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Construction Project Reveals Third Miami Circle
Tuesday, April 30, 2013
A third circle made up of post holes cut into bedrock by the Tequesta has been uncovered near the mouth of the Miami River. The first Miami Circle, thought to be a 2,000-year-old religious site, has been listed as a National Historic Landmark. The second circle, called the Royal Palm Circle, may have been used as a dwelling. The third circle was found nearby, beneath fill brought in by Henry Flagler to develop the city's downtown area in the early twentieth century. A well and foundations of the Royal Palm Hotel, built by Flagler in 1897, have also been uncovered.MIAMI, FLORIDA—
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Anglo-Saxon Jewelry Box
Wrecks of the Pacific Theater
The Environmental Cost of Empire
One Ring to Bind Them
Cosmic Rays and Australopithecines
Bison Bone Mystery
Rome's Earliest Fort
A Parisian Plague
Finding Lost African Homelands
Neanderthal Necklace
Slime Molds and Roman Roads
Open House
World Roundup
Civil War booze, world's oldest pretzels, Austria's war camels, coral tombs of the Pacific, and a 2.8-million-year-old human
Styling hair in Bronze Age Wales
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Hotel Torenzicht
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Amsterdam for a whole week - Part 2
... and built a causeway to the mainland. Now it is just a pretty little village with some interesting houses and a cute little museum with a display of local costumes.
Back to Amsterdam by return bus and a walk home from near the Central Station. Window shopping is usually as far as we get but a pretty little pendant left out of the window at me and we had to get one. We ended up designing the size material and tulip colour and will pick it up on Saturday. Dinner was ...
... 6 year olds…. They play soccer on Monday and Wednesday and are still crazy about watching it on TV (we watched Barcelona-Madrid El Clasico together and they knew 80% of the players). Only a matter of time before we are getting season tickets at Ajax…..! Kat is also doing super well. In her first year running her own company, she's really hit the ground running and os loving it. She was at Nike for 6 months as the Organization Capabilities Director and has done ...
Day 28: London to Amsterdam
... back in the coffee shop buying two more joints! Each! In their state, they decided they wanted another one, and that they should also take one each back for Bruce and Audrey so they could all sit around as a happy family sharing them at breakfast tomorrow. Top idea fellas, they'd surely be up for that! This time they smoked their joints in a different area of the shop, but halfway through they were kicked outside as apparently they weren't allowed to smoke in that area. So at about ...
Shtop! Thish Blog ishn't ready yet...
... standards, not being first populated until the 1200's when the locals first dammed the Amstel River. The town that grew up around it became the Amsteller Dam and then Amsterdam. Being close to the sea and in a relatively protected little spot, a huge merchant navy developed and soon intrepid merchants were setting off for all points around the globe. It was a Dutch navigator that first mapped the coast of Australia and named Van Diemans land. much of India and ...
Our luck ran out....
... had cleared up a bit so we headed toward the Slave Lodge (museum). A few other stops along the way, a bit to eat and then we headed back to the dryness of the hotel lobby. A few hours later, we were a bit dryer and decided to just head to the airport and eat dinner there rather than run the risk of getting caught in more showers.
The rest of the night was uneventful. We took off at 11:30 pm and flew overnight to Amsterdam.

subset: commoncrawl


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Happy Hour at Pracna on Main
Built in 1890, Pracna on Main Boasts quite an impressive history: It is the oldest restaurant on the oldest street in Minneapolis. Ye olde hotspot features excellent river views, a gorgeous patio (which is still a little too cold to enjoy right now, alas), and close proximity to St. Anthony Main Movie Theater, making it an ideal spot for a date-night nightcap or icebreaker. Though a decidedly American bar in menu selections, the dark wood and old feel of the decor prompt comparisons to an Irish pub. Friendly servers offer around 20 different beers on tap, and those taps become $3 a pint every day between the magical hours of 9 p.m. to 1 a.m. Some consider the bar walkable from Dinkytown, though if you're traveling via vehicle, finding a lot or some street parking in front shouldn't be a problem.
Starts: May 6. Daily, 2009
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VIEWS: 4,433 PAGES: 8 CATEGORY: Business Services POSTED ON: 11/4/2009 Public Domain
This Janitorial Services Contract is made between a company and a contractor for the provision of janitorial services to the company at a specified location. The contract contains the material terms and conditions of the agreement including the specific services to be provided, the payment details, and the term of the agreement. It contains numerous standard provisions that are commonly included in these types of agreements and may be customized to fit the specific needs of the parties. This should be used by company and a janitor when entering into an agreement for janitorial services.
This Janitorial Services Contract is made between a company and a contractor for the provision of janitorial services to the company at a specified location. The contract contains the material terms and conditions of the agreement including the specific services to be provided, the payment details, and the term of the agreement. It contains numerous standard provisions that are commonly included in these types of agreements and may be customized to fit the specific needs of the parties. This should be used by company and a janitor when entering into an agreement for janitorial services. JANITORIAL SERVICES AGREEMENT THIS JANITORIAL SERVICES AGREEMENT (the "Agreement"), made this ____ day of _________, 2_____, by and between _______________ ("Contractor") located at _____________________________and ___________ ("Company"), located at _________________________ hereinafter referred to as (the "Parties"). WHEREAS, Contractor provides janitorial services; AND WHEREAS, Company desires to obtain the janitorial services of Contractor pursuant to the terms and conditions herein. IN CONSIDERATION of the promises and other good and valuable consideration set forth, the parties agree as follows: I. SERVICES 1. Contractor and Company hereby agree that Contractor shall provide janitorial services to Company (the "Services") for the building(s)/warehouse located at _________________________ (the "Premises"). 2. Contractor shall provide the Services to the Company which are more particularly described in Schedule "A" annexed hereto. II. TERM 1. The term shall commence on the ____ day of __________, 2_____ (the "Commencement Date") and shall continue for a period of ( ) years (the "Term"). 2. Company may request to extend the Term ("Extension Term") by providing written notice to Contractor no less than ______ ( ) days prior to the expiration of the Term. The request shall include the length of the extension and any modifications to the terms and conditions contained herein. Contractor shall have ______ (__) days to accept Company's request . © Copyright 2011 Docstoc Inc. registered document proprietary, copy not 2 III. CONTRACTOR RESPONSIBLITIES 1. Contractor hereby agrees as follows: A. Contractor shall commence the Services contemplated herein on the Commencement Date and shall perform all of the Services diligently and using its best efforts to conform to local standards and practice; B. Contractor shall provide Company a mutually agreed upon [MONTHLY OR WEEKLY] work schedule for the Services to be performed; and C. Contractor shall at all times keep employed an adequate number of staff to perform the Services contemplated herein. . IV. FEES 1. Company shall pay Contractor the sum of __________ ($__________) Dollars (the "Fees") per month for the Services to be performed 2. All Fees are due and payable within ____ (__) days of Company's receipt of Contractor's monthly invoice. All invoices will include a detailed description of the Services performed for which payment is being requested. Company has the right to request additional reports and supporting information from Contractor as deemed reasonably necessary by Company to audit Contractor's performance. Contractor shall provide all additional reports and information requested by Company within ____ (___) days of the written request. V. MODIFICATIONS TO AGREEMENT 1. The Parties agree that Company shall have the right to add or delete buildings/warehouses and/or modify this Agreement upon _____ ( ) days written notice to Contractor. Any modifications must be agreed to by both Parties. VI. CONTRACTOR'S EMPLOYEES 1. Contractor shall use its own staff, employees and sub-contractors in respect to the Services to be performed. 2. The Parties acknowledge that the relationship between Company and Contractor is that of an independent contractor and Contractor alone has exclusive control and supervision of its staff, employees and sub-contractors. Nothing herein shall be construed to mean that Contractor or any of its staff, employees or sub-contractors are agents, employees or representatives of Company. 3. Contractor shall at all times ensure that industry accepted safety standards and practices are followed while performing the Services. Contractor shall at all times © Copyright 2011 Docstoc Inc. registered document proprietary, copy not 3 comply with all ordinances, regulations, and other legal requirements and obligations of the applicable jurisdiction while performing the Services. VII. EQUIPMENT AND SUPPLIES 1. Company shall provide all supplies necessary for performance of the Services which shall include, but not be limited to [INSERT ALL SUPPLIES COMPANY WILL PROVIDE, TRASH BAGS, PAPER TOWELS, TOILET PAPER, ETC.]. 2. Contractor shall supply all of the necessary equipment and tools for performance of the Services, which shall include, but not be limited to, floor waxers, brooms, mops, buckets and cleaners. 3. Company shall provide a designated area on the Premises for storage of Contractors equipment, tools and supplies. VIII. INSURANCE 1. Contractor shall at all times maintain liability insurance coverage to cover any claims for injuries to person or damages to property which may arise in connection with the performance of the Services by Contractor, its staff, employees and sub-contractors. The policy shall be in an amount of not less than ____________ ($________) Dollars. Contractor shall also provide worker's compensation insurance for its staff, employees and sub-contractors. Company may at any time request that Contractor provide proof of liability and worker's compensation insurance and any other certificates or documents reflecting that Contractor is in full compliance with all legal requirements and obligations for companies providing janitorial services in the State of _____________. IX. NON-EXCLUSIVITY 1. The Parties acknowledge that the Services to be performed hereunder are non- exclusive and that Company shall have the right to contract with a third party for the same or similar services for which Contractor is engaged, and similarly Contractor is free to enter into contracts to perform the same or similar services for third parties. © Copyright 2011 Docstoc Inc. registered document proprietary, copy not 4 X. EVENTS OF DEFAULT 1. In the event of non-performance by either Party of any of its obligations contained herein, the non-defaulting Party shall serve the defaulting Party with written notice of the alleged breach. The defaulting Party shall have _____ (___) days from the date of such notice to cure and rectify any non-performance or non-compliance. Upon the correction of any non-performance or non-compliance by the defaulting Party, the Agreement shall continue for the duration of the Term or Extension Term. In the event that the defaulting Party fails to correct any non-performance or non-compliance issue within the time frame set out above, the non-defaulting Party may terminate this Agreement immediately and without further notice. XI. CONFIDENTIALITY 1. Contractor shall treat all information relating to Company, its business practices and, any other information that Contractor may become aware of that is proprietary to Company, as confidential and shall not disclose any such confidential information to any third party except as may be reasonably required pursuant to this Agreement or as required by court order. XII. INDEMNIFICATION 1. Contractor shall indemnify and hold Company, its subsidiaries, officers, directors, employees and agents harmless from and against any loss, liability, damage, including but not limited to, any reasonable attorney fees and court costs which may arise out of or in connection with the Services performed hereunder, save and except for the gross negligence and wilful misconduct of Company, its subsidiaries, officers, directors, employees and agents. 2. Contractor shall at all times be solely responsible and liable for any damages caused by its staff, employees or sub-contractors to any of the Company's equipment, property or fixtures and shall make all repairs and/or replacements for any such damage without cost to Company. XIII. GENERAL PROVISIONS 1. Neither Party may assign any obligations hereunder to any third party without the prior written consent of the other Party. 2. This Agreement and the work schedules referred to herein constitute the entire agreement between the Parties and supersede all prior agreements, representations, warranties, statements, promises, information, arrangements and understandings, whether oral or written, express or implied, with respect to the subject matter hereof. 3. This Agreement may be executed in any number of counterparts, each of which shall be deemed to be an original and all of which taken together shall be deemed to constitute © Copyright 2011 Docstoc Inc. registered document proprietary, copy not 5 one and the same instrument. This Agreement may be executed and delivered by electronic facsimile transmission with the same force and effect as if it were executed and delivered by the Parties simultaneously in the presence of one another. 4. Any notices to be delivered pursuant to this Agreement shall be delivered to: in the case of Company to: Attention: Fax: (___) in the case of Contractor to: Attention: Fax: (___) and, in each case, mailed by ordinary mail, postage prepaid, or delivered to that address; and, if mailed as aforesaid, any such notice shall have been deemed to have been given on the fifth (5th) business day following that on which the letter containing the notice was posted. If any notice is given by electronic communication, such notice shall be deemed to be delivered on the day of transmittal thereof if given during normal business hours of the recipient and on the next business day if given after normal business hours. Any party may change its address for service from time to time by notice given in accordance with the foregoing. 5. In the event a court of competent jurisdiction finds any provision of this Agreement invalid or unenforceable, the remaining terms shall remain in full force and effect. 6. This Agreement shall be governed in accordance with the laws of the State of ___________. The parties hereby irrevocably submit to the jurisdiction of the courts of the State of located in County. In any suit or arbitration regarding the Agreement, the prevailing party shall be entitled to reasonable attorneys' fees and costs. © Copyright 2011 Docstoc Inc. registered document proprietary, copy not 6 IN WITNESS WHEREOF THE PARTIES HERETO HAVE DULY EXECUTED THIS CONTRACT AS OF THE DAY AND YEAR FIRST WRITTEN ABOVE. [COMPANY] Name: Title: [CONTRACTOR] Name: Title: © Copyright 2011 Docstoc Inc. registered document proprietary, copy not 7
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Mountain biking to access stellar fly fishing? Yes please! [Read More]


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Town Square
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SCHOOLS & KIDSMP school board OKs teacher layoffs
Teachers in the Menlo Park City School District could start seeing preliminary layoff notices in their inboxes this week....Original post made on Mar. 11, 2010
Last comment by Publius, on Mar 16, 2010 at 10:35 am | 27 comments | 3001 Views
SCHOOLS & KIDSHigh school board OKs security cameras
The Sequoia high school district's Board of Trustees has unanimously approved the installation of surveillance cameras in...Original post made on Mar. 15, 2010
There are no comments yet. 2081 Views
MENLO PARKDowntown parking: Change brewing
A consulting firm outlined possible changes to downtown parking rules in Menlo Park at a meeting attended by some 35 residents...Original post made on Mar. 12, 2010
Last comment by Julie, on Mar 15, 2010 at 9:23 pm | 3 comments | 1314 Views
AROUND TOWNRich Gordon elected board president
Rich Gordon, a 13-year member of the San Mateo County Board of Supervisors and a resident of unincorporated Menlo Park, was...Original post made on Jan. 13, 2010
Last comment by R.GORDON, on Mar 15, 2010 at 8:00 pm | 8 comments | 1462 Views
SCHOOLS & KIDSMP district will take all Tinsley students next year
The Menlo Park City School District voted to enroll its full allotment of Tinsley transfer students from the Ravenswood school...Original post made on Mar. 12, 2010
Last comment by Resident, on Mar 15, 2010 at 2:22 pm | 11 comments | 2070 Views
AROUND TOWNKepler's Bestsellers - Week of 3/8/10
Hardcover Fiction 1 Murder In The Palais Royal Cara Black 2 Help Kathryn Stockett 3 Man From Beijing Henning Mankell 4 3...Original post made by Pam Grange on Mar. 15, 2010
There are no comments yet. 1296 Views
PORTOLA VALLEYDeputies' ploy nets burglary suspects in Westridge neighborhood of Portola Valley
Alert observation followed by a little deception -- it was all in a day's work for sheriff's deputies patrolling the Westridge...Original post made on Mar. 11, 2010
Last comment by Luc, on Mar 15, 2010 at 1:42 pm | 2 comments | 2358 Views
OTHER TOPICSReport of mountain lion near Woodside
A mountain lion was spotted in the Teague Hill Open Space Preserve near Woodside this morning (March 12), the third time...Original post made on Mar. 15, 2010
Last comment by bob, on Mar 15, 2010 at 12:11 pm | 1 comment | 1498 Views
OTHER TOPICSWorld Affairs Council program: International Humanitarian Law
From: Ralph Kuiper <email@example.com> To: WAC Consolidate 3 <firstname.lastname@example.com> Sent: Sun, March 14,...Original post made by Poster on Mar. 14, 2010
There are no comments yet. 2057 Views
OTHER TOPICSHow to dispose of prescription drugs
Dont flush prescription drugs down the toilet or drain unless the label or accompanying patient information specifically...Original post made by Poster on Mar. 14, 2010
There are no comments yet. 1643 Views
SCHOOLS & KIDSTeam 100 from Woodside High School wins FIRST Robotics San Diego Regional
Our Woodside High School robotics team won the FIRST Robotics Regional in San Diego Saturday, March 6. There were 49 teams...Original post made by Becky Waschura on Mar. 14, 2010
There are no comments yet. 1752 Views
SPORTSBoys Varsity Baseball: M-A Evens Season Record with 9-4 Victory over San Mateo
Menlo-Atherton took full advantage of six walks, four hit batsmen, and four San Mateo errors to bury the Bearcats 9-4 on...Original post made by Bob Moriarty, Team Parent on Mar. 14, 2010
There are no comments yet. 1888 Views
OTHER TOPICSCounty holds free H1N1 clinics
San Mateo County officials will hold several free H1N1 vaccination clinics through the end of the month after high turnouts...Original post made by Correspondent on Mar. 14, 2010
There are no comments yet. 1578 Views
MENLO PARKGrant will help police enforce alcohol law
A $6,000 grant from the state will enable Menlo Park police to employ underage decoys to try to bait people into buying alcohol...Original post made on Mar. 10, 2010
Last comment by under 21, on Mar 13, 2010 at 6:06 pm | 11 comments | 246 Views
SCHOOLS & KIDSEverest, Sequoia district dance to familiar tune
Everest Public High School representatives have rejected a new offer of facilities from Sequoia Union High School District...Original post made on Mar. 9, 2010
Last comment by jim watson, on Mar 13, 2010 at 3:30 pm | 5 comments | 1973 Views
SCHOOLS & KIDSMy View: Our so-called lives -- Is techno-chatter replacing relationships?
About the author: Samantha Bergeson of Portola Valley is a freshman at Menlo School in Atherton. Read the full...Original post made on Mar. 12, 2010
Last comment by Marice George, on Mar 13, 2010 at 9:26 am | 2 comments | 1783 Views
OTHER TOPICSThe bell tolls for Steve Jobs' Jackling house
A San Mateo County judge has given Apple Corp. chief executive Steve Jobs the green light to demolish the Jackling house,...Original post made on Mar. 12, 2010
Last comment by R.GORDON, on Mar 13, 2010 at 9:19 am | 6 comments | 1732 Views
SCHOOLS & KIDSAt Woodside High: Murder mystery drives musical comedy
Fans of just about every theater genre should find something to like in Woodside High School's upcoming production of "Curtains,"...Original post made on Mar. 12, 2010
Last comment by Dan B., on Mar 12, 2010 at 11:59 pm | 1 comment | 1773 Views
SCHOOLS & KIDSRobotics Team 100 Hoping to Repeat San Diego Win at Silicon Valley Regional March 19-20
Laura Rhodes, mentor, FIRST Robotics Team 100, submitted this story. The aspiring young scientists and engineers of Team...Original post made by Laura Rhodes on Mar. 12, 2010
There are no comments yet. 1782 Views
AROUND TOWNThursday meeting on downtown parking issues: Loosen time restrictions? Install meters?
People interested in the parking situation in downtown Menlo Park are invited to attend a meeting Thursday, March 11, at...Original post made on Mar. 10, 2010
Last comment by MP shopper, on Mar 11, 2010 at 10:23 pm | 9 comments | 1602 Views
Calave wine bar opens in Palo Alto
By Elena Kadvany | 9 comments | 2,181 views
Constitution Guarantees Nationwide Right to Same-Sex Marriage
By Chandrama Anderson | 33 comments | 1,629 views
Family Outing - Inside Out
By Cheryl Bac | 0 comments | 447 views


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A teleconference or teleseminar is the live exchange and mass articulation of information among several persons and machines remote from one another but ...http://en.wikipedia.org/wiki/Teleconference
Teleconferencing lets large companies work more effectively. Learn different ways of teleconferencing, the equipment you need and read teleconferencing ...http://communication.howstuffworks.com/teleconferencing.htm
A teleconference is a telephone or video meeting between participants in two or more locations. Teleconferences are similar to telephone calls, ...http://www.fhwa.dot.gov/REPORTS/PITTD/teleconf.htm
... retail, government, non-profit and communications fields all enjoy our meet-me, reservationless and web-scheduled audio voice teleconferencing services.http://www.freeconference.com/
LiveOffice Teleconferencing & Reliable toll free conference call services for conference calls on-demand with competitive conference calling rates.http://teleconference.liveoffice.com/


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BBQ Chicken Dinner Fundraiser
From May 17, 2014 1:00 pm until May 17, 2014 5:00 pm Save to calendar
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Child Price: $5 - Hot Dog, baked beans, baked potato, salad, roll, beverage, and dessert
There will be a couple of raffles, a gift card tree and a Chinese Auction with items such as Thirty One, Pink Zebra, Pampered C...hef, Signature Homestyles, Tastefully Simple, a baking basket, a wine basket, a bucket with car wash items, a couple children's baskets, hand crafted wood item(s)...just to name a few!
Take-out orders will be available. There will be 350 chicken dinners and once they're gone, they're gone...So Get your Ticket NOW! by calling 740-310-4991 or through Epworth Center, LogoTech Signs, Longhorn Steakhouse, or Real Life Community of Faith
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Spurs great Tim Duncan one win away from fifth title - 0 Comments
MIAMI — Gregg Popovich didn't want to talk about milestones, not on this night.
Neither, for that matter, did his power forward who on Thursday became the both all-time NBA playoff leader in minutes played and in playoff double-doubles.
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Vic Christopher
Assistant General Manager, Tri-City ValleyCats
Yearly Archive for 2009
Rensselaer County Chamber's 109th Annual Dinner
On Wednesday night, I attended the Rensselaer County Regional Chamber of Commerce's annual dinner at The Franklin Plaza in downtown Troy. Five weeks ago, the ValleyCats hosted a meet-and-greet with our new coaching staff at the Plaza. It was excellent affair and so was last night's event, thanks to Mike Fusco and the top-notch Franklin […] [Read More]
Governor Paterson is a doll
On Monday, Jun. 29, the ValleyCats take on the Vermont Lake Monsters at 7pm. The first 1,200 fans will receive a ceramic hand carved, hand painted 7-inch Governor David Paterson Bobblehead doll as part of We Love NY Night. You can purchase your tickets on the ValleyCats website. I think the likeness is pretty […] [Read More]
The Desmond Albany Idol
I was asked to join The Desmond Hotel for their quarterly employee meeting as a judge in their "Albany Idol" talent competition. This isn't the first time I have been asked to judge something. Jeff Buell at the City of Troy had me judge chowder at last year's Chowderfest. I enjoy chowder, […] [Read More]
Team Building
As I mentioned earlier, we have 10 full-time staffers here in the front office of the ValleyCats. As in any small business, it is paramount that we work well together. We frequently participate in off-site team building exercises, sometimes for a good cause and other times to collectively improve our brand. Our common connection is […] [Read More]
Minor League Baseball Misconceptions
I can't tell you how many times I've been asked, "So, when are you going to make it to the Major Leagues?" A common misconception is that Minor League Baseball executives are working their way up the ladder to the big leagues, along with the players. As my ValleyCats counterparts will tell you, Minor League […] [Read More]
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T-Pain @TPAIN
Tweet: "Listen. If you don't like me. That's fine. But don't try to hold a full on conversation with me if thats the case. Need followers much?"
T-Pain puts his Twitter haters on blast.
(Photo: David Livingston/Getty Images)
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MoriahJovan posted Chapter 45 to Dunham
The Proviso
Knox Hilliard's uncle killed his father to marry his mother and gain control of the family's Fortune 100...
Moriah Jovan writes what her imaginary friends tell her to write. Thus far, they have shown up in the novels THE PROVISO, STAY, and MAGDALENE, "Twenty-Dollar Rag," DUNHAM, PASO DOBLE and WE WERE GODS, published by B10 Mediaworx, and will, most likely, continue to order her around until she hits on the right drug and dosage.
Fortunately, her husband is very understanding of all the other people in her life and her children have no need of their own imaginary friends since they know all of mommy's.
Moriah has a bachelor's in creative writing and journalism from the University of Missouri at Kansas City, and in 2011, she was a panelist at the Writer's Digest conference and the Sunstone Symposium. She is a flagrant manufacturer and dealer of the meth known as "ebooks."
Description: It's 1780. The Americans are losing their desperate fight for independence from the most powerful nation on Earth. Britain's navy is crushing outposts up and down the eastern seaboard and the Americans' pitiful navy consists mostly of small-vessel...
Knox Hilliard's uncle killed his father to marry his mother and gain control of the family's Fortune 100...
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Mu opioid receptors have previously been shown to be altered in people with affective disorders who died as a result of suicide. We wished to determine whether these changes were more widespread and independent of psychiatric diagnoses.
Mu receptor levels were determined using [3 H]DAMGO binding in BA24 from 51 control subjects; 38 people with schizophrenia (12 suicides); 20 people with major depressive disorder (15 suicides); 13 people with bipolar disorder (5 suicides) and 9 people who had no history of psychiatric disorders but who died as a result of suicide. Mu receptor levels were further determined in BA9 and caudate-putamen from 38 people with schizophrenia and 20 control subjects using [3 H]DAMGO binding and, in all three regions, using Western blots. Data was analysed using one-way ANOVAs with Bonferroni's Multiple Comparison Test or, where data either didn't approximate to a binomial distribution or the sample size was too small to determine distribution, a Kruskal-Wallis test with Dunn's Multiple Comparison Test.
[3 H]DAMGO binding density was lower in people who had died as a result of suicide (p<0.01). People with schizophrenia who had died as a result of suicide had lower binding than control subjects (p<0.001), whilst people with bipolar disorder (non- suicide) had higher levels of binding (p<0.05). [3 H]DAMGO binding densities, but not mu protein levels, were significantly decreased in BA9 from people with schizophrenia who died as a result of suicide (p<0.01).
Overall these data suggest that mu opioid receptor availability is decreased in the brains of people with schizophrenia who died as a result of suicide, which would be consistent with increased levels of endogenous ligands occupying these receptors.
Although the dynamics that contribute to a person dying by suicide are multifactorial, highly complex and vary between individuals [1], studies consistently suggest that psychiatric disorders confer an increased risk of suicide. For example, a Danish study found that nearly 50% of people who died as a result of suicide either had been or were psychiatric inpatients [2]. A retrospective study in Hong Kong discovered that 80% of the people who died by suicide had a psychiatric disorder, with 29% of these having a co-morbid diagnosis [3]. This study also showed the highest risk of suicide was in people with mood disorders (50%), with suicides in psychotic disorders showing a gender difference (females: 35.1%; males: 19.8%). Most significantly, the study showed that a psychiatric diagnosis had an adjusted odds ratio of 28.67 for risk of suicide.
The concept that psychiatric disorders significantly increase the risk of suicide is highlighted by a systematic literature review which showed that, of all factors studied, psychiatric disorders had the strongest association with suicide (all psychiatric disorders 91%; of which mood disorders accounted for 55% and co-morbid diagnoses 44%) [4]. A recent meta-analysis, which used data from both case–control studies and cohort studies, reported that the relative risk of suicide that could be attributed to any psychiatric disorder was 7.5% for males and 11.7% for females [5]. Together, these data demonstrate that there is an urgent need to understand the drives to suicidal tendencies in people with psychiatric illnesses; the ultimate goals of such investigations being to identify markers for such tendencies and/or to develop interventions to reduce this behaviour.
There is now a body of literature which supports the hypothesis that neurochemical changes in the brain are associated with suicidal behaviour, which include roles for the central serotonergic system [6], the signal transducer protein kinase C [7] and the glutamatergic system [8]. There are also studies linking the endogenous opioidergic system to the major psychiatric disorders [9,10] and suicide [11]. In the case of the mu opioid receptor (OPRM), it has been reported that these receptors are increased in the inferior frontal, cingulate, postcentral, medial temporal and lateral occipitotemporal gyri as well as the thalamus [12] of young (<41 years) people who had died as a result of suicide. OPRM were also reported to be increased in the frontal cortex and caudate [13] from people who had died as a result of suicide compared to those from age and sex matched control subjects. Significantly, one of these studies [12] only included people with depression among those who had died by suicide, whilst the second study predominantly consisted of people with undetermined diagnoses or depression (one subject had schizophrenia) [13]. Thus, it is not clear if there is an interaction between psychiatric disorder and OPRM in people who died by suicide. Finally, in a study that did not assign psychiatric diagnoses to the cohorts, it has been reported that OPRM in the prefrontal cortex from people who died by suicide bound agonists with a higher affinity than that for people who did not die by suicide. However, a decreased binding affinity was reported for the pre- and post-central gyri from people who died as a result of suicide [14].
Given the potential for changes in OPRM to be important in the predisposition of an individual to attempt suicide and the relative paucity of studies on OPRM, we decided to measure the density of OPRM using [3 H][D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) binding in BA24 from people with no history of psychiatric disorders (controls), people who had died by suicide and had no discernable history of psychiatric disorders (suicides) and people with schizophrenia, major depressive disorder (MDD) or bipolar disorder (BP); some of whom had died by suicide.
Following our initial studies we went on to measure [3 H]DAMGO binding in BA 9 and the caudate-putamen (CPu) from people with schizophrenia who had or had not died as a result of suicide and compared these data to those from age/sex matched controls (non-suicides) to begin to determine the extent of altered OPRM binding in these people. Finally, to determine if changes in radioligand binding were associated with changes in levels of OPRM protein, we used Western blotting and an anti-human OPRM antibody to measure protein levels in BA9, 24 and the caudate from the schizophrenia study cohorts.
Approval for this study was obtained from the Ethics Committee of the Victorian Institute of Forensic Medicine and the Mental Health Research and Ethics Committee of Melbourne Health (MHREC 2001.613). In all cases, tissue was obtained, post-mortem, after receiving written consent from the closest identifiable next-of kin. In compliance with State laws of the day, the source of the tissue collected up to 1999 was de-identified on completion of the case history review, tissue collected after 2002 was not de-identified. Tissues were provided by the Victorian Brain Bank Network as coded samples so that investigators cannot identify the donor.
Diagnostic evaluation
Case history reviews were conducted using the Diagnostic Instrument for Brain Studies (DIBS) [15,16], with a psychologist and psychiatrist reaching a diagnostic consensus using DSM-IV criteria [17]. The inter-rater reliability of this instrument is 94% (mean kappa = 0.88; 95% confidence interval = 0.7 – 1.0) [18]. Following the review, duration of illness (DOI: time from first contact with psychiatric services to death) was calculated and latest prescribed doses of psychoactive drugs converted to standardised drug doses [19] ( Additional file 1: Table S1). Importantly, CNS tissue was collected from cadavers refrigerated within 5 hours of being found, this has been shown to markedly slow autolysis processes and protein degradation [20]. The average time from discovery to autopsy was approximately 36 hours. To ensure preservation of the CNS molecular cytoarchitecture, samples were processed using a standardised procedure and rapidly frozen to −70°C after removal at autopsy [21]. In cases where death was witnessed, the post-mortem interval (PMI) was the time between death and autopsy. Where death was not witnessed, PMI was calculated as the interval mid-way between the donor last being seen alive and being found dead. Central pH was determined as described previously [22]; this being recognised as a good indicator of tissue and protein preservation [23].
Additional file 1. Table S1. Donor information related to the subjects from whom tissue was used for the measurement of markers for the mu opioid receptor. Description of data: Detailed information of the cohort from whom tissue was sourced for this study, includes demographic data.
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Cohort selection and tissue processing
Blocks of BA24 (ventral anterior cingulate gyrus, around the genu of the corpus callosum) were removed from the brains of 51 controls, 38 people with a diagnosis of schizophrenia (12 suicides), 20 people with a diagnosis of MDD (15 suicides), 13 people with a diagnosis of BP (5 suicides) and 9 suicides with no discernible history of a psychiatric disorder (see Table 1 for cohort summary and Additional file 1: Table S1 for details).
Table 1. A summary of the demographic and collection data (mean ± SEM) for the cohort used in the determination of markers for mu opioid receptors
Blocks of BA9 (lateral surface of the frontal lobe, including the middle frontal gyrus superior to the inferior frontal sulcus) and the CPu were excised from the brains of the 36 people (11 suicides) with schizophrenia (tissue unavailable from 2 people) and 20 control age and sex subjects.
The radioligand binding assays were conducted as single point saturation studies [24], which gives a good estimate of total available binding sites. Importantly, the concentration of radioligand used was sufficiently high to overcome the minor changes in radioligand binding affinity that occurs in human CNS tissue [25] ensuring this approach gives a good estimation of total binding sites within the tissue [26]. Thus, 5x 20 μm sections (3 total binding; TB, 2 non-specific binding; NS) were cut from each tissue block using a cryomicrotome (CM1800, Leica Microsystems) and thaw mounted onto gelatinised microscope slides. Approximately 100 mg of gray matter for use in Western blots was excised from the slices these sections were taken from and stored at −80°C.
Radioligand binding
3 H]DAMGO binding (3.3 x10-9 M) was measured in the presence (NS) and absence (TB) of 10-6 M naloxone; adapted from previous studies [12,27]. Sections were incubated in 50 x10-3 M Tris–HCl, pH7.4, for 1 hour. After incubation, sections were washed in ice-cold assay buffer, dipped in ice-cold water and thoroughly dried prior to being desiccated overnight in paraformaldehyde fumes. The sections, alongside 3 H]micro-scales™, were apposed to BAS-TR2025 imaging plates (14 days), which were scanned using a BAS5000 high resolution phosphoimager. The phosphoimage intensities were measured by comparison to the intensity of segments on 3 H]microscales™ using AIS image analysis software. Results were expressed as specific binding (TB minus NS) in dpm/mg estimated wet weight tissue equivalents (ETE) and converted to fmol/mg ETE [26].
Western blots
Homogenates were prepared from CNS tissue, at 5% w/v, in 10 x10-3 M Tris, (pH 7.4), containing 1% sodium dodecylsulphate and 1 x10-3 M fresh sodium orthovanadate and diluted in reducing buffer to give final protein concentrations of 1.0 mg/ml. Optimisation experiments showed that different protein concentrations from each region were within the linear range of the detection system. Thus the total protein loaded was; BA 9: 30 μg, BA 24: 10 μg and caudate: 40 μg. The proteins in each sample (run in duplicate) were separated using polyacrylamide gel electrophoresis (4% stacking gel, 10% running gel). They were transferred to nitrocellulose membranes overnight in Towbin transfer buffer [28]. Equal protein loading and transfer was confirmed by staining with 0.1% ponceau S in 3% trichloroacetic acid. Membranes were blocked for an hour at room temperature in Tris buffered saline with 1% Tween 20 (TTBS) and incubated with rabbit anti-OPRM antibody (1/2000 in TTBS) overnight at 4°C. This was followed by incubation with goat anti-rabbit antibody conjugated to horseradish peroxidise (1/2000 in TTBS) for an hour at room temperature. The antigenic reaction was visualised using enhanced chemiluminescence (ECL) and imaged using a Kodak Image Station 440CF. The sum intensity of the antigenic bands was measured using Kodak 1D software.
To control for inter-blot variability, an internal control (IC) sample [29], was run (in duplicate) on every gel, which were imaged so that the sum intensity of the IC fell within a previously established range. The sum intensities of all samples were standardised as a ratio of Internal Control (ratio IC).
All data sets were analysed using the D'Agostino & Pearson omnibus normality test to determine whether they approximated to Gaussian distribution. If the data was normally distributed, a Grubb's test was used to determine the presence of outliers. Due to small sample sizes, age, PMI and brain pH for the groups were compared using a Kruskal-Wallis test with Dunn's Multiple Comparison Test. Pearson product–moment correlations, assuming a straight line fit, were used to determine if there was a relationship between these factors and experimental data. Interpretation of Pearson product–moment correlations was assisted by published guidelines [30]; with an r value of 0.3 (r2 = 0.09) deemed to describe a typical relationship and a value of 0.5 (r2 = 0.25) describing a substantial relationship.
Where an association was found, the impact of the factors on the experimental data was determined by analysis of covariance (ANCOVA). For the radioligand binding data, a Student's unpaired t-test was used to determine whether there were changes in binding density with suicide. Due to the small samples sizes for some groups, a Kruskal-Wallis test with Dunn's Multiple Comparison Test, or a general linear model (GLM) which is most suitable for dealing with complex data sets were employed to identify variations in binding density between groups. Finally, in the schizophrenia/control cohort, one-way ANOVAs with Bonferroni's Multiple Comparison Test were used to identify variations in [3 H]DAMGO binding and OPRM protein levels between groups in each CNS region. GLMs and ANCOVAs were conducted using Minitab (Release 13.31, http://www.minitab.com webcite); all other statistical analyses were carried out using Graphpad Prism (version 5.04 for Windows, Graphpad Software, San Diego, California, USA).
Demographic and tissue collection data
The final recorded antipsychotic dose (chlorpromazine equivalents) and duration of illness in the people with schizophrenia were the only collection-related variables not normally distributed. There were significant variances associated with age (Kruskal-Wallis statistic = 30.80, p<0.0001) and pH (Kruskal-Wallis statistic = 27.87, p<0.001), but not PMI (Kruskal-Wallis statistic = 9.24, p = 0.24) between the groups. Post-hoc Dunns Multiple Comparison test revealed that the variance with age was due to the schizophrenia _ suicide group being younger than the control (p<0.01), MDD_non_suicide (p<0.05), BP_non_suicide (p<0.001) and the schizophrenia_non_suicide groups (p<0.01; Additional file 2: Table S2). Using the same approach, the variance with brain pH was found to be due to the tissue from the MDD_suicide group being higher than for the control (p<0.01) and schizophrenia_suicide (p<0.01) and schizophrenia_non_suicide (p<0.001) groups ( Additional file 2: Table S2).
Additional file 2 . Table S2. Correlations between demographic and tissue collection data and measures of mu opioid receptors. Description of data: Detailed information of the relationships between potential confounding factors and the experimental measures assessed in this study.
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[3 H]DAMGO Binding in BA24 and suicide
[3 H]DAMGO binding was homogenous across BA24 (see Figure 1a) therefore an integrated measure was taken across all cortical laminae. [3 H]DAMGO binding from people who had or had not died from suicide approximated to a Gaussian distribution and a Grubbs test revealed no outlying data. Significantly, there was a 16% decrease in the density of radioligand binding to OPRM in BA24 from people who had died by suicide (n = 41) compared to those who had not died by suicide (n = 81, p = 0.008, see Table 2 and Figure 1b).
Figure 1 . a) Representative autoradiograph of total [3 H]DAMGO binding in BA24. Insert shows non-specific binding. Results for the radioligand are represented graphically (mean ± SEM, fmole/mg ETE): b) Effect of suicide per se on levels of [3 H]DAMGO binding in BA24. c) Effect of psychiatric disorder and suicide on levels of [3 H]DAMGO binding in BA24.
Table 2. Levels of [3 H]DAMGO (mean ± SEM) binding in subjects with schizophrenia, major depressive disorder, bipolar disorder or no history of psychiatric disorder who did or did not die as a result of suicide
[3 H]DAMGO Binding in BA24, suicide and psychiatric diagnoses
To determine if the changes in OPRM in BA24 showed any suicide x psychiatric diagnosis, we divided the data according to suicide status within each clinical diagnoses. This showed changes in [3 H]DAMGO binding varied with suicide status (F7,114 = 4.16, p = 0.0004) and was due to a 34% increase in binding in the BP_non_suicide group and a 33% decrease in the schizophrenia _ suicide group (see Table 2 and Figure 1c).
Potential confounds
There were substantial correlations between [3 H]DAMGO binding and age in controls, MDD_non_suicide, MDD_suicide, BP_non_suicide and schizophrenia_suicide as well as between binding and pH in MDD and BP_suicide ( Additional file 2: Table S2). Furthermore, when [3 H]DAMGO binding was analysed with age and pH as cofactors, age (F1,89 = 23.12, p<0.0001) and pH (F1,89 = 3.91, p = 0.05) were shown to contribute to the variance associated with the binding data. However, even taking these factors into account, the suicide x psychiatric diagnoses differences were still strongly significant (F7,89 = 2.93, p = 0.008).
Mu opioid receptors and suicide in schizophrenia
[3 H]DAMGO binding
Whilst the primary focus of this study was the potential interactions between diagnoses x suicide, a sub-analyses of the BA 24 data revealed that there was a 33% decrease in binding in the schizophrenia_suicide group compared to the schizophrenia_non_suicide group (Table 2), prompting further study of the potential interactions between suicide x schizophrenia in other CNS regions. Subsequently, we measured [3 H]DAMGO binding in BA9 (Figure 2a) and CPu (Figure 2b) from people with schizophrenia_ suicide, schizophrenia_non_suicide and age matched controls only. These data were shown to approximate to a Gaussian distribution in both regions and Grubbs test revealed one outlier in the data in the CPu from people with schizophrenia (Z >2.82). For transparency, analyses are presented with and without that outlier. With the outlier, there was a significant variation in [3 H]DAMGO binding in the CPu with diagnoses (With outlier: F2,53 = 3.90, p = 0.026; without outlier: F2,52 = 4.66, p = 0.013). Binding in the schizophrenia _ suicide group was decreased by 36% compared to controls and 31% compared to schizophrenia_non_suicide (Table 2, Figure 2c).
Figure 2 . Representative autoradiographs of total [3 H]DAMGO binding in a) BA9 and b) caudate-putamen; inserts show non-specific binding. c) Effect of suicide on radioligand binding in these areas are represented graphically (mean ± SEM, fmole/mg ETE).
[3 H]DAMGO binding also varied with diagnoses in BA9 (F2,55 = 6.81, p = 0.0023), due to a 24% decrease in binding in the schizophrenia_suicide group compared to controls and 30% compared to schizophrenia_non_suicide (Table 2, Figure 2c). In neither region did [3 H]DAMGO binding in the schizophrenia_non_suicide differ from that in controls.
Potential confounds
Pearson product–moment correlations indicated that age, pH and DOI but not PMI or chlorpromazine equivalents, had significant associations with [3 H]DAMGO binding ( Additional file 2: Table S2). Significantly, reanalysing the schizophrenia cohort data with age and pH as covariates showed that although age (F1,161 = 25.48, p<0.0001) and pH (F1,161 = 5.64, p = 0.019) contributed to the variance in binding, the variation associated with suicide was still significant (F2,161 = 3.33, p = 0.038).
OPRM protein
The antigenic band measured in these studies had the predicted molecular weight for MOR and, in our hands, showed the expected rank order of intensity (reflecting OPRM levels) across different brain regions (i.e. thalamus > cortex > caudate; data not shown) [12,27]) and was present in thalamic membrane and cytosolic [31-33] but not nuclear fractions (Figure 3a).
Figure 3 . a) Western blot showing the levels of OPRM in (left to right): Molecular weight standards in lane 1, values given in kDa; membrane, nuclear and cytosol fractions, in lanes 2–4 respectively. Band measured in subsequent experiments identified by the arrow. b) Levels of OPRM protein in BA24, 9 and CPu from controls and people with schizophrenia who did or did not die as a result of suicide are represented graphically (mean ± SEM, ratio of internal control).
OPRM data showed a Gaussian distribution with no outliers. There was no variance in OPRM protein levels between diagnostic groups in any region (BA9: F2,55 = 0.644, p = 0.53; BA24: F2,55 = 0.953, p = 0.392; caudate: F2,53 = 1.22, p = 0.303; see Figure 3b).
Overall, there were no correlations between age, pH, DOI or chlorpromazine equivalents and OPRM levels ( Additional file 2: Table S2). However, there were substantive relationships between OPRM level and age (r2 = 0.4809) and pH (r2 = 0.3758) in BA9 from the schizophrenia_ suicide group. An ANCOVA revealed that neither age (F1,53 = 0.67, p = 0.417) nor pH (F1,53 = 2.12, p = 0.151) had a significant effect on OPRM levels in BA 9. Furthermore, taking these 2 factors into account did not affect the outcome of the analysis (F2,53 = 0.72, p = 0.49).
This study has shown decreased 3 H]DAMGO binding in BA24, BA9 and the CPu from people with schizophrenia who died by suicide which, in BA24, was first revealed as a significant decrease in 3 H]DAMGO binding in larger cohort where people who had died by suicide were compared to those who had not. Significantly there were no differences in 3 H]DAMGO binding in BA 24 from people with either MDD or BP who had died as a result of suicide, making this a specific association with suicide in schizophrenia. However, this study did show an increase in 3 H]DAMGO binding in BA24 from people with BP who did not died by suicide. Some caution is needed in the interpretation of this finding in BP because of the relatively small number of individuals making up this cohort, particularly given that lithium treatment has been shown to increase OPRM expression in rats [34]. Although this novel finding deserves further investigation, it was not pursued in this study as it was not central to investigating the interactions between suicide x psychiatric disease.
Our study showed that 3 H]DAMGO binding increased with age, replicating previous studies that have reported this relationship [12,13]. Significantly, although DAMGO binds to all OPRM splice variants with similar affinities [35] our data showed that levels of OPRM protein did not vary with age. Thus, our data is the first to indicate that the number of available 3 H]DAMGO binding sites increases with age in the absence of changes in levels of OPRM, which would be consistent with decreasing levels of endogenous ligand occupancy with age. This hypothesis is supported from previous data on endogenous opioids in rat CNS [36] and in pain responsiveness in humans [37]. Together, these data suggest levels of endogenous opioid ligands decrease with age.
We have shown that decreased 3 H]DAMGO binding is detectable across several CNS regions in people with schizophrenia who died as a result of suicide, even when the effects of confounding factors such as age and tissue pH are taken into account, with no difference in binding levels between controls and people with schizophrenia. Our data partly replicates a previous study that showed no changes in binding to OPRM in people with schizophrenia who "had died from….. natural causes" [38] but contrasts with another report showing decreased binding to OPRM in the caudate from people with the disorder [39]. The latter study used tissue from people who had died as a result of either suicide or pneumonia but did not explore whether there was an association between low levels of binding and suicide; thus, it is possible that the decrease reported was due to suicide. However, our finding of low 3 H]DAMGO binding density in suicide per se does not agree with previous studies which report increased OPRM binding in the inferior frontal, cingulate, postcentral, medial temporal and lateral occiptotemporal gyri as well as the thalamus of young suicides [12] and in the frontal cortex and caudate [13] from people who had died by suicide or unaltered levels of radioligand binding in prefrontal cortex and prepost central gyri from people who suicided [14]. Notably, in the earliest studies investigating suicide and OPRM, tissue was obtained from individuals who had died by suicide and predominantly had MDD or BP; thus, these data are in contrast with our findings of no difference in levels of OPRM binding in people with MDD or BP who had died by suicide. By contrast, our finding of no change in 3 H]DAMGO binding in people with mood disorders or no history of psychiatric illness who had died by suicide is consistent with a previous study [14], which did not report on psychiatric diagnoses.
As stated earlier, our findings of decreased 3 H]DAMGO binding with no changes in OPRM protein would be consistent with a change in receptor availability rather than receptor number. There are three scenarios that would account for this decrease in agonist binding without a change in overall levels of protein. Firstly, it is possible that there is a change in the confirmation of the receptor which affects the binding of the ligand. Secondly, the biochemical modifications that are required for the insertion of the receptor into the membrane could be aberrant, reducing the availability of the receptor but not affecting the overall number of receptors. Finally, the simplest explanation is that the decrease in binding is due to competition for the binding site between the radioligand and an endogenous ligand. The latter hypothesis has been argued previously in the report of altered affinity for 3 H]DAMGO in tissue from people who died as a result of suicide [14] as changes in binding affinities are usually due to competition from endogenous ligands [40,41]. More recently, a positron emission tomography (PET) study reported that people with high impulsivity scores released higher levels of endogenous opioids in response to stress [42], resulting in lower 11C]carfentanil non-displacable binding potential. Impulsivity has been proposed as a candidate endophenotype for suicidal behaviour [43]; thus, it is possible the decreased OPRM availability in people with schizophrenia who died by suicide is due to increased endogenous opioid levels in response to external stressors. Two such molecules, endomorphin 1 and endomorphin 2, have high affinities and selectivity for OPRM (see [44]) making them prime candidates for future studies.
As with most approaches to the study of the neurobiology of psychiatric disorders, this study has some potentially confounding factors. In particular, we and others [12,13] have shown that 3 H]DAMGO binding increases with age. In our study the changes in 3 H]DAMGO binding remained when the effect of age was taken into account using an ANCOVA. Another confound is that all people with psychiatric disorders received psychotropic drugs prior to death. Overall, the literature suggests that treatment of rodents with antipsychotics results in either a decrease in the levels of OPRM [45,46] or no change [47] although clozapine, which none of the subjects with schizophrenia received (see Additional file 1: Table S1 for details), has been reported to cause decreases [48]. Evidence against the change in 3 H]DAMGO binding in tissue from people with schizophrenia who died by suicide being related simply to the effects of antipsychotic drugs are i) people with schizophrenia who did or did not die by suicide had been treated with antipsychotic drugs, receiving similar levels of final recorded drug doses expressed as chlorpromazine equivalents (Mann Whitney U = 131.0, medians: schizophrenia_non_suicide = 1216, schizophrenia_suicide = 1418;) and the difference in 3 H]DAMGO binding is present in all regions between these groups, ii) some of the people with BP had received antipsychotic drugs before death but there was no change in 3 H]DAMGO binding in people with this disorder who had died by suicide compared to control subjects and an increase in the BP_non_suicide group.
One potential methodological confound in our study was that the cohort size of people with schizophrenia who died as a result of suicide was relatively small. However, a power analysis ( http://www.biomath.info/power/ttest.htm webcite) showed our study was appropriately powered in BA 9 and 24 (power level 0.8) but was underpowered for the mean differences observed in the CPu (9 additional non-suicide people were needed).
Our study is the first to suggest there is a decrease in the availability of 3 H]DAMGO binding sites in people with schizophrenia who died by suicide. Given the strong link between schizophrenia and the inclination to complete suicide [49], further studies to understand the consequences of changes in the opioid system and its relationship to suicide in schizophrenia are warranted. It is possible that these chemical changes, if detectable by neuroimaging or by a peripheral measure, could be used clinically to identify people with a disposition to suicide which would facilitate the implementation of appropriate interventions [50]. Similarly, our novel, preliminary data on increased 3 H]DAMGO binding in BP are intriguing and deserve further investigations. Both findings add to the long held argument that the opioid system plays an important role in the pathophysiology and/or outcomes of psychiatric disorders [51].
Competing interests
There are no competing financial interests in relation to the work described in this paper. TTM, GP & JN report no competing interests. The following authors have received remuneration in the past: ES received honorarium from Astra-Zeneca and travel support from GSK. BD received travel support from GSK, honorarium from Pfizer, Eli Lilly and MSD.
Authors' contributions
ES was involved in the conceptualisation, design, supervision of research and analysis of the project and had the primary responsibility for drafting the manuscript. TTM undertook the radioligand binding and Western blots in the schizophrenia cohort and had input into the manuscript. GP and JN were responsible for the radioligand binding in the suicide cohort and had input into the manuscript. BD was involved in the conceptualisation, design and analysis of the project as well as having significant input into the manuscript. All authors read and approved the final manuscript.
This study was supported by an NHMRC project grant (#509306) and Operational Infrastructure Support (OIS) from the Victorian State Government. Partial funding for the Kodak Image Station 440CF was provided by Equity Trustees. ES was the Royce Abbey Postdoctoral Fellow and TM was an Ian Scott scholar, both funded by the Australian Rotary Health Research Fund. ES is now an ARC Future Fellow (FT100100689). BD is a NH&MRC Senior Research Fellow (#APP1002240). The Victorian Brain Bank Network is supported by the Mental Health Research Institute, The Alfred, Victorian Forensic Institute of Medicine, The University of Melbourne and funded by Australia's National Health & Medical Research Council, Helen Macpherson Smith Trust, Parkinson's Victoria and Perpetual Philanthropic Services.
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Pre-publication history
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A collection of news and information related to Carlos Dunlap published by this site and its partners.
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Ravens wary of Bengals defensive line
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Injury updates: Jah Reid doubtful, Asa Jackson questionableStory | Sep 8, 2012 | 1:55 PMRavens offensive tackle Jah Reid is listed as doubtful on the final injury report of the week. Reid might as well be ruled out, though, considering that he hasn't practiced in weeks since aggravating a strained right calf for the second time since...
Local places third in world in equestrian eventStory | Sep 11, 2012 | 7:51 PMBits and pieces from the local sports scene. Christine Hocutt-Senteney does well in equestrian event in Texas: Burbank resident Christine Hocutt-Senteney captured the third-place Farnam Select All-Around Amateur title at the 2012 Adequan Select...
Opposing defenses are preying on the Eagles' careless ball security, and then laughing about itStory | Dec 14, 2012After Thursday night's debacle against the Bengals at Lincoln Financial Field, the Eagles now have 35 fumbles on the season. They have lost 21 of them. That leads the NFL. The next closest team in terms of fumbles lost is......


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Tags » Twizzler
The Frappuccino, And Other 20-Year-Old Foods
Happy birthday, Frappuccino! Starbucks' premier beverage – proof that a milkshake by any other name really would taste as sweet – just turned 20. That means that this year's incoming college freshmen never even lived in a world without the frap. 1,290 more words
#TwizzlerChallenge For Autism
We all took the ALS Ice BUcket Challenge last summer raising millions in the fight against ALS. Oh yes I took it with the help of elephants from the… 151 more words
The Twizzler Challenge Might be the Next Ice Bucket Challenge
Post from Will the real reality please stand up!
'………..by lassy in looking for marbels
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After the ALS Ice Bucket challenge raised more than… 139 more words
New trending GIF tagged movies winona ryder smirk...
New trending GIF tagged movies, winona ryder, smirk, heathers, twizzler via http://ift.tt/1m5RzBR
The Twizzler...
This is the fourth part in a series of blog posts about my experiences in Latvia serving with Camp Hope a few weeks ago.
In my last post ( 666 more words


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Last update
: Sun, Jan 1, 2012 at 12:52 AM.
1/1/12 by DW
Deep in templateland
New button on Roots page
I continue to log my work
"over there"
-- the two places are almost ready to meet up. But not yet.
A current
screen shot
of the Roots page. What's new is obvious -- the New Root button at the bottom of the list. It works.
Link counts


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Don't Know What to Say
First of all, DKWTS, I'm sorry for what you went through and I'm proud of you for not only having survived it but for having been strong enough to get help and work through your issues. It's entirely understandable that you have scars from your experience – after all, you went through some hellish shit! – but those scars aren't something to be ashamed of. They're proof that you've been through the shit and came out on the other side.
So far, you're doing the right thing for you: taking your time and establishing a level of comfort and trust with the women you're interested in and that's good. You've got every right to want sex on terms that make you the most comfortable and feel safe. From the sounds of things, it sounds like you're having two issues. The first is communication. It's a social trope that all guys are horny at all times and can't wait to bone. Sometimes when a guy wants to take things at a slower pace, this gets read by others as "not actually interested". Now you don't say where your limits are and depending on how you've set them, this may cause some confusion; if you're uncomfortable with, say, kissing or making out, it's understandable that – even when you say you want to take things slowly – that women may feel as though they're getting mixed signal, especially on the first couple date. If there's no sign of physical chemistry, even a good night kiss, then your date may think you're just not interested in her at all or that you're only interested in her as friends. If you're setting the bar towards heavy fooling around (mutual masturbation, oral, etc.) or penetrative sex, then it's a bit easier to explain.
Either way: you want to say more than just "I want to take things slowly". Frame it as being part of who you are, not anything about them. Say "Hey just so you know: I think you're amazingly hot and incredible and I'm attracted to you, but I really prefer to go slow when I'm with someone I really like. It's just my thing." And you leave it there. It's short, simple and respectful. It won't be a big deal because you're not treating it like a big deal, and others will take your lead. If they push for details, just reiterate: "Its just my thing". You don't need to explain or justify it. The women who are right for you will understand and respect that. When you do have that level of trust and comfort, you'll have established that attraction and you'll seem like you're just practicing restraint or are the sort of person who has a slow-burn rather than not being interested. When you know them better and feel comfortable with them, you can explain. But until then: "It's just my thing. It's just how I am. That's all. No big deal."
And by the by, DKWTS, thank whatever gods you believe in that you didn't sleep with that last girl you mentioned. Between tossing "faggot" out as an insult and berating you for having boundaries, you very clearly dodged a massive bullet. Her shitty attitude and behavior is on her, not on you. That woman volunteered her status as an asshole right up front and life is too damn short for dealing with (or dating) assholes.
But you see: that's the great thing about having boundaries: you filter out the assholes who don't respect them or try to push past them.
Good luck!


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On April 8th, 2015, the Wall Street Journal headline read "Bloomberg Criticizes New York Fracking Ban."
the "Never Mind" Saga
Managing Risks to Reap Rewards
With interest in clean, renewable energy on the rise, the biogas market is poised for a period of strong growth in the coming years. According to a 2012 Pike Research report, global revenue is projected to double from $17.2 billion in 2011 to $33.1 billion by 2022. But as new production comes on line, owners and operators should take into consideration not just the opportunity, but the risks as well.
The nation of South Africa has long been the leading economic power of Sub-Saharan Africa. At the present day, the nation generates some 90% of its electric power from coal, including a recently built new generation clean coal power station rated at over 4,000MW of output. Some of South Africa's thermal power stations operate courtesy of advanced air-cooled condenser technology that originated in the research of Engineering Professor Kroger at Stellenbosch University, located near Cape Town. The quest to secure future electric power has led South African officials to engage in discussions with energy officials in both Congo and Ethiopia.
Four Years Since Fukushima
The sad events of March 11, 2011 have indelibly marked our hearts and our planet. While the events at the Fukushima Daiichi nuclear power plant were unprecedented, for many, nuclear fuel is still felt to be the cleanest, most efficient source of electric power.
In many shale plays, the composition of the "flowback" from fracking sites are a complex combination of water, extraction chemicals, soluble and insoluble gases, oil, as well as proppants (small particulates, typically sand or ceramic).
Horizontal Drilling and Hydraulic Fracturing, represent technology developed in the 1980's, are responsible for extracting Shale Gas and Shale Oil [Unconventional Liquid Hydrocarbons] from previously non-productive shale formations about one mile below the surface.
Reworking Generation
The energy world does not stand still. It is remarkable how much has changed in the industry - and in perspectives - from just a decade ago when Energy Biz launched. It is all the more remarkable considering that major investments in electric power can have a 60-year life, or even longer.
Live in a rural area? Enjoy the serene and pristine beauty around you? Clearly, anyone would. Do you start sweating each time that low rumble begins and your house starts to tremor? If you live within a few miles of a fracking site, that shaky ground just might become one of the charms of your cozy, country home.
Execs Tout Gas Utilities' Outlook
Reworking Generation
11 Interviews You May have Missed in 2014
Unique and increasing demands from customers, regulation and technology are forcing utilities to flex in unprecedented ways. The resulting effects of these changes have real impact on the entire energy industry. In 2014, Energy Central mapped the industry's ebb and flow in a number of candid interviews with top utility CEOs and influencers.
The federal agency that regulates the interstate transportation of natural gas by pipeline from places of production to its local distribution in homes and businesses has proposed a new facility modernization policy.
Not a single national infrastructure project under consideration today will do more to advance North American energy independence than Keystone XL. If energy independence is in the national interest, than Keystone XL deserves to be approved.
The Challenge of Infrastructure Investment
The gas industry is investing in upgrading its network and working to find new ways to pay for the upgrades. That is a key focus of executives, says Scott Prochazka, CenterPoint Energy chief executive officer.
When Mexico reformed its energy markets to attract foreign investment, analysts focused bullish outlooks on natural gas and petroleum. And without a doubt, the country will attract billions in new fossil fuel projects.
Hydraulic fracturing, or fracking, was banned in New York state in 2008 to study its effects on the environment. Since then, there have been numerous debates on whether or not fracking would be beneficial for the state.
Hydraulic fracturing (fracking) is a hot topic. There has never been a middle ground on the technology; people are either for it or against it.
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Here's Another Persona Tease, Straight From The Midnight ChannelBy Ishaan . November 22, 2013 . 7:08am
Atlus have streamed another tease in their countdown to their Persona announcement this Sunday.
Atlus have streamed another tease in their countdown to their Persona announcement this Sunday.
Atlus are counting down to their big Persona reveal this weekend, and at midnight Japan-time last night, they streamed what appears to be a brief teaser.
Atlus' countdown to their Persona announcement this weekend is live now.
Sony's Play Community website recently spoke with Persona director Katsura Hashino, who teased upcoming series developments.
In a recent blog post, Persona team director and producer Katsura Hashino shared a little message of appreciation for fans on the series' official blog.
In addition to the upcoming Persona 3 movies, the game is also receiving a live-action stage play, just like Persona 4 before it.
At this year's summer Wonder Festival 2013 in Japan, a number of toys were debuted, based on Atlus' Shin Megami Tensei, Persona and Dragon's Crown games.
Atlus have announced their next Persona concert.
The first Persona 3 movie will be released in Japanese theatres this Fall, and is titled Persona 3 the Movie #1: Spring of Birth.
Atlus designer, Katsura Hashino, tosses out another mention of the next Persona game.
An official website update indicates that the Persona 3 movie project will consist of more than one film.
The PlayStation 3 eBook reader now has digital copies of the Persona 4 Official Art Book, Shigenori Soejima Art Works 2004-2010, and Catherine Official Visual & Scenario Collection ♀VENUS☆MODE♂.
It's a brand new day for Persona music, people. Atlus reached out to Apple and put the Persona 3 & Persona 4 Vocal Sound Selection CD on iTunes.
Two new figures of Aigis, based on her Persona 4: Arena appearance, are on the way.
For just about a month, you can get a digital copy of Catherine for $19.99 or Persona 3 Portable for $9.99.