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Native New Englanders David and Alison Lobron live in West Newton with their two children and are members of Congregation Dorshei Tzedek. They participated in Parenting Through a Jewish Lens this past fall at their synagogue and David answered some questions for us about his experience.
What prompted you to sign up for the course?
My wife and I took this course because we wanted to try Jewish learning together, and we were excited to do it with members of our community. We had taken an earlier Parenting Through a Jewish Lens/Ikkarim class in a different synagogue, and while that was enjoyable, we found that we got more out of it by taking it in our own community.
How did you manage the time commitment?
Since we took an evening class, free babysitting wasn't offered. We hired a babysitter for Sunday nights so we'd both be able to go. This worked out very well - we had a lot of conversations about the class together that would not have been possible if only one of us had gone.
What makes this a "Jewish" parenting course?
The study materials, plus the fact that we were taking it with members of our Jewish community. I found it very stimulating to talk about parenting in the context of Judaism, and to see how other people work with the challenges that we all face.
Can you share one or two insights from the course that have stayed with you?
The sessions on Israel and Jewish chosen-ness were quite interesting, because people had very different points of view. I think the Israel session was my favorite, even though we had some disagreements, because it was clearly on many people's minds. Our class really spanned the political spectrum.
What did the instructor say or do to create a safe and stimulating learning environment?
I like that the instructor challenged us a bit. Our congregation is a very liberal Reconstructionist one, and I found it refreshing to read some more conservative texts, e.g., those from Joseph Soloveitchik or Abraham Joshua Heschel. I don't think our instructor was out to change minds, but I think it helped people see alternative approaches to some topics.
How might Jewish wisdom inform choices you make as a parent?
We've gained a tremendous amount just by being part of a community. I feel much more resilient and supported than I would feel if we did not have community support.
We invite you join us on Sunday March 10th for Matzah Matters - a free event open to the entire community that includes learning and babysitting/kids holiday craft projects.
Wednesday, February 27, 2013
Thursday, February 14, 2013
To Learn and to Teach
Rabbi Sharon Clevenger, a recent addition to our Parenting Through a Jewish Lens faculty, is the middle-school rabbi at the Rashi School in Dedham and the mother of two young daughters.
Today in my Parenting Through a Jewish Lens class, I learned something so elegant and inspiring that I wish I had thought of! I can't wait to try it out in my own life. I pretty much come home to my husband after every class and say this very same thing. Which is exactly what the framers of this course want - they hope that the small and large "aha moments" inspired by exchanges among teachers and learners will lead to lives more meaningfully lived. I love and look forward to what I will learn each Sunday. And I am the instructor.
There is a prayer in the morning service called Ahava Raba. It thanks God for showing divine love for us (the Jewish people) by giving us the gift of Torah. It asks God to have mercy on us by giving us what we need to: "understand and discern, to heed, to learn and teach and to fulfill all that is in God's Torah with love." As a Jewish educator, I deeply appreciate the fact that "learn" comes before "teach." I walk into my classroom each Sunday, ready to guide my phenomenal students through the inspiring and challenging texts in our binders. Yet I also enter knowing that I will leave that room knowing far more than I knew at the start of class. I am there to learn and to teach.
Most of the participants in the class are relatively new to Jewish learning and a few are non-Jewish spouses. Therefore, each person brings wisdom and knowledge from his or her life. A typical class session might include students making references to renowned secular philosophers, American business law or Hindu religious and cultural traditions, not to mention the daily inspirations (and grinds) of parenting. This would be a great conversation on its own, but what amazes me is the way that it all ties together with the texts in the binder and helps frame a "Jewish lens." This class is definitely providing transformative Jewish learning for all of the learners, including me.
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I have a friend at work who was going to have both of her kids together at the same time for a very small window at Christmast time. She said her son was little the last time they had family pictures done and would I be willing to run at lunch and get a couple of shots for her at some spots they had already scoped out.
Here are just a few from yesterday.
Wednesday, December 29, 2010
Tuesday, December 21, 2010
Invitation Pictures
I have had the honor of knowing these 2 precious people for a few years now. So when they got engaged, we were all thrilled! Since she is from Brazil, they are getting married in Brazil. Part of their culture sends out their invites way earlier than we do. So while this isn't their real "engagement" session, we had to get some very specific pictures (and feel) for their invitations they need to get done this month.
Congratulations, you two! We love y'all!
Congratulations, you two! We love y'all!
Posted by Billie at 1:04 PM 1 comment:
Labels: Engagement/Wedding
Thursday, December 9, 2010
Baby Kyle @ 4 Months plus Family
It was time to do Baby Kyle's 4 month pictures, but we really wanted to work in some good family shots this time with it being this close to Christmas. So the 6 month shot will be all his, but for this one, I think we got some sweet pictures for the whole family.
We even got a great Christmas Card picuture, but it's for her to get to show not me :-)
Merry Christmas, Teague Family!
We even got a great Christmas Card picuture, but it's for her to get to show not me :-)
Merry Christmas, Teague Family!
Posted by Billie at 7:54 AM No comments:
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subset: commoncrawl


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I'm not planning on doing this often, but I wrote a part of the book today that was sort of fun, it being Christmas and all, and I wanted to share. So here is a snapshot from Snapshots of Who I Am. This is from Chapter 9, The Good Shepherd.
I am the good shepherd. The good shepherd lays down his life for the sheep. John 10:11
While shepherds watched their flocks at night
It's Christmas time. I've spent the past few days strategically placing decorations around my apartment. There are Santa Clauses and nutcrackers, snowmen and angels smiling at me from every nook and cranny. There are candles and evergreen wreaths giving off their seasonal scents. And there are dishes filled with chocolates and gum drops, which I have to keep my husband and dogs from emptying on a daily basis! I love all of these decorations because they are filled with meaning, not only from my childhood traditions but as centuries-old celebratory symbols. Evergreens are verdant reflections of the eternal life that Jesus came to bring. The traditional Christmas color, red, reminds us of the blood that Jesus shed so that we could have eternal life. Sparkling ornaments and twinkling lights dazzle our minds into remembering the beautiful star over Bethlehem that announced the Messiah's birth to a band of Eastern kings. And the gifts we give are a token to the gold, frankincense and myrrh given to the Christ child.
But it occurred to me earlier today that a particular group of characters from the Christmas story are absent from my seasonal decor. I've just been taking inventory and this is what I found: five Santas, four nutcrackers, three wreaths, a pair of angels, one snowman and a reindeer. I've got two mangers, one that's painted onto a plate, along with the words, "Unto us a child is born", and a beautiful Buckley Moss print of Mary, Joseph and baby Jesus, surrounded by some very calm looking sheep. I confess that I don't have any kings on camel-back, but I do have gifts to wrap up for my loved ones and yesterday I seriously contemplated buying a bar of soap that was "frankincense and myrrh" scented. So St. Nick, Frosty, Rudolph, Mary, Joseph, the angels and the wise men all made the cut, so who is missing? Shepherds. I've got all this cute, sparkling, scented seasonal stuff and there's not a shepherd to be found.
I confess, I'm feeling a little guilty. Here I am about to write about Jesus as our Good Shepherd, and I haven't got a single shepherd in my apartment, even though they played a really significant role in the story of Jesus' birth. Here's an excerpt from their Biblical cameo.
And there were shepherds living out in the fields nearby, keeping watch over their flocks at night. An angel of the Lord appeared to them, and the glory of the Lord shone around them, and they were terrified. But the angel said to them, "Do not be afraid. I bring you good news of great joy that will be for all the people. Today in the town of David a Savior has been born to you; he is Christ the Lord. This will be a sign to you: You will find a baby wrapped in cloths and lying in a manger." ... So they hurried off and found Mary and Joseph and the baby, who was lying in the manger...the shepherds returned, glorifying and praising God for all the things they had heard and seen, which were just as they had been told.
Luke 2:8-12,16,20
I do find it amusing that though the Heavenly Host found shepherds to be a worthy audience of the biggest news in world history, Christmas decoration manufacturers don't seem to find them worthy of playing a major role in our holiday décor. It's a whole lot easier to go out and find a cute Santa or sparkly star than a shepherd to put up on the mantle or hang from the tree. But I'm sort of thankful for this because it's an indication that even today shepherds are viewed in much the same regard as they were in Jesus' time. Dirty and smelly, they're just not the guys you want to have hanging around, especially when there are so many other attractive options.
But that is part of the beauty and truth about Jesus. He's not interested in being what is most attractive to the world, He's interested in being what is most needed. Maybe this is the reason that the Heavenly Host appeared to those trustworthy shepherds upon that midnight clear. If you go back and fill in some of the blanks from the Luke story, you find that after seeing baby Jesus nestled into that manger, the shepherds immediately went out and "spread the word concerning what had been told them about this child." (Luke 2:17) It is the nature of the Good Shepherd to take every opportunity to gather His sheep together. Even while He was still wrapped in swaddling clothes, He was tending His flock by entrusting men whose lives were devoted to the care of other living things to spread the good news of great joy for all people. What a position of honor for those lowly shepherds. Maybe they aren't decking people's halls as Christmas decorations, but on that night they were donned with the Lord's favor. They might not have been the world's first-pick, but God considered them worthy. Perhaps because they were men after His own heart- men used to seeking and saving the lost. Men whose lives were devoted to tending to the weak and the wounded. Men who knew every one of their sheep- from the tiniest lamb to the oldest and most infirm ewe. Men who risked their own lives for the security of their sheep. .
It wasn't random coincidence that the angels appeared in that field that night. God planned perfectly every minute detail of Jesus' birth and recorded many of those details in prophecies written centuries before. He could have told someone with a lot more influence. Someone shinier, someone more attractive. But He chose the shepherds. He chose some of His own kind.
As I look around my apartment, I am maybe a little more embarrassed than I was when I started writing this. I'm not against Santa or Frosty or Rudolph. Angels are beautiful and evergreens are filled with fragrance and significance. But my identity depends on the Shepherd. Yours does too. And fortunately, it is He who will guide us. Like any good Shepherd, He will lead us as we explore this vantage point of our identity in Christ. And I'm confident that as He does so, we will discover good news that is sure to fill us with comfort and joy.


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ok, older laptop...Fujitsu Lifebook S series...model 6010. 1 gig chip, 512 ram, 40 gig HD...pretty good shape for it's age. It will not boot 19 out of 20 distros into x. So far, I have it able to boot dsl and something I stumbled across called cd linux. It is still under development and doesn't even have a package manager yet...DSL isn't an option. this laptop is going to an underprivileged child and it needs a more new-user-friendly distro.
Got a clue when I tried to boot mandriva and it got to the start x point and then gave me this dialog box:
Your system supports multiple head configurations
What do you want to do
(at this point I was looking for the "Throw-it-against-the-wall" option.)
Use Xinerama extension
Configure all heads independently
configure only card "intel Corporationl82830 CGC [chipset graphic controller]" (pci:0:2:1)
configure only card "intel Corporationl82830 CGC [chipset graphic controller]" (pci:0:2:0)
I've used all of the choices and it just pukes and comes back to the command line. Any help with this would be appreciated. Some kid will love this little laptop...but I don't and won't use windows...it came with xp sp2 originally but has a wiped drive by the donor.
EDIT: Finally was able to get a test on x and this is the errors that came back.
(EE) intel)(0): sil164 not detected got 5: from DVO12C_E Slave 112
(EE) intel(0): tfp410 not detected got VID 1305: from DVO12C_E Slave 112.
Try to change some parameters.
Dude, I've changed every parameter, every way they can be changed and I still remain with a fairly decent door stop. Your help would be appreciated,


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Is your home a hazard?
We all like to feel safe in our homes. No one would expect to discover serious risks to their health when moving into a rented property, or to find that their new home could be unsafe for their children.
Yet, for one in six households across England, these threats aren't just a possibility – they're a reality.
Half a million children in unsafe homes
Our new research, A Nation of Renters, found that 740,000 households are living in privately rented homes containing Category 1 hazards – the most serious of problems, from severe damp to risk of explosion. With over a million families raising children in privately rented properties in England, more than half a million children can't feel safe in their home.
There are almost three times as many dangerous properties in the private rented sector than in social housing, exposing families to a host of dangers. To add insult to injury, these households are paying an average of £157 per week in rent to private landlords for their hazardous homes. This equates to a total of £5.6bn each year going into the pockets of landlords who are putting their tenants at risk.
Action on hazardous homes
We're glad that the Government has pledged to tackle England's housing crisis and are calling on them to introduce renter refunds so that rogue landlords can't continue to collect rent on dangerous properties. We also believe that national landlord registration could help to tackle the worst offenders and prevent them from putting families at risk.
We're campaigning for improvements to the rights of private renters through our settled and safe campaign – but changes to the law might take some time. In the meantime, you can take action if your home is a hazard:
- If your privately rented home is damp, dangerous or needs urgent repairs, contact your landlord and explain the problem and what you would like them to do to fix it. They should get in touch and tell you what they will do to make sure your home is safe.
- If your landlord doesn't respond or refuses to take action, contact your council and ask the environmental health team to inspect your home. If they agree that it is hazardous they can issue a notice telling your landlord what needs to be done and take action if they don't comply.
- Many private landlords are members of trade bodies that can support them to improve their practice. Check if your landlord is a member of one and, if they are, write to that trade body explaining the problem. They can contact the landlord and explain their responsibilities – and if the landlord still won't ensure your home is safe, might be able to take further action, like removing their accreditation.
- If you've tried all of this and are still concerned for your health and safety at home, help us to change the law by sharing your story on our private renting blog.
Get advice
If your home is hazardous and you need further advice, please visit our online advice pages, call the Citizens Advice consumer helpline on 03454 04 05 06 (9am – 5pm, Monday to Friday) or visit your local Citizens Advice.
Visit our website to find out more about our campaign for a settled and safe future for England's private renters.
- Category: Advice, Campaigns, Housing, Policy research
- Tagged: housing, private rented sector, private renting, Renters rights, Settled and safe
6 comments
Rent Rebel
The point made here about tenants not taking legal action is very pertinent. In the absence of funds to do this with, can I pls suggest that private tenants follow @AnthonyGoldLaw on Twitter for some especially helpful videos – which discuss repairing obligations among them. (I don't work for them – but I do seek to empower tenants)
Also, the point made here about retaliatory eviction must be noted! If the property is in severe disrepair, just go straight to the council and ask them to serve any notice – on the landlord – as they deem appropriate. Only with such a notice from the council can eviction be prevented (for 6 months from service of).
@Rent Rebel
Our Tory government contains individuals who are heavily investing in being private landlords, mostly using 'company' umbrellas to hide away from public scrutiny. They are also attacking Social Housing with a view to eliminating it entirely (Right to Buy + refusing new investment in it) It is therefore unrealistic to expect them to introduce legislation to curb their own profits and worst excesses, when they are making huge amounts of personal money from their activities. They are currently clearing London of poor people (50,000 families and rising – this is a true figure) so that they can make even more money from the vacated homes. Your descendents will suffer.
These practices were first highlighted by the activities of Peter Rachman – absolutely NOTHING has changed since then. Look it up, and be ashamed that it hasn't been stopped. By us, the voters.
The article is dangerous. At bureau level one of the first pieces of advice that would be given to a client in such circumstances would be to caution them that a private landlord, in the event of a client following the procedures noted, may well be to give notice. This eventuality is not even mentioned.
Simon Harris
It is telling that the one thing we are not advising tenants to do is seek legal advice and take action themselves. I suspect the reason is that most tenants cannot afford to do this since most disrepair cases were taken out of scope of legal aid. perhaps we might argue that it should be brought back into scope to deal with just these issues. Rights are all well and good but pretty useless if you can't enforce them.
I disagree with the comment "Many private landlords are members of trade bodies that can support them to improve their practice." letting agents may belong to a trade body but many landlords have individual properties and do not belong to anything.
I'm sorry but your talking rubbish. So what if they are part of a certain organisation, it don't stop them from ripping off innocent people


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IGN: "Resogun is very much like Super Stardust, right down to its twin-stick mechanics and twitch gameplay. But it's its own game, too, one that stands out as being different enough not to be a Stardust game in its own right. Either way, after playing it a few times, it quickly became clear that this game has the potential to suck scores of hours from your life, just like the Housemarque arcade shooters that came before it."
Woop woop... Super Stardust HD one of my favorite games. This looks just as fun!
Its like defenders meet astroid. Just need a bit of missle command then my dream game from the atari era is complete.
Stardust i managed to get in the top 100 leader board at one time when i got it for free after PSN was hacked. Went 3 playthrough without dieing and no bombs used to increase my multipliers.
I take this game over the next COD. I am serious. I had more fun with Stardust that 85% of AAA budget games i played this gen.


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Sigh. Where should I start? A lot of things have been happening ever since November came in. Guess the song should have been "Wake me up when November ends" instead of September.
Yah better werk b**tch!
Things have been not so right yet not so wrong at work. I've been quite happy with my daily routine until November came in and felt tired and almost getting sick of it. My work became as hell as I don't expected it to be. And I've no idea how and why. I think I need a new way to keep my interest towards my work. It's not usual to me that I easily get tired of the same ol' things I do every day. Because this is also the main reason I left my first and second company. Despite of bigger promotion chances in my first company- and despite of the inspiration I have. Guess I need a new way to enjoy my work every now and then to keep my butt working.
One way love sucks! No matter how true.
In October, my not-so-secret blog turned two, and it occurred to me that even after a year of not seeing him or even his shadow I still feel the same way- or even worse. I thought by moving to Cebu and keeping my self out of Manila would change everything including my heart. But no. It's not what happened and it's not happening. I feel so hopeless this feeling would sink and be buried somewhere in the islands of central Visayas. And it sucks that I am the only feeling this. It sucks that I don't have a tiny idea if I ever cross his mind, or if he's feeling the same way. I need someone to break this spell! And I can't wait when it'll happen!
Good bye Yolanda, don't see us again please!
Super typhoon Yolanda which international name's Haiyan hit the Visayas area in less than a month after the 7.5 magnitude earthquake shook the central part of the country. It broke not only houses and trees, it also broke so many hearts by taking away EVERYTHING including the lives of thousands people.
No country, not even as happy and strong as Filipinos deserve this catastrophe. Not especially when Christmas is just some few days away. But who to blame? It's a natural disaster which I believe even God didn't wish to happen. Mother Nature might be mad by how people abuse her, but let's not go deeper into that. Not only Philippines abuses her. Not only Filipinos are sinners. So let's not argue about how we live our lives, who we praise and what we believe in.
I'm thankful and heaven knows how much I am to see my sister and my relatives alive and kicking despite of the devastation. I'm also glad that my fellow Waraynons are striving so hard to live longer. And I am grateful for all the help that they are receiving, especially the help from other countries.
Christmas is just 25 days as I write. There might be no happy and colorful Christmas lights that'll light in every street and corner in some parts of Samar and Leyte. There might be no voices of children singing Christmas songs in each and every household. There might be no Christmas décor to remind them how Merry Christmas is. But I know there will be smiles on each and every face that brings tears to my eyes.
My heart and prayers belong to you fellow waray-waray's. May you have a gratifying Christmas and a wonderful New Year. Ma-tindog kita tanan! Waray mabul-iw!


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Is everyone in the know about vertical gardens? I have been admiring a few for months now while simultaneously trying to figure out what to do with the strip of wall in my backyard where the ivy just won't seem to grow.
The wall above is the exterior of Smog Shoppe which is around the corner from our house and I drive by often. Isn't it beautiful? It is adorned with Woolly Pockets which is the brainchild invention of Smog Shoppe owner Miguel Nelson and his lovely wife Sherry.
When I googled vertical gardens I discovered that a gentlemen by the name of Patrick Blanc actually invented them and I was in awe as I surfed around his site. He maps out patterns on the walls before planting to create living works of art. They are true masterpieces.
And so it came ot be that two Sundays ago 2E and I hit the local nursery and started planting a Woolly Wall all our own......
images 1&2: Woolly Pockets image 3: Patrick Blanc images 4&5: Mio


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Monali Churi is an Engineer turned MBA, turned into a Business Analyst by profession.Writing helps her vent out, makes her happy, the way no other thing does.Monali believes in the power of words, for words can inspire, educate, raise voice, draw a smile, trickle a tear and touch your heart. Monali also has lot of lines on her palm and people say that the more the number of lines you have,the more you think.This blog is just to jot down those thoughts and a justification to those lines. Apart from writing, she also loves clicking pics and believes that writing and photography are so much related.. While words draw the pictures, emoting pictures frame the words.Monali believes in life, as a beautiful journey, which comes in a package of good as well as bad, some sweetness and some sourness too..But still if you can enjoy the journey, more than the destination, then you gotta be the one...
Saturday, January 10, 2009
About Me
Posted by Monali S Churi at Saturday, January 10, 2009 2 comments


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My friends know about my bead addiction (it is hard to hide it!) and they sometimes bring me beads as a present when they have been abroad. Those are often beads that I would not choose myself. But the funny thing is that they inspire me to bead different things than I would normally do, which is great!
I named this necklace after the friend who brought me these tiny dagger beads. Thanks very much Sharon. And my darling niece Julia was willing to pose for this necklace.


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Friday Amelia turned two years old! She got a few presents from my family on her actual birthday including this new princess kitchen:
We had dinner out with my family at Boca Chica and dessert at Izzy's in St. Paul.
Saturday we had a little birthday party for Amelia at my parent's house and my family and Jason's family came. Both days I had Amelia dressed up in the same birthday dress she wore last year. It was a tad short so I threw some leggings on under it and it was pretty cute!
I was pretty impressed I got two years of use out of the dress since it is size 18-month!
Amelia was so funny opening her presents. She would just empty the bags but throwing everything over her shoulder:
Auntie Lisa (one of my best friends) started to fold tissue paper and after that Amelia brought her every single piece and helped her smooth it out so she could fold it. Kids pick up on every little thing!
Auntie Kimmie got Amelia her first trike and We took turns pushing and pulling her around on it and when I stopped to get her picture on it she made sure I knew she did not like that I stopped pulling her:
She can be so demanding sometimes. We definately are entering the "terrible twos."
Amelia had a big cake and cupcakes. She tried so hard to blow out her candle on her cupcake:
We had to give her a little help. After all the work blowing out the candle she didn't even want to eat it! She picked sprinkles off and that was about it!
Amelia got a lot of great stuff for her birthday including new clothes for this spring/summer that we are anxious to wear! Auntie Liz got her a little suitcase which was perfect to bring home all her new clothes in.
That's really all the pictures I have to share. I didn't get any of her with family members because after presents and lunch she dragged her Nana to her bed and pretty much asked for a nap. So, the guest of honor slept through the last half of her party which meant no pictures really!
Lisa, Kim and I went bowling with Amelia in the evening. We thought we could get Amelia in on the bowling but she pretty much just liked walking out to the lane and then back. We'll take her again and hopefully she'll be more interested in it since it won't be a totally new envoronment for her. That means we'll have go sometime soon!


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Renovation contract reviewed
BLOSSBURG — A contract from Quad 3 Architects to renovate rooms at the Blossburg Elementary School into new administrative office space was reviewed and discussed during the Southern Tioga School Board's meeting Monday.
The $28,500 contract includes design work for the new offices as well as bidding and construction of the project, and includes up to $4,000 in expenses.
Board Vice President Sean Bartlett asked about a clause he found in the contract that allowed the firm to increase costs if the project was not completed eight months from the date of the agreement.
"That seems to say if that is eight months from mid February, date of agreement, the costs can go up. It is supposed to be done in October, so it's kind of tight," he said.
Superintendent Sam Rotella said the administration would check on it.
The board also heard from business manager Kathy Ciaciulli about a bid for proposal that was advertised in December and January for copier and printer replacement and maintenance.
The district currently has 61 devices, and the new proposal would reduce the number to 21 for a price of $32,408 and a maximum of 2.5 million copies per year on a five-year lease plan.
Other bids received included Philips Office Supply, Eastern Managed Print Network, Office Equipment Source and Ricoh.
The process to conduct an analysis of the district's equipment began in October 2015, Ciaciulli said.
"It has been a long process. The majority (of the current equipment) are either out of lease or outright purchases. We were looking for a vendor who would take all 61 devices and consolidate them," she said. The lease would include one Canon wide-format printer and the rest would be Lanier, she added.
The board also heard from Rotella about a recommendation to approve a one-year agreement with Source 4 Teachers substitute teacher service with a 1.5 percent increase to their administration fee.
"My satisfaction has increased, but not to the point where I want to commit to a four-year agreement," Rotella said.
The board also:
• Heard about one change to the district's school year calendar for 2017-18, with one in-service day changed to May 7, which pushed the last day of school to May 31.
• Heard about an Intermediate Unit 17 annual agreement to administer next year's IDEA grant.
• Heard from Future Business Leaders of America students who want to see the district implement a new course on business and leadership studies, which they worked on and developed, to begin next year.


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BOSTON--(EON: Enhanced Online News)--Boston Private Financial Holdings, Inc. (NASDAQ: BPFH) will report fourth quarter and year end 2015 financial results after market close on Wednesday, January 27, 2016.
Management will hold a conference call at 8 a.m. Eastern Time on Thursday, January 28, 2016 to discuss the financial results, business highlights and outlook. To access the call:
Dial In #: (888) 317-6003
Elite Entry Number: 6359964
Replay Information:
Available from January 28 at 12 noon until February 5
Dial In #: (877) 344-7529
Conference Number: 10079628
The call will be simultaneously webcast and may be accessed on www.bostonprivatefinancial.com.
Boston Private Financial Holdings, Inc.
Boston Private Financial Holdings, Inc. is a national financial services organization that owns Wealth Management and Private Banking affiliates with offices in Boston, New York, Los Angeles, San Francisco, San Jose, Atlanta, Florida, Wisconsin, and Texas. The Company has a $7 billion Private Banking balance sheet, and manages over $27 billion of client assets.
The Company positions its affiliates to serve the high net worth marketplace with high quality products and services of unique appeal to private clients. The Company also provides strategic oversight and access to resources, both financial and intellectual, to support affiliate management, marketing, compliance and legal activities. (NASDAQ: BPFH)
For more information about BPFH, visit the Company's website at www.bostonprivatefinancial.com.


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24 June 2018
News and Views
Links and Services
Showcase Journal
The incidence of pediatric Crohn's disease has increased in northern Stockholm, find physicians in the October issue of Gut.
There have been reports of a possible increase of pediatric Crohns disease.
In this study, physicians from Stockholm, Sweden, assessed the incidence and characteristics of inflammatory bowel disease (IBD) in northern Stockholm between 1990 and 2001.
The team evaluated all records of patients aged 0 to 15 years with suspected IBD, from an area with a population of 180,000.
They searched patient files for relatives with IBD, and for concomitant autoimmune diseases.
The physicians found that a total of 152 children were diagnosed with IBD. This was an overall incidence of 7.4 children with IBD per 100,000. The incidence of Crohns disease (CD) was 4.9, ulcerative colitis (UC) 2.2, and indeterminate colitis 0.2.
The team found that, between 1990 and 2001, there was an increase in the incidence of CD, while the incidence of UC was almost unchanged. Overall, they identified a net increase in the occurrence of IBD.
The team also found that there was a male dominance of CD.
They determined that 14% of patients with CD and 11% of patients with had a first or second degree relative with IBD.
In addition, 18% of patients with CD and 10% of patients with UC had a concomitant autoimmune disease.
Dr Hildebrand's team concluded, "The incidence of CD has increased in northern Stockholm".
"The current incidence is higher than that reported from other areas".
"Our results suggest a shift in presentation and diagnosis from UC towards CD, but also a net increase in IBD".
"Concomitant autoimmune disorders and family history are common in pediatric IBD".
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A great name for this great little Turkish walnut and tahini sauce, Really good with barbecued veggies, lamb or fish, duck or guinea fowl as a sauce or as a dip with pitta and some crispy raw veg for dipping.
2 cloves garlic
150 g walnuts
3-4 tablespoons tahina paste
juice 1-2 lemons
4 tablespoons boiling water
40 g flat leaf parsley, about 5 tablespoons chopped leaves
5 tablespoons chopped
salt and freshly ground black pepper
1. Peel and crush the garlic and throw into a food processor with the walnuts and a pinch of salt and whiz until almost a paste has formed. I neither had the time, inclination or a big enough pestle and mortar to pound this by hand, which you are more than welcome to try if you do.
2. Scrape into a bowl and stir in the tahini. Squeeze in the juice of one of the lemons and enough boiling water to make a thick saucy consistency.
3. Remove leaves form parsley and chop. Stir the parsley into the sauce, check seasoning, adding more lemon juice if you fancy.


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Instead of heading for white water rafting in CDO, we decided to postpone it for this trip. We were exhausted and we've spent too much in Camiguin.
We headed to a college friend's house to spend the night before we return to Cebu. The only place that we could explore was the SM Mall near their village and the village itself.
The village is located in the uptown area and most of the places has just been built. The city itself is downtown which is too far so we just have to check on the homes built the its architecture and designs that we plan to copy on our future home.
This blog is also give my appreciation to one of my close friends in college, for a very warm accommodation. To Maui, Lloyd and her cute baby girl, thank you so much.


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A$AP Rocky recently paid Zane Lowe a visit, giving an exclusive interview for Apple's Beats 1 Radio slot. A$AP Rocky discussed his new album and working with rock and roll legend Rod Stewart, who also calls in to add to the discussion. The 26-year-old Harlem rapper and 70-year-old iconic singer discuss how their new single Everyday came about.
Watch and listen via the video above now.
Related story: Watch now: A$AP Rocky featuring Rod Stewart, Miguel and Mark Ronson


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Jacob J.
Lesson Details
Jacob teaches
- Teaches students:
- Ages 5+
- Teaching since:
- 2009
I teach both academic subjects and guitar lessons.
For guitar: I have been playing guitar for nearly 20 years, in styles including rock, blues, folk, and finger style. I love playing songs by Leo Kottke, Mumford and Sons, Chris Smither, Walk the Moon, The Killers, The Beatles, and roots music like bluegrass and folk. I like to keep things fun in lessons and focus on building my students love of music.
For academic lessons: I earned my Ph.D. in Sociology from Southern Illinois University in 2009, and taught at South Dakota State University in the Sociology & Rural Studies department since then. I have been teaching at the college level for nearly a decade, and love helping people learn! I can talk about difficult subjects in plain language, and help students who are just shy of the grade they want earn that "A." I have had many students who started out hopeless, but who were able to learn things they never thought they could, and get better grades than they ever imagined, with my help.
I have worked with all kinds of students, some with jobs and kids, some fresh out of high school, and some working on getting their graduate degree. Helping them learn, grow as people, and accomplish their goals has brought me joy and satisfaction as I watched them succeed. Many have gone on to earn a Master's or Doctoral degree, or earn jobs as police officers, business people, and more.
Ph.D. in SociologyAug 2004 - Dec 2009
4 ReviewsWrite a Review
Jacob is not only a fantastic teacher but an amazing mentor as well. He encouraged me to not just memorize the material but to engage with it on a deeper level. He also provided me and other students with essential advice on how to put together applications for graduate programs and his tips on how to put together a personal statement were a large reason why I was accepted into and received a scholarship for law school. I did and will continue to recommend Jacob to anyone!+ Read More
Really enjoyed lessons! Really wanted to learn songs by my favorite band and despite not being musically inclined, felt at ease and ready to learn. Jake was patient and really helped me be able to play my favorites.
Jacob got me from no guitar experience to playing several fun songs in no time. He was super friendly and nice and answered even my stupid questions. I highly recommend him to anybody.
Jacob J. has an amazing talent and ability as a teacher to explain things in ways that will make sense to a diverse group of students. He truly cares about each student and it shows in any class you take with him (in any field). Because of him I have pursued a field I never thought I would get into, which has helped me travel the world and has opened new opportunities for me, that I would have not had without the skills and knowledge I gained with him as teacher. Unless you do not like a fun and knowledgable teacher, I highly recommend Jacob J.+ Read More


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This was my favorite Disney Bound to make. My husband suggested we do something "scary" since we would be at Disney in October. So when he said Jack Skellington, I was thrilled! I LOVE Tim Burton and everything he does! But I knew it would be hard to find ANY thing that looked like Sally without just buying a costume. Hot Topic and Walmart both sold their own versions of Sally's dress, but Disney Bounding isn't about wearing costumes. So I decided to make my own skirt.
Jack Skellington
- Black & Grey Striped Shirt – Express – $8
- Black Joggers – Pac Sun – $50
- Jack Skellington Snapback – Spencer's – $10
- Black Chucks – Previously Owned
- Hand-Made Skirt
- Black Cami – Forever 21 – $1.50
- Black & White Belt – Previously Owned
- Jack Skellington Light Up Necklace – Walgreens – $1
- Mickey Mouse Ears – $4 (Hand-Made Bow)
- Black Toms – Previously Owned
Eventually, I may do a post on how I made my Sally skirt, but for now you can just look at how cute we look in our Jack and Sally outfits!


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Wi-Fi Offload and Distributed ANDSF
The Challenge
With continued growth in mobile data traffic, MNOs are exploring ways to relieve network congestion and optimise the user experience. While there are many ways to accomplish this, one such involves increased use of non-3GPP networks, such as Wi-Fi in conjunction with conventional 3GPP access networks. MNOs need a solution that delivers the optimal network connection seamlessly from those available, while optimising the user experience and taking a leading role in simplifying and facilitating user choice. MNOs need flexibility.
The Solution
GoS Networks' ANDSF solution adopts the innovative step of decomposing the functionality between the GoS Agent and GoS Manager. The GoS Agent acts as distributed ANDSF client that can be deployed on mobile devices (UE), while the GoS Manager provides co-ordination, policy administration, distribution and backend database functionality securely in the network, managing all active distributed ANDSF clients via the S14 reference point.
Read the complete Wi-Fi Offload Use Case...
- Seamlessly manage handover between 3GPP and non-3GPP access networks
- Offer dynamic network selection for different applications and traffic types
- Increase user satisfaction through offers, engagement and opt-in / opt-out policies
- Take a leadership position in offering user choice and enhanced experience
- Manage registration and authentication processes seamlessly on behalf of users
- Alleviate local congestion
- Optimise conditions for demanding applications
- Best-available connectivity services
- Manage roaming data access for users
- Offer flexible packages for enhanced data rates and increased data coverage
- Automatic registration with APs and other non-3GPP access networks


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Personal Website
Budget $30-250 USD
I'm hoping to create a register and log in for my website, using php.
log in: username
Register: First name
Last name
Many thanks!
Awarded to:
33 freelancers are bidding on average $74 for this job
Hi, please check the pmb for more details. thanks
Dear Sir; I can do this work for less than $5. I have already completed a work like this. I hope you will be fully satisfied with my work. Because i am a student of CSE department. Thank You.
Its simple task. please provide this opportunity to me
we are looking forward to take up the work and execute the project as per the specifications


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You can't believe everything you read
I recently received a letter.
My first thought was, if you have to put "This is not junk mail" right on the envelope, that's not a good sign. My second thought – call me a cynic – is that it probably was junk mail.
So I threw it on my table with my other unimportant mail. A few days later I opened it.
Of course, it was junk mail. Does that prove I'm a cynic, or just that you can't believe everything you read?


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We started Dylan on solids around the time he turned 5 months, at first with very diluted rice cereal once a day, and slowly adding less milk till he takes it as a smooth paste.
Last Friday, at his 6th month check and third immunisation jab, the ped said that we can start feeding him solids more regularly as he can afford to put on more weight.
So I steamed potatoes and mashed it through a sieve, enough to last for a week. Poor baby - within days he became badly constipated, and grandma had to "help" dislodge the hard pellets of baby poo out.
Yesterday was particularly concerning, cos I could tell that he was trying to push something out but nothing came out. The grandparents called Elroy (he was in the middle of his 18-hole game with my Dad), and asked if we should send him in to see the doc.
They tend to worry a lot - Dyl's granma and granpa, but I guess with nothing much else to do other than take care of their grandson I can't blame them for being overly concerned.
Here's a pic of them three after Mother's Day dinner:
So E and I prayed hard on our walk back from dinner with Jacinda and Kevin.
And lo and behold, the minute we got home, I spied an odd scent coming from our baby, and true enough, a diaper full of poo!
I read Lai Yong's chapter on Psalm 62, about what are some things we find most difficult to wait for.
He talks about what one soft-spoken farmer from Lisu shared. He said that it was most difficult to wait when all the grains in the store have been eaten, and the grains in the fields are still in their stalks, but are not ready to be harvested. So while waiting, they stay hungry, and wonder at how much longer before their tummies can be filled.
It made me think.
We often want to rush growing up, don't we. Not just for our babies but even for ourselves.
Little Dylan's digestive system is still slowing maturing, but of course his mouth and taste buds don't know that yet, happy is he chomping away at mommy's special mashed potatos. So he too, needs to wait, and allow a bit of waiting time before trying a range of other solid foods.
Similarly, only before God's presence can my soul be still, and be silent before my maker.
"My soul finds rest in God alone;
my salvation comes from him.
He alone is my rock and my salvation;
he is my fortress, I will never be shaken."
- Psalm 62:1-2
This week, I pray that we can gently wait for God to mature the digestive tract inside our baby boy, and be rested in our harried souls.


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This blog is my views on the world I live in and my views only. So in saying that, Don't take any of my views personal. But then again, if the shoe fits....
Wednesday, May 29, 2013
I am a Soldier and wouldn't want it anyother way
I was doing OK in Dallas. I grew to love Dallas. Whenever I retire, the Wifey and I plan on moving back to Dallas. But I was not satisfied with just being in Dallas and just being OK. I was in a job that I hated; even though the money was OK. But it seemed like I could never catch up. I was always behind...either in my advancement at my job or bills or whatever. It was like I didn't fit in there. When I finished College and went back into the Military in a reserve capacity, I got bit by the Military bug all over again.
So here I am. Back full time and loving every minute of it. I kick myself in the ass everyday because if I had not gotten out I would have been eligible to retire last year. But it is what it is.
I am older than most of my unit...Hell I'm four years older than my damn 1st SGT!! But that's OK. It is like I never left. I needed that little break I took to recharge.
For the first time in years, I look forward to going to work. Yes it is a lot of bullshit. Yes I will probably get deployed again..and yes some days after a long ass long 3 mile run, I ask myself just exactly what the fuck do I think I am doing, with my not so young anymore ass...but as of this minute? right now?
3 comments:
- Donnell Harvey said...
Hey Redd without sounding to effeminate I am happy you found your niche. You sound as if you are in a good place in your life. Civilians will never know the camaraderie of men deployed together in real world situation.
These guys become friends for life when one seperates oh brother how you miss it you miss the uniform the rank the power the women the travel. Man I could really go on but I won't, I'm glad I served I ended up with a good job a beautiful wife and 3 daughters we all live under the same roof an I think the Air Force made me a better man not to mention that sweet ass VA loan. Well that's it Redd have a good one stay happy yu are in a beautiful ar f the country. Peace
- May 30, 2013 at 3:16 AM
- Dirty Red said...
Thank's man. Your are very right. If a person has never been in the military;they have no clue how close we can get to one another. I am stationed in WA state now. It is very beautiful up here. But I have to say that it ain't Dallas!
- June 3, 2013 at 3:39 PM
- Reggie said...
Thank you for serving my brother!!
- June 24, 2013 at 7:14 PM


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Here comes the worrying, overanalyzing, and oh did I say worrying?
Well we started aquatic therapy last week with Mrs. Jenny and Sean LOVED it. He wasn't bothered as much with her making him use his right hand. He was smiling, laughing and most of all NO CRYING:) We are going to be going every Tuesday night now. I took pictures but I don't think Mrs. Jenny would want them posted hear:)
We also went and got Sean's 2 year old pictures taken this weekend. It was quite a day we took some and then had to stop and go play at the Rainforest Café and see all of the fish and go to the Disney store. But we eventually made it back down to the studio and got one of the cutest pictures ever. They are all cute but here are my two favorites.
We are meeting with the pedi on St. Pattys day to talk about all of the different blood tests that Sean is going to get done and I am going to talk to him about my concerns and that I want to be referred to a pedi. Neuro. to get there opinion on everything that is going on with Sean and all of the big WORDS the radiologist wrote. I just want to know the possible long term and short term effects....at least I hope they can explain all of them to me…..
I am big worrier so the more info I know is better than the unknown. Currently Sean is trying to work on jumping and of course building his muscle tone. We are also going to work on his sensory issues and try to not let him or have him freak out when there is stuff on his hands. He gets very upset when there is finger paint, frosting, syrup, icecream, chocolate, bubbles etc… on his hands or him. He also gets upset when some one bumps into him or if a ball bounces into him but laughs when he doesn't to someone else.
Currently I am reading up on this to see what I can do to help. I think he might need to get massages every night. This is probably enough for now I will update more when I know more next week. I am worried that seizures may start at anytime so I am constantly starring at him just because something may or may not happen. Are all seizures noticeable?
The Worrier Queen Bee


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Hi all,
i choose comperative Univeral Decreration of Human Right and Cairo Declearation of Human Right in Islam for my topic in Human Right class .This topic i am going to illustrate what is the different between these two topics.and what of the impact of CDRR from UDHR why the second (CDHR) created?. I am also have some analysis from each piont that i will raise up from two declearations of human right what are the respected statements for our new youngs human right should accept for human right in the world.This toptic i am also try to exlaine some articles form each human right and compear them with the same meaning and different.With the compearation i will try to reject the main piont as my view on some cases and situation for easy to understand. With this topic, i think it is going to have a benifit or shear some ideas from your comment to improve the knowlege better.Your comments are the key to adopt the experience learnt.
Khieu Chansath


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This from Saturday evening (and does it matter how we got here in the first place?):
Me: If this church were sailing over the bounding main, where would the poopdeck be?
Joan: This section of pews is going to have to be reconsecrated after we leave.
Me: I often find that to be true. I leave a trail of apostasy wherever I go. Like a snail. In fact, I am pretty much the apostolic snail.
Tivey: I believe that "Snail of Apostasy" is more poetic.
This from Sunday afternoon:
Me to Smack: Do you have homework to do when you get home?
Smack: No, I have no homework on weekends. But I get enough homework during the week to be well-educated. Why are you laughing?
Me: I'm sorry, I'm just laughing. Well, that's great, it means you can spend your weekends having fun.
Smack: I do have some homework on the weekend. I have my stretchy exercises for physical education. (Bends down and touches his hands to the ground.)
Me: Wow, I could never do that, even when I was your age. And now that I'm fifty there's no hope.
Smack: Well, the average age is seventy.
Me: (thoughtful pause) Oh, the average age of death.
Smack: Yes, so you have a long time to do your stretchy exercises (bends down again to the ground).
I've got a good twenty years!


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This movie
It's kinda like American Beauty meets Mean Girls.
Monday, October 20, 2008
I Love...
Posted by † Brycrasch at 2:30:00 AM
1 comment:
- Matthew said...
We watched that film in Cinema Survey last year... it's definitely a good one.
- October 20, 2008 at 2:40 AM
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Monday, October 4, 2010
Sri Lankan frogmouth (Batrachostomus moniliger)
Don't be mistaken in thinking the frogmouth is, as its name suggests, an amphibian. This animal is actually a bird found in the dense tropical forests of Sri Lanka and parts of India. The name frogmouth describes the bird's wide head and gaping mouth which it uses to catch insects. The females are often a red colour with white freckles, and the males are greyer with even more white freckles. They are nocturnal, meaning they only come out at night. During the day they sleep perfectly camouflaged upon forest branches, as a result they are notoriously difficult to spot.
Sri Lankan frogmouths build their nests in the forked branches of trees anywhere between 2 and 12 m off the ground. A very unusual characteristic of this bird is that it only lays one egg! The single white egg is incubated by both parents, with the male sitting on the egg during the day and the female at night. Once hatched the chick is looked after by the parents for three weeks before flying the nest.
The main threat to these birds is habitat loss. The forests they live in are being destroyed to make way for tea and crop plantations.
Things you can do?
1. Why not ask your parents to buy only sustainably managed tea and help protect the habitat of these birds and other forest dwelling wildlife.
2. Try making a home garden and growing some of your own vegetables. This is fun and easy to do and will help to reduce your demand for crops that damage the frogmouth's vulnerable habitat.
3. Try listening out for these birds after dark, if you here a descending series of "klurck klurck klurck" calls you've heard yourself a frogmouth!
» Find out how else you can help to protect Sri Lanka's endangered species
Illustration by Asia Hewapathirana
Posted by Rainforest Rescue International at 3:54 AM 1 comment:
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Behind on blogging as always but here's a quick little update.
I graduated from my massage school program on December 20th. I finished my license application last weekend and am now just waiting for it to be processed and approved. Once I'm licensed, I'll be applying to massage envy and doing in home massages (referral based) for personal clients. I'm also planning on getting a massage chair and traveling around to all the various waiting rooms I've sat in for Alexander's therapies. People in waiting rooms need massages. They just do. If I can get any of the businesses to pay me, great, if not, I'll just set out a tip jar and make sure no one leaves without my business card.
Kevin is still working at Fiserv. He's working on a big project right now that had him working odd hours before the holidays. He was able to take off a couple of days to go for training in Alexander's feeding protocols at Marcus Autism Center and he worked extra hard at home while I was super sick over Christmas and New Years.
Alexander, as noted above, just finished his feeding program (which was 8:30am-3:30pm 5 days a week) at Marcus Autism Center. He's finally getting all the calories and nutrients he needs by eating food instead of just drinking Pediasure all day. His current protocol is for 900 grams of pureed foods and 250 grams of nutrient rich drink but we'll be going back for outpatient visits and trying to work up to table texture on all 16 of his acquired foods. While he was in the feeding program he wasn't attending kindergarten or any other therapies outside of Marcus. We were able to schedule his feeding therapy sessions around the Language and Learning Clinic sessions there so he was able to continue in that and will be finishing up with that program in early February. He's made some progress in verbalizations but we're still working and waiting for him to really start talking. Today was Alexander's first day back at kindergarten and he didn't have any trouble getting back in the groove after his feeding therapy absences.
Jacob was shuffled around quite a bit while Alexander was in the feeding program and I was finishing massage school. He was able to continue in preschool the whole time, though. Today was his first day back after the Christmas break as well and he had a good day. He's picked up quite a few more words, it's just a matter of getting him to use them. I'm really looking forward to being able to work with him more specifically now that I'm done with school myself.
And here is a video of Alexander having a home "Marcus Meal." My mom took the video while she was here before Christmas for my graduation. Like she says, this was taken toward the end of the meal, and he wasn't cooperating as well as he usually does, but it does show him eating, and we're all super happy about that! (And really, I'm kind of dull and not doing the best job at my ABA therapy duties in this clip, but well, this is the video we got.)


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Enteric pathogens often export toxins that elicit diarrhea as a part of the etiology of disease, including toxins that affect cytoskeletal structure. Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated rtxA. Here we investigate the association of rtxA with the cell‐rounding effect. We find that V.cholerae exports a large toxin, RTX (repeats‐in‐toxin) toxin, to culture supernatant fluids and that this toxin is responsible for cell rounding. Furthermore, we find that cell rounding is not due to necrosis, suggesting that RTX toxin is not a typical member of the RTX family of pore‐forming toxins. Rather, RTX toxin causes depolymerization of actin stress fibers and covalent cross‐linking of cellular actin into dimers, trimers and higher multimers. This RTX toxin‐specific cross‐linking occurs in cells previously rounded with cytochalasin D, indicating that G‐actin is the toxin target. Although several models explain our observations, our simultaneous detection of actin cross‐linking and depolymerization points toward a novel mechanism of action for RTX toxin, distinguishing it from all other known toxins.
The disease cholera is most notorious for the six pandemics that swept Europe and the Americas during the 19th and early 20th centuries. Then, in 1961, cholera re‐emerged on a pandemic scale. In 1998, nearly 300 000 cases of cholera and >10 000 deaths in 74 countries were reported to the World Health Organization (WHO), nearly a 100% increase over the previous year (WHO, 1999).
The bacterium that causes cholera is the Gram‐negative pathogen Vibrio cholerae. After consumption of contaminated water or food by the host, V.cholerae colonizes the upper intestinal tract and begins to secrete the major virulence factor, the cholera toxin (CT). The action of CT causes extensive fluid loss from intestinal cells producing massive diarrhea such that patients eventually die from dehydration (Kaper et al., 1995). Although CT is clearly the most important factor in the disease cholera, CT‐deficient strains of V.cholerae still elicit mild to severe diarrhea and other reactogenic symptoms in human volunteers, indicating that other toxins are likely to contribute to the pathogenesis of the disease (Kaper et al., 1984; Tacket et al., 1993; Taylor et al., 1994; Coster et al., 1995).
CT is composed of two subunits encoded by the genes ctxA and ctxB, which are located on a transmissible filamentous phage (CTXΦ) (Waldor and Mekalanos, 1996). Sequence analysis of the regions neighboring the insertion site of the CTXΦ on the V.cholerae chromosome indicated the presence of a very large open reading frame (ORF) 693 bp downstream from the CTXΦ insertion site, but oriented in the reverse orientation to that of the ctx genes (see Figure 1A) (Lin et al., 1999). At 13 635 bp, this gene, rtxA, is the largest ORF on the V.cholerae genome (Heidelberg et al., 2000).
Analysis of the predicted amino acid sequence indicated that rtxA encodes a putative toxin of 4545 amino acids or 484 kDa, making it the largest single polypeptide toxin ever described. This toxin is a possible member of the RTX (repeats‐in‐toxin) family of pore‐forming toxins, based on the presence of consensus Ca2+‐binding repeat sequences at the C‐terminus of the protein and the linkage of the gene with a putative toxin activation gene and type I secretion apparatus (Coote, 1992; Lin et al., 1999).
Disruption of the toxin gene had no effect on the ability of V.cholerae to lyse red blood cells (Lin et al., 1999), a characteristic previously associated with a vacuolating toxin encoded by the unlinked hlyA gene (Alm et al., 1988; Rader and Murphy, 1988; Coelho et al., 2000; Mitra et al., 2000). Mutants in the rtxA gene were, however, defective in a different phenotype: the ability of El Tor and O139 V.cholerae strains to elicit rapid rounding of tissue culture cells (Lin et al., 1999). This activity was absent from certain classes of vaccine strains as well as classical biotype strains, and these defective strains were subsequently shown to have deletions in the rtx locus (Lin et al., 1999).
Here we characterize further the association of the gene rtxA with the ability of V.cholerae to induce cell rounding. We find that the RTX toxin does not disrupt membrane integrity and thus is not likely to be a pore‐forming toxin. Rather, cell rounding results from depolymerization of the actin stress fibers dependent upon the RTX toxin. Furthermore, we find that coincident with F‐actin disassembly, the cellular actin is covalently cross‐linked, representing a novel mechanism for a toxin to utilize to achieve actin depolymerization.
Characterization of cell rounding induced by V.cholerae Bah1P
The mutant strains constructed for these experiments, Bah1P and Bah2P, are derivatives of O1 El Tor E7946, a strain previously shown to induce cell rounding (Lin et al., 1999). The 'core' deletions of the CTXΦ genome present in both Bah1P and Bah2P remove the genes for CT and other factors reported to induce cellular responses (Figure 1A) (Taylor et al., 1994). A second chromosomal deletion within the gene hapA eliminates hemagglutinin/protease (HA/P), a protease that releases adherent tissue culture cells (Wu et al., 1996; Mel et al., 2000). The larger CTXΦ genome deletion in Bah2P also removes the 3′ end of the gene rtxA, disrupting the function of the RTX toxin (Figure 1A) (Lin et al., 1999).
In an assay for cell rounding, Bah1P, but not the rtxA mutant Bah2P, induced rounding of human laryngeal cells (HEp‐2) (Figure 1B). Similar rounding after addition of Bah1P was observed for all target cell lines that were tested, including HEp‐2 cells, A549 lung epithelial carcinoma cells, Henle 407 intestinal epithelial cells, L6 rat fibroblastoma cells, Chinese hamster ovary cells, Raw264.7 and J774 macrophages, and PtK2 kidney epithelial cells. In each case, cell rounding was dependent upon rtxA since the mutant strain Bah2P did not elicit cell rounding (data not shown).
Addition of chloramphenicol, an inhibitor of bacterial protein synthesis, to the tissue culture medium inhibited cell rounding, indicating that the RTX toxin produced by bacteria after exposure to cells is the result of de novo synthesis. In contrast, addition of cycloheximide, an inhibitor of eukaryotic protein synthesis, did not inhibit cell rounding, demonstrating that synthesis of new host proteins is not essential for this process (data not shown).
Taken together, these data demonstrate that after addition of washed bacteria to tissue culture cells, the RTX toxin is produced from the gene rtxA, leading to rounding of a broad range of cell types.
Membrane integrity is intact in RTX‐exposed cells
We first investigated whether the RTX toxin causes cell death through pore formation. If the RTX toxin is a pore‐forming toxin, as predicted by its association with the RTX family, then membrane integrity should be compromised after infection, leading to necrosis and leakage of cytoplasmic contents. However, in standard assays for cell lysis, HEp‐2 cells rounded by co‐incubation with Bah1P did not release either lactate dehydrogenase or sodium chromate51 into the culture medium at levels above mock infection (Figure 2A and B). In addition, cells excluded the fluorophore DEAD‐RED (Figure 2C) as well as other membrane‐impermeant dyes including trypan blue and propidium iodide (data not shown). These results indicate that cell membrane integrity remains intact in cells that are rounded by co‐incubation with Bah1P, and thus pore formation and necrosis cannot account for the observed cell rounding.
When bacteria were washed away 1 h post‐inoculation and the cells were subsequently maintained in the presence of gentamicin, the cells remained adherent to the surface and were biochemically active 24 h later as determined by reaction with 10% Alamar blue dye (data not shown). Taken together, these data demonstrate that V.cholerae expressing the RTX toxin does not cause rapid cell death.
Depolymerization of actin stress fibers depends on the RTX toxin
A common toxic mechanism that leads to rounding of a broad range of cell types without directly affecting cell viability is alteration of the polymerization state of the actin cytoskeleton (Steele‐Mortimer et al., 2000). To test whether the RTX toxin affects the state of actin polymerization, HEp‐2 cells at various stages of rounding were fixed and stained with fluorescently labeled phalloidin to visualize polymerized F‐actin (Figure 3). The actin stress fibers in HEp‐2 cells exposed to Bah1P are completely depolymerized. At 1 h, the actin within the rounding cells is found in dense phalloidin‐staining aggregates (Figure 3B) that were also observed in cells treated with cytochalasin D, a drug that inhibits formation of actin filaments (Figure 3D). After 2 h, the RTX‐treated cells ceased to bind phalloidin, indicating a total absence of polymerized F‐actin (Figure 3F and G). Depolymer ization of stress fibers is not observed in cells exposed to the rtxA mutant Bah2P (Figure 3C and H), demonstrating that disassembly of actin requires the RTX toxin.
Two other putative toxins of V.cholerae, zonal occludens toxin (ZOT) and the protease HA/P, have also been associated with rearrangements of F‐actin (Fasano et al., 1995; Wu et al., 1996). Since the RTX+ strain Bah1P used in our studies is deleted for both the zot and hapA genes, the cell biological effects we observe cannot be due to these other putative toxins. Thus, we conclude that the depolymerization of actin fibers is due to RTX toxin.
Rho in RTX‐exposed cell extracts is ADP‐ribosylated by exoenzyme C3
The amino acid stretch from amino acid 3376 to 3625 of the RTX toxin was found to have sequence similarity (22% identity, 40% similarity) to a region of Clostridium difficile toxin A (CdA) (Figure 4A), another very large toxin that causes depolymerization of actin (Fiorentini and Thelestam, 1991; Boquet et al., 1998). CdA is known to transfer a UDP‐glucose molecule to Thr37 of Rho and Thr35 of Rac and Cdc42, rendering these GTPases biologically inactive (Boquet et al., 1998). The modification protects Rho in cell extracts from in vitro ADP‐ribosylation by Clostridium botulinum exoenzyme C3, a toxin that specifically targets RhoA, B and C (Dillon et al., 1995; Just et al., 1995). Although the region of amino acid similarity between RtxA and CdA lies outside the catalytic domain of CdA (Faust et al., 1998), RTX‐exposed cells were tested for a CdA‐like modification of Rho.
Rho proteins in cell extracts from cells co‐incubated with Bah1P or Bah2P were not protected from in vitro ADP‐ribosylation by exoenzyme C3 (Figure 4). In contrast, control cells exposed to purified CdA toxin did show protection from exoenzyme C3. These data demonstrate that a CdA‐like modification of Rho is not responsible for actin depolymerization. In addition, a shift in electrophoretic mobility of Rho was not detected in Bah1P‐exposed cell extracts, suggesting that a different modification of Rho also did not occur, although these data do not rule out other effects on signaling through the Rho family GTPases.
Actin is modified in RTX‐exposed cells
Surprisingly, in the previous experiment, it was observed that a 42 kDa band was absent from Coomassie Blue‐stained SDS–polyacrylamide gels of cell extracts from RTX‐exposed cells as compared with control extracts (data not shown). This observation suggested that the target of the toxin may be actin itself.
Western blot analysis of cell extracts using anti‐actin antibody showed that virtually all of the monomeric actin in Bah1P‐exposed HEp‐2 cells was absent and replaced by larger immunologically cross‐reactive species (Figure 5). In cells rounded by exposure to either cytochalasin D or CdA, the size of actin was not altered, demonstrating that this effect is not a general consequence of F‐actin disassembly (Figure 5). Furthermore, cells exposed to Bah2P did not show the alteration in the size of actin, demonstrating that the effect is dependent upon rtxA. These data indicate that the actin has been altered directly in RTX toxin‐exposed cells.
Cellular actin is cross‐linked covalently in RTX‐exposed cells
Closer examination of the slowly migrating actin‐related species showed that they varied by 42 kDa intervals, suggesting that G‐actin monomers (42 kDa) were being cross‐linked stably in toxin‐treated cells (Figure 5). This RTX toxin‐induced 'actin laddering' on SDS–polyacryl amide gels was observed even when RTX toxin‐treated HEp‐2 cells were boiled in SDS–PAGE buffer for up to 10 min in the presence of 0.2 M dithiothreitol (DTT), 5% β‐mercaptoethanol, 6 M urea or 1% Triton X‐100, indicating that the cellular actin is cross‐linked by a non‐sulfhydryl bond (data not shown). The stability of the toxin‐induced actin cross‐linkage strongly suggests that these bonds are covalent.
To test this hypothesis, all the Triton‐soluble actin of Bah1P‐exposed HEp‐2 cell extracts was purified over a DNase I affinity column. The 84 kDa 'dimer' form was stable even after elution from the DNase I column in 50% formamide, further indicative of a covalent linkage (Figure 6). This form was purified further by excision from a 7.5% SDS–polyacrylamide preparative gel, digested to completion with trypsin and the resulting peptide fragments separated by HPLC. The amino acid sequences of 40 individual digestion products were determined. Thirty‐nine of the peptide sequences, representing 20 independent sequences, could be aligned directly with the amino acid sequence of human β‐actin (DDBJ/EMBL/GenBank accession No. NP 001092). Interestingly, the sole non‐actin peptide was probably derived from calnexin (DDBJ/EMBL/GenBank accession No. A46673) (Figure 6). Calnexin, a molecular chaperone that binds aggregated proteins, migrates on SDS–PAGE with an apparent mol. wt of 88 kDa, suggesting that it may co‐purify with the 84 kDa cross‐linked actin (Bergeron et al., 1994). However, the absence of another 42 kDa protein at a proportion equal to the actin species demonstrates that the 84 kDa form is a homodimer. These data confirm that exposure of HEp‐2 cells to V.cholerae expressing RTX toxin results in the covalent cross‐linking of cellular actin monomers to themselves.
The RTX toxin is exported to culture supernatant fluids
In our previous report, we stated that the RTX toxin activity was not present in culture supernatant fluids (Lin et al., 1999). However, given that rtxA is found adjacent to genes for a type I secretion apparatus, it is likely that the toxin is exported. To assess this problem more carefully, we switched to the strain KFV43. KFV43 is a derivative of the El Tor strain N16961 with a deletion in hapA (Fullner and Mekalanos, 1999). In a survey of wild‐type El Tor strains, it was noted that both cell rounding and cross‐linking after co‐incubation of HEp‐2 cells with the strain KFV43 tended to progress more rapidly under identical infection protocols than observed for Bah1P or its wild‐type parent E7946P (data not shown). This observation suggested that KFV43 is an overproducer of RTX toxin compared with Bah1P and that this strain might serve as a better source for detection of RTX toxin in culture supernatant fluids (data not shown).
Indeed, when sterile culture supernatant fluids from logarithmic phase cultures of KFV43 were mixed 1:1 with tissue culture media and added to cells, cell rounding was observed (data not shown). No activity was detected if the cultures were grown to stationary phase. Cell rounding activity in these supernatant fluids could be concentrated 300‐fold by ultrafiltration using a 100 kDa cut‐off cartridge and 300 kDa cut‐off membrane (data not shown). For comparison, concentrated supernatant fluids from CW128, an isogenic rtxA mutant of KFV43, were also prepared by an identical protocol.
These concentrated preparations were examined for the presence of the RTX toxin by western blotting using an antibody raised against a 138 kDa subfragment of RtxA that had been purified from Escherichia coli as a His6‐tagged protein. Numerous bands were detected by the RtxA‐specific antibody that were not detected in either the rtxA mutant preparation or by the pre‐immune serum. Several large bands consistent with the predicted full‐length size of RtxA were detected, but the majority of the protein appears as breakdown products of ∼115, 100 and 65 kDa (Figure 7).
When added to HEp‐2 cells, these preparations induce both cell rounding and cross‐linking of actin (Figure 8). The activities in these preparations were neutralized by pre‐incubation with the RtxA‐specific antibody, but not by pre‐immune sera or a CT‐specific antibody (Figure 8). Taken together, these data demonstrate that the RTX protein is exported to culture supernatants during logarithmic growth and that the RtxA protein (or one of its major breakdown products) represents the active RTX toxin moiety responsible for both cell rounding and actin cross‐linking.
G‐actin is a substrate for actin cross‐linking
The RTX toxin could either act on the monomeric G‐actin form, leading to depolymerization, or it could break F‐actin chains directly. To test whether G‐actin can serve as a substrate for the RTX toxin, the actin in HEp‐2 cells was depolymerized with cytochalasin D, similarly to that shown in Figure 3D, prior to addition of concentrated culture supernatant fluids. Although the cross‐linking activity was reduced by ∼50%, cells rounded with cytochalasin D do show cross‐linking of actin (Figure 9). This result indicates that G‐actin is a substrate for the RTX toxin activity.
In this report, we have presented both biochemical and cell biological evidence for the depolymerization of F‐actin stress fibers in a manner that produces a novel by‐product: a highly cross‐linked complex of actin dimers, trimers and higher multimers. We have shown that addition of live bacteria to cells initiates this process and that both actin depolymerization and actin cross‐linking depend on the presence of the gene rtxA. Furthermore, we have shown that toxin exported to the supernatant fluids also has these activities and that this toxin can be neutralized with an RtxA‐specific antibody.
Therefore, the mechanism by which the gene rtxA is linked to cell rounding is through production of the secreted RTX toxin, which then interacts with cells, leading to depolymerization of stress fibers and cross‐linking of actin. Preliminary peptide mapping of the cross‐linked products suggests that the cross‐link may occur between two peptide fragments in the N‐terminal portion of actin protein. However, extensive chemical characterization will be necessary before the exact nature of the cross‐link is known. Indeed, our current mapping strategy has not excluded the possibility that more than one chemical cross‐link can be formed. Thus, the actin in the higher cross‐linked forms may be a 'web' instead of a chain.
Numerous toxins and type III secreted effector proteins have now been reported that mediate depolymerization of actin through action on the Rho family GTPases, by either direct modification or indirect action through their roles as GTP exchange factors (GEFs) and GTP‐activating proteins (GAPs) (Lerm et al., 2000; Steele‐Mortimer et al., 2000). In our system, cross‐linking of actin was not observed when cells were rounded by CdA, a toxin that inactivates Rho, Rac and Cdc42 by the addition of UDP‐glucose (Boquet et al., 1998). Thus, down‐regulation of the Rho family GTPase alone does not lead to cross‐linking of actin. This result indicates that the RTX toxin is unlikely to act through the Rho GTPase signaling pathway.
To explain how the RTX toxin causes both actin depolymerization and cross‐linking, we favor a model more closely related to those proposed for the action of the clostridial binary toxins, including C.spiroforme toxin, C.botulinum C2, C.perfringens iota toxin and an ADP‐ribosyltransferase from C.difficile. These toxins all ADP‐ribosylate G‐actin at Arg177 (Boquet et al., 1998). The modified G‐actin is unable to undergo polymerization and therefore acts as a capping protein at the barbed end of the actin filament. The unmodified pointed end continues to depolymerize, leading to a disorganization of the cytoskeleton and rounding of the target cells (Boquet et al., 1998; Popoff, 1998). By analogy, cross‐linking of actin into dimers and higher order cross‐linked products would also deplete the pool of G‐actin available for polymerization at the barbed end of actin filaments. A shift in the equilibrium between G‐ and F‐actin would occur, ultimately resulting in F‐actin disassembly (Figure 10). In concordance with this proposed model, we have found that rounding of cells with cytochalasin D prior to addition of the RTX toxin does not disrupt cross‐linking of actin, indicating that the toxin does target monomeric G‐actin. Thus, we suggest that G‐actin is cross‐linked directly by the RTX toxin, removing it from the actin pool, and that the stress fibers then depolymerize as the dynamic equilibrium shifts.
This simple model, however, is not the sole viable model. We have been unable to detect in vitro cross‐linking activity when concentrated supernatant fluid containing RTX toxin is incubated with either purified actin or HEp‐2 cell lysates in the presence of ATP, GTP and/or divalent cations. Our inability to detect this activity may simply indicate that the reaction requires unknown cofactors or intracellular modification of the full‐length toxin. However, this experiment may also predict that the RTX toxin works by an indirect mechanism.
As an alternative model, the RTX toxin could stimulate activity of an endogenous cross‐linking protein (Figure 10). An attractive cellular target within this model are the transglutaminases (TGases), a family of enzymes that catalyze a Ca2+‐dependent acyl transfer reaction between a γ‐carboxyamide group of glutamine and ϵ‐amino group of lysine or other primary amines (Chen and Mehta, 1999). Such enzymes have been associated with cross‐linking of proteins involved in cell growth and differentiation, wound healing, receptor‐mediated endocytosis, apoptosis and the formation of plaques in Alzheimer's patients (Murthy et al., 1998; Chen and Mehta, 1999).
Both in vivo and in vitro studies have demonstrated that actin can serve as a substrate for TGases. For example, bacterial TGase has been used in vitro to introduce photo‐cross‐linking reagents at glutamine residues of actin for the study of actin structure (Hegyi et al., 1998; Kim et al., 1998).
Furthermore, it has been shown that cytoplasmic actin can be a substrate for TGase in vivo. The lysine derivative DALP (3‐[Nα[Nϵ‐[2′,4′‐dinitrophenyl]‐amino‐n‐hexanoyl]‐ l‐lysylamido]‐propane‐1‐ol) is a membrane‐permeant amine substrate for tissue TGase. In leukemia‐derived human cell lines HL‐60 and U937 that were induced to undergo apoptosis, cytoplasmic actin was labeled with DALP, indicating that actin is a target for TGase in these cell lines. Although the majority of DALP label attached to the 43 kDa form of actin, other higher species that reacted with an actin‐specific antibody were also labeled, although these were not identified as multimers of actin (Nemes et al., 1997). Thus, it is conceivable that the RTX toxin acts via a similar TGase in cells to cross‐link actin without inducing the cells to undergo apoptosis.
Other models for the activity of the RTX toxin can be formulated if it is presumed that F‐actin depolymerization and cross‐linking are not interconnected activities (Figure 10). For example, the RTX toxin could work indirectly to alter the intracellular environment or signal transduction pathways such that many processes are affected concurrently. A modulation of a signal molecule could affect the activity of the Rho GTPase signaling pathway leading to actin depolymerization while simultaneously activating the TGase or other cross‐linking protein. Thus, cross‐linking would not be the direct cause of the depolymerization, but rather a concurrent response to a common signal.
Similarly, cross‐linking and actin depolymerization activities could be separate activities mediated by different domains of the 484 kDa toxin protein. Both Salmonella typhimurium SptP and Pseudomonas aeruginosa ExoS function as enzymes as well as GAP proteins (Kaniga et al., 1996; Ganesan et al., 1998; Fu and Galán, 1999; Goehring et al., 1999; Pederson et al., 1999). In each case, these two activities are associated with different domains of the toxin. Validation of a dual activity model for the RTX toxin would require demonstration that actin cross‐linking and depolymerization of actin are separable activities. As yet, we have not observed rounding in the absence of cross‐linking or the reverse.
Regardless of the mechanism, it is clear that the RTX toxin has a dramatic effect on target cells in vitro. However, it remains to be shown whether the RTX toxin is an important factor in V.cholerae pathogenesis. Deletion of the ctxA and ctxB genes that encode CT created strains that are still pathogenic in human volunteer studies, albeit with milder symptoms. In some volunteer studies, a second toxin gene, including genes encoding HlyA (Tacket et al., 1993), HA/P (Benítez et al., 1999) or RTX toxin (Taylor et al., 1994), have also been eliminated prior to toxin administration to human volunteers. For each of these strains, mild symptoms still persisted. Thus, none of these toxins represent 'the reactogenic factor', but rather reactogenicity may reflect a combined contribution of each of these factors. Indeed, a strain deleted of all potential reactogenic factors has never been tested in human volunteers. Our demonstration of the dramatic cellular consequence of RTX exposure, coupled with recent evidence that both HA/P (Wu et al., 1996, 1998, 2000; Mel et al., 2000) and hemolysin (Coelho et al., 2000; Mitra et al., 2000) also have devastating cellular effects, suggests that such studies may be warranted. Such a multiple toxin mutant may lead finally to the production of a safe, live, oral vaccine that could be implemented worldwide to control the incidence of cholera.
Materials and methods
Bacterial strains
Bah1 and Bah2 are vaccine derivatives of the V.cholerae O1 Ogawa strain E7946 (Taylor et al., 1994). hapA was deleted from these strains to create Bah1P, Bah2P and E7946P, respectively, using the sacB‐based counterselectable plasmid pHapSal1 as previously described (Finkelstein et al., 1992; Fullner and Mekalanos, 1999). KFV43 is a ΔhapA derivative of the V.cholerae O1 Inaba strain N16961 (Fullner and Mekalanos, 1999). CW128 is a derivative of KFV43 with a 5.5 kb internal deletion in the rtxA gene that inactivates the activity of the RTX toxin (C.Walchle and J.J.Mekalanos, unpublished).
Cell rounding and actin depolymerization
Bacterial cultures, grown for 16 h in LB broth at 30°C and washed with phosphate‐buffered saline (PBS), were added to adherent cells (m.o.i. ∼200) covered with fresh RPMI 1640 medium containing 10 mM HEPES buffer, 2 mM glutamine and 10% fetal calf serum. For some experiments, 5 μg/ml chloramphenicol or 200 μg/ml cycloheximide was added 60 min prior to the addition of bacteria. Cell rounding was monitored with a Nikon Diaphot 200 inverted microscope equipped with a computer interface. For visualization of actin, cells grown on glass coverslips were stained with Molecular Probes Alexa‐488‐labeled phalloidin according to the manufacturer's instructions. Cells were counterstained with propidium iodide to facilitate location of cells in the field. Coverslips mounted with ProLong Antifade mounting solution (Molecular Probes, Inc.) were viewed with a Nikon Eclipse TE300 fluorescence microscope.
Cell lysis assays
For all cell lysis assays, cells were co‐incubated with PBS‐washed bacteria at an m.o.i. of ∼200. Assay for release of lactate dehydrogenase to the medium was carried out using the Promega CytoTox96 Non‐radioactive Cytotoxicity kit according to the manufacturer's instructions using HEp‐2 cells seeded into 96‐well dishes in phenol red‐free medium. For measurement of Cr51 release, non‐adhered HEp‐2 cells were mixed with sodium chromate51 and incubated at 37°C for 1 h. Cells loaded with Cr51 were washed three times in medium and then were seeded into a 96‐well dish. After 3 h, the cells had adhered to the surface. After cells were exposed to bacteria, Cr51 released to the medium was measure in a Wallac 1470 Wizard Automatic Gamma Counter. Membrane integrity analysis using the fluorophores SYTO10 and DEAD‐RED was carried out using reagents in the Molecular Probes Live/Dead Reduced Biohazard Viability/Cytotoxicity kit according to the manufacturer's instructions.
ADP‐ribosylation of cell extracts with exoenzyme C3
Labeling of cell extracts by C.botulinum exoenzyme C3 (List Laboratories) was carried out as described by Dillon et al. (1995). Subconfluent HEp‐2 cells seeded into 100 mm tissue culture dishes were co‐incubated with bacteria at an m.o.i. of ∼200 or 1 nM CdA (kindly provided by I.Castagliuolo and J.Lamont). Cell rounding in response to bacteria was allowed to proceed for 80 min while CdA‐exposed cells were processed after 6 h when cells appeared similar to cells rounded by Bah1P. Cells were washed in PBS, recovered by scraping and pelleted at 1000 g. Cells were resuspended in extract buffer [1% Triton X‐100, 20 mM Tris pH 7.5, 1 mM MgCl2, 125 mM NaCl, 1 mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml pepstatin A, 0.5 μg/ml leupeptin] and were frozen at −20°C. A 50 μg aliquot of protein extract was added to the reaction buffer (10 μM cold NAD, 250 nM [32P]NAD, 10 mM HEPES pH 8.0, 2 mM MgCl2, 1 mM DTT, 200 μM GTP, 10 mM thymidine, 15 mM isoniazid, 80 nM exoenzyme C3) and the reactions were incubated at 30°C for 1 h. Reactions were stopped by boiling in SDS–PAGE sample buffer for 5 min. Samples were loaded onto a 15% SDS–polyacrylamide gel, electrophoresed, and ADP‐ribosylated proteins were visualized by exposing the gel to the Bio‐Rad Model GS505 PhosphorImager.
Purification of actin and peptide sequencing
Purification of actin was based on the protocol of Goode et al. (1999). A total of 108 HEp‐2 cells seeded into 500 cm2 tissue culture plates were exposed to Bah1Δhap (m.o.i. ∼200) for 65 min. The cells were washed once in PBS, collected by scraping and pelleted by centrifugation at 1000 g. Pelleted cells were lysed in 5 ml of G‐buffer (10 mM Tris pH 7.5, 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM DTT) containing 1% Triton X‐100, and the Triton‐insoluble fraction was removed by centrifugation at 26 000 g. A 1 ml aliquot of the soluble fraction was loaded onto a 2 ml DNase I–Affigel 10 column. The column was washed with two column volumes of G‐buffer, two column volumes of G‐buffer with 0.2 M NH4Cl and two column volumes of G‐buffer with 10% deionized formamide, and was eluted with G‐buffer containing 50% formamide. Pooled eluants from five column runs were dialyzed into G‐buffer, concentrated with a Centricon‐30 microconcentrator, boiled in SDS–PAGE buffer and then loaded onto a preparative 7.5% SDS–polyacrylamide gel. Coomassie Blue‐stained proteins corresponding to the 42 and 84 kDa forms of actin were excised, rinsed in 50% acetonitrile and frozen at −20°C for HPLC analysis. Both forms were digested with trypsin and the sequence of the peptide fragments was determined at the Harvard Microchemistry Facility by microcapillary reverse‐phase HPLC non‐electrospray tandem mass spectrometry (μLC/MS/MS) on a Finnigan LCQ quadrupole ion trap mass spectrometer (LeRoy et al., 1998).
Concentrated supernatant preparations
Supernatants from four 500 ml flasks of KFV43 or CW128 culture grown in LB broth at 37°C to log phase (A600 = 0.3–0.5) were collected by centrifugation at 13 000 g. Pooled supernatants were filtered over a 0.22 μm Corning cellulose acetate filter unit and concentrated to 150 ml across an Amicon spiral‐wound ultrafiltration cartridge with a 100 kDa cut‐off. The retentate was supplemented in three changes with 1 l of TE (20 mM Tris pH 8.0, 1 mM EDTA, 0.2 mM PMSF). The supernatants were concentrated further in a 50 ml Amicon Spin‐cell apparatus fitted with a 300 kDa cut‐off filter with one supplementation of 50 ml of TE buffer to a final volume of 6.3 ml (300‐fold total concentration). Glycerol was added to 10% (v/v) and preparations were frozen and stored at −80°C in aliquots.
Western blot for actin laddering
A total of 5 × 105 toxin‐ or mock‐treated HEp‐2 cells were washed once with PBS, released in PBS with 0.1 mM EDTA, and recovered in RPMI 1640 medium by scraping. Cells were collected by centrifugation at 8000 g and resuspended in SDS–PAGE buffer containing 0.2 M DTT. These cell lysates were heated in boiling water for 5 min unless otherwise stated, electrophoresed in 8% SDS–polyacrylamide gels and then transferred to nitrocellulose. Antibody detection with 1:2000 polyclonal anti‐actin (Sigma) or monoclonal anti‐tubulin (Sigma) antibodies was performed using 1:2000 anti‐rabbit IgG–alkaline phosphatase (AP) conjugate as a secondary antibody (Boehringer Mannheim) and as a substrate for colorimetric development with 5‐bromo 4‐chloro indolyl phosphate p‐toluidine (Bachem) and nitroblue tetrazolium (Sigma) (Ausubel et al., 1990).
Production of RtxA138‐ and RTX‐specific antibody
pWL126 contains a 4 kb EcoRI–HindIII subfragment of the rtxA gene in pET22b (Novagen). A 138 kDa His‐tagged product, designated RtxA138, was purified from E.coli BL21(DE3)(pLysS) cells in the presence of 6 M urea on a nickel column by protocols recommended by the manufacturer (Novagen). A New England White rabbit was immunized with RtxA138 for the production of a polyclonal antiserum. For secondary boosts, the antigen was purified further by gel filtration chromatography or by electroelution. For antibody neutralization assays, the IgG1 fraction was isolated by passage of serum over a protein A–agarose column (Harlow and Lane, 1988).
Western blot for detection of RTX toxin
Concentrated culture supernatant fluids from KFV43 (RTX+) and CW128 (RTX−) were loaded in each lane of a 4–15% gradient SDS–polyacryl amide gel (Bio‐Rad). Gels were transferred in a Bio‐Rad Mini Trans‐Blot transfer cell at 350 mA for 2 h at 4°C. RTX toxin was detected using a 1:1000 dilution of serum and the Amersham ECL for western blotting kit.
We thank J.Lamont and I.Castagliuolo for providing purified toxin A, W.Lin for constructing plasmid pWL126, Cynthia Walchle for providing CW128, and the Harvard Microchemistry Facility for the peptide sequence analysis. T.Mitchison, E.Rubin, B.Guo, D.Higgins and J.Mogridge are thanked for their helpful suggestions. This work was supported by NIH grant AI‐18045 to J.J.M. K.J.F. was supported by an NRSA postdoctoral fellowship AI‐10395.
- Copyright © 2000 European Molecular Biology Organization


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Signing and saying, "Cookie, cookie, cookie," -- has been a sure-fire success for Isaac, when he is with Daddy and big brothers, and Mama, sometimes, it is really cute, but mostly I'll offer an apple or some grapes,...as it avoids getting Isaac into a cookie coma!!!
Of course these pictures were taken after school and Isaac was a wee bit tired after a busy day of coloring, and cutting, and pasting, and painting,...along with speech therapy and some occupational therapy, and the most important, playtime on the playground!!!
Preschool has been a wonderful resource and we're so thankful Isaac has transitioned so easily and has had so many successes:
1. Nap time: Isaac sleeps - actually sleeps at school - hurray!
2. Breakfast and lunch: - reportedly Isaac eats more breakfast than lunch, but that's about par, but he has been trying new foods and new textures - so another hurray!
3. Social: good eye contact, and such nice good-byes to his teachers.
4. Verbal: thru the roof!!!! The other day, Isaac walked by my office door, and said, "Hi Mama," as if he's been speaking as such for day one!!! Apraxia hasn't let him say it again,...but it was beautiful to hear,...and is an emerging skill....He, also, is appropriately responding to requests by saying, "OK!"
5. Potty: there has been success!!! Only six times and only at school, but who cares, success is success, and a this new skill will eventually be mastered!!!!!
6. Bedtime: getting our late night owl into bed at a more reasonable hour has been easier,...anything before 10:00 o'clock is utterly wonderful, and anything before 12:00 pm is a vast improvement!!
7. Routine: finally getting into one,...which again is an improvement, as before the only thing predictable about our daily routine is that there was nothing predictable about it.
Yep, our little Cookie-face guy is coming along nicely,...
Until I post again,...may God bless and keep you!


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(School Director, Instructor, & Dancer) is the Director of Ipswich Moving Company School of Dance and Assistant Director of the Ipswich Moving Company. She teaches classes for students who range in age from infant-toddlers in Creative Movement classes to elementary school students, teens, and adults. She has developed an audition-based performance group called the Youth Dance Ensemble.
Jenny also offers introductory dance classes at several local educational institutions, including the Ipswich Public Schools as well as private preschool and early elementary programs.
She has danced with Flipside Dance Theatre, Fresh Squeezed Dance, Fallen Flight Project, Julie Pike Edmond, Julie Ince Thompson, and Adrienne Mincz. These companies and choreographers have taken her from California to Europe to perform. Jenny has taught Aerial Dance at Perry Mansfield in Steamboat Springs, CO with Janet Craft. The Boston Globe described Jenny as "a beautifully lyrical and articulate dancer, who moves with a lush fluidity."
Jenny Carlson trained at the Imperial Academy of Dance in Connecticut and continued her studies at Emerson College, receiving a BFA in Dance. Jenny has been flying through the air for 18 years on rope, fabric, and steel frames, performing and choreographing aerial dances with Ipswich Moving Company.


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The rising complexity of distributed computer systems give reason to investigate self-organization mechanism to build systems that are self-managing in the sense of Autonomic and Organic Computing.
In this paper we propose the Artificial Hormone System (AHS) as a general approach to build self-organizing systems based on networked nodes. The Artificial Hormone System implements a similar information exchange between networked nodes like the human hormone system does between cells. The artificial hormone values are piggy-backed on messages to minimize communication overhead.
To show the efficiency of the mechanism even for large scale systems we implemented a simulation environment in Java to evaluate different optimization strategies. The evaluations show that local information is enough to meet global optimization criterion.
1st IFIP International Conference on Biologically Inspired Cooperative Computing - Biological Inspiration 2