themselves instantly felt more like something from an X-Men movie. And while I had no attachment to the comic book Mandarin, I couldn't help but feel disappointed that instead of the scary terror mastermind that had been built up, we were just stuck with the hammy Guy Pearce as the main villain.
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Recent Posts on MikeCann.net
***IMPORTANT*** THE YOUNG JURKS ***Like us on facebook***
Tuesday, May 19, 2009
2009 Massachusetts State House Medical Marijuana Hearing Part 1
Posted by MikeCann
Link to youtube
5/19/2009, Boston, MA
With Steven Epstein (MassCann/NORML), Bill Breault, and Mike Cann.
Steven Epstein talks about US Supreme court upholding states rights to medical marijuana and medical users in MA that need legal cannabis.
Bill Breault doesn't address the sick people that are denied medicine that helps them.
Mike Cann (me), I talk about my wrestling injury, medical marijuana, the anger I feel watching patients like Marci Duda denied their medicine of choice.
More videos from this hearing to be posted.
The ProblemAddicts
1 comment:
Nice testimony Mike Cann, it's featured today on front page of cannabisculture.com Marc Emery must be a fan of yours, too.ReplyDelete
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Norman and Nikki Hayes created Livvy's Love in remembrance of their beloved late daughter Olivia [read more]
Converse Inc. debuted their newest collaboration with rap superstar Wiz Khalifa this week. The collection will feature… Read More
Bride: Carla Murray Groom: Jamaal Lofton Date of Wedding:… Read More
The New Vintage: Real Men Rock Florals Some may say floral prints are for strictly for the ladies, but we beg to differ:… Read More
We are not "old Black" nor "new Black" we are ALL Black.
The London-bred designer knew by the age of 5 that she wanted to be a fashion designer. Correction: she was going to be… Read More
Sheer is a hot trend we saw on the runways for spring and summer, and it doesn't seem to be losing any steam for fall. Maybe… Read More
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Vernon Reid Talks 30 Yrs of the Black Rock Coalition
Justin Adu Explains African Amedia
Beautifully Brown Grace Jones
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Detroit's New Activist Generation
by Najja Parker
by JetMag.com Staff
by Shantell E. Jamison
by Associated Press


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At times in our lives, we all wear masks. Sometimes to try and hide from reality. Sometimes to pretend to be something we are not. This week, we hear about those masks from V, Scooby, The Warrior, Verse, Trey and more. Hosted this week by Little Jockey. Thanks for listening.
8/20/15 – The Streets
This week on Sending Messages, we bring you four stories about the streets, a common ground for young people searching for glory and status, an arena that often fosters struggle and regret. Listen to original pieces from Verse, Trey, The Woman, and Rico. Hosted by mentor Gabriella Huggins. Thanks for listening!
8/17/15 – Family
Today, we explore the idea of family, the bonds we are born into and create, the people to whom we are attached and often have no choice but to be attached to. Hear stories from Guero, Shaggy, Jude, Rico, Tunes, and Forgiven on this episode. Hosted by mentor Gabriella Huggins. Thanks for listening!
4/28/15 – Memories
They are the things that shape us. That frame our perspective of the world. Our memories. This episode, join Pallance as he takes you through several pieces, some funny, some sad. Poetry, interviews and narrative by Sotia, Shadow, The Architect, Eagle, Verse, Tunes and more. Thanks for listening!
2/14/15 – Valentines Day 2015
This episode, the guys want to talk about the love we give and the love that is given to us. It's the Valentines Day episode! Join Lil' Skimpy, Oxygen, Pallance, Tunes, Ghost and more. Hosted by Diefy. Thanks for listening!
2/2/2015 – How To Forgive
In our lives, often we need to ask for forgiveness, and we need to give it to others. Everyone has been on both sides of that act. This episode, we take a look at those stories. Pieces by Trial By Tears, Alias, Chance, Vanished, Snow, Sotia, Low Key, V, and Oxygen. Music by Risky & The Hatter as well as a song by Lil' Skimpy. Hosted by Defy. Thanks for listening.
12/20/2014 – Christmas 2014
Happy Holidays! This episode we bring you stories from Defy, Tortuga, Ghost, Cam, Blackford and more. New Years resolutions, poetry and Christmas memories. Thanks for listening! See you next year.
11/30/14 – Choices
This episode is all about the choices we have to make in our lives. How to decide and what the reprocussions of those decisions might be. Stories from SLS, Lyric, Pee Wee, The Architect, and Hope. This episode is hosted by Tortuga. Thanks for listening!
10/30/14 – Halloween 2014
Happy Halloween Everyone! This episode we take a look at legends and folklore, choosing legends that help explain the struggles we have gone through. Stories from JZapp, Defy, Ghost, Sotia, Blackford, an interview with Pallance and a song from Oxygen. Thanks for listening!
8/20/14 – Episode #49 – Betrayal
This episode is all about the two faced, the back stabbing, the loss of trust and belief. Betrayal. How it happens and what it does to us when we experience it. Stories, music and poetry this episode by V, Tiger, Majors, Chance, Lyric, Scarecrow, Spark Of Courage and more. Hosted by Blackford. Thanks for listening.


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Gene expression in blood was correlated with mercury levels in blood of 2- to 5-year-old boys with autism (AU) compared to age-matched typically developing (TD) control boys. This was done to address the possibility that the two groups might metabolize toxicants, such as mercury, differently. RNA was isolated from blood and gene expression assessed on whole genome Affymetrix Human U133 expression microarrays. Mercury levels were measured using an inductively coupled plasma mass spectrometer. Analysis of covariance (ANCOVA) was performed and partial correlations between gene expression and mercury levels were calculated, after correcting for age and batch effects. To reduce false positives, only genes shared by the ANCOVA models were analyzed. Of the 26 genes that correlated with mercury levels in both AU and TD boys, 11 were significantly different between the groups (P(Diagnosis*Mercury) ≤ 0.05). The expression of a large number of genes (n = 316) correlated with mercury levels in TD but not in AU boys (P ≤ 0.05), the most represented biological functions being cell death and cell morphology. Expression of 189 genes correlated with mercury levels in AU but not in TD boys (P ≤ 0.05), the most represented biological functions being cell morphology, amino acid metabolism, and antigen presentation. These data and those in our companion study on correlation of gene expression and lead levels show that AU and TD children display different correlations between transcript levels and low levels of mercury and lead. These findings might suggest different genetic transcriptional programs associated with mercury in AU compared to TD children.
The online version of this article (doi:10.1007/s12640-009-9137-7) contains supplementary material, which is available to authorized users.
Autism (AU) is a neurodevelopmental disorder characterized by impaired social interactions and impaired verbal and nonverbal communication as well as restricted, stereotyped patterns of behavior and interests. The etiology(ies) of autism remains elusive. The interactions of multiple genetic, environmental, and epigenetic factors are postulated to be involved (Ashwood et al. 2006; Hertz-Picciotto et al. 2006; London 2000). Exposures to heavy metals have received much attention because of their developmental neurotoxicity (WHO 1990, 1991; Tchounwou et al. 2003; Clarkson and Magos 2006). Mercury is a toxic and hazardous heavy metal that is ubiquitous in the environment, being found in fish, thermometers, dental amalgams, and in the atmosphere and affects multiple organ systems (WHO 1990, 1991; Tchounwou et al. 2003; Clarkson and Magos 2006; Counter and Buchanan 2004; Zahir et al. 2005). In animals, mercury binds to the endogenous tripeptide glutathione (GSH), and the resulting mercury–glutathione complex is metabolized by the ectoproteins gamma-glutamyl transpeptidase (GGT) and dipeptidase (Rice 2008; Newland et al. 2008; Guzzi and La Porta 2008). Mercury is then eliminated as GSH conjugates, GSH being produced by glutamyl-cysteine ligase (GCL) and conjugation by glutathione S-transferases (GST). The high affinity of mercury for sulfhydryl, and to a lesser degree hydroxyl, carboxyl, and phosphoryl groups of structural and functional proteins, is believed to be the biochemical basis for its harmful effects on biological systems (Clarkson and Magos 2006).
The element mercury exists in three species: elemental mercury (Hgo), inorganic mercury compounds (primarily mercuric chloride), and organic mercury (primarily methylmercury). Exposure to each mercury species can cause general as well as specific toxic effects in children and adults (Clarkson and Magos 2006; Counter and Buchanan 2004). For example, methylmercury derived mostly from fish consumption passes through the blood–brain barrier as an l-cysteine complex via the l-amino acid transporter (Clarkson and Magos 2006; Mokrzan et al. 1995). The mechanisms by which inorganic mercury damages human brain, however, are poorly understood. The different mercury species have different absorption, distribution, metabolism, excretion, and toxicity (Guzzi and La Porta 2008; Ercal et al. 2001; Quig 1998). In addition, the effect of a toxicant can be influenced by another substance, so-called "mixture toxicity" (Rice 2008; Newland et al. 2008; McCarty and Borgert 2006a, b).
The development of whole genome microarrays has made it possible to characterize how the entire transcriptome of cells or organisms responds to changes in external or internal environments. Measurements of gene expression (RNA) levels provide a "transcriptional signature" of an individual's genetic, physiological, and environmental status. The relationship between levels of xenobiotics and transcriptional signatures measured in blood can be used to provide mechanistic information about toxic exposures (Yoon et al. 2008).
The objective of the current study was to analyze the entire transcriptome in similar aged typically developing (TD) and AU boys to identify those genes whose expression level in blood correlated with relatively low total mercury (Hg) levels in blood. Moreover, the correlations of RNA levels with Hg levels might differ in AU compared to TD boys. Lastly, we postulated that differences in RNA expression signatures for AU compared to TD boys would occur not only for Hg but would also be different for other xenobiotics including lead (Tian et al. 2009, accompanying paper). This study begins to address these hypotheses.
Subjects were participants in the CHARGE study (Hertz-Picciotto et al. 2006). These children were assessed at the UC Davis Medical Investigations of Neurodevelopmental Disorders (M.I.N.D.) Institute. Each child was evaluated using standardized clinical assessments. AU cases were assessed using the Autism Diagnostic Interview–Revised (ADI-R) (Le Couteur et al. 2003; Lord et al. 1994, 1997) and the Autism Diagnostic Observation Schedules (ADOS) (Lord et al. 2000, 2003). For this study of gene expression versus Hg levels, we analyzed boys with AU (n = 33, mean age 45.3 with a range of 31–60 months) and age-matched TD control boys (n = 51, mean age 43.3 with a range of 28–57 months). Only subjects for whom the Affymetrix expression data was generated in 2007–2008 were analyzed because Affymetrix arrays processed prior to those times were done using a different protocol which resulted in significant technical variation. Only boys were analyzed in our study because there were few girls who had both Affymetrix expression data and blood Hg data. The subjects used in our companion study on correlation between gene expression and blood lead levels are different from the ones used in this study. Parents provided informed consent for all subjects (Tian et al. 2009). The study was approved by the Institutional Review Board at University of California Davis Medical Center and was conducted in accordance with the Declaration of Helsinki.
Total blood Hg was measured on an Agilent 7500i Inductively Coupled Plasma Mass Spectrometer (ICP-MS) (Agilent, Palo Alto, CA) in the UCD Department of Civil and Environmental Engineering. Detailed methods are included in our previous publication (Hertz-Picciotto et al. 2009). Blood Hg concentrations were log2-transformed due to the skewed distribution over a wide range of values (Fig. 2). The normality of the distributions was assessed by performing the Kolmogorov–Smirnov test in Partek Genomics Suite 6.4. The detection limit for Hg was 0.02 μg/l (Hertz-Picciotto et al. 2009), with the (slightly) lower detection limit of 0.01 μg/l (limit of barely detected). Log2-transformation of the Hg levels was performed to produce a more linear distribution of the values and to match the log2-transformation of gene expression (see below). For log2-transformation of the Hg data, Hg levels below detection levels were assigned a value of 0.009 μg/l. The value of 0.009 was selected to be slightly lower than the lower limit of Hg detection of 0.01 μg/l in order to not create "outlying" values, which can artificially influence the correlation coefficient.
Whole blood was collected and total RNA was isolated using PAXgene tubes and kits (Qiagen, Valencia, CA). RNA concentration and purity were checked using a Nanodrop ND-1000 spectrophotometer, and integrity was checked using the Agilent 2100 Bioanalyzer (260/280>2.0; 28S/18S>1.5; RIN>8).
Human whole genome U133 Plus 2.0 GeneChip microarrays (Affymetrix; Santa Clara, CA), surveying over 54,000 probe sets, were used. The 54,000 probe sets represent over 38,500 potential human genes (Affymetrix Manual). The standard Affymetrix protocol was followed for the sample labeling, hybridization, and image scanning.
Raw gene expression values from Affymetrix.cel files were imported into Partek Genomics Suit, version 6.4, release 6.09.0422 (Partek Inc., St. Louis, MI). Probe summarization and probe set normalization were performed using the GC-RMA algorithm (Wu and Irizarry 2004), including GC-RMA background correction, Quantile Normalization, log2-transformation, and Median Polish probe set summarization.
Analysis of covariances (ANCOVAs) were performed in Partek Genomics Suite, version 6.4, release 6.09.0422 (Partek Inc., St. Louis, MI). We estimated the partial correlations between log2-transformed gene expression and log2-transformed Hg levels while removing the effects of age and microarray technical variation (batch) (Eisenhart 1947). Partial correlation coefficients describe the linear relation between two variables (in our case, gene expression and Hg level) while controlling for the effects of one or more additional variables (Morrison 1976). Correlations are a measure of linear association. A positive partial correlation between Hg level and gene expression indicates that gene expression is increasing when Hg levels are increasing. Similarly, a negative partial correlation indicates that gene expression is decreasing when Hg levels are increasing. We performed the ANCOVA on several models (see below). We overlapped these gene lists (Fig. 1) to reduce the large false discovery rates due to multiple comparisons between thousands of genes. The following ANCOVA models were used.
Log2(Gene Expression) is a function of the following factors: Log2Hg (continuous), Age (continuous), and Batch (categorical). Genes with a P(Log2(Hg)) ≤ 0.05 in this model would be associated with the left circle in Fig. 1.
Log2(Gene Expression) is a function of the following factors: Log2Hg (continuous), Age (continuous), and Batch (categorical). Genes with a P(Log2(Hg)) ≤ 0.05 in this model would be associated with the right circle in Fig. 1.
Log2(Gene Expression) is a function of the following factors: Log2Hg (continuous), Age (continuous), Batch (categorical), Diagnosis (binary categorical), and interaction Diagnosis*Log2Hg.
Genes with a P(Diagnosis*Log2Hg) ≤ 0.05 would be associated with the top circle in Fig. 1.
Batch in all three models is a random effect. We focused on gene lists that came from the combined analyses to minimize the false discovery rate (Fig. 1, intersections labeled A–D).
Gene ontology annotation was obtained from the Affymetrix NetAffix database (www.affymetrix.com). Pathway analysis was performed using Ingenuity Pathways Analysis (IPA) (Ingenuity Systems®, www.ingenuity.com). A dataset containing gene identifiers (called focus molecules) from each of the lists (A, B, C, or D) was uploaded into the program. Each identifier was mapped to its corresponding gene object and overlaid onto a global molecular network developed from information contained in the Ingenuity knowledge base. The Bio Functional Analysis identified the biological functions and/or diseases that were most significant to the dataset. Canonical Pathway Analysis identified the pathways from the IPA library of canonical pathways that were most significant to the dataset. The significance of the association between the dataset and canonical pathways was assessed by calculating the ratio of the number of genes from the dataset that map to the pathway divided by the total number of molecules that exist in the canonical pathway. A Right-tailed Fischer's Exact Test was then used to calculate a P-value determining the probability that each biological function and/or disease assigned to that dataset is due to chance alone. A P < 0.05 was considered to be statistically significant for over-representation of the molecules in a given process. Thus, over-represented functional or pathway processes are processes which have more focus molecules than expected by chance. It should be noted that for lists A and B (Figs. 1, ,3),3), due to the small number of genes, pathway over-representation is not possible. In this case, IPA was used to map the genes into their corresponding pathways and biofunctions. Genomatix software (http://www.genomatix.com) was utilized to identify over-represented transcription factor binding sites in each of the four gene lists (A–D, Fig. 1).
Chromosome and cytoband enrichment for each of the gene lists (lists A, B, C, and D) as well as functional annotation of the probe sets in the enriched chromosomes was performed in DAVID (http://david.abcc.ncifcrf.gov/tools.jsp). A modified Fisher Exact P-value (the threshold of EASE score) for gene enrichment analysis was used. We considered the default P ≤ 0.1 to be considered strongly enriched in the annotation categories. A default value of 2 for the threshold of the minimum gene counts belonging to an annotation term was adopted. Visual representation of the statistically enriched chromosomes was done using Partek and was presented in Supplementary figures 1–3.
Total blood Hg levels ranged from below the lower detection limit (1 sample with [Hg] < 0.01 μg/l) to 4.3 μg/l in the TD group, and from below the lower detection limit (4 samples with [Hg] < 0.01 μg/l) to 3.0 μg/l in the AU group (Fig. 2). The distribution of the linear Hg levels was significantly different from the normal distribution (Kolmogorov–Smirnov test: P-value = 3.73E−04, 7.58E−03, and 6.28E−05 for TD, AU, and TD + AU, respectively). However, the distribution of the log2-transformed Hg levels followed a normal distribution (Kolmogorov–Smirnov test: P = 0.90, 0.97, and 0.94 for TD, AU, and TD + AU, respectively). There was no significant difference in Hg levels between the TD and AU groups (P = 0.42 and P = 0.07, two-sample unpaired t-test, unequal variances on the linear Hg levels and the log2-transformed Hg levels, respectively). The mean (±SD) Hg level was 0.60 (±0.82) and 0.46 (±0.73) μg/l for TD and AU, respectively. The geometric mean Hg level was 0.29 and 0.16 μg/l for TD and AU, respectively. The median was 0.30 and 0.16 μg/l for TD and AU, respectively. The inter-quartile range was 0.14–0.67 and 0.07–0.54 μg/l for TD and AU, respectively. The correlative analysis of gene expression and Hg levels were performed on log2-transformed data.
ANCOVA analyses, in which the TD boys and AU boys were modeled separately, revealed 1,293 and 1,276 probe sets, respectively. The expression levels for these genes significantly correlated with Hg levels (P ≤ 0.05, Fig. 3, bottom left and right circles). Similarly, an ANCOVA analysis, in which TD and AU boys were combined, revealed 1,113 genes that correlated with Hg levels, with P ≤ 0.05 for the interaction Diagnosis (TD, AU)*log2Hg (Fig. 3, top circle). No genes passed a False-Discovery Rate filter in any of the models. Therefore, to reduce the false discovery inherent to microarray studies, we focused our analyses on the genes that were identified in more than one of the analytical models (intersections in Fig. 3, denoted as lists A–D). This approach reduced the numbers of genes approximately 4–6 times.
There were 15 probe sets whose expression correlated with Hg levels in both the AU and TD groups, and for which the correlation coefficients did not significantly differ between the two groups (Fig. 3, list A). Figure 4 shows a heat map of the partial correlation coefficients with Hg levels for each of the 15 probe sets (rows) in the TD and AU groups (columns). Most of the probe sets showed same sign correlations with Hg levels both in the TD and AU groups. Exceptions were LOC10012902 and TXNDC17 (refer to abbreviation list for full gene name), which showed different sign correlations with Hg in the TD and AU groups. The P-values for their interaction (Diagnoses*log2Hg) were 0.06 and 0.14 which are higher than the statistical cutoff of P ≤ 0.05. This is why they were not grouped into list B (most contrasting genes). The 15 probe sets (Supplementary Table 1) represent 10 annotated genes which are involved mainly in cell death (CADM1, MSH6, SSTR3, TXNDC17, P-value range 9.92E−04 to 4.40E−02), signaling (TXNDC17, SSTR3, FEZ2), the immune response (PXDN, CADM1), and response to oxidative stress (PXDN) (Table 1). Analysis of the promoter sequences of these genes using Genomatix software revealed binding site sequences for the transcription factor SP1F family (P = 0.032, minimum 86% of sequences). The target genes are MSH6, SSTR3, PXDN, and LMLN. Chromosome 2 showed significant enrichment with 4 out of the 15 probe sets (Table 2, Supplementary Fig. 1).
There were 11 probe sets, whose expression correlated with Hg levels but in opposite directions for TD compared to AU (Fig. 3, list B). Figure 5 shows a heat map of the partial correlation coefficients with Hg levels for each of the 11 probe sets (rows) in the TD and AU groups (columns). The majority of the genes show positive correlations with Hg levels in the TD group but negative correlation with Hg levels in the AU group. The 11 probe sets (Supplementary Table 1) represent seven annotated genes associated with cellular assembly and organization (NTRK3, FLNB, NCAPD3, P-value range 3.31E−04 to 2.91E−02), cellular compromise (NTRK3, FLNB, P-value range 6.62E−04 to 1.32E−03), small molecule biochemistry (KRT1, NTRK3, P-value range 9.92E−04 to 3.07E−02), vitamin and mineral metabolism (KRT1, P-value = 9.92E−04), and cell death (NTRK3, P-value = 1.35E−02) (Table 1). Analysis of the promoter sequences of these genes using Genomatix software revealed binding site sequences for the transcription factor MYBL family (P = 0.0069, minimum 86% of sequences) and the AP1R family (P = 0.0289, minimum of 86% of sequences). The target genes are FLNB and NTRK3. No significant chromosome nor cytoband enrichment (at P ≤ 0.1) was observed.
There were 316 probe sets (Supplementary Table 1) whose expression correlated with Hg levels in the TD group but not in the AU group (Fig. 3, list C). Of these, 96 were positively correlated with Hg levels and 220 were negatively correlated (Supplementary Table 1). Figure 6 shows a heat map of the ten highest positive and ten lowest negative partial correlation coefficients with Hg levels in the TD group compared to their partial correlation coefficients in the AU group. The most represented molecular and cellular functions were cell death (37 molecules, P-value range 6.09E−04 to 4.98E−02), cell morphology (27 molecules, P-value range 9.68E−04 to 4.55E−02), gene expression (39 molecules, P-value range 2.67E−03 to 3.43E−02), cellular assembly and organization (29 molecules, P-value range 5.34E−03 to 4.98E−02), and cell cycle (17 molecules, P-value range 5.39E−03 to 4.55E−02) (Table 1). Analysis of the promoter sequences of these genes using Genomatix software revealed binding site sequences for the transcription factor ETSF family (P = 0.072, minimum 86% of sequences). Chromosomes 19, 7, and 13 showed significant enrichment with 30, 20, and 10 out of the 316 probe sets, respectively (Table 2, Supplementary Fig. 2). Functional annotation of the genes on chromosome 19 showed that they are involved in nucleic acid binding (43.3%, P = 0.01) and protein binding (60%, P = 0.09), whereas the genes on chromosome 7 have protein serine/threonine kinase activity (13%, P = 0.05). Functional annotation of the genes on chromosome 13 did not show significant over-representation of molecular functions (at P ≤ 0.1). Cytoband enrichment was noted for 22q12.1 (3/316, P = 0.03), 18q12-q21 (2/316, P = 0.04), 12q24.33 (3/316, P = 0.06), and 19q13.4 (4/316, P = 0.08).
There were 189 probe sets (Supplementary Table 1) whose expression correlated with Hg levels in the AU group but not in the TD group (Fig. 3, list D). Of these, 62 were positively correlated with Hg levels and 127 were negatively correlated (Supplementary Table 1). Figure 7 shows a heat map of the ten highest positive and ten lowest negative partial correlation coefficients with Hg levels in the AU group compared to their partial correlation coefficients in the TD group. The most represented molecular and cellular functions for the 189 probe sets (Table 2) were cell morphology (DIRAS1, PIP5K3, ANK1, CAPZB, ASAP1, RRBP1, P-value range 1.64E−04 to 4.39E−02), amino acid metabolism (GGT1, SLC25A22, SLC7A8, P-value range 7.33E−03 to 3.56E−02), antigen presentation (PPT2, NLRP1, NOD1, SLC11A1, HLA-DQA1, and HLA-DQB1, P-value range 7.44E−03 to 1.77E−02), carbohydrate metabolism (PIP5K3, SLC35B4, P-value range 7.44E−03 to 2.95E−02), and cell cycle (SASS6, PIP5K3, AMACR, P-value range 7.44E−03 to 2.22E−02). Analysis of the promoter sequences of these genes using Genomatix software revealed binding site sequences for the transcription factor KLF family (P = 0.00015, minimum 86% of sequences). Chromosomes 1 and 8 showed significant enrichment with 25 and 11 out of the 189 probe sets, respectively (Table 2, Supplementary Fig. 3). Functional annotation of the genes on chromosome 1 showed that they are involved in heat shock protein binding (8%, P = 0.07), whereas the genes on chromosome 8 are involved in protein kinase activity (11%, P = 0.1). Cytoband enrichment was noted for 1p33 (3/189, P = 0.02) and 6p21.3 (5/189, P = 0.05).
Finally, we performed an analysis of all genes whose expression correlated with Hg levels regardless of diagnostic group or sign of correlation (all four gene lists combined—A–D). The rationale for doing this was to ask whether there were genes in all four lists that were associated with the same functional networks. The IPA analysis revealed that the top molecular and cellular functions of the genes correlating with Hg levels were cell morphology (29 molecules, P-value range 1.19E−03 to 4.01E−02), cell death (24 molecules, P-value range 2.35E−03 to 4.60E−02), cellular assembly and organization (25 molecules, P-value range 3.87E−03 to 4.01E−02), cellular movement (8 molecules, P-value range 3.87E−03 to 4.988E−02), and cellular development (15 molecules, P-value range 5.72E−03 to 4.49E−02). The top disease and disorder biofunctions correlating with Hg levels were cancer (30 molecules, P-value range 6.03E−04 to 4.98E−02), hematological disease (14 molecules, P-value range 6.02E−04 to 4.89E−02), genetic disorder (185 molecules, P-value range 1.81E−03 to 3.98E−02), neurological disease (103 molecules, P-value range 2.35E−03 to 3.98E−02), and skeletal and muscular disorders (11 molecules, P-value range 1.04E−02 to 3.98E−02). The top canonical pathways correlating with Hg levels were GABA receptor signaling (4/55 molecules, P-value 1.16E−02), lysine degradation (7/234 molecules, P-value 2.74E−02), thyroid cancer signaling (3/45 molecules, P-value 4.04E−02), and cyanoamino acid metabolism (2/64 molecules, P-value 4.98E−02). Figure 8 shows the IPA network with the highest score. It is notable that many genes are regulated in this network, but that the Hsp70, Hsp90, proteasome, NFκB, Raf, and caspase nodes themselves are not regulated.
To gain more insight into the associations between transcript levels and circulating Hg levels in the TD and the AU children, and to evaluate the biological significance of the differences between the TD and AU boys, we compared the genes that correlated with Hg levels only in the TD group (List C), and genes that correlated with Hg levels only in the AU group (List D). We split each gene list into two lists, based on the direction of the correlation coefficients—into a list of genes whose expression positively correlates with Hg levels and into a list of genes whose expression negatively correlates with Hg levels. The IPA canonical pathways over-represented in at least one of the four categories (TD (+) and TD (−) from List C, and AU (+) and AU (−) from List D, signs represent the direction of the partial correlation) are shown in Fig. 9. The TGF-beta signaling pathway (Fig. (Fig.10)10) and molecular mechanisms of cancer (P = 3.48E−02, 2/86 and P = 3.66E−02, 4/370, respectively) are over-represented in the positively correlating genes between Hg level and gene expression in the TD group. Lysine degradation (P = 3.63E−02, 4/234) was over-represented in the negatively correlating genes between Hg level and gene expression in the TD group. p38 MAPK signaling and α-adrenergic signaling were over-represented in the positively correlating genes between Hg level and gene expression in the AU group.
Though some of the genes and pathways are similar, the major finding of the study is that almost all of the genes that correlate with circulating Hg levels in AU boys are different from TD boys, and almost all of the genes that correlate with circulating Hg levels in TD boys are different from AU boys. These transcriptional differences were observed even though the Hg levels were low and not significantly different between the groups in our study. Since the data reported are only correlations, no conclusions can be drawn as to whether Hg plays any role in the pathogenesis of autism based upon the current results. Given the low Hg levels and the fact that this is a cross-sectional study, no cause and effect relationship should be drawn from the current data. Though the data can be interpreted in several ways, we suggest that the different transcriptional programs associated with Hg in AU compared to TD subjects may be related to the genetic differences in the two groups of children.
The major finding of this study is that very few genes that correlated with Hg levels in AU subjects also correlated with Hg levels in TD subjects. There were only 15 genes that correlated with Hg levels in both the TD and AU groups—and that were not significantly different between the two groups. These genes were involved in apoptosis, the immune response, and response to oxidative stress. Moreover, there were only 11 genes whose expression correlated inversely with Hg levels in AU compared to TD subjects—that is genes correlating with Hg levels in both groups but in opposite directions. These genes were involved in pathways contributing to neuronal development and neuronal survival and cell death. Notably, one gene (FLNB, filamin B, beta (actin binding protein 278)), which was positively correlated with Hg levels in TD and negatively correlated in AU subjects, is involved in anchoring and biogenesis of cytoskeleton and organization of actin filaments (Xu et al. 1998; Tu et al. 2003; Leedman et al. 1993). A number of studies show that most heavy metals, including zinc, arsenic, mercury, and cadmium, can disrupt actin and microtubules in intact cells (Templeton 2000).
The analysis of all genes correlating with Hg levels in all AU and TD subjects showed a number of common networks. Figure 8 is a good example where different genes from the different groups are regulated within the same network. This could suggest that similar functions might be accomplished with different genes in AU compared to TD, and that homeostasis is maintained in spite of the genetic differences of AU and TD subjects.
Thus, most of the genes whose expression correlated with Hg levels in the TD group were different from those whose expression correlated with Hg levels in the AU group. For the TD subjects, the top biological function for the genes that correlated with Hg levels was cell death. Some of these genes were involved in apoptosis of neurons (NFATC4, BAD, BDNF) (Graef et al. 2003; Ottilie et al. 1997; Hoey et al. 1995; Botta et al. 2004; Kerschensteiner et al. 1999; Mamounas et al. 1995), survival of neurons, recovering and regeneration of axons (BDNF, RTN4) (Kerschensteiner et al. 1999; GrandPre et al. 2000), apoptosis of pre-T lymphocytes (BCL2L11) (Bouillet et al. 1999), survival of pro-B lymphocytes, and survival of memory B cells (BRAF, BCL2L11, BDNF, TCF3) (Oliver et al. 2004; Fischer et al. 2007). It is interesting that most of these correlations are negative. Chronic exposure to low concentrations of heavy metals, including mercury, causes immune dysfunction related in large part to apoptosis of leukocytes (Pollard and Hultman 1997; Zelikoff and Gardner 1996; Laiosa et al. 2007; Madureira et al. 2007; Ziemba et al. 2005; Mondal et al. 2005; McCabe et al. 2003, 2005; Cunha et al. 2004; Sarmento et al. 2004; Kuo and Lin-Shiau 2004; Colombo et al. 2004; Field et al. 2003). Both inorganic and organic mercurials cause human T-cell apoptosis with mitochondria being the target organelle for the induction of cell death (Shenker et al. 1999). In addition, microarray analysis of Hg-induced changes in gene expression in human liver carcinoma (HepG2) cells revealed genes involved in apoptosis and metabolic regulation with significant effects on genes involved in immune system pathways (Ayensu and Tchounwou 2006). However, the levels of Hg applied to the cells were much higher than the Hg levels of the subjects in our study.
The second major biological function for genes that correlated with Hg levels in TD subjects was cell morphology and cell death. Mercury-induced cell death can be apoptotic or nonapoptotic. The nonapoptotic cell death produced by mercury is associated with a specific cellular morphology (Pollard et al. 1997, 2000). Mercury-induced apoptosis results from dys-regulated signal transduction that leads to inactivation of enzymes, generation of free radicals and peroxides, protein conformation changes, and inhibition of transport processes which disrupt cell membrane permeability (Tchounwou et al. 2003; Clarkson and Magos 2006; Counter and Buchanan 2004; Zahir et al. 2005). Genes associated with transmembrane potential and permeabilization of mitochondria correlated with Hg levels in TD subjects in our study. Mercury-induced membrane changes of mitochondria associated with glutathione imbalance and antioxidant defenses have been reported in TD versus AU transformed lymphoblastoid lines (James et al. 2009). Mercury increases mitochondrial permeability, which is characterized both by a decline in transmembrane potential and intracellular pH, as well as the generation of reactive oxygen species and a decline in T-cell GSH content (Shenker et al. 2000). In our study, Granulysin (GNLY) showed a positive correlation with Hg levels in the TD group. GNLY is a cytolytic and pro-inflammatory molecule, which contributes to mitochondrial damage (Krensky and Clayberger 2009). B-cell lymphoma 2-like protein 11 (apoptosis facilitator) (BCL2L11) showed a negative correlation with Hg in the TD group in our study. BCL2L11 (also known as BIM) is a BH3-only protein from the Bcl-2 family (Youle and Strasser 2008), whose family members are the main regulators of programmed cell death via the mitochondrial (intrinsic) apoptotic pathway. Interactions between pro- and anti-apoptotic proteins of the Bcl-2 family decide the fate of cells in response to stress signals. BCL2L11 is essential for hematopoietic homeostasis and thymocyte negative selection, and suppresses autoimmunity (Youle and Strasser 2008).
Some of the cell morphology genes that correlated with Hg levels in TD subjects also have functions within the central nervous system. These included genes that affect neuronal survival (ABL1 (+), BDNF (−)), sprouting and projection of axons (BDNF (−), RTN4 (−)), plasticity of the synapse (BDNF (−), NFATC4 (+), PMCH (−)), and remodeling of axons (SFRP1 (−)) (Kerschensteiner et al. 1999; GrandPre et al. 2000; Gascon et al. 2007; DeFreitas et al. 2001; Mamounas et al. 2000; Fournier et al. 2001; Seil 1998; Groth and Mermelstein 2003; Krylova et al. 2002). It is possible that mercury affects these genes in the brain as well as the blood, and this might relate to neurotoxicity observed following exposure to much higher levels of mercury (Rice 2008; Guzzi and La Porta 2008).
We observed a negative correlation between GCLM and Hg levels in TD, but no correlation in AU. GCLM is a member of the glutamate-cysteine ligase complex, which is the first rate-limiting enzyme of the glutathione synthesis, and is the major route of mercury detoxification.
James et al. (2009) observed a reduced glutathione reserve capacity in both cytosol and mitochondria in lymphoblastoid cells derived from children with autism when compared to children without autism. Glutathione reserve capacity is an indicator of systemic redox status that can be used to assess and treat individuals at risk of oxidative stress-related pathology (Jones 2006a, b). In our study, we observed a negative partial correlation between GCLM transcript abundance and Hg levels in the TD group, while there was no significant correlation in the AU group. GCLM is transcriptionally regulated in human HepG2 cells (Lee et al. 2006). We also observed a negative partial correlation between GGT1 transcript abundance and Hg levels in the AU group, while there was no significant correlation in the TD group (see section "Genes Whose Expression Correlates with Hg Levels in AU but not in TD Boys (List D)"). GGT1 is involved in the utilization of l-glutamate, l-cysteine, and glutathione metabolism. It is involved in the generation of l-glutamate, which is a substrate for glutathione synthesis. Thus, if there is negative regulation of this gene, there may be less substrate for the glutathione biosynthesis pathway, which may affect the redox status. Overall, our studies of transcript abundance in blood of AU and TD boys are difficult to relate to the in vitro studies of AU and TD lymphoblastoid cell lines except to say that both approaches show associations between genes in the glutathione pathways and Hg levels.
CDKN2AIPNL and ZMYM6 negatively correlated with Hg levels in this study and both were downregulated with mercury treatment in human liver carcinoma (HepG2) cells (Ayensu and Tchounwou 2006). ALDH3B1, RPS9, SUB1, and VPS25 also were negatively correlated with Hg in TD subjects in this study and were downregulated in Hg-treated yeast (Jin et al. 2008).
Genes whose expression correlated similarly with Hg levels both in TD and AU (List A) and whose expression correlated with Hg levels only in the AU group (List D) have transcription factor (TF) binding sites for SP1 and Kruppel-like factor (KLF)-like TFs, respectively. KLF6, which is an Sp1-like zinc finger transcription factor, is a transactivator of inducible nitric oxide synthase (iNOS) (Warke et al. 2003), which produces NO and modulates inflammatory signals and oxidative stress (Mungrue et al. 2003).
One of the top biological functions in the list of genes that correlated with Hg levels in AU but not TD boys was amino acid metabolism. The genes included gamma-glutamyltransferase (GGT1/2/3; probe 244179_x_at); as well as solute carrier family 25 (mitochondrial carrier: glutamate), member 22 (SLC25A22 (−)), and solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 (SLC7A8 (−)) which are involved in the transport of l-amino acids (Pineda et al. 1999). SLC7A8, which had negative partial correlation with Hg levels in the AU group in our study, was upregulated in mercury-treated yeast cells (Jin et al. 2008).
FAF1 (Fas-associated factor 1) expression was positively correlated with Hg levels in AU but not TD subjects. Fas is a member of the tumor necrosis receptor factor family, which can induce apoptosis when activated by Fas-ligand binding (Chu et al. 1995). Apoptosis induced by Fas plays an important role in the development and function of the immune system (Lowin et al. 1994; Nagata 1994). Genetic studies in mice show that defects in Fas-mediated apoptosis resulted in abnormal development and function in the immune system (Takahashi et al. 1994). FAF-1 also inhibits the NF-κB activation induced by various stimuli, including tumor necrosis factor-α, interleukin-1β, and lipopolysaccharide (Park et al. 2004). NF-κB is a central regulator of genes involved in the immune response, inflammatory response, and apoptosis (Beg and Baltimore 1996; Pahl 1999; Baldwin 1996).
CHCHD4 (negatively correlated with Hg level in AU), ELOVL1, SLC7A8, and Cxorf34 (positively correlated with Hg level in AU) responded to mercury in yeast in the same direction as in our AU subjects. NTHL1 (positively correlated with Hg level in AU) was downregulated in yeast after mercury treatment, whereas MYBBP1A (positively correlated in AU) was up- or downregulated depending on the treatment dose (Jin et al. 2008).
HLA-DQ, an αβ heterodimer of the MHC Class II type which is a cell surface molecule found on antigen presenting cells, presents antigens to T-lymphocytes (Germain and Margulies 1993; Janeway et al. 1997; Kwok and Nepom 1998). The α and β chains are encoded by HLA-DQA1 and HLA-DQB1, respectively, and both the α- and β-chains vary greatly in the population. Different DQ isoforms can bind to and present different antigens to T-cells. Several lines of evidence suggest that mercury toxicity depends on the mercury species, dosage, individuality, and the type of MHC association (Ayensu et al. 2004; Hanley et al. 1997, 2002). Moreover, chronic exposure to low concentrations of heavy metals such as mercury results in immune dysfunction (Ayensu et al. 2004; Hanley et al. 1997, 2002). Immune dysregulation has been observed in autism, and has been postulated to play a role in the development and pathogenesis of autism in some cases (Warren et al. 1996, 1997; Singh and Rivas 2004; Ashwood et al. 2006; Gupta et al. 1996). In this study, we found that HLA-DQA1 was negatively correlated, whereas HLA-DQB1 was positively correlated with Hg levels in AU but not TD subjects. They both were downregulated in response to mercury in human liver carcinoma (HepG2) cells (Ayensu and Tchounwou 2006).
There is a different association between transcript level and circulating Hg levels in the TD and AU subjects that involves genes from the TGF-β signaling pathway. TGF-β signaling regulates cell growth, cell differentiation, cellular homeostasis, apoptosis, and immune responses. TGF-β binds to and activates the TGF-β receptor, which then activates the SMAD family of transcriptional regulators. SMAD2 and SMAD3 mediate TGF-β and activin signals, while SMAD1, SMAD5, and SMAD8 act downstream of bone morphogenetic proteins (BMPs) (Zhang and Derynck 1999; ten Dijke et al. 2000; Massague and Wotton 2000). SMADs dimerize with SMAD4 and translocate to the nucleus where the complex modulates transcription of target genes. In our study, we found bone morphogenetic protein receptor type 2 (serine/threonine kinase) (BMPR2) and SMAD2 to be positively correlated with Hg levels in TD but not in AU subjects. Notably, Ashwood et al. (2008) observed decreased TGF-β1 plasma levels in children with autism spectrum disorders when compared to typically developing controls.
Hg levels in children from the CHARGE study with and without autism have been studied in detail (Hertz-Picciotto et al. 2009). The Hg levels in the TD and AU groups were quite similar in this sample to those in the larger CHARGE sample: means of 0.60 μg/l for TDs in both groups, and means of 0.46 and 0.49 μg/l for the AU/ASD children in this subsample and the larger study (Hertz-Picciotto et al. 2009). Thus, the sample of subjects in this study is representative of the CHARGE study as a whole. None of the samples used in this study had Hg levels higher than the EPA reference level of 5.8 μg/l. The median Hg concentration and inter-quartile range were comparable to those of the U.S. population of children ages 1–5 years in 1999–2002 (McDowell et al. 2004). It is notable, therefore, that the gene expression differences described here occurred in the EPA allowable range. This is consistent with previously reported dose-dependent effects of Hg (Tchounwou et al. 2003; Clarkson and Magos 2006; Counter and Buchanan 2004; Zahir et al. 2005) and with a dose-dependent transcriptional response in human liver carcinoma cell lines treated with mercury (Ayensu and Tchounwou 2006). It is important to emphasize that acute intoxication with much higher mercury concentrations or even chronic exposure to much higher Hg concentrations might produce different transcript associations.
Samples collected from children ages 2–5 in this study do not directly address the role of Hg as a possible causal factor for autism which likely begins in utero or shortly after birth. In addition, it is important to emphasize that one cannot infer cause and effect from the data presented in this study. Our data shows that there are associations between transcript levels and circulating Hg levels which are different in the AU compared to the TD group. It is possible that these low levels of Hg cause changes of gene expression in the white blood cells that could affect the function of immune cells that could play a role in the pathogenesis of autism. Alternatively, and perhaps more likely, the altered transcriptomic associations with low levels of Hg in AU may have no functional effect on immune cells, brain, or other organs. Further studies on protein levels of the genes whose expression was associated with mercury levels should be conducted. Protein levels may or may not reflect RNA levels, particularly in stressed cells. Again, correlation does not mean causality and extreme caution should be exercised when interpreting the correlations reported here. In addition, since this is a one-time study, the correlation of an individual gene might not be replicated in future studies due to stochasticity.
The P-values which were used to derive the gene lists indicate statistical but not biological significance. It is unclear what level of correlation of gene expression and Hg levels represents biological significance. Correlation is also affected by "outlying" values. One way of reducing this effect is by log-transforming the data as was done here. Also, the somewhat unequal sample sizes may affect the calculation of the P-values and thus lead to some skewing of the data. The strengths of the correlations were not large for most of the identified genes. Therefore, caution should be exercised regarding the correlation of gene expression with Hg level for a given gene.
No gene reported here passed a multiple comparison correction for statistical significance. We addressed this problem in part by focusing on genes common to at least two of the ANCOVAs. However, the finding that so few genes correlated with Hg levels in both AU and TD subjects implies that multiple comparisons had no impact on the major conclusion of this study: AU and TD children have different associations between transcript level and circulating Hg levels. Increasing multiple comparisons would have made it likely that there would be many transcripts that correlated with Hg and were common to the AU and TD groups.
This study evaluated a single sample of blood where only 5% of the body burden of Hg is found (Burbacher et al. 2005; Stinson et al. 1989). The different Hg species were not measured (Goldman and Shannon 2001; Clarkson 2002). It is likely that different Hg species induce specific genes and affect specific enzymes that mediate Hg species specific toxicity (Clarkson and Magos 2006; Aschner et al. 2006).
The observed correlation between Hg levels and gene expression might be due to other factors that were not included in our ANCOVA models. Different diets of AU compared to TD subjects might affect gene expression. For example, fish is a major source of organic mercury intake and in the larger CHARGE study sample (Hertz-Picciotto et al. 2009) accounted for the differences in mercury levels between subjects. Fish contain n-3 polyunsaturated fatty acids, which are potent regulators of gene expression (Price et al. 2000). It is possible that some of the associations observed in this study are confounded by n-3 polyunsaturated fatty acids levels or other molecules found in fish.
Below are the links to the electronic supplementary material.
The authors thank the CHARGE study staff for recruitment, assessment, and data collection and the many families who participated. We also thank the UCD genomics core for processing the samples. This study was supported by NIH grant (I P01 ES11269) (1 R01 ES015359), the U.S. Environmental Protection Agency through the Science to ARCHIVE Results (STAR) program (R829388, R833292), a Cure Autism Now (Autism Speaks) grant, a UC Discovery (Industry-University Cooperative Research Program) grant, and an award from the Clinical and Translational Science Center (CTSC) and MIND Institute at UCD. The authors declare they have no competing financial interests.
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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VANCOUVER, Oct. 16 /PRNewswire-FirstCall/ - Beanstalk Capital Ltd. ("Beanstalk") is pleased to announce that it has entered into an arm's length arrangement agreement dated October 15, 2007 (the "Arrangement Agreement") with iCo Therapeutics Inc. ("iCo"). The proposed transaction (the "Arrangement"), when completed, will constitute Beanstalk's "Qualifying Transaction" as a capital pool company. Under the Arrangement, iCo will amalgamate with a wholly-owned subsidiary of Beanstalk and all of the issued and outstanding securities of iCo, including options and warrants, will be taken up by Beanstalk and paid for by the issuance of equivalent Beanstalk securities on a one-for-one basis. The amalgamated company, which will carry on the business currently conducted by iCo, will be a wholly-owned subsidiary of Beanstalk.
Beanstalk currently has 5,500,000 common shares, 250,000 warrants and 605,000 options issued and outstanding and iCo currently has 13,723,053 common shares, 1,638,708 warrants and 1,290,000 options issued and outstanding. Prior to the closing of the Arrangement (but after the financings described below), Beanstalk will consolidate its common shares on the basis of one share for each 2.8 shares currently outstanding. Following the closing of the Arrangement, current iCo shareholders will hold approximately 77.8% of the issued and outstanding Beanstalk Common Shares, current Beanstalk shareholders will hold approximately 11.1% of the issued and outstanding Beanstalk Common Shares and investors in the private placement described below will hold approximately 11.1% of the issued and outstanding Beanstalk Common Shares.
Under the terms of the Arrangement Agreement, Beanstalk has agreed to
use commercially reasonable efforts to complete an equity financing raising
a minimum of $1,802,500 prior to or concurrent with the closing of the
Arrangement Agreement. On October 15, 2007, B
|SOURCE Beanstalk Capital Ltd.
Copyright©2007 PR Newswire.
All rights reserved


text: (Photo by Bob Levey/Getty Images) [ Syracuse Wins Texas Bowl [Photo Gallery] By Keeler in the Morning December 28, 2013 Terrel Hunt scrambled 12 yards for a touchdown with 1 minute 14 seconds left to lift Syracuse to a 21-17 win over Minnesota in the Texas Bowl in Houston... Read More|Comment Category: Morning Show, Sports Tags: ncaa football, orange, syracuse, texas bowl Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


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Students Grades 1 to 6 requiring help with reading, homework, and study skills receive individualized help for one hour per week - Wednesdays or Saturdays. Free program. Registration required....
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Join us for stories, songs and crafts!
Sat Jan 02
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We read a new book every month and meet to discuss the funniest, spookiest, most mind-boggling, and suspenseful details followed by fun activities and cool prizes! To register, call 416 393 7610. Pick up a copy of this month'...
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I was thinking it would be a good idea in case the epidural doesn't take or something.. Plus it might give the hubby an idea what's going to happen and what to expect (along with me)...
Birthing or Lamaze classes even if planning on epidural?
I was thinking it would be a good idea in case the epidural doesn't take or something.. Plus it might give the hubby an idea what's going to happen and what to expect (along with me)...
Comments (9)
Your delivery hospital usually offers a day course that introduces terms and techniques. Even if you plan to be medicated, it would behoove you to at least go to this class, rather than have a crash course while you're in the delivery room. The class is taught by medical staff, so you'll be able to ask them questions, and they'll give you helpful tips, not only about the labor, but about pre and post delivery. Most often, your SO or designated delivery coach is invited to attend, which can be helpful to them in learning ways to help you be more comfortable during labor, and what to expect.
Even if you think you don't have any questions, others will be sure to bring questions up that maybe you hadn't thought of already, and hearing the answers always helps! I highly recommend it!
Just an FYI - you don't have to tolerate not being given an epi until 4 cm - that is an outdated practice. Current recommendations from both ACOG and the association of anethesiologists is that women be given the epi whenever they are in enough pain to request it.
Someone trying to deny me my epi due to arbitrary measurements is one of my greatest anxiety points - I have a whole folder full of the research supporting placement prior to 4cm if anyone is interested. :) They don't make men with kidney stones wait to a certain point before treating them - it is bull and crap that they might try to deny a woman pain relief.
Also thank you for asking this question - I'm in the same boat as you and still debating a class. I keep deciding against it but then wondering what I'm missing.
Married 3/2004 ~ had our little girl in April 4th 2013! She was born deaf and we are learning a lot from her about hearing loss and we are learning to sign with her. She has changed our world in the best possible way!
Thanks ladies.. Im going to see what is offered around where I live. (But really hoping the epidural works)


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Let's Not Laugh (Too Much) At #SochiProblems: Just Say 'No' To Poverty Porn
In Defense Of Sochi 'Whining'
AU PACT Welcomes Sojourners Lisa Sharon Harper
Dumpster Diving As An Act Of Faith
For the World's Poor, Violence Is an Everyday Threat
Caleb is a father in Africa. He works hard as a night watchman, and he and his wife save from their small income with the dream of sending their daughter to college. But the family's dreams are destroyed when the police arrest Caleb on a random sweep for a robbery he had nothing to do with. This is not to say that the evidence against him was flimsy; there is no evidence against him whatsoever. The police needed to show an arrest had been made, and Caleb was an easy target … because he was poor.
Once in police custody, Caleb is viciously beaten. He is shaken down for bribes. And then, he is thrown in jail and charged with a capital offense. He is given no indication of when he might have a chance to prove his innocence – and even if he were, Caleb can't afford a lawyer to help him. His family struggles to hang on without him.
What is perhaps most stunning about Caleb's story is not the brutality (though it certainly is brutal), the singular unfairness of it all (though it is dramatically and utterly unjust), the hopelessness (though the story is obviously devastating). No, what is most stunning is just how ordinary Caleb's story is.
What's Wrong With Inequality
AUDIO: StandUp w/PeteDominick - Jim Wallis: How To Win The "War On Poverty"
5 Reasons Why Jesus People Ought Oppose The Death Penalty
As I stated yesterday, I believe that America's justice system is broken and in need of desperate repair. One of those areas is the practice of putting our citizens to death, something I believe that all Jesus People should resoundingly oppose.
When I was a conservative evangelical, I was a huge supporter of capital punishment for all of the standard reasons. I even had a quick response when folks correctly brought up the hypocrisy of being against abortion while simultaneously being pro-death penalty, a position I previously argued you can't hold and still call yourself "pro-life."
However, when I decided to follow Jesus instead of simply being a Christian who paid him hollow worship while conveniently ignoring the red words, I was forced to abandon my support of the death penalty (and abandon my support of violence in general) as part of Following Jesus 101.
While America's broken justice system is a complex issue, perhaps the first area we can fix is by abolishing the death penalty in all 50 states. Here's why I think Jesus People should be leading the charge on this issue:


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A short documentary exploring the history and character of Great Britain's oldest working commercial bakery, established in the early 16th century and still going strong in Plymouth on the Barbican.
This old business is full of character, with rooms filled with vintage ornaments and old baking equipment.
Song - 'Behind The Seas' by Possimiste (Free Music Archive)
Filmed and Edited by Ritchie Hunt
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It just wouldn't be a Tarantino film without him.
Just don't do it.
If you click here, there's an embedded video that for the 'Ghostbusters' theme song. I swear to God.
Say your prayers and it might happen sooner.
Get ready to feel really, really old.
He was heads above the rest.
Let's go get drunk.
These space ladies should have the right to compete too.
Now you can live Oprah's nightmare with Tom Cruise all up in your face.
It's scheduled to come out in fall 2012. Come on, Mayans. Do your thing!
The 'Spartacus: Blood and Sand' star has passed away.
Born to be a flop.
If I see so much as one mo-cap pingpong ball within fifteen feet of Denzel, so help me God, I'm burning down that set.
Arnold just wants to act, man.
I think the survivors will envy the dead in this scenario.
Holla at yo' boi this weekend.
In all fairness, Jews have been making movies about Nazis for years…
But will it conflict with his regular gig on 'Two and a Half Men'?
You know your balls taste great when an ice cream is named in their honor. Congratulations, Alec Baldwin!
Damn you, Dexter. You've pulled me back in.
If that's not a mo-cap suit, but rather a costume, then the costume designer ought to be executed where he or she stands.
Obviously she's not a reader.
Who ever they pick to play him, they'll have to skinny him up in post.
Seriously. How many kids are you friends with?
We're about to find out that Ventnor Avenue isn't really a place, per se, but more like a state of mind.
Now you say his name three times and he appears as a crassly commercial version of himself played by Russell Brand.
Prepare the body double!
Will he wear a fat suit?
Oops. I meant you SHOULD worry.
You can go ahead and pick your favorite 'Arrested Development' quote and put it right here.