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7 Bible Verses about Being Busy
Philippians 4:6-7 ESV / 27 helpful votes
Do not be anxious about anything, but in everything by prayer and supplication with thanksgiving let your requests be made known to God. And the peace of God, which surpasses all understanding, will guard your hearts and your minds in Christ Jesus.
1 Peter 5:7 ESV / 25 helpful votes
Casting all your anxieties on him, because he cares for you.
Romans 8:6 ESV / 18 helpful votes
For to set the mind on the flesh is death, but to set the mind on the Spirit is life and peace.
Colossians 2:8 ESV / 7 helpful votes
See to it that no one takes you captive by philosophy and empty deceit, according to human tradition, according to the elemental spirits of the world, and not according to Christ.
Jeremiah 10:23 ESV / 5 helpful votes
I know, O Lord, that the way of man is not in himself, that it is not in man who walks to direct his steps.
Leviticus 1:1-17 ESV / 4 helpful votes
The Lord called Moses and spoke to him from the tent of meeting, saying, “Speak to the people of Israel and say to them, When any one of you brings an offering to the Lord, you shall bring your offering of livestock from the herd or from the flock. “If his offering is a burnt offering from the herd, he shall offer a male without blemish. He shall bring it to the entrance of the tent of meeting, that he may be accepted before the Lord. He shall lay his hand on the head of the burnt offering, and it shall be accepted for him to make atonement for him. Then he shall kill the bull before the Lord, and Aaron's sons the priests shall bring the blood and throw the blood against the sides of the altar that is at the entrance of the tent of meeting. ...
Ecclesiastes 3:11 ESV / 3 helpful votes
He has made everything beautiful in its time. Also, he has put eternity into man's heart, yet so that he cannot find out what God has done from the beginning to the end.
Suggest a Verse
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Visit the Bible online to search for words if you don’t know the specific passage your’re looking for.
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BISC219/F12: Lab 3
From OpenWetWare
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== Assignment ==
== Assignment ==
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Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.
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Remember to check the [http://www.openwetware.org/wiki/BISC219/F12:Assignments Assignment section] of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab. [http://www.openwetware.org/index.php?title=BISC219/F11:_Assignment_Series2_Linkage_Testing_Crosses&action=edit Lab 3 homework link]
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==Links to Labs& Project Info==
==Links to Labs& Project Info==
'''Series1:'''<BR>
'''Series1:'''<BR>
Revision as of 10:43, 16 August 2012
Lab 3: Forward Genetics Project: Linkage Analysis
Now that you have found evidence of a homozygous recessive gene defect that is associated with a phenotype alteration from wild type (true breeding mutant), you will determine on which chromosome this defective gene is located. First, you need to determine whether or not the mutation is x-linked and, if not, on which of the five autosomes (possible linkage groups) it is found. This task is a prerequisite to mapping the mutation (locating where on a particular chromosome the mutation is likely to be found). Linkage testing is accomplished by determining the segregation behavior of your unmapped mutation relative to standard reference markers (e.g., mutations whose locations are already known). Recall that unlinked genes will segregate independently (your basic dihybrid inheritance as first observed by Gregor Mendel) whereas mutations associated with linked genes will not.
In practice, linkage tests are performed using the following steps (where "d" (dpy) represents your recessive mutant tested with reference marker "u" (unc)). The markers d and u must be visually distinguishable. Since homozygous mutant males usually will not mate, the desired double heterozygote is constructed by mating males that are heterozygous for your dpy mutation [wild type for all other genes including the reference mutation (d/+;+/+)] with hermaphrodites that are homozygous for the reference mutation unc (+/+; u/u) and have no dpy mutation. The genotypes of the F1 hybrids will be (+/d;u/+) and (+/+;u/+). We are only interested in the double heterozygote (+/d;u/+). The F1 hybrids containing only u are not useful. To select the (+/d;u/+) heterozygotes, we let 2 F1's self fertilize on two separate plates (two animal on each plate for a total of 4 worms). We score the progeny of the F1 individuals (the F2) for linkage. Only F1 worms which produce d/d homozygotes in the F2 generation are scored, since those are the F1 (+/d;u/+). You should find d/d homozygotes on 50% of the plates. Why?
F2 progeny of each class are counted in the (+/d;u/+) plates: wild-type (+/+;+/+); d (d/d;+/+); u (+/+;u/u) and du double (d/d;u/u). If assortment is independent, progeny will be: 9/16 wild type; 3/16 d, 3/16 u; 1/16 du (that is the 9:3:3:1 ratio)!
On the other hand, if the markers are closely linked double homozygotes (d u/d u) will occur only through a very rare recombination event; therefore, you are not likely to observe the double mutant class.
To Do Today:
1. For linkage testing set up four different crosses. Each cross will contain 3 heterozygous males (d/+; +/+) from the cross you initiated using your mutant Dpy worms. Make sure that these are the only animals that you transfer from that plate by transferring the males to a transfer plate and letting them crawl around for a minute - away from any contaminating worms - then pick a second time to the mating plate.
2. Each heterozygous (d/+; +/+) male will be mated to three L4 hermaphrodites that are homozygous for one of 4 known unc(+/+; u/u) mutations on a mating plate. The strains and their reference mutations are:
Chromosome Strain Phenotype
Chromosome 1 unc-13 coiler
Chromosome 2 unc-52 immobile
Chromosome 3 unc-32 coiler
Chromosome 4 unc-17 coiler
3. Label your four plates with your PURPLE Sharpie. With the genotype of the strain - for example: +/+; unc-13/unc-13 (H) X d/+; +/+ (M) with your initials and date.
4. Incubate all of the worms at 23°C for 3 days in your team's worm box.
3-4 days after lab:
1. For linkage testing, transfer 2 wild type cross progeny (potentially heterozygous for both traits) that are L4 stage hermaphrodites from each of your 4 crosses to each of 2 new plates per cross for a total of 8 plates.
2. Label your 8 (4 sets of duplicates) plates with your PURPLE Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?
3. Incubate all worms at 23°C until the next lab period.
Assignment
Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab. Lab 3 homework link
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Yu Lab
From OpenWetWare
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List of [[Yu_Lab:Publication|publications]].
List of [[Yu_Lab:Publication|publications]].
Assistant Professor
Assistant Professor
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Center for Cell Analysis and Modeling and Department of Genetics and Developmental Biology
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Center for Cell Analysis and Modeling
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Department of Genetics and Developmental Biology
email: [mailto:jyu@uchc.edu jyu@uchc.edu ]
email: [mailto:jyu@uchc.edu jyu@uchc.edu ]
phone: (860) 679-7680
phone: (860) 679-7680
Revision as of 12:50, 21 February 2007
Welcome to our lab's wiki page. We are part of the Center for Cell Analysis and Modeling at University of Connecticut Health Center.
Contents
People
Ji Yu
List of publications.
Assistant Professor
Center for Cell Analysis and Modeling
Department of Genetics and Developmental Biology
email: jyu@uchc.edu
phone: (860) 679-7680
B.S. Tsinghua University
Ph.D University of Texas at Austin
Amal Kasry
Postdoc
email: kasry@uchc.edu
phone: (860) 679-7609
B.S. Cairo University
M.Sc. Ain-Shams University
Ph.D Johannes Gutenberg University & Max Planck Institute for Polymer Research
Research
One Molecule at a Time
A general aim of our group is to develop new single molecule experiments to study processes in live cells. For the past decade, single molecule techniques of various kinds have greatly extended the toolbox of biochemists for studying biomolecules in vitro. However, in order to truly understand the activity of biomolecules, it is also imperative to put them back into the environment of live cells and watch them from there -- hence our goal. We will keep sharing with you our thrills of adventuring into this very young field, by keep updating this web page.
CCAM
UCHC
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User:Etienne Robillard/Notebook/NK603
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Contents
NK603: Bioremediation or eugenic science ?
• I often see this word at a numerous places but rarely a sound explanation to the inherent motivation of adopting bioremediation for synthetic biology, a subset of genetic engineering using live organisms as the media for creating new and artificial life forms.
• Back in World-War 2, were the germans developing the eugenic science or were they "experimenting" new bioremediation techniques with organophosphate gases?
• Is thus bioremediation a singular aspect of eugenics, especially for devices developed for the E.coli K-12 strain, a prokariote organism ?
• What are the real objectives of Synthetic biology beyond inducing cancerous cells prematurely to mouses fed with genetically-modified corn ?
• Is cancer detection over cancer prevention the culprit and main concern of Synthia design ?
• Is not human based experimentation strictly forbidden by some public authority ?
• If so, is not bioremediation a futile term to hiden eugenics into a philantropist hat?
• Or is the people not the only authority which should encompass and lead scientific research from the hands of the governments and corporations?
• Would the germans have approved bioremediation, assuming they knew Hitler was preparing a large-scale genocide ?
• Why did the mouses developed severe mutations over a long-term period as they were fed corn grown for mass-consumption ?
• Were the scientists who developed the strain of GE corn (NK603) knowledgable or were the mutations caused from mistakes in the protocol ?
Feel free to add your comments here or using the talk page!
Common food products suspected to contains GE corn.
• canola oil (chips)
• corn syrup (marshmallows)
• corn itself (?)
• hydrogenated vegetable oil (?)
References
"We make our world significant by the courage of our questions, and by the depth of our answers." ~Carl Sagan
"Pour faciliter économiquement la sélection du maïs, Monsanto a utilisé et maintenu au sein des plants GM gène marqueur de résistance à un antibiotique appelé NPTII (néomycine phosphotransferase II). Ce dernier produit dans la cellule végétale une protéine qui induit une résistance à la Kanamycine, antibiotique bien connu." 1
See also
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Hagel says U.S.-Armenia defense relationship sound
PanARMENIAN.Net - In response to a direct inquiry by Senate Armed Services Committee Chairman Carl Levin (D-MI), President Obama's nominee to serve as Secretary of Defense, former Senator Chuck Hagel, called for the expansion of U.S.-Armenia defense relations, reported the Armenian National Committee of America (ANCA).
"We would like to thank Chairman Levin for drawing attention, during this especially closely watched Senate confirmation process, to the importance of growth in the U.S.-Armenia defense relationship," said ANCA Executive Director Aram Hamparian. "We share Senator Hagel's view that there is much room for the development of these ties, and look forward, should he be confirmed, to engaging with the Department of Defense on this matter."
In response to a written inquiry by Chairman Levin, Senator Hagel explained "The U.S.-Armenia defense relationship is sound. As with all relationships, there is room to grow and areas where we can strengthen our cooperation and partnership." He went on to note that, if confirmed, "I would continue to engage Armenian leaders to strengthen existing areas of engagement and identify new areas of cooperation that support Armenia's defense reforms, especially its peacekeeping brigade, and continue its ability to deploy in coalition operations."
In the days leading up to the Hagel confirmation hearing, ANCA activists across America, including those in Chairman Levin’s state of Michigan, urged their legislators to engage the nominee on a range of issues of concern to the Armenian American community.
Of special concern were statements by Hagel opposing official U.S. affirmation of the Armenian Genocide. "What happened in 1915, as one United States Senator, I think the better way to deal with this is to leave it open to historians and others to decide what happened and why," then-Senator Hagel told a group of Armenian reporters during a trip to Armenia in 2005. "The fact is that this region needs to move forward," Hagel added. "We need to find a lasting, just peace between Turkey and Armenia and the other nations of this region. I am not sure that by going back and dealing with that in some way that causes one side or the other to be put in difficult spot, helps move the peace process forward."
Hamparian told Commentary magazine in December that the ANCA objected to the argument that official U.S. recognition of the genocide would hinder peace between Turkey and Armenia. "As much as Erdogan and his allies might like, the ‘lasting, just peace between Turkey and Armenia’ that Chuck Hagel seeks cannot be built on Genocide denial. The U.S. and the international community must set an example by condemning the Armenian Genocide — and speaking out against all genocides, wherever and whenever they occur," said Hamparian.
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A meeting between Edward Nalbandian and Azerbaijani FM Elmar Mammadyarov will be held on the sidelines of the event.
Bergen began by recounting his 1997 meeting with Bin Laden in Afghanistan after a long process of negotiations.
Arman Kirakosyan urged Azerbaijan to accept suggestions of Minsk Group to achieve a breakthrough in Karabakh settlement.
Taner Yildiz: countries investing in construction of Turkey-based NPPs will refrain from mentioning the Armenian Genocide in future.
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Serj Tankian to president: you have not answered my questions
PanARMENIAN.Net - A System of a Down rock band frontman Serj Tankian, who earlier urged Serzh Sargsyan to return power to people, responded to a letter the reelected president sent him Tuesday, February 26.
"Dear Mr. President,
I am honored that you have responded to my letter.
I actually wasn't expecting a response, given the harsh criticism I conveyed.
The fact that you have done so is encouraging.
But with respect, you have not answered any of my questions nor addressed any of the issues I brought up in my letter directly.
I think you have done a great job at securing Armenia's bordersand dealing with the extremely sensitive and difficult situation presented by the realities after the Karabakh war.
If you remember, I told you that in person and commended your efforts in that regard.
That said, security cannot be the scapegoat to diffuse attention from the inequities and injustices in our homeland.
Republicans in the U.S. have done that for too many elections and no one seems to buy it anymore.
I, too, feel responsible to future generations for what we leave behind.
That is the reason for my speaking out, getting involved with mining and environmental abuse in Armenia (Teghut) and encouraging further farm subsidies to render our nation more self sufficient.
Citizens across Armenia are protesting the outcome of the elections and the injustice inherent in the political establishment. Please listen to their complaints. Listen to the striking students and don't let the schools or police shut out their voices from our democracy. They are the future of Armenia after all.
Corruption, injustice, emigration, lawlessness and falsified elections. These ills have emptied our country of its citizens more than mines and bombs.
What are you going to do about them? Are you going to reform the system?
Dear Mr. President, please institute the rule of law once and for all so that people feel respected in the eyes of the law and the courts. The constitution and the laws of Armenia are fine. It is their execution that is lacking. We are all tired of hearing about investors turned away, robbed of their investments in Armenia, political pressure used for personal gain, media manipulation and consolidation for political means, injustice in the courts, and on and on.
Most people feel that it will take a generational change to alter the political culture in Armenia, to rid ourselves of the overt corruption and abuse.
You can make that happen now!
In your letter you have recognized the need for that change and seem to suggest that you are willing to take substantive steps in that regard so that we don't have to wait another 20 years for us to arrive to true freedom.
For that, your people will be eternally grateful Serzh.
An equitable nation is a strong nation, where people are not only proud of their history, but also proud of their present.
I agree that we should all work together toward a better future for Armenia.
I appreciate the warm wishes to my father. I too, send my regards and love to your family and hope to meet your grandson Serj sometime," Tankian said in his letter.
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Possible ways to advance the peaceful settlement of Nagorno Karabakh conflict were in the focus of the discussion.
A meeting between Edward Nalbandian and Azerbaijani FM Elmar Mammadyarov will be held on the sidelines of the event.
Bergen began by recounting his 1997 meeting with Bin Laden in Afghanistan after a long process of negotiations.
Arman Kirakosyan urged Azerbaijan to accept suggestions of Minsk Group to achieve a breakthrough in Karabakh settlement.
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[170] superb horseman. At the outbreak of the war, he received a commission as captain of a band of picked rangers, working in conjunction with the main operations of the Confederate armies, but unhampered by specific instructions from a superior. He was rapidly promoted. As colonel of a partisan band he was a continual menace to the Federal trains, and moved with such rapidity as oftentimes to create the impression that several bodies of mounted troops were in the field instead of but one. Falling upon an isolated column of army wagons at dawn, he would strike a Federal Camp thirty miles away by twilight of the same day. His men were picked by their leader with great care, and although there is reason to believe that Southern writers surrounded these troopers with a halo of romance, there is no disputing that they were brave, daring, and self-sacrificing.
Ashby himself was looked upon by many officers and men in the Union armies as a purely mythical character. It was said that no such man existed, and that the feats accredited to Ashby's rangers were in reality the work of several separate forces. Much of the mystery surrounding this officer was due to his beautiful white horse, strong, swift, and a splendid jumper. He and his horse, standing alone on a hill or ridge, would draw the Union troops on. When the latter had reached a point where capture seemed assured, Ashby would slowly mount and canter leisurely out of sight. When his pursuers reached the spot where he had last been seen, Ashby and his white charger would again be observed on the crest of a still more distant hill.
Only once during his spectacular career in the Confederate army was Ashby outwitted and captured, but even then he made his escape before being taken a mile by his captors — a detachment of the First Michigan Cavalry.
The Confederate leader was surrounded before he was aware of the presence of the Union troops, and the latter were within fifty rods of him when he saw several of them pushing
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Murder and suicide.
--Martin Porter and Emily Scull arrived recently in New Orleans, La, in the same ship, from Europe, and have resided since then in that city as man and wife. On the night of the 5th instant they had an altercation, when Porter drew a revolver and shot Emily dead. He then put the pistol to his own head and blew his brains out.
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[22] When he had said this, he breathed on them, and said to them, "Receive the Holy Spirit!
This work is licensed under a Creative Commons Attribution-ShareAlike 3.0 United States License.
An XML version of this text is available for download, with the additional restriction that you offer Perseus any modifications you make. Perseus provides credit for all accepted changes, storing new additions in a versioning system.
load focus Latin (Saint Jerome, Bible Foundation and On-Line Book Initiative)
load focus Greek (Brooke Foss Westcott, Fenton John Anthony Hort, 1885)
hide Places (automatically extracted)
View a map of the most frequently mentioned places in this document.
Visualize the most frequently mentioned Pleiades ancient places in this text.
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Citation URN: urn:cts:greekLit:tlg0031.tlg004.perseus-eng1:20.22
Document URN: urn:cts:greekLit:tlg0031.tlg004.perseus-eng1
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Google Users Forced To Country Specific TLD
Jun 18, 2012 • 8:53 am | (20) by | Filed Under Google Search Engine
There are dozens of complaints in the Google Web Search Help forums where users who are based outside of the United States are being forced to use their country specific TLDs.
For example, users in Germany are forced to go to Google.de, even though they want to use Google.com. Same with users in the UK, they are forced to use Google.co.uk, even though they want to use Google.com.
One person said the solution is to switch your browser to Chrome and then it might start working.
One user said:
Today morning I had small health issue and was trying to find EN information about it from google.com and google was redirecting me to my local country google page which is terrible user experience I was clicking google.com link at the bottom of the page, removed my cookies still not able to access google.com, not able to find any information which I was looking for.
I'm using google more than 8 years and what I can say that all this igoogle, +1, redirects to local google version is total waist of user time, you should make everything simple and fast.
I personally experienced this issue yesterday morning at Frankfurt airport. But I honestly thought that is the behavior, I just didn't know better, since 99.999% of my searchers are done in the US.
Is this a bug or is Google changing how they handle this behavior?
Forum discussion at Google Web Search Help.
Previous story: Hangout With Google's Top Contributors on Google+
blog comments powered by Disqus
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CMD sent two reporters to track ALEC in Oklahoma
Click here to help support our future investigations.
Bart Stupak
From SourceWatch
Jump to: navigation, search
Bart Stupak previously served the 1st Congressional district of Michigan
Bart T. Stupak, a Democrat, is a former U.S. Representative for the 1st Congressional district of Michigan, having served 1993 to 2011.[1]
Contents
Record and controversies
General information about important bills and votes for can be found in Congresspedia's articles on legislation. You can add information you find on how Bart Stupak voted by clicking the "[edit]" link to the right and typing it in. Remember to cite your sources!
Iraq War
Stupak voted against the Authorization for Use of Military Force Against Iraq Resolution of 2002 that started the Iraq War.[2]
For more information see the chart of U.S. House of Representatives votes on the Iraq War.
Environmental record
For more information on environmental legislation, see the Energy and Environment Policy Portal
Pays wife for campaign work
In October 2006, the Sunlight Foundation, a Washington-based non-profit organization which advocates better transparency in government, reported that Stupak’s campaign committee paid his wife $49,500 to serve as the campaign’s treasurer during 2005-2006. [1]
Bio
Background
Born February 29, 1952,Stupak earned his Associate's degree from Northwestern Michigan College, a community college in Traverse City in 1972. He earned his Bachelor's degree in Criminal Justice from Saginaw Valley State College in 1977, graduating magna cum laude, and he earned a J.D. from Thomas M. Cooley Law School in Lansing, Michigan in 1981.
Stupak began his career in public service as an Escanaba, Michigan police officer in 1972. He later served as a Michigan State Police officer from 1973 to 1984. Stupak served as a Michigan State Representative from 1989-90, representing Menominee, Delta, and Dickinson counties.
Congressional Career
Stupak was first elected to the House in 1992.
2006 elections
In 2006, the Republicans nominated Don Hooper to face Stupak in his November 2006 bid for reelection. (See U.S. congressional elections in 2006) [2] Stupak retained his seat.
Positions and views
Stupak is especially known for his severe mistrust of Accutane, an anti-acne drug made by Hoffmann-La Roche. He believes unadvertised psychological side effects from the drug drove his teenage son, B.J., to commit suicide. B.J. Stupak, a student popular amongst his peers and a football player at Menominee High School, shot himself in the head on May 14, 2000 hours after his junior prom [3].
He is currently one of several strongly pro-life Democrats in the House (others include Tim Holden, James Oberstar, and Dan Boren); his 2004 congressional campaign was endorsed by the National Right to Life Committee.
Stupak easily defeated Republican Don Hooper of Iron River in the 2002 and 2004 elections.
2010 elections
In April 2010, Stupak announced that he would retire. The House seat was won by Republican Dan Benishek in the fall election.[1]
Money in politics
This section contains links to – and feeds from – money in politics databases. <crpcontribdata>cid=N00004196&cycle=2006</crpcontribdata>
Links to more campaign contribution information for Bart Stupak
from the Center for Responsive Politics' OpenSecrets.org site.
Fundraising profile: 2006 election cycle Career totals
Top contributors by organization/corporation: 2006 election cycle Career totals
Top contributors by industry: 2006 election cycle Career totals
Committees and affiliations
Committees
• House Committee on Energy and Commerce
• Subcommittee on Environment and Hazardous Materials
• Subcommittee on Oversight and Investigations - Ranking Minority Member
• Subcommittee on Telecommunications and the Internet
Committee assignments in the 109th Congress (2005-2006)
• House Committee on Energy and Commerce
• Subcommittee on Environment and Hazardous Materials
• Subcommittee on Oversight and Investigations - Ranking Minority Member
• Subcommittee on Telecommunications and the Internet
Coalitions and caucuses
• Caucus on Unfunded Mandates
• Co-Chair, Law Enforcement Caucus
• Congressional Auto Parts Caucus
• Congressional Automotive Caucus
• Congressional Boating Caucus
• Congressional Grace Caucus
• Co-Chair, Congressional Northern Border Caucus
• Congressional Travel and Tourism Caucus
• Democratic Homeland Security Task Force
• Democratic Regional Whip
• Democratic Study Group
• New Democrat Coalition
• Older Americans Caucus
Boards and other affiliations
• National Committeeman, Boy Scouts of America
• Member, Eagles Club
• Member, Elks Club
• Member, Knights of Columbus
• National Guard & Reserve Components Congressional Members Organization
• Member, Sons of the American Legion
• Member, State Employees Retirement Association
• Member, Wildlife Unlimited
More background data
Wikipedia also has an article on Bart Stupak. This article may use content from the Wikipedia article under the terms of the GFDL.
Articles and resources
References
1. 1.0 1.1 Bart Stupak profile, The Washington Post, accessed January 2011.
2. Roll call vote, Authorization for Use of Military Force Against Iraq Resolution of 2002.
Local blogs and discussion sites
Personal tools
Namespaces
Variants
Actions
Navigation
How To
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Google AdSense
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Cameras Are His New Drug
Mötley Crüe bassist Nikki Sixx photographs the real.
via, leica
Picture of the Day
Michael Jang
Picture of the Day
Alec Soth, Comfort Inn 2005
Los Angeles Gang and Prison Photo Archive
Recently, a two-album collection containing 400 images of Los Angeles gang and prison photos taken between 1977 and 1993 sold for $45,000. Pete Brook of Prison Photography writes about the the journey the collection took to market, and ponders the difference between moneyed collectors and preservationists.
[Read more]
Everybody Street
Excited for this film by Cheryl Dunn, whenever it comes out… no release date has been set.
Art Tonight in New York
JOSH SMITH
May 15 – June 20, 2013
Home Alone 2
208 Forsyth Street
NY, NY 10002
[Read more]
Picture of the Day
James Cooper
Picture of the Day
Jean Gaumy
Picture of the Day & The Weekly Round-Up
Roni Horn
This Week…
We saw Rob Pruitt’s ‘Last Panda’
Charles Ramsey made internet headlines
We stopped by an opening at The Hole
JR went to Rikers
and
Alena Blohm was our Muse
Picture of the Day
David Benjamin Sherry
Saguaro Field, Tucson, Arizona, 2013
Picture of the Day
Terry Richardson
Picture of the Day
Roe Ethridge
The Journey is The Destination
A behind-the-scenes look at 10 years of Ryan McGinley’s photography. The Ryan McGinley Purple Book is available with the latest issue of Purple FASHION Magazine (Issue 19).
Picture of the Day
Dave Schubert
Powell St., 1995
Picture of the Day & The Weekly Round-Up
Simen Johan
This Week…
We interviewed Mark Ryden
We looked through San Francisco photography
We introduced Yung Lenox
This photo of a falcon in the hospital was cool
and
Amber Anderson was our Muse
25 Grams: Chances With Wolves
25 Grams is a feature that culls pictures from some of our favorite instagram feeds.
Chances With Wolves are a New York-based DJ collective. They can be heard on East Village Radio on Monday’s from 6-8pm.
They can be followed on instagram at @chanceswithwolves
Picture of the Day
Matt Jones
The Largest Falcon Hospital in the World
Is located in Abu Dhabi.
San Francisco Photography
Travis Jensen, Andrea Sonnenberg, and Ted Pushinsky at Guerrero Gallery. On view through May 4th.
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Debian Galleon Media Server
From NAS-Central Buffalo - The Linkstation Wiki
(Difference between revisions)
Jump to: navigation, search
(Step 2 – Load packages)
(Step 2 – Load packages)
Line 157: Line 157:
-
5. Start the VNC server with this command. Note that the command will come back with a host name followed by colon and a number. This will likely be a :1. (Remember this screen number).
+
5. Start the VNC server as a regular user with this command. Note that the command will come back with a host name followed by colon and a number. This will likely be a :1. (Remember this screen number).
vncserver
vncserver
Revision as of 22:52, 28 April 2006
Contents
Background
Galleon is a free open source media server that typically runs on a home computer and can serve your media collection, Internet content, and applications to your TV using your TiVo© DVR. This article describes how to install the Galleon server and the required packages used to run the server on your Kurobox.
For a complete description of Galleon’s capabilities, refer to http://www.galleon.tv
Performance
The following instructions have been tested on a Kurobox HG with the Debian, Java, and Galleon versions listed below. When using a wired Ethernet connection to a Series 2 TiVo©, near real-time .tivo file transfers are possible on the “high” quality setting of the DVR. Lower quality video transfers, the MP3 features, and other Galleon applications not requiring high data bandwidth should run well on the Kurobox HG.
Setting Up
These instructions assume a fresh Debian installation on a newly installed hard drive. Skip to Step 2 – Load Packages if Debian is currently loaded on your Kurobox. Previous distributions of Debian or other distributions have not been tested. Proceed at your own risk.
Get copies of the following files before you start. You will need to FTP these files to the Kurobox from another host during the installation process.
For the Kurobox HG: Debian/Linux Distro
debian-sarge-2.6.16.9-KUROHG-20060422.tgz from http://genbako.vodapone.com/debian-2.6.16.9/
OR
For the Kurobox: Debian/Linux Distro
debian-sarge-2.6.16.9-KUROBOX-20060422.tgz from http://genbako.vodapone.com/debian-2.6.16.9/
Java JRE for the 32 bit pSeries/J2SE 5.0
Registration with the IBM developers site is required before downloading
ibm-java2-jre-50-linux-ppc.tgz from http://www-128.ibm.com/developerworks/java/jdk/linux/download.html
Galleon 2.3.0 – TiVo Media Server
galleon-unix-2.3.0.zip from http://www.galleon.tv
Step 1 – Build Debian on the Kurobox
http://www.kurobox.com/mwiki/index.php/Debian_install is an excellent article for installing Debian on the Kurobox. The article is accurate but some common pitfalls to avoid are:
1. If the newly installed hard drive ever had a Linux or Windows partition, it confuses the Kurobox and it attempts to boot from it. The partitions need to be wiped out. One option is to install the hard drive in a PC and boot from a floppy. Run fdisk.exe and list the partitions. Delete any and all partitions found. Otherwise, wipe the partitions from another Linux box. Also, even though the partitions are wiped from another box, run the mfdisk –e /dev/hda step in the article listed above anyway.
2. Replace the debian_2004_12_26_dist.tgz file with the appropriate Debian/Linux distribution listed above.
3. Static IP addresses are preferred. Use ipconfig on a Windows box to get your gateway IP.
4. For etc/resolve.conf, log into your router’s web interface to get your ISP’s DNS IP address.
5. For etc/hosts.deny, comment out the ALL:ALL line (i.e. #ALL:ALL) otherwise you will be locked out.
6. Don’t forget to create a regular user (adduser). It will be used for running VNC and Galleon.
Follow all steps in the article including the first boot. If you can successfully get through the first boot, go to the next step.
Step 2 – Load packages
http://linkstationwiki.org/Articles/Debian is the base page for loading many Debian packages. What follows below is an adaptation of those articles suitable for Galleon. They should be loaded in the order described below. Note the comments for each step. Also note not all steps in the articles are used.
DebianKeyPackages – Install key packages.
1. Login (telnet) into the Kurbox and su to root
2. Install webget, GNU C compiler, bzip, deborphan, localepurge, logrotate, automatic script builder, unzip, zip, automatic makefile, autotools, GNU C library, libtools, and debfoster.
apt-get install wget gcc bzip2 deborphan localepurge logrotate autoconf unzip zip automake autotools-dev libc6-dev libtool debfoster
3. Don’t do any additional steps listed in Linkstation wiki for the DebianKeyPackages.
DebianSamba - Set up a Windows-readable file share using Samba.
1. Install Samba as described in the article.
DebianWebmin - Install and use Webmin to remotely administer your Kurobox.
1. Install Webmin and its related packages. This will install the core features, the CPAN interface (for installing Perl modules), a java-based file manager, and firewall (iptables) manager.
apt-get install webmin webmin-core webmin-cpan webmin-filemanager webmin-inetd webmin-logrotate webmin-firewall
2. For the Samba installation:
apt-get install webmin-samba
3. Edit /etc/webmin/miniserv.conf to allow your IP address (under the “allow” line). Note that this is the host that will communicate with the Kurobox, not the Kurobox’s IP address.
4. Stop and restart Webmin with the commands:
/etc/webmin/stop
/etc/webmin/start
5. Create share directories as root on /mnt with:
mkdir /mnt/share
mkdir /mnt/share/Recordings
6. Open the directories up with the following commands. It does make the directory wide open but leave it that way for now. It will make setup easier. Tighten up permission later if you would like.
chmod 777 /mnt/share
chmod 777 /mnt/share/Recordings
7. Use a browser from another host and point to https://<kuro’s IP address>:10000. Login as root.
Follow the Configuring Samba: directions in the article if you like. The Webmin interface is really straightforward. Try sharing the /mnt/share directory listed above. Use a PC to see if the share is visible from another host.
8. The Samba sharing and Webmin isn’t a necessary step for Galleon but it’s handy if you want to manage files from a Windows host.
DebianVNC - Set up a Virtual Network Computing (VNC) Server
1. Download the free Real VNC viewer from http://www.realvnc.com if you currently don’t have a copy of it. This viewer runs on a remote desktop and allows access the Kurobox iceWM window manger.
2. Install the VNC server, remote desktop and related packages. Use the command:
apt-get install vnc4server xfonts-base icewm menu grun iceme icewm-themes iceconf icemc icepref xfe
3. Although not required, grab a copy of Firefox. It will be used later to check out the VNC server.
apt-get install mozilla-firefox
4. Configure a VNC password. This setting is unique for each user account on the Kurobox. Do this step as a regular user (the one that will run Galleon). Although not required, consider using your regular user password for the VNC password. Configure the VNC password with the command:
vncpasswd
5. Start the VNC server as a regular user with this command. Note that the command will come back with a host name followed by colon and a number. This will likely be a :1. (Remember this screen number).
vncserver
6. From a VNC viewer running on another host, specify the IP address of the Kurobox followed by :1. For example:
192.168.0.15:1
The article directions specify a port address accessible by a web browser. Using the vncviwer program and the :1 screen number usually results in a faster screen refresh.
7. What starts up is a virtual iceWM window manger that you can use for Galleon’s GUI later. Just for fun, start up Firefox with the web browser button in the window manager.
8. Exit out of the window manager and kill the vncserver with:
vncserver –kill :1
Step 3 – Load Java
DebianJava
1. A manual installation of the Java JRE is needed. The apt-get install java-package fakeroot never seemed to work properly as described in the wiki. su to root and do the following:
cd /
mkdir opt
chmod 777 opt
cd /opt
2. Move the ibm-java2-jre-50-linux-ppc.tgz file into the /opt directory and type the following command:
tar –xvfz ibm-java2-jre-50-linux-ppc.tgz
The installation will be tested later when environment variables are set up.
3. Disable IPv6. This is a requirement for running the Galleon server. As root, edit /etc/modprobe.d/aliases and change
alias net-pf-10 ipv6
to
alias net-pf-10 off
#alias net-pf-10 ipv6
4. Reboot the Kurobox with the command:
shutdown –r now
Step 4 – Load Galleon
1. If a Galleon server is currently running on another host on your network (i.e. another Linux box or PC), stop the server on the other host. Attempting to run two Galleon servers on the network appeared to cause problems for the Kurobox. This is an unconfirmed problem. Help on http://www.galleon.tv states that a headless Galleon server can be accessed by invoking the GUI plus IP address of the server on another host. This appeared to cause many problems with both the Kurobox server and the TiVo DVR itself. As a precaution, stop any Galleon servers on the network and don’t attempt the headless GUI access until Galleon is properly running on the Kurobox. It’s easier to troubleshoot one problem at a time. Access to the Kurobox Galleon server will be performed using the vncserver and GUI run from the Kurobux as described in the steps below.
2. Log out and log back in as a regular user. Don’t install Galleon as root.
3. From the regular users’s home directory, type the following command:
mkdir galleon
cd galleon
4. FTP the galleon-unix-2.3.0.zip file into the galleon directory and type:
unzip galleon-unix-2.3.0.zip
In the galleon directory you should see the apps, bin, conf, data, hme, lib, and so forth directories from the zip extraction.
5. Move into the galleon/bin directory:
cd ~/galleon/bin
6. Create a file called galleon_startup containing the following lines. Substitute the DISPLAY environment variable with your Kurobox IP:screen number. The PATH and JAVA_HOME variables are set where Java is installed.
PATH=/usr/local/bin:/usr/bin:/bin:/usr/bin/X11:/usr/games:/opt/ibm-java2-ppc-50/jre/bin
export PATH
JAVA_HOME=/opt/ibm-java2-ppc-50/jre
export JAVA_HOME
DISPLAY=192.168.0.15:1
export DISPLAY
7. Make the files executable:
chmod +x galleon_startup run.sh gui.sh
Step 5 – First-Time Start of Galleon
1. If the VNC server isn’t running, start it by the command below. Look for the :# (typically :1) display number after invoking the command. If the number is different, adjust the DISPLAY environment variable in galleon/bin/galleon_startup accordingly. Starting the VNC server is a requirement only for the first time startup of the Galleon server. Subsequent startups of the Galleon server do not require VNC. Loading the VNC server appears to supply certain x11 modules required by Galleon for its initial setup.
vncserver
2. In the galleon/bin directory, type the command to set the environment variables:
./galleon_startup
3. Next start the server with:
./run.sh
4. Once the message saying “Galleon is ready.” is displayed, set the process in the background by:
<ctrl>-Z
bg
5. Next fire up the Galleon GUI from the bin directory with:
./gui.sh
6. Invoke a vncviewer from another host to view your iceWM scrren. This takes a while but the Galleon logo and configuration windows should show up within a minute or two on your vncviewer program window.
7. Complete the configuration process using the GUI. Refer to the “Configure” instructions located in http://www.galleon.tv for additional details. The TiVo© should now see your Galleon server on the Kurobox. The /mnt/share/Recordings directory (created and opened up to 777 earlier in this article) should be a good location for Galleon’s ToGo Recordings directory.
8. Once the Galleon setup is complete, just exit the gui and kill the vncserver with the following command. The vncsever doesn’t need to run any longer.
vncserver –kill :1
Step 6 – Subsequent Startups of Galleon
1. The Galleon server will continue to run until the telnet or ssh window used to invoke ./run.sh is closed. When the window is closed, a hangup signal is sent to Galleon and the server is stopped.
2. A slight modification to the ./run.sh command above can be used to avoid this:
nohup ./run.sh &
The nohup and ampersand allows the telnet or ssh window to close without killing the Galleon server. When using nohup, stdout messages go to a log file called nohup.out.
Personal tools
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Connexions
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You are here: Home » Content » Safety measures concerning electricity
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Name: Safety measures concerning electricity
ID: m23761
Language: English (en)
Subject: Science and Technology
License: Creative Commons Attribution License CC-BY 3.0
Authors: Siyavula Uploaders (support@siyavula.org.za)
Copyright Holders: Siyavula Uploaders (support@siyavula.org.za)
Maintainers: Siyavula Uploaders (support@siyavula.org.za)
Latest version: 1.1 (history)
First publication date: May 22, 2009 9:43 am -0500
Last revision to module: May 22, 2009 9:46 am -0500
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Version: 1.1 May 22, 2009 9:46 am -0500 by Siyavula Uploaders
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If you wish to help EDRI promote digital rights, please consider making a private donation.
EDPS: Data Retention Directive fails to meet data protection requirements
1 June, 2011
»
This article is also available in:
Deutsch: EDSB: Richtlinie zur Vorratdatenspeicherung wird Datenschutzanforderun...
Peter Hustinx, the European Data Protection Supervisor (EDPS) adopted, on 31 May 2011, an opinion on the European Commission's Evaluation Report on the Data Retention Directive submitted on 18 April 2011 to the Council and the European Parliament.
The EDPS has several times expressed his concerns related to the necessity for retaining data on such a large scale in view of the rights to privacy and data protection and has called for a clear demonstration that such a measure is necessary and proportionate.
On the basis of the Commissions' Evaluation Report, the EDPS has drawn the conclusion that the Data Retention Directive does not meet the requirements set out by the rights to privacy and data protection, primarily because the necessity for data retention has not been sufficiently demonstrated. Hustinx also believes that data retention could be regulated in a less privacy-intrusive way and that the Directive lacks foreseeability.
"Although the Commission has clearly put much effort into collecting information from the Member States, the quantitative and qualitative information provided by the Member States is not sufficient to draw a positive conclusion on the need for data retention as it has been developed in the Directive. Further investigation of necessity and proportionality is therefore required, and in particular the examination of alternative, less privacy-intrusive means" says the EDPS.
Also, in Hustinx's opinion, the present Directive leaves too much room for Member States to decide on the purposes for which the data may be used, on who can access the data and under which conditions.
Therefore, the EDPS calls on the Commission to seriously consider "all options in the impact assessment including the possibility of repealing the Directive". An eventual future data retention directive should be considered only if the necessity of data retention, "supported and regulated by the EU, could be sufficiently demonstrated, which includes a careful consideration of alternative measures."
The respective directive should be exhaustive (with a clear and precise purpose), proportionate (without going beyond what is necessary), comprehensive and should "genuinely harmonise rules on the obligation to retain data, as well as on the access and further use of the data by competent authorities."
EDPS - Press Release (31.05.2011)
http://europa.eu/rapid/pressReleasesAction.do?reference=EDPS/11/6&...
Opinion of the European Data Protection Supervisor on the Evaluation report from the Commission to the Council and the European Parliament on the Data Retention Directive (31.05.2011)
http://www.edps.europa.eu/EDPSWEB/webdav/site/mySite/shared/Documents/...,
EDRi-gram: Top 10 misleading statements of the European Commission on data retention (20.04.2011)
http://www.edri.org/edrigram/number9.8/data-retention-evaluation
EDRi-gram: Data retention in EU Council Meeting (18.05.2011)
http://www.edri.org/edrigram/number9.10/data-retention-eu-council
Syndicate:
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7th Battalion, Tennessee Cavalry (Bennett's) (Confederate)Edit This Page
From FamilySearch Wiki
United States U.S. Military Tennessee Tennessee Military Tennessee in the Civil War 7th Battalion, Tennessee Cavalry (Bennett's)
Contents
Brief History
This regiment was organized at Camp Jim Davis, Macon County, Tennessee, in November, 1861, with six companies. In June, 1862, it merged into Barteau's 2nd Tennessee Cavalry which later became 22nd (Barteau's) Tennessee Cavalry Regiment. [1]
Companies in this Regiment with the Counties of Origin
Men often enlisted in a company recruited in the counties where they lived though not always. After many battles, companies might be combined because so many men were killed or wounded. However if you are unsure which company your ancestor was in, try the company recruited in his county first.
The information about the companies is from Tennesseans in the Civil War, (accessed 19 Nov 2011).
The Civil War Soldiers and Sailors database lists 543 men on its roster for this unit. Roster.
Other Sources
• Beginning United States Civil War Research gives steps for finding information about a Civil War soldier or sailor. It covers the major records that should be used. Additional records are described in 'Tennessee in the Civil War' and 'United States Civil War, 1861 to 1865' (see below).
• National Park Service, The Civil War Soldiers and Sailors System, is searchable by soldier's name and state. It contains basic facts about soldiers on both sides of the Civil War, a list of regiments, descriptions of significant battles, sources of the information, and suggestions for where to find additional information.
• Tennessee in the Civil War describes many Confederate and Union sources, specifically for Tennessee, and how to find them.. These include compiled service records, pension records, rosters, cemetery records, Internet databases, published books, etc.
• United States Civil War, 1861 to 1865 describes and explains United States and Confederate States records, rather than state records, and how to find them. These include veterans’ censuses, compiled service records, pension records, rosters, cemetery records, Internet databases, published books, etc.
• The 7th Tennessee Cavalry Battalion, Muster Roll for Company F, Later known officially as Company G, 22nd Tennessee Cavalry, (accessed 29 Dec 2011).
• Confederate Military Rosters Online, 7th Cavalry Battalion, (Bennett's Battalion), (accessed 26 Feb 2012). Transcribed by Linda Rives, extracted fromHancock's Diary or A History of the Second Tennessee Confederate Cavalry, published in 1887. Links to military history and company rolls.
References
1. National Park Service, The Civil War Soldiers and Sailors System, (accessed 6 December 2010).
Need additional research help? Contact our research help specialists.
Need wiki, indexing, or website help? Contact our product teams.
Did you find this article helpful?
You're invited to explain your rating on the discussion page (you must be signed in).
• This page was last modified on 4 January 2013, at 03:24.
• This page has been accessed 342 times.
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Alleghany County, VirginiaEdit This Page
From FamilySearch Wiki
Revision as of 21:38, 3 April 2012 by Cottrells (Talk | contribs)
This article is about a county on the mid Virginia-West Virginia border. For other uses, see Alleghany.
Alleghany County, Virginia
Map
Location in the state of Virginia
Location of Virginia in the U.S.
Facts
Founded January 5, 1822
County Seat Covington
Courthouse
United States Virginia Alleghany County
Southwest Virginia county in the Shenandoah Valley region.
Contents
County Courthouse
Alleghany County, Virginia Courthouse
Allegheny County Courthouse
P.O.Box 670
266 West Main Street
Covington, VA 24426-0670
• Clerk Circuit Court has marriage records from 1845, divorce, probate, court and land records from 1822
Beginning Dates for Alleghany County, Virginia Government Records
Birth Marriage Death Census Land Probate
History
Allegheny Mountain Range, Spruce Knob
The county was named after the Alleghany Mountains.[1]
Parent County
1822--Allegany County was created 5 January 1822 from Bath, Botetourt and Monroe Counties. Monroe County is now in West Virginia.
County seat: Covington [2]
Boundary Changes
Record Loss
Places/Localities
Populated Places
Neighboring Counties
Resources
Research Guides
African Americans
Cemeteries
For a detailed list, including addresses, phone numbers, and external links, see Alleghany County, Virginia Cemeteries.
The following is a list of cemeteries in Alleghany County:[3]
• Alleghany Memorial Park
• Carter Cemetery
• Central Church Cemetery
• Crown Hill Cemetery
• Helmintoller Cemetery
• Hoke Cemetery
• Hooks Cemetery
• Humphries Cemetery
• Lewis Tunnel Cemetery
• Mallow Cemetery
• Mount Carmel Cemetery
• Mountain View Cemetery
• Oakland Cemetery
• Rose Hill Cemetery
• Simmons Cemetery
• Smith Cemetery
• Walton Memorial Cemetery
Census
Historical populations
Census Pop. %±
1830 2,816
1840 2,749 −2.4%
1850 3,515 27.9%
1860 6,765 92.5%
1870 3,674 −45.7%
1880 5,586 52.0%
1890 9,283 66.2%
1900 16,330 75.9%
1910 14,173 −13.2%
1920 15,332 8.2%
1930 20,188 31.7%
1940 22,688 12.4%
1950 23,139 2.0%
1960 12,128 −47.6%
1970 12,461 2.7%
1980 14,333 15.0%
1990 13,176 −8.1%
2000 12,926 −1.9%
Source: "American FactFinder". United States Census Bureau.
To access census records online, see Virginia Census.
1785
1840
• Douthat, James L. 1840 Alleghany County, Virginia Census. Signal Mountain, Tenn.: Mountain Press. Free online surname index and purchase details at Mountain Press website.
1860
• Perkins, Louise M. 1860 Alleghany County, Virginia Census. Signal Mountain, Tenn.: Mountain Press. Free online surname index and purchase details at Mountain Press website.
1890 Union Veterans
Church
Court
Genealogy
Several genealogies have been published about Alleghany County families. To view a list, visit Alleghany County, Virginia Genealogy.
Land
Local Histories
• Alleghany County, Virginia: Its Resources and Industries. 1907. Digital version at Google Books (full-view).
• McAllister, Hugh Maffitt. Historical Sketch of Alleghany County, Virginia. Richmond: n.p., 1910. Available at FHL US/CAN Fiche 6015385; digital version at Ancestry ($).
• McAllister, W.A. "Pioneer Days in Alleghany County," The Virginia Magazine of History and Biography, Vol. 10, No. 2 (Oct., 1902), pp. 183-187; Vol. 10, No. 3 (Jan. 1902):254-257. Digital version at JSTOR ($).
• Morton, Oren Frederic. A Centennial History of Alleghany County, Virginia. Dayton, Va.: J.K. Ruebush Co., 1923. Available at FHL US/CAN Book 975.581 H2m and FHL US/CAN Film 599242 Item 1; digital version at Ancestry ($).
Maps
Military
Revolutionary War
• A Census of Pensioners for Revolutionary or Military Services: With their Names, Ages, and Places of Residence, as Returned by the Marshalls of the Several Judicial Districts, Under the Act for Taking the Sixth Census]. 1841. Digital versions at U.S. Census Bureau and Google Books et. al. 1967 reprint: FHL Collection 973 X2pc 1840. [See Virginia, Western District, Allegany County on page 133.]
War of 1812
• List of Pensioners on the Roll, January 1, 1883; Giving the Name of Each Pensioner, the Cause for Why Pensioned, the Post-Office Address, the Rate of Pension Per Month, and the Date of Original Allowance... Washington, D.C.: Governnment Printing Office, 1883. FHL Collection 973 M2Lp v. 5; digital versions at Google Books and Internet Archive. [See Vol. 5, Virginia, Alleghany County, p. 60.]
Civil War
Civil War service men from Alleghany County served in various regiments. Within a regiment, companies were often comprised of men who lived in a smaller geographical area within the county parameters. Listed below are regiments with whom men from Alleghany County served. Because Confederate units often reorganized, this list might not be complete.
The following Regiments are mentioned in A Centennial History of Alleghany County, Virginia, by Oren Frederic Morton FHL book 975.581 H2m
- 2nd Regiment, Virginia Cavalry (Confederate), A Centennial History of Alleghany County, Virginia, only lists the names of five men who were from Alleghany County.
- 10th Regiment, Virginia Infantry (Confederate)
- 11th Regiment, Virginia Infantry (Confederate)
- 22nd Regiment, Virginia Infantry (Confederate)
- 27th Regiment, Virginia Infantry (Confederate)
- 59th Regiment, Virginia Infantry (Confederate)
- 60th Regiment, Virginia Infantry (Confederate)
However, A Centennial History of Alleghany County, Virginia, also lists additional names of Alleghany Companies such as the Alleghany Rough Artillery (Capt. John C. Carpenter's Co.) , Alleghany Light Infantry (Co. A, 27th Regiment) afterwards called Alleghany Artillery, Alleghany Reserves (Co. C, 10th Battalion, Virginia Reserves), Alleghany Rifles (Co. C, 27th Regiment)
Civil War Sources
Virginia, Civil War Service Records of Confederate Soldiers 1861-1865
Virginia, Civil War Service Records of Union Soldiers 1861-1865
• Morton, Oren F., A Centennial History of Alleghany County, Virginia, (J. K. Ruebush Company, 1923). (pp. 156-172), FHL book 975.581 H2m
Naturalization
Newspapers
Occupations
Petitions
• Eckenrode, H.J. Virginia State Library: A Calendar of Legislative Petitions Arranged by Counties Accomac - Bedford. Richmond, Va.: Davis Bottom, Superintendent of Public Printing, 1908. Digital version at Google Books. [Alleghany County petitions (1820-1861) are described on pp. 87-95.]
Private Papers
Probate
Taxation
How can Virginia tax lists help me?
• [1774] Tithables, 1774, Alleghany Highlands Genealogical Society Newsletter, Vol. 7, No. 2 (Apr. 1998).
Vital Records
Birth
• Fridley, Beth. Alleghany County, Virginia Births, 1853-96 [database on-line]. Provo, UT, USA: The Generations Network, Inc., 1999. Available at Ancestry ($).
Societies and Libraries
Family History Centers
Websites
Genealogy courses: Learn how to research from an expert in Fun Five Minute Genealogy Videos.
References
1. Wikipedia Contributors, "Allegheny Mountains," in Wikipedia: The Free Encyclopedia, http://en.wikipedia.org/wiki/Allegheny_Mountains, accessed 13 January 2012.
2. The Handybook for Genealogists: United States of America, 10th ed. (Draper, UT: Everton Publishers, 2002).
3. USGS Map, www.trails.com
Need additional research help? Contact our research help specialists.
Need wiki, indexing, or website help? Contact our product teams.
Did you find this article helpful?
You're invited to explain your rating on the discussion page (you must be signed in).
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Mason County, KentuckyEdit This Page
From FamilySearch Wiki
United States Kentucky Mason County
Guide to Mason County Kentucky genealogy. Birth records, marriage records, death records, census records, family history, and military records.
Kentucky
Online Records
Mason County, Kentucky
Map
Location in the state of Kentucky
Location of Kentucky in the U.S.
Facts
Founded November 5, 1788
County Seat Maysville
Courthouse
Adopt-a-wiki page
This page adopted by:
KYGenWeb Project
who welcome you to contribute.
and its representative
Mason Co. KYGenWeb
Adopt a page today
Contents
County Courthouse
Beginning Dates for Mason County, Kentucky Government Records
Birth*
Marriage
Death*
Census
Deeds
Wills
1852
1789
1852
1810
1789
1790
*Many years between 1852 and 1911 are missing
Mason County, Kentucky Courthouse.JPG
Mason County Courthouse
219 Stanley Reed Street
Maysville, KY 41056-0234
Phone: 606.564.3341[1]
History
Parent County
1788--Mason County was created 5 November 1788 from Bourbon County.
County seat: Maysville [2]
Boundary Changes
See an interactive map of Mason County boundary changes.
Record Loss
Places/Localities
Populated Places
Bates Manley (hist.) Old Washington South Ripley
Charlestown Bottom (hist.) Marshall Orangeburg Springdale
Country Club Heights Mays Lick Plumville Taylors Mill
Dexter (hist.) Maysville Rectorville Washington
Dover Minerva Sardis Weedonia
Fernleaf Moranburg Shannon
Helena Mount Gilead Shawnee Hill
Lewisburg Murphysville Somo
Neighboring Counties
Resources
Kymason.png
African American Research
The following have information concerning African American research. Both should be used:
Biographies
Cemeteries
Kentucky cemetery records often identify birth, death, relationship, and military information, as well as religious affiliation.
Census
Historical populations
Census Pop.
17902,729
180012,182346.4%
181012,4592.3%
182013,5889.1%
183016,19919.2%
184015,719−3.0%
185018,34416.7%
186018,222−0.7%
187018,126−0.5%
188020,46912.9%
189020,7731.5%
190020,446−1.6%
191018,611−9.0%
192017,760−4.6%
193018,8626.2%
194019,0661.1%
195018,486−3.0%
196018,454−0.2%
197017,273−6.4%
198017,7652.8%
199016,666−6.2%
200016,8000.8%
http://ukcc.uky.edu/~census/21161.txt
For tips on accessing Mason County, Kentucky census records online, see: Kentucky Census.
1790 - Lost, but a substitute is available, see Taxation.
Church
Court
Genealogy
It is anticipated that this bibliography will eventually identify all known family histories published about residents of this county. Use this list to:
• Locate publications about direct ancestors
• Find the most updated accounts of an ancestor's family
• Identify publications, to quote Elizabeth Shown Mills, about an ancestor's "FAN Club" [Friends, Associates, and Neighbors]
Bibliography
• [Anderson] Black, William. "Stokes-Anderson of Lunenburg County," in John Bennett Boddie, ed., Historical Southern Families, Vol. 2 (Redwood City, Calif., 1958), 217-225.
• [Anderson] Darnel, Michael R. "Which James Anderson Married Elizabeth Ligon?" The Virginia Genealogist, Vol. 39, No. 3 (Jul.-Sep. 1995):163-170. Digital version at American Ancestors ($). FHL Book 975.5 B2vg v. 39 (1995).
• [Asbury] Holtzclaw, B.C. "Asbury of Westmoreland County, Virginia," Virginia Genealogical Society Quarterly, Vol. 5, No. 3 (Jul. 1967):55-65; Vol. 5, No. 4 (Oct. 1967):77-87; Vol. 6, No. 1 (Jan. 1968):10-26. Digital version at Ancestry ($).
• [Beeler] Pappas, Carolyn H. "Christopher Beeler, 1705-1775," The Virginia Genealogist, Vol. 43, No. 4 (Oct.-Dec. 1999):243-255; Vol. 44, No. 1 (Jan.-Mar. 2000):18-30; Vol. 44, No. 2 (Apr.-Jun. 2000):115-125. Digital version at American Ancestors ($). FHL Book 975.5 B2vg.
• [Bell] Ball, Helen A. Bell Family: The Bells of Mason County, Kentucky, Charles Bell and Daniel Bell, Including Descendants and Some Related Families. East Lansing, Mich.: H.A. Ball, 1981. FHL Book 929.273 B413bh
• [Collier] Boss, Lula Reed. Captain Thomas Collier of Charlotte County, Virginia, Including Records of Mason County, Kentucky. MSS. Microfilmed 1956. FHL Film 157151 Item 2
• [Day] DeVerter, Ruth Hendricks. The Day and Hendrix(cks) Families: Including Poe and Allied Lines: Bucks County, Pennsylvania; Baltimore and Cecil Counties, Maryland; Bracken and Mason Counties, Kentucky; Brown and Clermont County, Ohio; Wayne and Hendricks Counties, Indiana. Multi-volume. Baytown, Texas: R.H. DeVerter, 1963. FHL Book 929.273 H384d v. 3
• [Flinn] Taylor, Evelyn M. The Family of John Flinn (abt 1793) - Rebecca Reeves (abt 1795) of Mason County, Kentucky and Putnam County, Indiana. Salt Lake City, Utah: Taylor, R.J.Jr. Foundation, 1997. FHL Book 929.273 F646t; digital version at FamilySearch Books Online .
• [Heflin] Heflin, Donald L. Reuben Heflin of Somewhere in Virginia to Mason, Fleming & Mason counties, KY, 1785-1863. Georgetown, Ky.: D.L. Heflin, 1995. FHL Book 929.273 H361hdd
• [Levi] Pearson, Spencer. Of Turkeys and Eagles. Corpus Christi, Texas: S. Pearson, 1998. FHL Book 929.273 P318p
• [McCracken] Everett, Jeanne McCracken. McCracken Family Records: Descendants of John McCracken and Elizabeth Wood 1784-1984: John McCracken & Elizabeth Wood were Married 1784 in Frederick, Maryland; Resided 1788-1817 in Mason County, Kentucky; and after 1817 in Daviess County, Indiana. Typescript. Microfilmed 1990. FHL Film beg. 1674175 Item 9.
• [Marshall] Buford, Marcus Bainbridge. A Genealogy of the Buford Family in America, With Records of a Number of Allied Families. San Francisco, Calif., 1903. Digital version at Internet Archive - free.
• [Marshall] Prewitt, John Marshall. Marshall of King George Co., Va. and Mason Co., Ky: Berry -- Kercheval -- Roy and Connected Families. Cincinnati, Ohio: J.M. Prewitt, 2002. FHL Book 929.273 M355pj
• [Matthews] Matthews, Mitchell Dudley. Walter Matthews (1845-1930) of May's Lick, Mason County, Kentucky and His Descendants. 1968. FHL Book 929.273 A1 no. 353
• [Owens] Owens, David Hatfield. Owens Family of Virginia (Richmond, King George, Stafford, Prince William, Fauquier Counties) and Kentucky (Mason, Lewis, Fleming, Bracken, Mercer, Boyle Counties). Ann Arbor, Mich.: D.H. Owens, 1991. FHL Book 929.273 Ow2od 1991
• [Prichard] Prichard, Paul Preston. The Prichard Family: History and Genealogy of the Descendants, Antecedents and Collateral Relatives of Harmon and Nancy Purcell Prichard of Mason County, Kentucky. El Paso, Texas: P.P. Prichard, 1976. FHL Book 929.273 P931r
• [Randolph] Randolph, Blanche Willie Grace and Deborah Ann Kraemer Small. The Randolphs of Prince William County, Virginia. Little Rock, Arkansas: B. Randolph, 1979. FHL Collection
• [Randolph] Small, Deborah Ann Kraemer and Blanche Willie Grace Randolph. The Randolphs of Prince William County, Virginia, Volume II. Winter Park, Florida: D. Small, 1989. FHL Collection
• [Randolph] Raski, Arma, James Randolph, and Glenita Guthrie. Additions & Corrections to Vol. II, the Randolphs of Prince William Co., VA by Deborah K. SmallFHL 6075877
• [Reeves] Taylor, Evelyn M. The Family of Samuel Reeves, Sr. (1770/1780) - 1850: of Mason County, Kentucky and Putnam County, Indiana. Salt Lake City, Utah: E.M. Taylor, 1997. FHL Book 929.273 R259t
• [Robinson] Robinson, John Bunyan. The Adin Robinson Family and Collaterals. Libertyville, Ill., 1904. FHL Film 856108 Item 4; digital version at Google Books.
• [Rust] Rust, Ellsworth Marshall. Rust of Virginia: Genealogical and Biographical Sketches of the Descendants of William Rust, 1654-1940. Washington, D.C.: Rust, 1940. FHL Book 929.273 R922re.
• [Tarvin] Torrey Alice Ellenor Herndon and Ronald Brennan. Tarvin Family Connections: Including Cowgill and Cracraft Families of Mason County and Campbell Co., Ky. Wilder, Ky.: R. Brennan, 1977. FHL Book 929.273 T179ta
• [Walton] Walton, Matt Savage. Chart, Direct Line of Descent and Other Documents Pertaining to the Walton Family of Mason County and Lexington, Kentucky. Typescript, 1940s. FHL Book 929.273 W173wm
• [Walton] Walton, John. "Job Walton of Fleming County, Kentucky," National Genealogical Society Quarterly, Vol. 52, No. 1 (Mar. 1964):15-24. FHL Collection 973 B2ng v. 52
Land
Local Histories
Maps
Military
Civil War
Regiments. Service men in Mason County, Kentucky served in various regiments. Men often joined a company (within a regiment) that originated in their county. Listed below are companies that were specifically formed in Mason County, Kentucky:
- 6th Regiment, Kentucky Cavalry (Confederate). Company H.
- 9th Regiment, Kentucky Cavalry (Confederate). Company H.
Miscellaneous
Newspapers
Obituaries
Probate
Taxation
• 1790 - Heinemann, Charles B. "First Census" of Kentucky 1790. Washington, D.C.: n.p., 1940. 1981 reprint: FHL US/CAN 976.9 X2ph 1981; digital version at Kentucky Kinfolks. ["1 volume of 1790 was used." (p. 2)]
• 1800 - 1800 Tax List of Mason County, Kentucky is included in Clift's Second Census of Kentucky 1800,[3] digitized by Ancestry ($).
Vital Records
Birth
• 1852-1861 - Mason County Birth Index 1852-1861. Batch C517731 at FamilySearch - free.[4]
• 1874 - Mason County Birth Index 1874. Batch C517732 at FamilySearch - free.[4]
Marriage
• 1789-1805 - Mason County Marriage Index 1789-1805. Batch M539871 at FamilySearch - free.[4]
• 1806-1819 - Mason County Marriage Index 1806-1819. Batch M539872 at FamilySearch - free.[4]
• 1820-1837 - Mason County Marriage Index 1820-1837. Batch M539873 at FamilySearch - free.[4]
• 1852-1855 - Mason County Marriage Index 1852-1855. Batch M539876 at FamilySearch - free.[4]
• 1852-1860 - Mason County Marriage Index 1852-1860. Batch M539875 at FamilySearch - free.[4]
• 1852-1861 - Mason County Marriage Index 1852-1861. Batch M517731 at FamilySearch - free.[4]
• 1859-1864 - Mason County Marriage Index 1859-1864. Batch M539878 at FamilySearch - free.[4]
• 1864-1866 - Mason County Marriage Index 1864-1866. Batch M539879 at FamilySearch - free.[4]
• 1866-1872 - Mason County Marriage Index 1866-1872. Batch M539881 at FamilySearch - free.[4]
• 1866-1897 - Mason County Marriage Index 1866-1897. Batch M539887 at FamilySearch - free.[4]
• 1866-1949 - Mason County Marriage Index 1866-1949. Batch M539886 at FamilySearch - free.[4]
• 1872-1879 - Mason County Marriage Index 1872-1879. Batch M539882 at FamilySearch - free.[4]
• 1879-1885 - Mason County Marriage Index 1879-1885. Batch M539883 at FamilySearch - free.[4]
• 1885-1892 - Mason County Marriage Index 1885-1892. Batch M539884 at FamilySearch - free.[4]
• 1892-1896 - Mason County Marriage Index 1892-1896. Batch M539885 at FamilySearch - free.[4]
Unsorted
Societies and Libraries
Family History Centers
Web Sites
References
1. Handybook for Genealogists: United States of America, 10th ed. (Draper, Utah: Everton Pub., 2002), Mason County, Kentucky page 273, At various libraries (WorldCat); FHL Book 973 D27e 2002.
2. The Handybook for Genealogists: United States of America,10th ed. (Draper, UT:Everton Publishers, 2002).
3. G. Glenn Clift, Second Census of Kentucky 1800: A Privately Compiled and Published Enumeration of Tax Payers Appearing in the 79 Manuscript Volumes Extant of Tax Lists of the 42 Counties of Kentucky in Existence in 1800 (1954; reprint, Baltimore, Md.: Genealogical Publishing Co., Inc., 2005).
4. 4.00 4.01 4.02 4.03 4.04 4.05 4.06 4.07 4.08 4.09 4.10 4.11 4.12 4.13 4.14 4.15 4.16 Genealogical Society of Utah, Parish and Vital Records List (July 1998). Microfiche. Digital version at https://familysearch.org/learn/wiki/en/images/7/75/Igikentuckymz.pdf.
Need additional research help? Contact our research help specialists.
Need wiki, indexing, or website help? Contact our product teams.
Did you find this article helpful?
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• This page was last modified on 29 March 2013, at 20:41.
• This page has been accessed 4,303 times.
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For the half-year to 30 June 2013, the IPKat's regular team is supplemented by contributions from guest bloggers Stefano Barazza, Matthias Lamping and Jeff John Roberts.
Two of our regular Kats are currently on blogging sabbaticals. They are Birgit Clark and Catherine Lee.
Sunday, 17 February 2013
IP blogging: a couple of ethical issues
Information received from anonymous sources
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Recently, a member of the blogging team received an anonymous communication suggesting that sweeping changes are being considered in relation to the organisation and marking of the EQE (that's the European Qualifying Examination taken by aspiring patent attorneys). Occasionally, information of this nature is received from anonymous correspondents, who hope that the IPKat will publish an unconfirmed and unverifiable story. If the IPKat does not know who the information has come from, no judgment can be made as to its authenticity.
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Information of a personal nature
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source: josm/trunk/data/ar.lang @ 4019
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source: josm/trunk/data/de.lang @ 4260
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• Property svn:mime-type set to application/octet-stream
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Agarose gel electrophoresis
From OpenWetWare
Revision as of 18:49, 25 August 2005 by ClarkeS (Talk | contribs)
Jump to: navigation, search
Contents
Endy Lab
Pouring agarose gels
Purpose: To prepare gels for gel electrophoresis.
Materials
• gel box, gel tray, comb
all in gel room
• 1% agarose in 1X TAE
premade, premelted bottles available in Peltier incubator; 564D
• Ethidium Bromide (abbreviated EtBr)
in gel room, upper right hand shelf
• 100 ml glass bottle or beaker
prewarmed in incubator or water bath for a few minutes
Procedure
1. Push gel tray down into pouring box. The rubber gasket ends of the tray should form a seal with the sides of the box.
2. Place comb of appropriate width, size, and number into niche at end of the tray.
3. Pour agarose solution, 35 ml for small gels or 70ml for large gels, into prewarmed bottle. The marks on the side of the bottle should be a sufficient guide.
4. Add 0.2 μl of EtBr per 35 ml 1% agarose. EtBr is extremely carcinogenic - be careful with it. (Wear nitrile gloves; dispose of tips properly into EtBr waste container; don't expose any part of the pipette other than the tip).
• Note that our EtBr is currently diluted to an unknown (but low) concentration. More EtBr may need to be added to achieve the same signal.
5. Swirl bottle or mix with pipette tip to mix in EtBr. Pour the mixture into the tray. Rinse out the bottle with hot water.
6. Let agarose sit for ~15 minutes to solidify. After it has set a bit (enough so that you can walk with it), you can set it in the cold room to chill further and solidify completely, saving a few minutes.
7. When gel has solidified, remove the comb, lifting straight up. Lift the gel tray and rotate it 90°, so that the ends of the gel are now exposed to the ends of the gel box.
Running agarose gels
Materials
Procedure
1. Add enough 1X TAE to fill the reservoirs at both ends of the gel box and cover the surface of the gel--the gel should be immersed. (There are bottles of 1X TAE in the gel room on the right side of the bench. If they're empty, 1X TAE is above the sink in 564D.)
2. Load prepared ladder (+dye +loading buffer). Typically, the ladder solution is at 1 μg per 12 μl, but you can change the concentration or total mass. The mass of ladder is important to know if you need to quantify your bands by comparison with the ladder bands.
Load ladder in left-most lane and sometimes right-most lane if you want to and have the space.
3. Use 2 μL loading dye per 10 μL of sample. Some lab members use dyed loading buffer; others use clear sucrose solution. The dye makes the sample easier to see when loading, but may interfere with seeing bands later.
4. Load samples left to right.
The capacity of the 8 well, 1.5mm wide well is approximately 45 μL. The capacity of the 15 well, 1.5mm well is approximately 15 μL.
5. Place gel box cover on gel box such that your samples will run towards the positive, red electrode. Make sure that the cables from the cover are connected to the power supply correctly.
6. Turn on the power supply and run your gel at ~85 V for 1 hr 20 mins (voltage and time values can vary). Use the timer to enable automatic shutoff of your gel.
7. Verify that bubbles are rising from the electrodes once you start your gel to ensure your gel is running properly.
Knight lab
Casting agarose gels
We precast our gels.
Materials
Procedure
1. Add 300mL 1X TAE to a 500 mL bottle.
2. Measure out sufficient agarose to cast either a 1% (3 g) or 1.5% (4.5 g) gel.
3. Add the agarose to the TAE buffer in the 500 mL bottle.
4. Swirl to mix.
5. Microwave bottle with loosened cap on high until the gel starts to bubble and is transparent.
This generally takes just over two minutes for 300 mL. If you microwave too long, the gel will bubble over causing a big mess and you will need to start over.
6. Remove from microwave and let cool by either sitting on bench top or adding stir bar and placing on stir plate.
The advantage of the stir plate is that, if you forget about your gel for a while, it is less likely to solidify accidentally.
If you are in a hurry, you can place the bottle in a beaker of room temperature water on the stir plate to speed the cooling process significantly.
7. While gel is cooling, assemble casting trays and gel combs and verify that the trays are level.
8. Once gel is cooled so that it can be touched comfortably with your gloved hand, add 15 μL Ethidium Bromide (final concentration of 0.5 μg/mL).
9. Pour gel into casting trays.
The height of the gel will depend on how much you wish to load. Diagnostic gels can be reasonably shallow since typically 10 μL volumes are loaded. For gel purifications, the gel should be deeper to enable loading of large sample volumes.
10. Let gels sit until they are solidified.
Gels are solid when they are cloudy in appearance and firm to the touch.
11. Gels may be used immediately. Alternatively, gels may be individually sealed in 6 x 10 inch polyethylene bags, labelled with initials, date and percentage and stored at 4 °C.
It is a judgement call as to whether a gel is too old to be used. If it takes on a shrivelled appearance, don't use it. If there is lots of condensation on the bag, only use it if your intended experiment isn't critical.
Running agarose gels
Materials
Procedure
1. Take a gel from the 4°C fridge.
If the number of gels is getting low, cast more gels as described above.
2. Place your gel in gel box.
3. Add 1X TAE buffer to gel box such that buffer just covers the top of the gel.
4. Remove comb.
5. Add 10 μL ethidium bromide stock solution to the running buffer well near the positive terminal.
6. Load 12 μL prepared ladder
Typically load ladder in left-most lane and sometimes right-most lane as well depending on whether you have the space.
7. Use 2 μL loading dye per 10 μL of sample.
8. Load samples left to right.
The capacity of the 8 well, 1.5mm wide well is approximately 45 μL. The capacity of the 15 well, 1.5mm well is approximately 15 μL.
9. Place gel box cover on gel box such that your samples will run towards the positive, red electrode.
10. Run your gel at ~85 volts for 1 hr 20 mins. Use the timer to enable automatic shutoff of your gel.
If you are in a hurry the gel can be run faster at ~95 volts for less than an hour.
11. Verify that bubbles are rising from the electrodes once you start your gel to ensure your gel is running properly.
Visualizing agarose gels
Note that this procedure is under development
Materials
• FluorChem 8800 gel imager
Procedure
1. Remove gel from gel box shaking gently to allow residual buffer to fall back into gel box.
2. Place in middle of UV box inside gel imager (you can leave the gel in gel tray).
3. Close the door and turn on reflective white light button.
4. In gel imager software, click the "Acquire" button such that gel displays on screen.
5. Adjust gel position on UV box so that the entire gel is within the frame.
6. Close the door, turn off reflective white light and turn on transilluminating UV light.
7. In gel imager software, click "Acquire image" button to capture gel image to the screen.
It is occasionally necessary to adjust exposure time to improved image.
8. Increase filtering if bands are difficult to see.
9. Annotate gel as necessary.
10. Save a copy of gel picture in your user folder.
11. Print.
12. Remove gel and dispose in ethidium bromide waste container.
13. Wipe down UV box if necessary.
Interpreting results
If you are getting unexpected bands on your gel you may want to look at the Common Agarose Gel Issues
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Help Wikitravel grow by contributing to an article! Learn how.
Muslim sites and events
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Islam is the world's second most prolific religion, second only to Christianity. Several sites built in the name of Islam are on the UNESCO World Heritage List. The annual Muslim pilgrimage to Mecca, the Hajj, is one of the largest human migrations.
As Muslim congregations have had a significant role in most communities where they are present, a traveller will learn much from visiting a local mosque, regardless of belief.
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
3401.0 - Overseas Arrivals and Departures, 1969
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Continued by: Overseas Arrivals and Departures, Australia
Short summary of monthly data by category of movement. For visitors arriving and residents departing short-term, the intended length of stay, purpose of journey, and principal destination (departures) or country of usual residence (arrivals). For settler arrivals -- region of birth.
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Australian Bureau of Statistics
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Private sector house approvals fall in July
ABS Building Approvals show that the total number of dwellings approved rose 1.0% in July 2011, in seasonally adjusted terms, after falling 3.6% in June.
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An alternative to the hand searching gold standard: validating methodological search filters using relative recall
Margaret Sampson*, Li Zhang, Andra Morrison, Nicholas J Barrowman, Tammy J Clifford, Robert W Platt, Terry P Klassen and David Moher
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Research article
Do the mutations of C1GALT1C1 gene play important roles in the genetic susceptibility to Chinese IgA nephropathy?
Gui-Sen Li1,2,3, Guang-Jun Nie1, Hong Zhang1*, Ji-Cheng LV1, Yan Shen3 and Hai-Yan Wang1
Author Affiliations
1 Renal Division, Department of Internal Medicine, Peking University First Hospital, and Peking University Institute of Nephrology, Beijing 100034, PR China
2 Renal Division, Sichuan Medical Science Academy & Sichuan Provincial People's Hospital, Chengdu 610072, PR China
3 Chinese National Human Genome Center, Beijing 100176, PR China
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BMC Medical Genetics 2009, 10:101 doi:10.1186/1471-2350-10-101
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2350/10/101
Received:7 November 2008
Accepted:24 September 2009
Published:24 September 2009
© 2009 Li et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
The deficiency of β1,3 galactose in hinge region of IgA1 molecule played a pivotal role in pathogenesis of IgA nephropathy (IgAN). Cosmc, encoded by C1GALT1C1 gene, was indispensable to β1,3 galactosylation of IgA1. We designed a serial study to investigate the relationship between the mutations of C1GALT1C1 gene and the genetic susceptibility to IgAN.
Methods
Nine hundred and thirty-eight subjects, including 661 patients with IgAN and 277 healthy controls were enrolled in the study. Firstly, single nucleotide polymorphisms (SNPs) in the promoter region of C1GALT1C1 gene were screened. Then the c.-347-190G>A was analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) for further case-control association analysis. Secondly the somatic mutations of DNAs from peripheral blood B lymphocytes were detected in 15 patients and 7 normal controls.
Results
No significant association was observed between the different alleles or genotypes of c.-347-190G>A and IgAN. The patients with different genotypes of C1GALT1C1 gene did not significantly associate with clinical manifestations, including hematuria, proteinuria, and serum creatinine of patients with IgAN. There was no somatic mutation detected in total 202 clones of 22 individuals.
Conclusion
The c.-347-190G>A polymorphism and the somatic mutation of encoding region of C1GALT1C1 gene were not significantly related to the genetic susceptibility to IgAN in Northern Chinese population.
Background
IgA nephropathy (IgAN), which is the most common glomerulonephritis and a leading cause for end-stage renal disease (ESRD) worldwide, is characterized by presence of IgA1 deposit in the glomerular mesangium. In recent years, aberrant glycosylations of IgA1 molecule in patients with IgAN were reported [1,2] and were considered as the most important pathogenic mechanism of IgAN [3-5]. Previous studies had demonstrated that circulating and glomerular deposited IgA1 in patients with IgAN showed deficiency of β1,3 galactose in the hinge glycopeptides [2,4,6]. The deficiency of hinge-region glycosylation of serum IgA1 showed an increased tendency to self-aggregation and/or the increased binding to circulating glycoproteins and enhanced reaction with specific IgG antibodies directed against IgA1 hinge O-glycans. These IgA aggregates could escape the clearance by hepatic receptors for asialoglycoproteins [4]. Others' and our previous studies demonstrated that IgA1 with deficiency of hinge-region β1,3 galactose had a higher binding capacity and stronger biologic effects to cultured human mesangial cells, leading to accumulation and/or prolonged deposit of IgA within the mesangium [7-9]. The β1,3 galactose deficiency of serum IgA1 were closely associated with renal pathologic phenotypes of IgAN [10]. Therefore, deficiency of hinge-region β1,3 galactosylation of IgA1 molecule might play a pivotal role in the pathogenesis of IgAN.
The mechanisms contributed to aberrant galactosylation of IgA1 molecule in patients with IgAN were still unclear. In fact, the core 1 structure Galβ1→3GalNAcα1- Ser/Thr (T antigen) was synthesized from GalNAcα1-R (Tn antigen) by the action of core 1 β3-galactosyltransferase (C1β3Gal-T). The coding gene of C1β3Gal-T was C1GALT1. However, there was no apparent disparity of C1GALT1 expression among normal controls, non-IgAN glomerulonephritis, and IgA nephropathy [11]. Further studies revealed that the C1β3Gal-T activity required expression of a molecular chaperone designated Cosmc (core 1β3-Gal-T-specific molecular chaperone) [12]. C1GALT1C1 (OMIM*300611), the coding gene for Cosmc [12,13], was mapped to chromosome Xq23, included 3 exons and spanned about 4 kb [12,13]. Mutations of C1GALT1C1 could impressively changed the enzyme activity of C1β3Gal-T [12,14]. But our previous study in patients with IgAN revealed that there was only one mutation (c.393T>A) detected in the coding region of the C1GALT1C1 gene [15]. The minor allele frequency (MAF) of c.393T>A was only 6.90% [15]. In additionally, a previous study had revealed that the conservative amino-acid substitution (Asp131→Glu) which derived from the c.393T>A mutation gave normal C1β3Gal-T activity [14]. However, other two somatic mutations of C1GALT1C1 gene contributed to the aberrant galactosylation of Tn syndrome [14]. Malycha et al [16] found that none of C1GALT1C1 mutations played important roles in the pathogenesis of IgAN in a rencent study. It was unclear whether there were any losses of function mutations or somatic mutations of C1GALT1C1 gene in Chinese IgAN patients.
We hypothesized that the mutations of C1GALT1C1 gene could influence its activities in two pathways: mutations in the promoter region or somatic mutations in the coding region. To prove the validity of the hypothesis, firstly, we screened the mutations in the C1GALT1C1 gene in the promoter region and used a case-control association analysis to test the relationship of the polymorphisms and the susceptibility or clinical manifestations of IgAN. Secondly, somatic mutations of C1GALT1C1 gene were detected in patients with IgAN.
Methods
Subjects
A total of unrelated 938 northern Chinese were involved in this study, including 661 patients with IgAN proved by renal biopsy, and 277 geography-matched healthy controls with normal urine analysis and blood pressure. Patients with Henoch-schonlein purpura, systemic lupus erythematosus, and chronic hepatic diseases were excluded by detailed clinical and laboratory examinations. The mean ages were 31.2 ± 11.4 (patients) and 28.9 ± 8.2 (the controls) years. There were 82 female subjects in the control group and 280 female subjects in the IgAN patient group. Twenty-two individuals (15 patients who were recently diagnosed as primary IgAN and 7 controls) were selected for somatic mutation detection.
Clinical data of patients with IgAN, including age, course of kidney disease before renal biopsy, as well as blood pressure and the level of urine protein excretion at the time of renal biopsy, were collected. At the same time, the renal function was evaluated, including serum creatinine and estimated glomerular filtration rate (eGFR) calculated by the Modification of Diet in Renal Disease abbreviated equation [17].
The protocol for this study was approved by medical ethics committee of Peking University, and informed written consents for the study were obtained from all participants.
SNPs Discovery and Genotyping
Genomic DNA of subjects was extracted from the EDTA-anticoagulated whole blood samples by salting out procedure [18]. The C1GALT1C1 gene was located in the chromosome X. Reference sequence of C1GALT1C1 gene (OMIM*300611, Version: NC_000023.9) was obtained from National Center for Biotechnology Information (NCBI) Gene database http://www.ncbi.nlm.nih.gov/entrez webcite. Fifty-eight alleles from chromosome X were screened in all of the 46 individuals, including 27 unrelated patients with IgAN (6 females) and 19 unrelated healthy controls (6 females). The polymerase chain reaction (PCR) amplification regions included 5' untranslated regions and the upstream 1 kb from transcriptional initiation site. PCR primers were designed by Primer3 program [19]. Target sequences were amplified by PCR from 50 ng genomic DNA in 20 μl of final reaction volume. DNA sequencing was performed on an ABI PRISM 3700 automated sequencer. The results of sequencing were analyzed by Phred/Phrap/Consed suite of software [20]. The SNPs were described according to the Human Genome Variation Society (HGVS) nomenclature guidelines [21].
One single nucleotide polymorphism (SNP), c.-347-190G>A (rs3810744), was detected in the promoter region of the C1GALT1C1 gene. The MAF of c.-347-190G>A (rs3810744) was 48.48%. So the SNP was genotyped for further association analysis in all 938 subjects by the standard PCR-restriction fragment length polymorphism procedures. The genomic DNA samples were amplified by PCR using the following primers, forward: 5'-ACGCAGGGGTACATCAGAGAA-3', reverse: 5'-TGACCAGGCTGTTCTAGCTG-3'. The products of 420 base pairs (bps) were digested by restriction endonuclease Hpy8I (Fermentas International Inc., Hanover, USA). The genotypes weren't detected in three controls and one patient. Forty PCR products were sequenced for accuracy confirmation of PCR-restriction fragment length polymorphism analysis.
B lymphocyte DNA extraction and PCR amplification
Peripheral blood B lymphocytes from 22 participants (15 patients and 7 controls) were isolated by using lymphocyte isolation sterile solution (Amersham Biosciences, Uppsala, Sweden) and CD19 magnetic beads (Dynal Biotech ASA, OSLO, Norway), and then DNA of B lymphocytes was extracted by salting out procedure [18]. The whole coding region was amplified by PCR with following primers, forward: 5'-GTTGTTGCAAAGTTCCAGTTTA-3', reverse: 5'-TTATACCAGTGCCACCAAATTA-3'. The length of PCR production was 1314 bps. B lymphocyte DNA (50 ng) was amplified in a final reaction volume of 50 μl, containing 10 pmol of each primer and 2U Pyrobest DNA polymerase (Takara Biotechnology, Japan). Touchdown PCR procedure was used as followings: the initial denaturation at 95°C was followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 68°Cfor 35 sec (the temperature decrease 0.5°C each circle), and elongation at 72°C for 100 sec, then 18 cycles of denaturation at 95°C for 30 sec, annealing at 53°C for 35 sec, elongation at 72°C for 100 sec, finally elongation at 72°C for 7 min.
Gene cloning and somatic mutation detection
PCR products from B lymphocyte DNA of 22 individuals were subcloned into PGEM-T vector (Promega Corporation, Madison, WI, USA) after purification and adding adenine to them. Then ligation productions were transformed to Ecoli Top 10 competent cells and cultured in Luria-Bertani (LB) solid medium for 14 hours at 37°C. More than 8 clones per individual were randomly selected and amplified in LB liquid medium for 14 hours at 37°C. Plasmids were extracted and digested with PST1 restriction enzyme (Promega Corporation, Madison, WI, USA) to verify the insertion of PCR productions. Total 202 clones, including 8 to 10 clones per individual, were directly sequenced to detect somatic mutation.
Statistical Analysis
Observed genotype frequencies in female subjects for all case and control groups were tested for Hardy-Weinberg equilibrium using χ2 tests with 1 df. Data were expressed as percentages or mean ± standard deviation. Pearson's χ2 was used for categorical data. Continuous variables were tested in each group for normal distribution using the Kolmogorov-Smirnov test for one variable. Differences of the means between two groups were tested with Student's t test. The means among the three groups were compared by ANOVA analysis. Statistical analysis was performed by SPSS 10.0 program (SPSS Inc., USA). All tests were two-sided and a P value of less than 0.05 was considered statistically significant.
Results
Detection of polymorphisms and association study
One SNP, c.-347-190G>A (rs3810744) was detected in the promoter region. And then an association analysis was performed in the cases and controls for the SNP. The frequencies of alleles and genotypes were presented in table 1. The difference in allele frequencies between male and female controls were not significant (A allele: 0.467 vs. 0.500, P = 0.533). No significant associations were observed between alleles and IgAN, whether in total (P = 0.121), in male (P = 0.684), or in female samples (P = 0.085). The association between genotypes of c.-347-190G>A and patients with IgAN was significant only in female population. The frequency of GG/GA genotype was higher in patients than in controls (0.781 vs. 0.645, P = 0.033).
Table 1. Distributions of c.-347-190G>A Polymorphism in C1GALT1C1 Gene
C1GALT1C1 gene c.-347-190G>A polymorphism and clinical manifestations or prognosis in patients with IgAN
Clinical characteristics of patients with IgAN at the time of renal biopsy were listed in Table 2. The patients were divided into two groups according to their genders. When clinical parameters of two male groups with different genotypes were compared, there was not significant difference in age, course of disease, incidence of gross hematuria or high blood pressure, urine protein excretion in 24 hours, serum creatinine concentration, and serum levels of IgA. These clinical parameters also did not differ significantly among the three female groups with different genotypes.
Table 2. General clinical parameters of IgAN patients with different c.-347-190G>A genotypes of C1GALT1C1 gene
Somatic mutation detection of B lymphocyte DNA
Although more than 8 clones per individual were sequenced for the whole coding region, neither new mutation nor new polymorphism except c.393T>A (rs17261572) was detected in total 202 clones from B lymphocyte DNA in 22 individuals (15 patients and 7 controls). The A allele of c.393T>A was only detected in each clone in two patients with IgAN and one male control. One of the two patients was male and another one was a female with AA homozygote. The results were completely consistent with the sequencing results derived from the previous genomic DNA.
Discussion
IgA nephropathy, the most common primary glomerulonephritis, was considered as a polygenic and multifactorial disorder. There were extensive evidences suggested the genetic components were involved in the susceptibility and progression of IgAN [5,22-24]. The pathogenesis of IgAN was still indistinct, as far as we knew. Fortunately, more and more evidences suggested that deficient β1,3 galactosylation of hinge region of IgA1 molecule played an important role in the pathogenesis of IgAN in recent years [4,5].
The galactosylation of GalNAcα1-R in hinge region of IgA1 molecule depended on the activity of C1β3Gal-T. Intriguingly, patients with IgAN had normal expression of C1GALT1 gene and decreased expression of C1GALT1C1 gene [11]. Furthermore, diseases resulted from deficiency of β1,3 galactose, such as Tn syndrome, weren't a result of decreased expression of C1GALT1 gene, but resulted from the mutations of C1GALT1C1 gene[14]. These results suggested that it was rather the variants of C1GALT1C1 gene in influencing the galactosylation of IgA1 hinge-region than the variants of C1GALT1 gene. It implies that the variants of C1GALT1C1 gene could contribute to susceptibility of IgAN by influencing β1,3 galactosylation of IgA1 molecule. Our previous study revealed that there was only one SNP, c.393T>A (rs17261572), in coding region of C1GALT1C1 [15]. It was a nonsynonymous SNP. The MAF of c.393T>A was only 0.069. A previous study revealed that the mutations weren't important for the European IgAN patients [16]. Were the mutations (including somatic mutation) in the promoter region important in the pathogenesis of IgAN in China? Therefore, we designed a study to test the hypothesis.
We firstly screened the polymorphisms of C1GALT1C1 gene in promoter region. One SNP, c.-347-190G>A was detected. And its MAF was 48.48%. Therefore, association between the c.-347-190G>A polymorphism and IgAN was explored in a case-control association study in a large population sampled from the Northern Chinese. The association analysis revealed that there was no significant difference of the alleles between the controls and the IgAN patients in total samples or in two sub-group samples divided by genders. There was only a weak association between the genotypes of c.-347-190G>A (GG/GA) and IgAN detected in female cases. But the positive association wasn't replicated in male sample simultaneously. The C1GALT1C1 gene located on chromosome X, so the effect of polymorphisms would be influenced by the inactivity of sex chromosome. The positive association might not demonstrate a truly causal association between the SNP and IgAN. These results suggested that polymorphisms of C1GALT1C1 gene might not be related to the susceptibility of IgAN.
In present study, we further analyzed the association between the SNP of C1GALT1C1 gene and clinical parameters of IgAN. The results revealed that there was no significant difference of blood pressure, proteinuria, and renal function among the IgAN patients with different genotypes. These data suggested that the genotypes of the C1GALT1C1 gene did not influence the clinical manifestations of IgAN.
In previous studies of Tn syndrome, three somatic mutations of C1GALT1C1 gene were identified in two patients. The three somatic mutations, c.202C>T, c.393T>A and c.454G>A, were all in the coding region of C1GALT1C1 [14]. Except the c.393T>A mutation, both of the other two somatic mutations could impressively inhibit chaperone activity and lead to inactivation of C1β3Gal-T, and the expression of autoimmune Tn antigen on blood cells of all lineages[14]. Galactosylation deficiency was already proved in patients with IgAN. Does somatic mutation exist in C1GALT1C1 gene in patient with IgAN too?
In order to prove this hypothesis, we furthermore performed a somatic mutation screening in the patients with IgAN. DNAs from B lymphocytes where IgA molecule was produced were isolated from 22 individuals. And then the coding region of C1GALT1C1 gene was amplified, cloned and sequenced. Except the c.393T>A, no other mutations were detected. The mutation, c.393T>A, was only found in the patients whose mutations were demonstrated in genomic DNA by routinely sequencing. Furthermore, c.393T>A was proved not to be a somatic mutation in these IgAN patients. The result indicated that the variation of coding region of C1GALT1C1 gene might be of little importance in the processing of aberrant glycosylation of IgA1 molecule in patients with IgAN. Mutations in other regions of C1GALT1C1 gene, which may influence the glycosylation process of IgA1 molecule, were needed to be clarified in patients with IgAN. In fact, in a recent study, Malychaet al [16] detected mutations in whole blood DNA and in B cell DNA separately in a relative small European sample. They didn't found any important mutations of C1GALT1C1 gene in patients with IgAN. These results suggested that the C1GALT1C1 gene might influence the susceptibility to IgAN by an alternative pathway if it was important for IgAN.
Conclusion
Our results suggested that the mutation (including somatic mutation) of C1GALT1C1 gene did not significantly contribute to the genetic susceptibility or clinical manifestations of IgA nephropathy in Chinese population.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Both GSL and GJN carried out the molecular genetic studies and GSL performed the statistical analysis and drafted the manuscript. GSL, HZ, YS and HYW participated in its design. HZ, YS and HYW participated in its coordination. JCL and HZ participated in the acquisition of data and follow-up. HZ and HYW revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by National Nature Science Foundation (30670981), the Capital Medical Science Foundation (2003-2001) and the Foundation of Ministry of Education, People's Republic of China (985-2-007-113) to H. Z.
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Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2350/10/101/prepub
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Research article
Targeted high throughput sequencing in clinical cancer Settings: formaldehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and tumor heterogeneity
Martin Kerick1, Melanie Isau1,2, Bernd Timmermann1, Holger Sültmann3, Ralf Herwig1, Sylvia Krobitsch1, Georg Schaefer4,5, Irmgard Verdorfer5,6, Georg Bartsch4, Helmut Klocker4, Hans Lehrach1 and Michal R Schweiger1*
Author affiliations
1 Max Planck Institute for Molecular Genetics, Ihnestr. 63-73, 14195 Berlin, Germany
2 Free University Berlin, Department of Biology, Chemistry and Pharmacy, Takustrasse 3, 14195 Berlin, Germany
3 Unit Cancer Genome Research, DKFZ German Cancer Research Center and National Center for Tumor Diseases, Im Neuenheimer Feld 460, 69120 Heidelberg, Germany
4 Innsbruck Medical University, Department of Urology, Anichstr. 35, A 6020 Innsbruck, Austria
5 Innsbruck Medical University, Department of Pathology, Muellerstr. 40, A-6020 Innsbruck, Austria
6 Innsbruck Medical University, Department for Medical Genetics, Molecular and Clinical Pharmacology, Division of Human Genetics, Schöpfstraße 4, 6020 Innsbruck, Austria
For all author emails, please log on.
Citation and License
BMC Medical Genomics 2011, 4:68 doi:10.1186/1755-8794-4-68
Published: 29 September 2011
Abstract
Background
Massively parallel sequencing technologies have brought an enormous increase in sequencing throughput. However, these technologies need to be further improved with regard to reproducibility and applicability to clinical samples and settings.
Methods
Using identification of genetic variations in prostate cancer as an example we address three crucial challenges in the field of targeted re-sequencing: Small nucleotide variation (SNV) detection in samples of formalin-fixed paraffin embedded (FFPE) tissue material, minimal amount of input sample and sampling in view of tissue heterogeneity.
Results
We show that FFPE tissue material can supplement for fresh frozen tissues for the detection of SNVs and that solution-based enrichment experiments can be accomplished with small amounts of DNA with only minimal effects on enrichment uniformity and data variance.
Finally, we address the question whether the heterogeneity of a tumor is reflected by different genetic alterations, e.g. different foci of a tumor display different genomic patterns. We show that the tumor heterogeneity plays an important role for the detection of copy number variations.
Conclusions
The application of high throughput sequencing technologies in cancer genomics opens up a new dimension for the identification of disease mechanisms. In particular the ability to use small amounts of FFPE samples available from surgical tumor resections and histopathological examinations facilitates the collection of precious tissue materials. However, care needs to be taken in regard to the locations of the biopsies, which can have an influence on the prediction of copy number variations. Bearing these technological challenges in mind will significantly improve many large-scale sequencing studies and will - in the long term - result in a more reliable prediction of individual cancer therapies.
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Research article
Sputum smear negative pulmonary tuberculosis: sensitivity and specificity of diagnostic algorithm
Hedwiga F Swai1*, Ferdinand M Mugusi2 and Jessie K Mbwambo3
Author Affiliations
1 Department of Internal Medicine Muhimbili National Hospital, Dar-es-salaam, +255 Tanzania
2 Department of Internal Medicine Muhimbili University of Health and Allied Sciences. Dar-es-salaam, +255 Tanzania
3 Department of Psychiatry Muhimbili National Hospital. Dar-es-salaam, +255 Tanzania
For all author emails, please log on.
BMC Research Notes 2011, 4:475 doi:10.1186/1756-0500-4-475
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1756-0500/4/475
Received:14 March 2011
Accepted:1 November 2011
Published:1 November 2011
© 2011 Swai et al; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
The diagnosis of pulmonary tuberculosis in patients with Human Immunodeficiency Virus (HIV) is complicated by the increased presence of sputum smear negative tuberculosis. Diagnosis of smear negative pulmonary tuberculosis is made by an algorithm recommended by the National Tuberculosis and Leprosy Programme that uses symptoms, signs and laboratory results.
The objective of this study is to determine the sensitivity and specificity of the tuberculosis treatment algorithm used for the diagnosis of sputum smear negative pulmonary tuberculosis.
Methods
A cross-section study with prospective enrollment of patients was conducted in Dar-es-Salaam Tanzania. For patients with sputum smear negative, sputum was sent for culture. All consenting recruited patients were counseled and tested for HIV. Patients were evaluated using the National Tuberculosis and Leprosy Programme guidelines and those fulfilling the criteria of having active pulmonary tuberculosis were started on anti tuberculosis therapy. Remaining patients were provided appropriate therapy. A chest X-ray, mantoux test, and Full Blood Picture were done for each patient. The sensitivity and specificity of the recommended algorithm was calculated. Predictors of sputum culture positive were determined using multivariate analysis.
Results
During the study, 467 subjects were enrolled. Of those, 318 (68.1%) were HIV positive, 127 (27.2%) had sputum culture positive for Mycobacteria Tuberculosis, of whom 66 (51.9%) were correctly treated with anti-Tuberculosis drugs and 61 (48.1%) were missed and did not get anti-Tuberculosis drugs. Of the 286 subjects with sputum culture negative, 107 (37.4%) were incorrectly treated with anti-Tuberculosis drugs. The diagnostic algorithm for smear negative pulmonary tuberculosis had a sensitivity and specificity of 38.1% and 74.5% respectively. The presence of a dry cough, a high respiratory rate, a low eosinophil count, a mixed type of anaemia and presence of a cavity were found to be predictive of smear negative but culture positive pulmonary tuberculosis.
Conclusion
The current practices of establishing pulmonary tuberculosis diagnosis are not sensitive and specific enough to establish the diagnosis of Acid Fast Bacilli smear negative pulmonary tuberculosis and over treat people with no pulmonary tuberculosis.
Keywords:
Sputum smear negative; Human Immunodeficiency Virus; Symptoms
Background
There has been a sharp rise in the incidence of pulmonary tuberculosis (PTB) worldwide since the mid 1980's, particularly in the Sub-Saharan African region. This has been attributed mainly to the appearance and wide spread of Human Immunodeficiency Virus (HIV) infection on the continent [1-3]. For the diagnosis of PTB the detection of Acid Fast Bacilli (AFB) in expectorated sputum is still crucial, especially in developing countries of Sub-Saharan Africa, where other facilities including sputum culture for Mycobacterium Tuberculosis (MTB) are unavailable or are prohibitively expensive. When AFB is detected in sputum, the diagnosis of PTB is certain. However diagnostic problem start when patients with suspected PTB have a negative sputum smear [4]. It has always been recognized that a proportion of patients are sputum smear negative using the Ziehl-Nelseen (ZN) stain, the commonly used stain in most laboratories in the region to detect AFB in sputum. This is a simple, rapid and cheap test but lacks sensitivity of a single sputum test [4]. About 5000 bacilli per milliliter of sputum must be present for it to be positive. However it has been reported that multiple sputum tests in a good laboratory can give a sensitivity of about 90% [5]. Sputum smear using ZN stain for AFB seems to be even less sensitive in patients with HIV associated PTB. With the sharp rise of PTB in countries which are worst affected by the HIV epidemics, the number of patients with suspected PTB who are sputum smear negative has increased [5].
Chest radiography is not always helpful in smear negative patients. The radiographic distinction between active and inactive tuberculosis can be difficult and appearance may be atypical due to other infections in HIV positive patients [4]. In fact, substantial numbers of patients are treated for tuberculosis without definitive diagnostic criteria [5]. With the advent of HIV associated tuberculosis with more frequent smear negative tuberculosis, the role of culture in TB control programs may need to be reassessed [4]. In countries where resources are limited, and where the use of chest X-rays may be inadequate due to the cost as well as atypical presentation found in HIV infected patients, clinical and/or laboratory characteristics which are able to identify smear negative but culture positive PTB are required. The Tanzania National TB and Leprosy Programme uses a smear negative PTB diagnostic algorithm adopted from the World Health Organization (WHO) (Figure 1) [6].
Figure 1. Flowchart on the diagnosis of pulmonary TB in children above 6 years and adults.
This study was conducted with the aim of assessing the sensitivity of the current recommended algorithm for the diagnosis of sputum smear negative PTB.
Methods
A cross-sectional study with prospective enrollment was conducted at Muhimbili National Hospital (MNH), a university teaching and national referral hospital, and at out-patient tuberculosis clinics at the Infectious Disease Clinic (IDC), Mwananyamala, Temeke and Ilala district hospitals, from September 2000 to December 2000. All these hospitals are located in the city of Dar es Salaam. The city accounts for over 26% of all new tuberculosis cases reported each year in Tanzania [7].
Adult male and female patients aged 18 to 75 years, presenting with chronic cough (≥2 weeks); who were three times sputum smear-negative for AFBs (ZN stain); and who gave a written informed consent to participate in the study and for HIV testing were included into the study. Patients with known tuberculosis or who had PTB in the past, on anti-TB for treatment or prophylaxis; those with known chronic respiratory diseases (e.g. bronchial asthma, chronic obstructive pulmonary disease, bronchiectasis), those with misplaed HIV results, contaminated cultue results and those with heart failure were excluded. The study protocol was approved by the MUHAS Ethical Review Committee.
Study procedures
A detailed medical history and physical examination was done by a study clinician and the findings were recorded on a clinical record form. The investigators did not interfere in the treatment of these patients. The treatment centre followed the diagnostic algorithm for smear negative. Laboratory tests included a complete blood count (coulter counter model, manufacturer, city and country) which included estimation of haemoglobin, red blood cell count and indices; and white blood cell count both total and differential. A peripheral blood smear for assessment of red cell morphology was also made. Erythrocyte sedimentation rate (ESR) was set using the Westergren method within 2 hrs of drawing blood.
HIV testing was done according to the Tanzania National AIDS guidelines. Each patient received pre- and post-test counseling and the HIV test was done using a dual ELISA algorithm. Sera which were non-reactive on first ELISA were considered HIV antibody negative, and those reactive on first ELISA were retested by a second ELISA based on a different test principle. Sera reactive on both ELISA tests were considered HIV-positive. Samples with discordant test results were confirmed by Western blot (WB) and western blot interpretation was done according to the WHO criteria [8].
Patients who came to the clinic with symptoms suggestive of PTB had their sputum examined. Those who were three times smear negative were consequently selected and asked to bring one more sputum sample which was sent to the Tuberculosis Reference Laboratory at MNH for AFB culture (Löwenstein-Jensen culture media).
Smears were considered positive if AFBs were seen on smear from any of the three sputum samples. Patients found to have sputum smear positive were treated for tuberculosis according to the National Tuberculosis and Leprosy program treatment guidelines. Those found to have sputum smear negative for AFB and who consented were enrolled into the study. A chest x-ray was ordered for those who were found to be HIV positive. If the chest x-ray results were abnormal, the patient was considered to have sputum smear negative PTB, and started on anti-TB medications. The rest were treated with broad-spectrum antibiotics. All enrolled patients were requested to stay at the clinic for a month for follow up. Two weeks later, patients on broad spectrum antibiotics were evaluated again by doing sputum smear and chest radiograph. Those found to have smear negative sputum but had symptoms still suggestive of PTB were treated as smear negative PTB; others were treated accordingly.
Two weeks later we came back to review treatment of clinician of which others were given ant TB and others were treated for other respiratory problems. Because they followed NTLP diagnostic algorithm, all of them had a chest X-ray done, and all of them were reviewed and reported using a structured format by two independent radiologists. In case of disagreement in their initial independent reporting, they reviewed the radiographs together and resolved the disagreement by consensus.
Sample size and data analyisis
Power calculations
This study was part of another study on that aimed to investigate/examine sputum smear negative but culture positive PTB the association with HIV.
Assuming a sensitivity of 50% and specificity of 75%, a sample of size 413 subjects would give a 95% confidence interval of plus/minus 0.048% for sensitivity and plus/minus 0.042% for specificity. This is a reasonable amount of precision for the given sample size.
Data were analyzed using Statistical Package for Social Sciences (SPSS) and EPI Info. Pearson chi-square test was used for comparison of categorical data and a student t-test was used for continuous data. Logistic regression analysis was applied and the direct effects of the predictors were assessed by their 95% confidence intervals. A p-value of < 0.05 was considered to be statistically significant.
Sensitivty and specificity of the diagnostic algorithm was calculated using the following formulas:
Positive and negative predictive value was calculated using the following formlas
Results
Over the course of the study, 467 patients were enrolled. Of those enrolled, 318 (68.1%) were HIV positive. Sputum was culture positive for MTB in 27.2% (127/467) of patients; samples of 11.1% (52/467) patients were reported to have been contaminated. In the remaining 61.7% (288/467) patients sputum was culture negative for MTB at 8 weeks. Of the 467 study subjects, 68.1% (318/467) were HIV positive. Two study subjects whose culture results were negative had their HIV results misplaced. These two, together with the 52 subjects whose culture results were contaminated were excluded from further analysis. Of the 413 samples analyzed, 30.8% (127/413) were MTB culture positive.
There was a high proportion of PTB patients who were not treated as well as a high proportion of patients without TB who were treated with anti TB [Table 1]. As observed the diagnostic procedure at the clinics had a sensitivity of 38.1% and a specificity of 74.5%. The positive predictive and the negative predictive diagnostic value were 52% and 62.5% respectively [Table 2].
Table 1. Diagnosis made using culture results and Treatment given
Table 2. Sensitivity and specificity of the diagnostic procedure of patients with smear negative
Of those who were presumptively diagnosed to have TB, the diagnosis of TB was established in HIV negative (58.1%) more than positive subjects (48.8%) [Table 3].
Table 3. Diagnosis made and Treatment given by subjects' sputum culture results and HIV sero-status.
Using an unadjusted logistic regression model characteristics which predicts smear negative culture positive were determined. Matted lymph node, tachypnoea (RR > 20), presence of a cavity, mixed type of anaemia, were strong predictors of PTB culture positivity. Eosinophilia was also found to be associated with a 50% less chance of being sputum culture positive [Table 4].
Table 4. Unadjusted bivariate logistic regression analysis of clinical characteristics predictive of smear negative culture positive PTB
In an attempt to develop supplemental method for diagnosing smear negative pulmonary tuberculosis, forward stepwise multiple logistic regression analysis of the data was done to establish clinical and laboratory characteristics that predict the presence of sputum culture positive. Using this analysis, high respiratory rates, low eosinophil counts, mixed type of anaemia and the presence of cavities on X-rays were predictors of smear negative but culture positive[Table 5].
Table 5. Multivariate analysis of clinical characteristics predictive of smear negative culture positive PTB
Discussion
This study showed that of those found to have a negative result for AFB, a significant proportion (27.2%) had sputum culture positive for MTB. Therefore our data indicate that smears did not detect PTB in a very large proportion of patients. Sputum culture being the gold standard for the diagnosis of Tuberculosis disease [9] shows that sputum smear is not a very sensitive tool in the diagnosis of PTB. This has been shown by other studies where sensitivity has been described to be between 51% to 53.3% [10,11]. One of the reasons for low sensitivity is reported to be due to the fact that 104/ml are required for AFB to be seen using smear microscopy [4].
Although the gold standard for the diagnosis of Tuberculosis involves the isolation and identification of Mycobacterium Tuberculosis (MTB) using cultures [9], the cost and facilities of doing cultures are prohibitive in most developing countries. Sputum smear microscopy remains the main diagnostic tool for PTB that allows initiation of treatment and monitoring of patient progress [11,12]. As sputum smear and microscopy is not a very sensitive tool in the diagnosis of PTB, presumptive diagnosis is usually made based on an algorithm of clinical and radiological criteria. This is commonly termed as AFB negative PTB [9,13]. In some cases when sputum smears are negative but the patient has clinical features highly suggestive of PTB, broad-spectrum antibiotics are recommended for at lest 10-14 days and sputum smears repeated. If the patient's condition does not improve while sputum smear remains negative, a chest radiograph is done and if found to be abnormal, a presumptive diagnosis of PTB is made and the patient is started on anti-Tuberculosis treatment as AFB negative PTB [9]. In this study patients whose sputum smears were AFB negative, were evaluated using the above algorithm by the treating doctors at the clinics or hospital. A presumptive diagnosis of AFB sputum smear negative PTB was made in 41.8% (173) of all study subjects, and patients were started on anti-TB treatment as recommended by the Tanzania NTLP. The remaining 58.2% (240) patients were assumed to have other forms of respiratory diseases and were treated accordingly.
Less than half (38.1%) of those who were presumed to have active TB and started on Anti TB actually had TB by sputum culture results. More than 60% of these patients they received 8-months of treatment despite having a negative culture results. This is similar to what has been reported before in Malawi were it was reproted that 40% of smear negative had TB confirmed microbiologically after taking Broncho alveolar larvage [14]. The treatment of individuals without tuberculosis adds to the cost of the TB programs in most developing countries. Likewise about 48% (61) of patients who had active tuberculosis by the results of sputum culture were missed and they received inappropriate treatment, leaving them vulnerable to developing severe disease as well as remaining source of TB infection in the community.
The current diagnostic algorithm leading to the establishment of the diagnosis of AFB smear negative PTB is inefficient; it over-diagnoses PTB and misses a lot of people with active disease. Instituting a more sensitive diagnostic tool will prevent the unnecessary cost of treating individuals who do not have TB and at the same time it will prevent the further spread of TB. This emphasizes the need of culture and the need of further research in order to identify a better diagnostic tool for diagnosis of AFB negative PTB.
In an attempt to improve on the diagnostic algorithm, the study looked at the clinical presentation of the patients to identify clinical laboratory and radiological features that are associated with smer negative PTB and which can be used to predict PTB in patients with symptoms suggestive of PTB. A multivariate analysis showed the following features to be highly predictive of AFB negative but culture positive PTB; low eosinophil counts, a mixed type of anaemia and the presence of cavities on chest radiographs. Low eosinphil seems to be an incidental finding Further studies have to be done to confirm this findings
Limitation in the current study is the inclusion of patients with cough of more than two weeks in which there may be inclusion of patients with simple chest infection that sometime may be complicated with cough for 2-3 weeks. This may be a selection bias that may explain the low sensitivity and specificity of the diagnostic algorithm.
Another limitation is the method of sputum delivery, which is delivered by the patient himself, may have affected the results as some might bring saliva.
We could not be certain that the algorithm was followed at all times because resechers were not involved in the management of these patients rather we evaluated the treatment gien to patients by the attending clinicians. In Tanzania National TB and Leprosy programme is well organized and the algorithm is well adhered by the District TB and Leprosy Coordinators and all workers of the NTLP who were the attending clinicians in this study
Conclusion
• The current procedures of establishing AFB negative PTB are not sensitive enough to establish the diagnosis of active tuberculosis. They under-diagnose PTB and over treat people without PTB.
• The presence of a dry cough, a high respiratory rate, a low eosinophil count, a mixed type of anaemia and presence of a cavity were found to be predictive of smear negative but culture positive PTB.
Consent
The study protocol was approved by the MNH Ethical Review Committee.
Written consent was obtained. To those who were not able to write oral consent was obtained
The strength of our study is that it evaluates very well the dignostic algorithm.
Recommendations
1. We do not recommend adhering to the diagnostic algorithm.
2. A much more sensitive diagnostic algorithm for smear negative pulmonary tuberculosis should be developed to be able to identify those individuals who are actually sputum culture positive for AFB.
List of Abbreviations
HIV: Human Immunodeficiency Virus; PTB: Pulmonary Tuberculosis; AFB: Acid Fast bacilli; MTB: Mycobacterium Tuberculosis Mycobacterium Tuberculosis; NTLP: National Tuberculosis and Leprosy Proagramme; ZN: Ziehl-Nelseen; IDC: Infectious Disease Clinic; TB: Tuberculosis; Anti-TB: Anti-tuberculosis drugs; RR: Respiratory rate; MNH: Muhimbili National Hospital; WHO: World Health Organization; ELIZA: Enzyme Immunosorbent assay; SPSS: Statistical Package for Social Sciences; OR: Odds ratio.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FM participated in the design of the study, proof read the manuscript and performed statistical analysis. HS participated in the design of the study, collect data, drafted the manuscript, and performed statistical analysis. JM participated in the statistical analysis and proof read of the manuscript. All authors' read and approved the manuscript.
Acknowledgements
The study was supported by the Muhimbili University of Health and allied Sciences. We wish to thank Dr Kazema and Dr Kimaro (X-ray and imaging specialists) who read all the X-rays. We thank and Mr Ngowi and Mr Shogolo (Laboratory technicians) for storing and working on the sputum samples. I thank doctors who assisted in data collection and last but not list I wish to thank Dr Makwaya (Biostatician) for assisting in sample size calculation and data analysis.
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Switch to glossy paper with this issue.
[no title indexed] (Table of Contents)
Batman / cover / 1 page (report information)
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The Winged Dragon [War of the Dragons: Part Three] (Table of Contents)
Batman / comic story / 22 pages (report information)
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Pencils:
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Genre:
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Characters:
V: King Snake, Lynx, Silver Monkey; GS: Nightwing, Huntress
Indexer Notes
Story continued from Robin (DC, 1993 series) #17.
[no title indexed] (Table of Contents)
Detective Comments / letters page / 2 pages (report information)
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Script:
Scott Peterson
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Typeset
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1. 0. [no title indexed]
Batman
2. 1. The Winged Dragon [War of the Dragons: Part Three]
Batman
3. 2. [no title indexed]
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This issue was most recently modified by:
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User talk:Black bart
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Bibliography: Earthclan
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Title: Earthclan
Author: David Brin
Year: 1987
Type: OMNIBUS
Storylen: /2,3
Series: Uplift
Language: English
ISFDB Record Number: 136342
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Copyright (c) 1995-2011 Al von Ruff.
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Molecules 2006, 11(6), 469-477; doi:10.3390/11060469
Article
Synthesis of New Bis-1,2,4-Triazole Derivatives
Department of Chemistry, Giresun Faculty of Arts and Sciences, Karadeniz Technical University, 28049, Giresun, Turkey
* Author to whom correspondence should be addressed.
Received: 10 May 2006; in revised form: 20 June 2006 / Accepted: 21 June 2006 / Published: 21 June 2006
Download PDF Full-Text [77 KB, uploaded 20 June 2008 16:50 CEST]
Abstract: A series of new 1,2/1,3-bis[o-(N-methylidenamino-3-aryl-5-phenyl-4H-1,2,4-triazole-4-yl)phenoxy]ethane/propane derivatives 4 were prepared in good yields bytreatment of 4-amino-3-aryl-5-phenyl-4H-1,2,4-triazoles 2 with certain bis-aldehydes 1.Compounds 4 were reduced with NaBH4 to afford the corresponding 1,2/1,3-bis[o-(N-methylamino-3-aryl-5-phenyl-4H-1,2,4-triazole-4-yl)phenoxy]ethane/propane derivatives5. All new compounds were characterized by IR, 1H-NMR, 13C-NMR and mass spectraldata.
Keywords: 4-Amino-4H-1; 2; 4-triazoles; bis-1; 2; 4-triazoles; bis-Schiff bases; bis- aldehydes
Article Statistics
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Cite This Article
MDPI and ACS Style
Bekircan, O.; Bektas, H. Synthesis of New Bis-1,2,4-Triazole Derivatives. Molecules 2006, 11, 469-477.
AMA Style
Bekircan O, Bektas H. Synthesis of New Bis-1,2,4-Triazole Derivatives. Molecules. 2006; 11(6):469-477.
Chicago/Turabian Style
Bekircan, Olcay; Bektas, Hakan. 2006. "Synthesis of New Bis-1,2,4-Triazole Derivatives." Molecules 11, no. 6: 469-477.
Molecules EISSN 1420-3049 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
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Cancers 2011, 3(2), 2243-2254; doi:10.3390/cancers3022243
Review
Targeted Therapy for Biliary Tract Cancer
1 Department of Internal Medicine, Medical Oncology, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo, 181-8611, Japan 2 Department of Hepatobiliary and Pancreatic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
* Author to whom correspondence should be addressed.
Received: 4 March 2011; in revised form: 10 April 2011 / Accepted: 13 April 2011 / Published: 3 May 2011
(This article belongs to the Special Issue Cancer Diagnosis and Targeted Therapy)
Download PDF Full-Text [229 KB, uploaded 3 May 2011 14:30 CEST]
Abstract: It is necessary to establish effective chemotherapy to improve the survival of patients with biliary tract cancer, because most of these patients are unsuitable candidates for surgery, and even patients undergoing curative surgery often have recurrence. Recently, the combination of cisplatin plus gemcitabine was reported to show survival benefits over gemcitabine alone in randomized clinical trials conducted in the United Kingdom and Japan. Thus, the combination of cisplatin plus gemcitabine is now recognized as the standard therapy for unresectable biliary tract cancer. One of the next issues that need to be addressed is whether molecular targeted agents might also be effective against biliary tract cancer. Although some targeted agents have been investigated as monotherapy for first-line chemotherapy, none were found to exert satisfactory efficacy. On the other hand, monoclonal antibodies such as bevacizumab and cetuximab have also been investigated in combination with a gemcitabine-based regimen and have been demonstrated to show promising activity. Furthermore, clinical trials using new targeted agents for biliary tract cancer are also proposed. This cancer is a relatively rare and heterogeneous tumor consisting of cholangiocarcinoma and gallbladder carcinoma. Therefore, a large randomized clinical trial is necessary to confirm the efficacy of chemotherapy, and international collaboration is important.
Keywords: biliary tract cancer; chemotherapy; molecular targeted agent
Article Statistics
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MDPI and ACS Style
Furuse, J.; Okusaka, T. Targeted Therapy for Biliary Tract Cancer. Cancers 2011, 3, 2243-2254.
AMA Style
Furuse J, Okusaka T. Targeted Therapy for Biliary Tract Cancer. Cancers. 2011; 3(2):2243-2254.
Chicago/Turabian Style
Furuse, Junji; Okusaka, Takuji. 2011. "Targeted Therapy for Biliary Tract Cancer." Cancers 3, no. 2: 2243-2254.
Cancers EISSN 2072-6694 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
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Nano Express
Tunable resonance transmission modes in hybrid heterostructures based on porous silicon
Karina S Pérez1*, J O Estevez1, Antonio Méndez-Blas1, Jesús Arriaga1, Gabriela Palestino2 and Miguel E Mora-Ramos3
Author affiliations
1 Instituto de Física, Universidad Autónoma de Puebla, A.P. J-48, Puebla, 72570, México
2 Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, Alvaro Obregón 64, San Luis Potosí, 78000, México
3 Facultad de Ciencias, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Cuernavaca, CP 62209, Morelos, México
For all author emails, please log on.
Citation and License
Nanoscale Research Letters 2012, 7:392 doi:10.1186/1556-276X-7-392
The electronic version of this article is the complete one and can be found online at: http://www.nanoscalereslett.com/content/7/1/392
Received:30 April 2012
Accepted:13 July 2012
Published:13 July 2012
© 2012 Pérez et al.; licensee Springer.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
In this work, we report the experimental results and theoretical analysis of strong localization of resonance transmission modes generated by hybrid periodic/quasiperiodic heterostructures (HHs) based on porous silicon. The HHs are formed by stacking a quasiperiodic Fibonacci (FN) substructure between two distributed Bragg reflectors (DBRs). FN substructure defines the number of strong localized modes that can be tunable at any given wavelength and be unfolded when a partial periodicity condition is imposed. These structures show interesting properties for biomaterials research, biosensor applications and basic studies of adsorption of organic molecules. We also demonstrate the sensitivity of HHs to material infiltration.
Keywords:
Fibonacci substructure; Porous silicon; Heterostructures
Background
Photonic crystals are attractive optical materials to control and manipulate the flow of light. A periodic dielectric system (multilayered), typically consisting of two alternated dielectric materials with periodic variation of refractive index (n), is the simplest photonic crystal (PC) [1]. The propagation of electromagnetic radiation in PCs is forbidden in specific wavelength ranges (photonic band gaps or PBGs) because the light wave is scattered at the layers’ interfaces, so the multiple-scattered waves interfere destructively into the material [2]. The behavior of light in a periodic scattering media can be described by Bloch states [3]. In addition, localized modes can appear into the PBGs by breaking the periodicity of the dielectric multilayer, i.e., by introducing a defect into a PC [4] that allows a narrow range of light wave frequencies to propagate through the whole structure. Physically, the defect is a single layer with different optical parameters (refractive index or thickness) or a completely different multilayer substructure [5]. Novel applications to optical devices, such as all-optical circuit, dielectric mirrors, Fabry-Perot filters, distributed feedback lasers, etc., have been proposed for the above-mentioned structures with localized modes. However, not only PCs based on periodic or periodical structures with defects are of interest but also deterministic aperiodic systems or quasicrystals because of their unexpected optical features [6-10]. The quasicrystals can be considered as a class of complex dielectric structures between ordered crystals and fully random structures. These structures show PBGs, but they are non-periodic multilayer structures. The quasicrystal structures are formed of layers with optical parameters that obey deterministic rules [11]. The Fibonacci and Thue-Morse mathematical sequences are two examples of numbers generated by deterministic rules. In order to associate these kinds of sequences to multilayer structures, it is necessary to define the so-called generators, i.e., initial layers with specific values of their optical properties.
Important applications for quasicrystal-type structures, such as band-edge lasing [12], optical frequency-selective filters [13], efficient nonlinear filters [14], bistability [15], and switching [16], have been suggested. In optical sensor applications based on PCs, the sensitivity is associated with the capacity for binding analyte molecules to the surface of the layers [17]. Porous silicon (PSi) has a great capacity of binding molecules at its surface due to its large specific superficial area (≥ 200 m2/cm3) [18]. The biocompatibility of the PSi [19] makes it a promising material to be used as a biosensor. PSi is a nanostructured material [20] considered as a mix of silicon and air with effective optical parameters, and its optical and structural features allow the fabrication of complex PCs [21-23]. Since PSi is obtained by electrochemical etching, and the porosity is directly related to the refractive index [24], it is possible to control its optical parameters by controlling the thickness and porosity by means of time and applied current during the process, respectively [25]. These features allow the fabrication of several types of one-dimensional (1D) PCs and the introduction of complex defect layers into a periodic multilayer structure. The strong confinement of electromagnetic fields within the engineered defect layers is an advantage offered by PCs because it is highly dependent on the refractive indices and thickness of each constituent layer; any change in these parameters is reflected as a change in the optical response. It is possible to achieve a spectral shift of the localized modes when a slight change of refractive index in some layers or on the whole structure is induced. Such displacements could be obtained by natural or thermal oxidation of the PSi structure or by introducing into the pores some specific substances. This advantage can be exploited particularly in biosensing applications due to its high sensitivity requirements compared to other sensors, which only use the weak evanescent field for sensing [26]. It is possible to obtain small spectral shifts in the reflectance or transmittance measures by introducing solutions or analytes into the pores of PSi, which can be monitored with exceptional precision. Numerous works have been published based on this idea, but only the simplest PSi structures (i. e. monolayers, distributed Bragg reflectors (DBRs), and several types of filters) have been used to study different molecular species as proteins [27,28], DNA [29], solvents [30], neurons [31], etc. However, the optical features of PSi complex multilayer structures have not been explored widely for their application in the biosensing area. From this perspective, our interest lies on the fabrication of a highly efficient photonic structure for biosensing purposes. To achieve this goal, in this work, the fabrication of hybrid heterostructures (HHs) based on PSi is proposed. The HHs are a complex combination of the features of periodic and quasiperiodic photonic structures. The study of hybrid heterostructures has been approached in previous works by other authors but only in the theoretical aspect, and they not consider PSi nanostructures [32,33]. It is the first experimental study of HHs based on Fibonacci (FN) sequences.
Methods
The HHs are formed by stacking a FN substructure between two DBRs in the sequence (DBR)N−(FN)M−(DBR)N[34]. The DBRs are formed by a periodic arrangement of two alternated layers, A and BN times. The FN sequences are generated by the recursion relation FM=FM−1 + FM−2where M represents the order of the sequence ( M=2,3,4,…). It is possible to generate dielectric multilayered structures that follow the FN mathematical sequence of any order by choosing F0=C and F1=CD where C and D are two different layers. For example, F2=CDCF3=CDCCDF4=CDCCDCDC, and so on. The HHs present strongly localized transmission modes as a function of the design parameters and can be localized over a wide range of frequencies. The HHs based on PSi studied in this work were obtained by electrochemical etching from boron-doped silicon wafers (100)-oriented and 0.007 to 0.013 Ω cm resistivity. A small piece of silicon wafer was used as substrate for etching in an HF (40%) and ethanol (99.98%) solution in a volumetric ratio of 1:1. More details about the process can be found in reference [5]. In order to calculate the refractive index of each layer for a given current density, we use the effective medium approximation of the Bruggeman’s model [35]:
(1)
This model provides the complex dielectric constant of PSi (εPSi) as a function of the dielectric constants of silicon (εSi) and air (εair), as well as porosity fp. The values of fpwere calculated by the gravimetrical method. As the PSi layers consist of only two optical media, the εPSi value is intermediate between the εSi and εair values, weighed by the volume fraction 1−fpand fp, respectively, (in Equation 1 we take εair=1). Solving for εPSiin Equation 1, we obtain
(2)
Cauchy model is useful to know the refractive index (n) and the extinction coefficient ( k) for dielectric materials (with exponential absorption), far from the absorption bands [36], by the equations
(3)
(4)
Note that this model is defined by five parameters: abcd, and e. These parameters are adjusted to experimental values of n and k for crystalline silicon from reference [37]. For example, in Figure 1n and k are plotted for two different values of porosity (low and high) which correspond to the design parameters for the A and B layers (40% and 77% of porosity, respectively) of the DBRs mentioned above. In all the simulations, we consider Equations 3 and 4.
Figure 1. Optical parametersnandkvs wavelength. The values for layers with low and high porosity (40% and 77%, respectively) were calculated using the Bruggeman and Cauchy models.
For a λ value around 1.0 μm, the refractive index and thickness for layer A is 1.4 and 178.5 nm, and for layer B, 2.5 and 100 nm, respectively. The refractive indices for C and D layers are 1.6 and 2.2, and their thicknesses are 156.25 and 113.64 nm, respectively. In all these layers, the optical thickness nd, where n is the refractive index and d is the physical thickness, has the constant value λ/4 = 250 nm. A schematic of the structure with the sequence (DBR)4−(FN)4−(DBR)4is shown in Figure 2, in which it is possible to observe the formation of two optical modes corresponding to λ/2 defects.
Figure 2. Schematic of the HHs. The number of periods for DBRs is N = 4, and the order of the Fibonacci substructure is M = 4. Resonances will be observed in reflectance spectra due to the λ/2 layer defects.
On the basis of this idea, structures with different order of the FN substructure were designed to show resonances in the IR region. These resonances can be seen experimentally like narrow transmission bands in a reflectance spectrum. When the FN substructure is third, fourth, or fifth order (M = 3, 4, 5…), it presents one, two, or three resonance transmission modes, respectively. The number of resonances is in direct analogy with the FN sequence (see Table 1). N = 4 in DBRs substructures was kept constant for all the structures.
Table 1. Fibonacci order, substructure, and sequence
The reflectivity measurements of 1D photonic crystals based on PSi structures were carried out in an Agilent spectrophotometer (Cary 5000 UV-VIS-NIR, Agilent Technologies, Santa Clara, CA, USA), with the specular reflectance accessory (VASRA). All the spectra were measured at an angle of incidence of 20°. Reflectivity measurements were carried out with a p-polarized beam. The experimental results were compared with those given by the theory.
Theoretical model
To model the propagation of light in these systems, we used the transfer matrix method [38]. If we consider an electromagnetic (EM) wave propagating in the structure with propagation constant k= k + kz, there are two independent EM modes: transverse-magnetic (TM) modes and transverse electric (TE) modes. The electric (magnetic) field for the TE (TM) mode is perpendicular to the plane defined by the wave vector and the direction of periodicity. Using the transfer matrix formalism, we can relate the amplitudes of the fields and in the jth layer of the system to the amplitudes of the field in the ( j + 1)− thlayer according to
(5)
where is the amplitude of the wave in the layer j, with polarization μ( μ = sp) traveling to the right (left). For the case considered in this work, the total transfer matrix of the system can be written as a product of matrices of the type [39]
(6)
where for p-polarization and q=(kjz) for s-polarization; ϕj = kjzdjkj = (ω/ c) njkjzis the component of the wave vector along the growth direction of the system in the jth layer given by ; and is the complex refractive index. The reflectivity of the system is given in terms of the matrix elements of the total transfer matrix according to . We have implemented a realistic transfer matrix approach by considering the wavelength dependence of the refractive index as well as the optical absorption. Absorption is a very important parameter, especially when considering the visible region of the electromagnetic spectrum.
Results and discussion
In Figure 3, we present the optical reflectivity measurements of three HHs (solid line). In all cases, strongly localized transmission modes can be seen. The third-order FN substructure (Figure 3a) (DBR)4−(FN)3−(DBR)4 presents one localized transmission mode at 988 nm with 19.7 nm of full width at half-maximum (FWHM). The localized mode is produced by the two adjacent λ/4 layers, in this case, two C layers in the FN substructure. The HH based on the fourth-order FN substructure (Figure 3b) presents two localized modes at 977 and 1,112.4 nm with a FWHM of 15.7 and 22.7 nm, respectively. The first localized transmission mode is produced again by two C layers in the FN substructure. However, the second defect is produced by a C layer from an FN substructure adjacent to an A layer from the DBR substructure. Even though the refractive index and the thickness for both layers are different, their optical thickness is the same ( nd= λ/4), so the λ/2 condition is kept. Furthermore, in this case, the condition of periodicity is met before and after the defect. In Figure 3c, the fifth order of the FN substructure between the DBRs produces three resonant modes at 934.5, 1,036.3, and 1,160.8 nm with FWHMs of 13.2, 14.8, and 23.6, respectively. In this case, all the localized modes are due to three pairs of C layers in the FN substructure. For HHs corresponding to the upper order of FN substructures, similar defects are found, and the number of defects follows the numerical Fibonacci’s sequence (see Table 1). The optical modes can be designed to appear at almost any wavelength because they depend on the optical thickness, i.e., we can design specific PSi structures with the correct refractive indices and thicknesses to match any electromagnetic region. Theoretical simulations of the same HHs are also plotted in Figure 3 (dotted line). In these simulations, we took the values of refractive index and thickness mentioned in the ‘Methods’ section but considering ±0.05 deviation in the refractive index values. From this figure, the excellent agreement with the experimental results can be seen, which gives us certainty on the theoretical modeling. This model takes into account the n( λ) and k( λ) dependence for each layer.
Figure 3. Reflectance measurements of the HHs and their theoretical simulation. The FN substructure was (a) third order, (b) fourth order, and (c) fifth order.
Figure 4 shows a high-resolution scanning electron microscopy (HRSEM) image of a HH with a FN substructure of fourth order; in this particular sample, there are five periods for the DBRs. Three zones can be seen clearly: the top and bottom zones correspond to DBRs, and the middle one corresponds to the FN substructure. The dark and clear zones are due to layers with high (low) and low (high) porosities (refractive index value), respectively. The lower contrast in the layers of FN substructure is due to the low contrast in the refractive index of their constituent layers compared to DBR layers. Measures of the thickness of the layer’s in this and other HRSEM images showed an excellent agreement between the observed thickness values and those calculated by the gravimetrical procedure for each layer.
Figure 4. HRSEM of a HHs with the sequence(DBR)5(FN)4(DBR)5. Dark and clear layers correspond to high and low porosities, respectively.
In order to show the effect of two specific modifications on the HHs, we chose the structure (DBR)4−(FN)4−(DBR)4 shown in Figure 3b. The first modification consists of adding or removing layers from the FN substructure; in any case, the Fibonacci sequence is lost. However, the spectral positions where the resonances appear can be changed by this modification. In Figure 5, the effect of removing some layers from the FN substructure can be observed. The original structure from Figure 3b is shown again in Figure 5a for reference. From the (DBR)4−(FN)4−(DBR)4structure, the two last layers from the FN substructure were removed, and the resulting structure is now (DBR)4−(CDCCDC)−(DBR)4. In Figure 5b, it can be seen that the two resonances are localized at 960.7 and 1,143.6 nm with FWHMs of 17.4 and 28.6, respectively, so the range between the resonances has increased to 182.9 nm, the range being 135.4 nm before modifying the FN sequence (Figure 5a). Moreover, the addition of a pair of DC layers to the end of the FN substructure results in the structure (DBR)4−(FN)4DC−(DBR)4. Figure 5c shows the resulting reflectance spectra after the last modification. Now, the optical resonances appear at 1,013.9 and 1,118.4 nm, so the new range between the two resonances is 101.5 nm, i.e., the difference of the two resonances’ spectral position has decreased, compared to original HHs. This result can be explained taking into account the interaction between the defect modes. The larger the physical distance between the defect layers, the weaker is the interaction of the eigenmodes so that they can appear at the nearest frequencies when there is a very long distance between defects layers. On the other hand, when the distance between defects is short, the interaction of the eigenmodes increases, but they cannot appear at the same energy or frequency in the optical spectra. Consequently, the localized modes in the reflectance spectra appear more separated [40]. These important features prove the flexibility of the structures and their capacity to localize the resonant modes at almost any wavelength as a function of the design parameters. This tunability can be exploited in optical devices, particularly in the biosensing area.
Figure 5. Reflectance of modified HHs in the FN substructure.(a) Reference structure. (b) The last two layers of the FN substructure have been removed. (c) A pair of DC layers has been added to the end of the FN substructure.
The second modification to the structure was carried out by imposing a partial periodicity on the structure. The unit cell is now formed by a DBR and a FN substructure. In order to observe the split of the resonances, a DBR substructure was added to the end of the whole structure. In this way, FN substructures are always seen as defects. Applying this modification to the structure and using the appropriated optical parameters, it is possible to observe the unfolded resonance mode. The number of splitting modes from each resonance modes depends on the number of times that the unit cell is repeated in the whole structure. For example, repeating two times the unit cell in the structure (DBR)4−(FN)4−(DBR)4, we obtain (DBR)4−(FN)4−(DBR)4−(FN)4−(DBR)4, and the resulting reflectance spectra of the new structure (see Figure 6) show twofold of each resonance mode that appears in the original structure in Figure 3b. A threefold splitting can be obtained by repeating three times the unit cell; fourfold splitting would correspond to four times, and so on.
Figure 6. Reflectance spectra of HHs with a unit cell repeated twice. The structure has two resonant modes unfolded.
The effective refractive index in PSi is the result of a homogeneous mixture of air and silicon, so any material infiltration into the pores displaces off part of the air. Consequently, a red- or blueshift could be expected in the optical reflectance spectra due to a partial change of the optical parameters. The same effect is expected in monolayers and multilayers or even in more complex PSi structures. So, red- or blueshift can be monitored in order to estimate the sensitivity of the structures. To achieve this in multilayered structures, it is preferable to have strong localized modes in order to follow more easily the spectral displacements. The HHs developed in this work have the strong localized modes needed, but they are observed in very complex structures. However, we want to demonstrate that these complex structures make them more sensitive to material infiltration, in particular, when biological molecules are placed into the pores of HHs based on PSi. In order to know the feasibility of the HHs to be used as biosensors, 3-aminopropyltriethoxysilane (APTES) molecule was attached to the internal surface of the structure [41]. To do this, it is necessary to follow a specific process described here: (1) HHs based on PSi were thermally stabilized at 900°C under oxygen flow; (2) APTES silanization was done in a 5% solution with toluene during 1.5 h; (3) the samples were rinsed with toluene and dried under nitrogen flow, and finally, (4) samples were baked in an oven at 110°C for 15 min. The procedure to silane’s modification is well described elsewhere [42].
In Figure 7, the experimental reflectance spectra for the infiltration process can be observed (solid line). A blue- or redshift of the (DBR)4−(FN)4−(DBR)4HHs in the three stages of the procedure can be seen clearly. Figure 7a shows the reflectance spectra of the sample as prepared. The thermal stabilization of the samples in oxygen atmosphere produces a partial transformation of the silicon filaments to silicon dioxide. The refractive index of silicon dioxide is lower with respect to silicon; therefore, a decrease in the effective refractive index of the layers [43] in the HHs can be detected by a large displacement of the resonant modes to short wavelengths. This blueshift is observed in Figure 7b and compared with a sample as prepared in Figure 7a. Moreover, the infiltration of biological molecules into the pores produces a redshift because the APTES molecules displace the air from the pores. The redshift of resonances can be easily detected by optical reflectance. As can be seen in Figure 7c, a redshift of 44.3 nm was obtained with respect to the oxidized sample (Figure 7b). Even by using low concentration of APTES (approximately 5%), the spectral displacements are larger than the previously reported ones in PSi structures that were infiltrated with several molecular species [44,45]. This effect can be attributed to the large specific surface area of the PSi that allows us to have a large quantity of available sites for chemical binding. This idea is based on the saturation curves obtained for APTES in micro- and mesoporous structures reported in reference [46] and confirmed in our samples (not shown for brevity). In order to compare the sensitivity of the APTES infiltration in HHs and the structure given in reference [45], we have calculated the ΔE (in electron volt) from the spectral position of the resonance modes before and after the APTES infiltration. In that reference, ΔE∼ 0.27 eV for structures designed at 830 nm. In our case, ΔE=0.053 eV for λ=1.0 μm, according to the pore size criteria given above. An important characteristic that the HHs offer is the possibility to generate strong localized transmission modes that allow us to better monitor their spectral displacements even at a low concentration of analyte.
Figure 7. Experimental reflectance data of the (DBR)4−(FN)4−(DBR)4HHs. Solid line: (a) As obtained , (b) thermally oxidized at 900°C, and (c) after APTES infiltration. Dashed line, theoretical simulation.
Theoretical simulation is also presented in Figure 7 (dashed line). In this case, unlike the first simulations and as a result of changes in the effective refractive index produced by thermal oxidation and APTES infiltration, we introduced a constant Δni in order to reproduce the best possible experimental reflectance, where i corresponds to layers A, B, C and D. The values of Δni were adjusted, taking into account that the silicon dioxide grows at the expense of silicon (reducing the effective refractive index) and APTES displaces the air from the pores (increasing the effective refractive index). We found that the values of Δni induced by the APTES attachment on the oxidized layers is ≤0.09 for each layer. Figure 7 shows a good agreement between the experimental and calculated spectra. The simulation of the optical spectra, based on experimental data of the HHs structures with the molecule infiltration, could give us a quantitative analysis method for material infiltration even at a very low concentration.
Conclusions
In conclusion, we have been able to demonstrate the fabrication of hybrid heterostructures consisting of dielectric multilayers of distributed Bragg reflectors and Fibonacci type. Even considering the complexity of the HHs and the multiple factors involved in PSi formation, we obtain a very high quality and reproducibility in PSi multilayers in our experimental setup. The possibility to localize resonant modes and tuning them has been proven. Unfolding of resonant modes can be generated by repeating periodically the hybrid structure. The theoretical model is in good agreement with the experimental data, so it could be used to estimate changes in optical responses of chemically modified structures. Such hybrid heterostructures can be very promising in the field of optoelectronics, optical communications [47], and biosensors [48].
Competing interests
The authors declare that they have no competing interests.
Authors’ contribution
KP carried out all the experimental processes, optical characterization and drafted the manuscript. AM participated in performing the process of porous silicon, analyzed the characterization data and drafted the manuscript. JOE and JA developed the theoretical calculation programs. GP designed the infiltration protocol of biological molecules into porous silicon. MEM made the initial theoretical model of heterostructures based on porous silicon. All authors read and approved the final manuscript.
Acknowledgements
This work has been partially supported by CONACyT under project No. 101486 and by PROMEP “Red Temática”. SEM images: Instituto Potosino de Investigación Científica y Tecnológica A. C. (IPICYT).
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Compare Projects
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Ads at the Top of Google AdWords Results
Jul 20, 2004 • 12:55 pm | (3) by | Filed Under Google AdWords
Why do some Google AdWords ads show at the top of the Google search results and some show only on the right? This question was the first question asked and answered to the AdWordsRep at the Search Engine Watch forums.
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The only ads that can show above the main serps at the top of Google.com are the ones that meet both of the following criteria: (1) be reviewed and approved, and (2) meet an additional performance bar that focuses on relevancy - rather than what you're paying
The AdWordsRep further clarifies:
Ads on the right are positioned by virtue of two factors, measured equally. These factors are Maximum CPC and CTR. Max CPC x CTR = your rank number. And it is your rank number as compared to your competitors rank numbers that determines position.
Ads going to the top, however, weigh CTR (which is a measure of the relevance of your ads to users) more heavily than CPC. And rather than Maximum CPC, it is the actual CPC that matters.
Previous story: Home Page PageRank 0 But Inner Page PageRank Constant
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Search Pulse 14: Search Holidays, New Years, Google Update, Blog Tips, Social Links, Paid Reviews, Ask.com X, AdWords & More
Jan 3, 2007 • 8:25 am | (0) by | Filed Under Search Pulse Podcast
The fourteenth edition of the Search Pulse has now been archived. We were able to discuss in detail about half of the topics I wanted. This week, we talked about the holiday season and the new year. We argued about a possible Google update. We also debated on the blog tip Google is offering. We looked at social links and gave some tips with that. Overall, we had some pretty good discussions that may be of use to you in the 2007 year. The topics we covered are listed below, in order of priority (based on search community buzz). You can download the MP3 file here and listen at your convenience.
Topics We Covered:
1. Happy 2007 New Year From The Search Engine Industry
2. 2006 Holiday Season Search Logos
3. Open To Suggestions: Things To Improve, Change or Remove in 2007
4. Yahoo!'s Holiday Gifts for 2006
5. WebmasterWorld Threads of 2006
6. Google Data Refresh - Rankings Fluctuations Before Christmas
7. Google's Data Refresh; Any Patterns or Commonalities?
8. Blog Like Searches Bring Up Special One Box Result For Google's Blogger
9. When Will Google Begin Devaluing Social Links; Such As Digg.com, Yahoo! Answers & del.icio.us?
10. Paid Blog Reviews: ReviewMe & PayPerPost
11. Do Larger Web Sites Require More Links Than Smaller Sites To Rank Well?
12. If You Don't Have a Duplicate Content Problem; Don't Fix It
13. Ask X Allows Users To Interact More With Search Results
14. Screen Shot Of Quality Score Metric in AdWords Console
15. Fighting Search Spam With PhraseRank: The Latest Google Patent Buzz
Lightening Round:
1. Wikiasari, Community Search Engine; New Search Engine by Wikipedia Founder
2. Losing Trust in Google's Webmaster Central?
3. Google Calculator Breaks On New Years
4. Google AdSense Competitive Ad Filter Not Working?
5. Google's AdSense Team Fixes Ad Filter Bug
6. Publishers Noticing Yahoo!'s New Compliance Manager At Work
7. Top Factors in PPC & AdWords That Determine Your Click Through Rate in Google
8. Dynamic Keyword Insertion In Yahoo!'s New Panama PPC Engine
9. Dynamic Keyword Insertion in Your URLs With Google AdWords
10. Panama Edit Bulk Keyword Page Needs A Yahoo! Touch Up
11. Google May Add Referrer Data To Web Crawl Error Reports
12. How Does Google Crawl Pages & Index Them?
13. Google Changes AdSense Feedback & Violation Form
14. Need Content Removed From Google Quickly? Expedited Google Content Removal
15. Google AdWords Updates Quality Scores At Least Monthly
16. Google & Search Engines Do Not Mind Bad HTML When Crawling
17. Encouraging Clicks of AdSense Referral Products is Allowed
18. Open Directory Project (DMOZ) Coming Back From the Dead?
19. Making Bidding Mistakes at Google AdWords ($0.10 Vs. $10.00)
20. Sharing Some 2006 Stats From Search Engine Roundtable
21. PPC Managers Laugh Off Google's Traffic Estimator
22. Google Drops Ads Automator for New AdWords Campaigns
The topic list is in order of how I wanted the conversation to go. I felt that these were the most talked about and discussed topics in the search community. Again, you can listen to the MP3 file here.
Next week we will not be having a show.
Past Shows: - December 19, 2006: Thirteenth Edition of the Search Pulse Live: Google Link Building, Duplicate Content, CSS Tricks, Images Near AdSense, Yahoo! Directory Tag, Here Comes Panama, & More - December 12, 2006: Twelfth Edition of the Search Pulse Live: SES Recap, Versioning SEO, Contact Google, Yahoo, Google, Click Fraud, Duplicate Content, AdWords, Microsoft & More - December 5, 2006: Eleventh Edition of the Search Pulse Live: Google Spam Tools, Danny & SEW, Chris Boggs & SEW, Ask AskCity, MSN Bot, Yahoo Topics & Much More - November 28, 2006: Tenth Edition of the Search Pulse Live: Turkey Day, Google Spam, SES Chicago, MSN Update, AdWords Denial, Holiday Gifts, Yahoo Jobs & More - November 21, 2006: Ninth Edition of the Search Pulse Live: PubCon Vegas, Hefner's Sky Villa, Google Update, Yahoo's Compliance, Microsoft's Banning, AdWords Quality, Sitemaps Protocol & More - November 7, 2006: Eighth Edition of the Search Pulse Live: Google AdWords Update, MSN Search Update, PageRank Update, SEO, PubCon Coverage, Firefox, JotSpot, Yahoo! Eggs, & More - October 31, 2006: Seventh Edition of Search Pulse Live: Halloween, Yahoo's Panama, Did-It, Ask.com Mobile, MSN Links, Eric Schmidt, 30 Point Penality, & More - October 10, 2006: Sixth Edition of Search Pulse Live: Tim Mayer on NOODP Tag, GooTube, Google News Porn, SEO, PageRank, Pinging, Code Search, SearchMash, Yahoo Panama UK, Mobile AdSense & More - October 4, 2006: Fifth Edition of Search Pulse Live: Google Maps Bug, PageRank Update, 8th BDay, Links, Matt Cutts, Daniel Read, Ask.com, adCenter, Apax, & More - September 26, 2006: Fourth Edition of Search Pulse Live: Google.com Rankings Anywhere, Live.com Fixed, Canada Not Welcome, Danny's B-Day, Yahoo! Ad Growth, Google Product Search Changes & More - September 19, 2006: Third Edition of Search Pulse Live: Live.com, AdWords Test, Intuit & Google, Yahoo! Ads, Supplementals, Belgian Cache, OneBox Spam, Adam Lasnik on Links - September 12, 2006: Second Edition of Search Pulse Live: Google News Archive, Sitelinks, AdWords Preview, Sullivan PubCon, ChaCha & More... - September 6, 2006: First Edition of Search Pulse Live: Sullivan Leaves SEW, AdSense Lawsuit, Yahoo Blog Search, Keywords in URLs, New URLs, Navigational Search, Google Apps & More
Previous story: Google Sorting Ad Groups by Clicks Bug
blog comments powered by Disqus
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Place:Udora, Ontario, Ontario, Canada
Watchers
NameUdora
TypeVillage
Coordinates44.257°N 79.183°W
Located inOntario, Ontario, Canada ( - 1974)
Also located inDurham, Ontario, Canada (1974 - present)
See alsoScott, Ontario, Ontario, Canadatownship in which Udora was officially located until 1974
Georgina, York, Ontario, Canadatownship adjacent to Udora
Uxbridge, Durham, Ontario, Canadamunicipality in which Udora located since 1974
Udora is a "compact rural community" on the border between Scott Township in Ontario County and Georgina Township in York County. In 1970s both Ontario County and York County went through a major municipal reorganization. Scott Township merged with its neighbouring township to the south to become Uxbridge Township within Durham Region, and York County split in half with the northern half (which contained Georgina Township) becoming York Region.
History
the text in this section is copied from an article in Wikipedia
Udora is a small rural community in Ontario, Canada. It has a population estimated to be around 500 and is situated in the most south-eastern part of Georgina, split between York Region and Durham Region. The town was originally known as Snoddon Corners and was the location of the Snoddon Hotel. Some of the descendents of the Hotel's namesake still live in Udora. The name "Udora" was thought to have come about when a town meeting was taken place to select a name for the post office in 1862. The chairman asked the people attending for options. He looked at Dora Brethour and asked, "What about you Dora?” Whether or not this is actually true is unknown, but it is what is rumored.
In the 1950s, the Independent Toronto Estonian Women’s Association purchased land in the north-west side of Udora, divided the land into 150 subdivided lots for summer cottages to Estonians in Toronto and named the grounds Jõekääru, which means River Bend in English, beautifully named because Pefferlaw River runs through the grounds. Local street names in the grounds are also in the native Estonian. With the cottages also came the Estonian Children's Camp, which is still active to date as an Estonian language immersion camp for part of the summer.
Highway 48 (which links Markham to Port Bolster) lies to the north while Highway 12 linking to Whitby and Orillia, lies to the east. Ravenshoe Road gridlocks with Victoria Road and Durham Regional Road 1. Area code 705 is bounded to the north while the south of Udora is in Area code 905. The Canadian National Railway runs north of Udora, having its nearest train station in Pefferlaw.
Udora is located about 10 km South of Port Bolster, at Lake Simcoe. About 20 km S/E of Sutton, about 25 to 30 km SW of Beaverton and Orillia, west of Lindsay, north of Uxbridge, about 50 km north of Whitby, about 80 km north of Toronto and NE of Newmarket, Ontario.
In the centre of Udora (or downtown) on the main road (Victoria), there is a General Store, where General Delivered mail can be picked up and lottery tickets purchased along with local homegrown foods. There is also a UPI full serve gas station and convenience store directly next door.
West of Victoria lies the below mentioned Town Hall along with a baseball diamond, playground and basketball / tennis (badminton) court. In the winter, the court also hosts a small skate rink for kids.
Research Tips
The primary source for basic documents (vital statistics, land records, wills) for people who lived in the Province of Ontario is the Archives of Ontario, 134 Ian Macdonald Blvd, Toronto, Ontario, Canada M7A 2C5.
Early Records
Civil registration did not begin in the province until 1869. Before then there may be church records of baptisms and burials. For the most part these are still held by the denomination who recorded them. Copies of marriage records made pre-1869 had to be sent by individual clergymen to the registrar of the county in which the marriage took place. These marriage records are available through Ontario Archives, on micorfilm through LDS libraries, and on paid and unpaid websites, but because they were copied at the registrars' offices, they cannot be considered a primary source.
Vital Records after 1869
Birth, marriage and death registrations are not open to the public until a specific number of years after the event occurred. Births to 1914 are now available [October 2012]; dates for marriages and deaths are later. Birth and death registration was not universally carried out in the early years after its adoption. Deaths were more apt to be reported than births for several years. The more rural the area, the less likely it would be that these happenings were reported to the authorities.
Images and indexes of civil registrations for the "viewable" years can be found on paid websites, and indexes only on FamilySearch. The latest year published is not yet available online. The FamilySearch Wiki on Ontario Vital Records explains how these records are organized and their availability.
Land Records and Wills
Information on how to access land records and wills is best sought on the Archives of Ontario website. An ancestor's land holding might be found on Canadian County Atlas Digital Project if he was in occupancy circa 1878.
Association for the Preservation of Ontario Land Registry Office Documents (APOLROD). A list of Land Registry Offices for all Counties of Ontario.
Censuses
The original censuses are in the hands of Library and Archives Canada. All of the original census (1851-1911) images are online with the exception of that for 1861. Not all of them are indexed. Later censuses are not yet available. Census divisions were redrawn as the population increased and more land was inhabited.
Other websites, some paid and some free, also provide Canadian census originals and/or indexes online. One can view censuses on microfilm at the Archives of Ontario or at big libraries throughout Canada.
E-books and Books
• The Internet Archive, particularly texts from Canadian universities, can contain interesting material
• Our Roots is a Canadian website similar to The Internet Archive
• Global Genealogy is an online bookshop specializing in Ontario material who will ship anywhere in the world.
=== Some websites with more local information on Ontario County ===
This page uses content from the English Wikipedia. The original content was at Udora, Ontario. The list of authors can be seen in the page history. As with WeRelate, the content of Wikipedia is available under the Creative Commons Attribution/Share-Alike License.
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The Conversation Keeps Evolving
Doug Kaye's announced a new, hybrid business model (PDF) for the Conversations Network. Doug's vision has been that the Conversations Network would be non-profit, but that has proven to place limitations on the network that limit its ability to scale, build tools, and grow channels like IT Conversations.
As a result, Doug is moving to a hybrid business model. The Conversations Network will continue as a non-profit. It will own audio and preserve it, have a license to the tools that make it work, and serve as a home for many new channels.
Doug and Michael Geoghehan have formed a new company called GigaVox that will be for-profit, own software, pay employees, and run some channels. GigaVox will license software to the Conversations Network. Nothing in the Conversations Network (including money and assets) will flow back to GigaVox.
You can listen to Doug explain all this. I'm excited to see Doug evolving this model to find out what works.
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Tell me more ×
Answers OnStartups is a question and answer site for entrepreneurs looking to start or run a new business. It's 100% free, no registration required.
I am an 18-year old individual, in my first year of university. At the beginning of the university year, I had an idea for a website that I followed up on and have, at this point, created a product that could be finished within a short period of time. The target audience would be students of any kind and I have shown the idea to several students at my campus and their responses were very positive.
As for my concerns: So far I have been the only person doing anything with this project. I have seen everywhere that it is highly recommended to have at least a co-founder and I really feel the need for some outside ideas and some motivation boosts. I have tried looking around my university (so far only people that I actually know) but nobody seems to really 'have the spark'.
Furthermore, the kind of site I am working on, if it is launched, would not generate profit at an early stage but it does have the potential to be highly profitable. Sadly, I do not have very extensive knowledge about the business side or the legal side of things. Should I worry about investments, business partners or any of that now? Do I need to register trademarks for my website's (company's?) name? When do I register my site as a company?
Any tips would be greatly appreciated. Thanks! :)
share|improve this question
3 Answers
up vote 5 down vote accepted
Move forward. The probability of success is low but you at least will learn something that will be useful for you next projects. At this point I will not worry about investments not trademarks but worry more about how to sell that product to expected audience.
share|improve this answer
I would focus on one thing at a time, either your studies completely or the business idea. Given your age, and I mean this with the utmost respect, the business is unlikely to be successful. I would concentrate on your studies first-most. Studying is a lot easier at your age, I found that I didn't have the right skill-set for starting a business until I reached about 30.
That's not to necessarily say you don't have them now, it's just your academic studies get you into your first job and it's the domain your work in during your 20's that often is the one your first successful business is based on.
share|improve this answer
If this is an affordable risk for you, then finish the site.
You'll learn more by seeing whether, and how people use your site than by asking them whether they like it. It's less work, too.
If finishing the site means months of work, then first simplify to a kick-ass half, and finish something in the next two weeks.
The business stuff is worth worrying about when, and only when, you can see there's a business bursting to emerge. Trust me, when you hit that stage, you'll be highly motivated to learn fast.
share|improve this answer
Your Answer
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Not the answer you're looking for? Browse other questions tagged or ask your own question.
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Fedora on the Kurobox
From NAS-Central Buffalo - The Linkstation Wiki
Revision as of 17:45, 23 March 2006 by Kuropotato (Talk)
Jump to: navigation, search
This is the official home of Fedora on the Kurobox. You will find links on how to install Fedora on the Kurobox and various HOWTOs.
Install Fedora (coming soon)
What can you run on the Kuro with Fedora
Personal tools
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TeraStationPro
From NAS-Central Buffalo - The Linkstation Wiki
Revision as of 04:06, 14 January 2007 by Entropy (Talk)
Jump to: navigation, search
Contents
Background
In March 2006 Buffalo relased a new version of the TeraStation.
It is supposed to feature more current hardware like S-ATA disk.
For now please refer to the official site.
Hardware
See hardwarePro for the new mainboard.
Where to upload Software?
If you have anything (Firmwares, Custom Firmwares, useful software packages) that might be useful for other Tera/Tera HS/TeraPro-users then send mindbender a private message in the forums. He will provide you with the needed information to upload files to
http://downloads.linkstationwiki.net/terastation/uploads/
All linkstationwiki-admins & itimpi have full access and administrate the terastation-section:
http://downloads.linkstationwiki.net/terastation/
Firmware
Official
v1.01
The TeraStation Pro can be a member of Active Directory Enviroment of a Windows200x network.
To enable this Buffalo has released a beta firmware for this feature.
This firmware can be downloaded from http://de.buffalotech.com/downloads/TSPro-1.01-0.51-withAD.zip
And it uses an known password
v1.03
Some newly purchased units are coming out with a version 1.03 firmware. This firmware seems to have some issues, including the inability to set Active Directory settings(stays greyed out even after checking the AD checkbox, apparently not all 1.03 users are having this issue). Flashing with the 1.01 linked above seems to fix this issue.
In late June I had trouble with my TS Pro. Buffalo tech support suggested reinstalling the firmware. My box was at 1.03 but only 1.01 was available on the Buffalo website. I inquired and was told by Buffalo tech support 1.03 created more issues than it solved and was pulled for rework. My TS Pro subsequently died and I did a warranty exchange with the distributor (Dell). The replacement box contained 1.01, not 1.03.
Buffalo tech support does not recommend using 1.03.
v1.04
Buffalo has a Beta 1.04 firmware available on their (originally temporary) FTP site. According to Buffalo this should fix the issues that some users have been having when joining to their AD domains. It still does not appear to fix the name resolution issue(can connect to the device using AD users by connecting to "\\IPaddress" but not "\\Name").
-It seems that download link does not working, If someone have this firmware please let me know and I will provide hosting for it.
-It's working right now, but it seems go down intermittently. If someone has more reliable storage that would be great. Also, they have a new one out today: TSPro-1.04-0.01-CharFix. It is 1.04-0.01 instead of 1.04-0.00. Probably some sort of minor revision. --Wastedlife 05:09, 4 October 2006 (CEST)
Non-Official Firmware
The following firmware files have been provided by Entropy. There is always an inherent risk associated with updating your system. If your system breaks, you own both pieces. If you have feedback on the firmware, please leave a message on the Talk Page.
Terastation Pro Firmware by Entropy
Version Base Image Status Released Includes
1.01-1 Buffalo US 1.01-0.51-withAD Stable 2006-May-20 dropbear v??
user_nfs v??
1.01-2 (test2) Buffalo US 1.01-0.51-withAD TEST 2007-Jan-13 less-358
md5*sum/sha*sum from busybox-0.60.5
openssh-3.5p1
rsync-2.6.9
strace-4.4.93
unfs3-0.9.16
user_nfs v??
Release Notes 1-01-2
Please test this version and let me know if there are any problems. The highlights of this version is that it replaces dropbear with openssh for compatibility reasons and includes a NFS v3 daemon so that files larger than 2G can be written to the Terastation via NFS. Most of the additional tools for this package are built using the Denx ELDK
• Once you have flashed this image to your TSPro, you can ssh into the box using the admin account and whatever password you setup when you configured the box. The admin user has been added as a sudoer, so you can execute "sudo -s" if you want/need to use the root account.
• Anytime the box is reflashed, the ssh host keys are regenerated.
• If you are attempting to get public key authentication working, remember, all of the users' home directories are in /home, so if you setup public key authentication for one, it is for all because /home/.ssh is shared among all users. I would suggest changing your users home directory to /mnt/array1/home/USER (where USER is the username) so that your ssh keys are kept across firmware upgrades.
• I created a link so that if you create a directory called /mnt/array1/web, you can serve files from that directory using your Terastation. I wouldn't exactly recommend putting it on the Internet though! Unfortunately, the master configuration does not allow for indexes or overrides, so you'll have to change the /etc/apache/httpd.conf file if you want directory listings. (Remember, you lose these changes when you reflash the box).
Known Issues 1.01-2 (test2)
1. None so far.
Entropy's firmware worked great for me. I am having couple of issues it might be my inexpertise.
1. Couldnt get the Pulic key authentication working. Tried using the tips on QandA.
2. NFS - i was able to mount both on mac and linux and i tried to copy a file little over than 2GB and got error.
I would really appreciate any help.
Thanks a Million
- Nav
Entropy could you tell me briefly how i can get V3 UNFS into the firmware. I will give it a shot. Appreciate your help.
- Nav
Thanks a lot for the info. It seems little bit out of my skills.
-- Naveen
Entropy, great work. Thanks a million. Here is my contribution, hope it helps someone. TeraStation Pro and NFS Walkthough for OSX and OSX Server.
- Hays
Talk: Hacking the TeraStation Pro
Follow this link: Talk:TeraStationPro what is the root password for TeraStation Pro for the non-official firmware
Using the serial console
Serial console TsP
Miscellaneous
Joining Windows Domains
One problem is that the TeraStation will not allow you to join a domain that begins with a number. I had to rename my Windows domain because it began with a number. What was the programmer at Buffalo thinking? -Mike 2006 May 25
If you have a PDC with leading number(s) running send an Email to the helpdesk@buffalo-technology.ie. They can help you. - Joergl 2006 July 6
Have Terastation Pro 1TB. I can join it to my Small Business Server 2003 Active Directory, just fine. The network sees the TS-Pro just fine, but the user accounts are not updated on the TS. I have tried working with buffalo tech support, but the just keep telling me it is a Active Directory problem, that my AD is setup wrong.
I explained to them that AD in a SBS network has to be created in a exact patteren or it will not work right. This is created during setup. Explained to them that if I join the TS as a workgroup and not part of the domain then it works correctly. This points to an issues with TS and not my PDC. The TS is not retreiving the AD information correctly. I did flash to 1.03 no good.
Have found other posts elsewhere with the same problem in a SBS network. Has anybody been able to join to the SBS 2003 AD? Bryan July 2006
Is it realy not possible to join a domain which isn't in mixed mode? Thaek Sept 2006
Here is how I joined a native 2003 domain with a Pro 1TB:
0) Configure your proper domain settings under the web config (even if it errors out on join)
1) Connected a serial cable.
2) http://your_Terastation_IP/cgi-bin/task.cgi?task=console¶m=on
3) Login as admin
4) Become root by entering sudo -s as a command and the admin password
5) /etc/init.d/smb restart
6) vi /etc/hosts
7) add the following: your.primary.DC.address FQDN.of.primary.DC (example: 192.168.1.24 DC1)
8) /lib/ld.so.1 --library-path /usr/local/samba3/ /usr/local/samba3/net ads join -U Your_Domain_Admin_Account@Your.Domain.Qualifier
9) Enter the domain admin account password.
10) If you are still having problems connecting to the shares, make sure the time is correct (mine never gets it right) and repeat steps 5 - 9.
With that, you should be off to the races. I am not sure why, but after a botched upgrade, I have manually do all of those settings each time I reboot the Terastation. But after I get it connected, it works great. Hope this helps.
--Aaron Dec. 27, 2006
Samba Performance?
Seems that I am the only one having problems with the bad samba performance from Windows clients (when having a few 1000 files in a directory). I assume that it has to do with the case insensitivity needed for Windows clients.
Is there a simple hack resolving this?
--Mbob 10:24, 3 October 2006 (CEST)
Localization of This Page
Notice! :This page of Original is English version.
TeraStation Pro(日本語) - Japanese
Help
Walk Though
TeraStation Pro and NFS Walkthough for OSX and OSX Server
Personal tools
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Influence of Polyethylene Pipe on the Quality of Water in a Water Distribution System
Beata Kowalska, Dariusz Kowalski, Marian Kwietniewski, Anna Musz
Abstract
In the past two decades economic considerations and very good mechanical parameters of polyethylene pipes contributed to more and more common use of the pipes for construction and repair of water distribution systems all over world. This paper presents the results of research into parameters of water collected from a polyethylene pipeline which was a part of an operational water supply network. We determined Total Organic Carbon (TOC) content and the content of organic compounds in the examined water samples using a multichannel gas chromatograph (Trace Ultra Thermo) coupled with a mass spectrometer (Polaris Q). We identified organic compounds bounded with antioxidants, added to polyethylene in the process of pipe production, in the water samples.
Full Text: PDF DOI: 10.5539/jsd.v6n2p1
This work is licensed under a Creative Commons Attribution 3.0 License.
Journal of Sustainable Development ISSN 1913-9063 (Print) ISSN 1913-9071 (Online)
Copyright © Canadian Center of Science and Education
To make sure that you can receive messages from us, please add the 'ccsenet.org' domain to your e-mail 'safe list'. If you do not receive e-mail in your 'inbox', check your 'bulk mail' or 'junk mail' folders.
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Thursday, June 22, 2006
I and the Bird #26
The latest I and the Bird is now available at The Hawk Owl's Nest. Go there to read about the sides for the IATB World Cup. (Unlike the FIFA World Cup, you don't have to take your pants off to visit Patrick's blog.)
The next issue of I and the Bird (on July 6) will be its first anniversary. For that occasion, it is returning to its original host and founder, 10,000 Birds, for a themed issue. Mike is calling for posts on one (or more) of three questions: why do you bird, why do you blog, and why do you blog about birds. For details, see here.
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{
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"uncompressed_offset": 74487156,
"url": "dotnetkicks.com/stories/8429/SubSonic_Download_Helper_SQL_Script",
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"warc_url": "http://dotnetkicks.com/stories/8429/SubSonic_Download_Helper_SQL_Script"
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Error!
Success!
SubSonic - Download Helper SQL Script
0
kicks
SubSonic - Download Helper SQL Script (Unpublished)
I find I frequently employ a standard set of my own conventions for each new database table I'm creating. Combine that with making the use of SubSonic conventions and you're prime for a free, reusable, helper sql script to save you time.
Kicked By:
Drop Kicked By:
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"url": "genomebiology.com/2009/10/4/307/abstract",
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"warc_filename": "<urn:uuid:4e5f5b8f-0760-4dba-9f3f-6897170f0950>",
"warc_url": "http://genomebiology.com/2009/10/4/307/abstract"
}
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Meeting report
Evolving proteins at Darwin's bicentenary
John W Pinney and Michael PH Stumpf*
Author Affiliations
Centre for Bioinformatics, Division of Molecular Biosciences, Imperial College London, Wolfson Building, London SW7 2AZ, UK
For all author emails, please log on.
Genome Biology 2009, 10:307 doi:10.1186/gb-2009-10-4-307
Published: 17 April 2009
Abstract
A report of the Biochemical Society/Wellcome Trust meeting 'Protein Evolution - Sequences, Structures and Systems', Hinxton, UK, 26-27 January 2009.
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{
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"url": "journals.tdl.org/icce/index.php/icce/article/view/2671/0",
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"warc_filename": "<urn:uuid:4e5f5b8f-0760-4dba-9f3f-6897170f0950>",
"warc_url": "http://journals.tdl.org/icce/index.php/icce/article/view/2671/0"
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LAND RECLAMATION AND GROIN-BUILDING IN THE TIDAL PLATS
Hele Focken Erchinger
Abstract
Along the North Sea eoast of Germany there are two large areas where land reclamation work in the tidal flats is being carried out. One is on the coast of Schleswig- Holstem and the other in Ostfriesland, on the coast and along the estuary of the river Ems near the border with the Netherlands. Conditions and working methods for land reclamation in tidal flats as well as the development of new groin constructions on the Ostfriesian coast are described below.
Keywords
land reclamation; tidal flats; groin; groin construction
Full Text: PDF
This work is licensed under a Creative Commons Attribution 3.0 License.
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{
"content_type": "text/html",
"provenance": "cccc-CC-MAIN-2013-20-0000.json.gz:16872",
"uncompressed_offset": 138102568,
"url": "journals.tdl.org/icce/index.php/icce/article/view/3071",
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"warc_url": "http://journals.tdl.org/icce/index.php/icce/article/view/3071"
}
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INTERACTION OF WAVES AND A TURBULENT CURRENT
J.D.A. van Hoften, S. Karaki
Abstract
An experimental investigation was made to study wave-current interaction. Wave amplitude attenuation was measured along a laboratory wave channel to compare wave dissipation with and without flow. Mean, wave, and turbulent velocities were also measured to determine the modifications of the flow imposed by the gravity waves propogating with the current. The process of energy transfer in the wave current system was studied. Energy was found to be extracted from the waves, diffused downward and dissipated by an increase in bottom shear stress.
Keywords
current; turbulent current
Full Text: PDF
This work is licensed under a Creative Commons Attribution 3.0 License.
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{
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"uncompressed_offset": 150357493,
"url": "listarchives.libreoffice.org/global/users/msg20876.html",
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[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]
Re: [libreoffice-users] Re: Is 3.5.4 ready for business users?
Felmon Davis wrote:
I am curious: what happens if one just changes the file extension from .doc to .docx?
You divide by zero and a black hole appears. ;-)
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"url": "myedmondsnews.com/2012/12/boy-scouts-offering-christmas-tree-recycling-this-week/",
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Boy Scouts offering Christmas tree recycling this week
The Boy Scouts will be at the following Edmonds-area locations to receive your tree starting this Tuesday:
Troop 61
Safeway, 23632 Highway 99, Edmonds
Jan. 1 (Tuesday) noon – 4 p.m.
Jan. 5 (Saturday) 10 a.m. – 4 p.m.
Jan. 6 (Sunday) noon – 4 p.m.
Troop 300
Westgate Elementary, 9601 – 220th St. S.W., Edmonds
and
Top Foods
21900 Highway 99 S, Edmonds
Jan. 5 from 9 a.m.-4 p.m. and Jan 6 from 10 a.m.-3 p.m.
Troop 312
PCC Natural Markets , 9803 Edmonds Way, Edmonds
and
QFC
196th St SW & 76th Ave W, Lynnwood
Jan. 5 and Jan. 6 from 8:30 a.m.-4:30 p.m.
Residents can also visit www.dryneedles.com and use the interactive map of Southwest Snohomish County to find any Boy Scout tree-cycle activity near them.
All decorations, tinsel, tree stands and nails must be removed from the tree in order to be recycled. Flocked trees are not accepted. Donations are appreciated.
To encourage constructive community dialogue, all commenters must use their real names, first and last. Comments from users with names that don't comply with this policy will be removed. Thank you for your cooperation.
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{
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Tag Archives: politics
Manifest Sense
Mark Henderson’s The Geek Manifesto is a remarkable book. Though many of its themes are not new, it is difficult to imagine such a book being published as recently as five years ago. The Geek Manifesto provides a timely analysis … Continue reading
Posted in Science & Politics | Tagged , , | 5 Comments
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{
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Thayer Lab
From OpenWetWare
Jump to: navigation, search
Contents
Mathew J. Thayer, Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology
Oregon Health & Science University
3181 SW Sam Jackson Park Road
Portland OR 97239
(503)494-2447
thayerm@ohsu.edu
Education:
California State University, Chico. BA: Microbiology and Chemistry (1982)
University of Southern California. PhD: Microbiology (1988)
RESEARCH INTERESTS
My laboratory uses somatic cell and molecular genetic approaches to identify and characterize genetic alterations found in tumor cells that induce abnormal cellular phenotypes. By utilizing this approach, my lab has identified a previously unknown chromosomal abnormality that is associated with certain chromosomal rearrangements. This chromosomal phenotype is characterized by a delay in mitotic chromosome condensation, a delay in the chromosome replication timing, and significant chromosomal instability. Chromosomes with this phenotype are common in tumor derived cell lines and in primary tumors. Furthermore, we have found that exposing cells to ionizing radiation generates chromosomes with this phenotype. Our findings support a model in which the chromosomal instability found in tumor cells, and in cells exposed to ionizing radiation, stems from a defect in the replication timing of certain chromosomal rearrangements. Recently, we developed a chromosome engineering strategy that allows us to generate chromosomes with this delayed replication and condensation phenotype in an efficient and reproducible manner. Our findings indicate that ~5% of all random chromosome translocations display this abnormal phenotype. In addition, on certain balanced translocations only one of the derivative chromosomes displays the phenotype, indicating that a cis-acting mechanism is responsible for this abnormal chromosomal phenotype. We are currently using ‘chromosome engineering’ strategies, combined with somatic cell and molecular genetic approaches, to generate and characterize chromosomes with this delayed replication and condensation phenotype. The long-term goal of these studies is to define the molecular mechanisms responsible for chromosomal instability, one of the most common types of genetic instabilities found in cancer cells.
Chromosomes with DRT/DMC
Cancer cells differ from their normal cellular counterparts in many important characteristics, including growth factor independence, resistance to apoptotic signals, loss of differentiation, and decreased drug sensitivity. Not surprisingly, genetic alterations occur in most, if not all cancer cells, and are thought to lie at the heart of these phenotypic alterations. The genetic changes found in cancer cells are typically of two types: dominant, thought to occur in proto-oncogenes; and recessive, thought to occur in tumor suppressor genes. The dominant type of alteration typically results in a gain of function, and the recessive type of alteration typically results in loss of function. Furthermore, it has been argued that an underlying genomic instability is present in cancer cells and is required for the generation of the multiple mutations that are thought to underlie cancer. In support of this hypothesis, molecular analysis of individual tumors often identifies multiple genetic changes, including chromosomal translocations, deletions, insertions, gene amplifications, as well as point mutations. It has been estimated that every colorectal carcinoma cell contains approximately 11,000 distinct genetic alterations, present in both coding and non-coding DNA sequences. More recently, it has been shown that breast and colorectal cancers contain on average 90 mutant protein-coding genes. Determining the mechanisms responsible for the generation of these large numbers of mutations is an important goal of current cancer research. Chromosome rearrangements represent a common type of genetic alteration, and are found in nearly every type of cancer (Mitelman et al., 2004). The presence of recurring chromosome abnormalities in specific cancers occurs with remarkable specificity. Recent surveys have identified more than 600 balanced and 1,500 unbalanced recurrent chromosomal aberrations among different neoplastic disorders (Mitelman, 2000; Mitelman et al., 1998; Mitelman et al., 2006). These recurrent genetic alterations have been the focus of intensive molecular analysis for many years, with over 300 genes being implicated in various neoplasia-associated chromosomal rearrangements (Mitelman et al., 2004). Furthermore, these recurrent structural alterations can assist in the diagnosis and sub-classification of a malignant disease and in the selection of the appropriate treatment.
Fig. 1: Delayed Mitotic Chromosome Condensation (DMC)
In addition to these recurrent chromosomal alterations, most solid tumors also contain many non-recurrent chromosomal changes, with >100,000 independent alterations having been documented (Mitelman et al., 2006). These non-recurrent changes are seen as simple balanced or unbalanced translocations, intra-chromosomal rearrangements (deletions, inversion, or duplications), or as marker chromosomes and gene amplifications. Unfortunately, the mechanisms responsible for the generation of these numerous chromosomal alterations and the molecular and phenotypic changes associated with the majority of these alterations remain undefined. My lab has characterized an abnormal chromosomal phenotype that is associated with certain tumor-derived and radiation induced chromosome alterations. These chromosomes display a significant delay in mitotic chromosome condensation (DMC), associated with a delay in the mitosis-specific phosphorylation of histone H3, and a 2-3 hour delay in the replication timing (DRT) of the entire chromosome. In addition, chromosomes with DRT/DMC participate in frequent secondary translocations and rearrangements, indicating that cells containing these chromosomes display CIN. The results that we have obtained during the characterization of chromosomes with DRT/DMC support a model in which the genetic instability found in tumor cells and in cells exposed to ionizing radiation stems from a defect in the replication timing of certain chromosomal rearrangements, and that this abnormal replication is regulated by a cis-acting mechanism. Individual cells can contain multiple chromosomes with the DMC phenotype, and the DMC chromosomes within a given cell can display a wide range in the extent of mitotic chromosome condensation (Fig. 1C-F). Furthermore, chromosomes with the more extreme under-condensed appearance lack the mitosis-specific phosphorylation of histone H3 on serine 10 (Fig. 1A and B) usually initiated in late G2 and completed just prior to nuclear envelope breakdown and the formation of prophase chromosomes. These observations suggest that the more extreme under-condensed chromosomes retain an “interphase state” of condensation in cells that contain fully phosphorylated and condensed metaphase chromosomes.
Engineering DRT/DMC
We have developed a chromosome engineering system that allows us to: 1) target the genome in a random fashion, 2) create reciprocal chromosome translocations at defined locations, 3) generate the same translocations in multiple independent events using two distinct mechanisms, and 4) characterize the chromosomes both before and after the translocation events. The chromosome engineering strategy that we are employing is based on the Cre/loxP site-specific recombinase system to generate inter-chromosomal translocations. Because the Cre/loxP system is relatively inefficient at generating inter-chromosomal translocations (<1 x 10-3), we are using reconstitution of a selectable marker to isolate the cells that undergo Cre-mediated recombination. A schematic representation of this strategy is shown in Fig. 2.
Personal tools
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Quotation added by staff
Why not add this quote to your bookmarks?
What is a hero without love for mankind. Lessing, Doris
This quote is about heroes and heroism · Search on Google Books to find all references and sources for this quotation.
A bit about Lessing, Doris ...
Doris Lessing, CH, OBE (born October 22, 1919), is a British writer, born Doris May Taylor in Kermanshah, Persia (Iran).
These people bookmarked this quote:
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It's easy! Just pick the product you like and click-through to buy it from trusted partners of Quotations Book. We hope you like these personalized gifts as much as we do.
Make and then buy your OWN fantastic personalized gift from this quote
There are two mistakes, one comes from the heart, the harder comes from the mind. Tadj Abelkader
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212 - The Extra Degree
The one extra degree makes the difference. This simple analogy reflects the ultimate definition of excellence. Because it's the one extra degree of effort, in business and life, that can separate the good from the great. This powerful book by S.L. Parker and Mac Anderson gives great examples, great quotes and great stories to illustrate the 212° concept. A warning - once you read it, it will be hard to forget. Your company will have a target for everything you do ... 212°
Click here to buy this »
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Your Morning Dump… Where we don’t need one guy doing it all
RedsArmyAdmin May 7, 2011 Uncategorized 3 Comments
Every morning, we compile the links of the day and dump them here… highlighting the big storyline. Because there's nothing quite as satisfying as a good morning dump.
Doc: “You’re down 2-0 and you want to look inside yourself to see what you can do better. We do it as a coaching staff, and you do it individually as players. And then hopefully we do it as a team in a game. I think the risk whenever you’re down in anything is that you want go inside yourself and do it yourself, and that never works."
CSNNE: Celtics Talk: What they said Friday at practice
Doc's right. It's natural. The team is in a hole and we're all looking for someone to put the team on his back and carry them to a win. But that's the exact kind of basketball that will doom the Celtics.
Think of when the Celtics are playing their absolute best. Think of the characteristics of that team. The ball is whipped around the floor… guys are setting picks, getting open, making the right pass to the right player for open shots. When the Celtics are rolling, you'll see a Rondo drive on one possession, a Pierce step back on another, a Ray 3 on the break after that, and KG finding someone with an interior pass for an easy dunk. When the C's are clicking, everyone is touching the ball… everyone is getting after it on defense… and the constant barrage becomes too much for the other team to handle.
If the Celtics go out there tonight and play hero-ball, they will lose. If Paul Pierce TRIES to carry this team… the C's will go down in a ball of flames. But if Paul Pierce gets hot within the flow of a good, crisp offense… THAT'S when he'll do some team-carrying.
It has to be a team effort. It can't be anything else.
Related links: Herald: Pierce feels better about chances
On Page 2: Don't go writing the C's obituary just yet
“We’re taking their best shot,” Garnett said. “I still don’t think we’ve played our best basketball and we’ve got to do that. We can’t just come out here and talk about it. We’re not on the white sands of the beach no more. We’re back in the jungle. Hopefully that’ll do some good for us.”
So, here they are backed up against that adverse wall and to top it off they have a plethora of injured bodies that include Rajon Rondo’s bad back, Pierce’s strained Achilles and Allen’s bruised stomach.
It’s tempting to begin writing the obituary and maybe that’s the reality, but bury this team? No way. Not after everything they’ve been through over the last four years. From building their own impromptu super-team and seeing that vision to its completion, to the Garnett injury of 2009 and the soul-crushing loss to the Lakers in Game 7. There is simply too much history behind them, no matter what the objective mind tells you about their chances.
WEEI: Is this the Celtics last stand?
All you gotta do is win tonight. Nothing else matters.
Excellent piece (as usual) by Paul Flannery. Required reading, I'd say.
Related links: Celtics Big 3 facing last chance at greatness
The rest of the links:
CSNNE: Celtics out of lifelines, must win Game 3 | Pierce #2 most influential athlete on Twitter | WEEI: Ainge: I believe Shaq is playing in Game 3 | Pierce returns to practice: "I feel good" | Garnett: An all in mentality for Celtics | ESPN Boston: Shaq's impact | West heating up for C's | Video: What role will Shaq play? | Herald: Tonight is the only game that matters | Is the Celtics comeback possible? You betcha | Globe: Calm in the face of a storm | Home to the year's biggest game | Mazz: I think its over | It could be a huge help | Heat: No big changes planned
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davdav
About Me
• About davdav
Location
Egypt | Forum editor
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Windows ,linux
Mobile Phone
N900
• Signature
N900 : 1.0 GHZ + Debian LXDE + Nitdroid
linux, windows and mac
nokia 3200>nokia 7610 >nokia n81 >nokia e71>nokia n97>NOKIA N900
Press the thanks button if i helped
And yes i live in Egypt
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All times are GMT -4. The time now is 04:02 AM.
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}
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San Francisco Mapping Party Mar2009
From OpenStreetMap Wiki
Jump to: navigation, search
A mapping party in San Francisco, California
Contents
What are mapping parties?
Mapping parties are events where anyone can come and participate in the OpenStreetMap project. OpenStretMap is a free, open source map that can be contributed, edited and used by anyone anywhere. Mapping parties are social events where experienced and new mappers can meet to share and learn more about the project. The events are generally held in a public place, and allow time for discussion, mapping and editing. The event is open to all.
It's fun. It's free. You can help.
More about Mapping parties
Saturday: 11am - 4pm, Sunday: 1pm-5pm
11:00 am: Introduction to OpenStreetMap for beginners.
11:30 am-2:30pm/3pm: Mapping time (walking/biking/driving)
2:00pm -4:00 pm: Uploading and Editing Map Data with refreshments and discussion.
4:00pm: Social event- drinks and discussion for whoever is interested, at a nearby eatery
Sunday (later times): Same Schedule
• If you own a GPS or laptop computer, please bring it along. If not, we will have GPS units to loan.
Venue
San Francisco Public Library Noe Valley Branch, 451 Jersey St. San Francisco, CA 94114 http://sfpl.org/librarylocations/branches/noevalley.htm
Transportation: BART to 24th St/Mission, Muni: 24, 48, J
Mapping objectives
To add more points-of-interest to the San Francisco map as well as tackle areas that still need mapping.
Mapping Party Suggestions
Please feel free to suggest locations and goals for this mapping event as well as future events.
Some ideas:
• Shops, restaurants etc, and the general extent of the commercial district along 24th St.
• GNIS data for schools, parks and churches has been uploaded recently, and needs fleshing out (churches need religion+denomination, etc)
• Some of the streets up the east slope of twin peaks may be steps, or absent.
• To the south, the Glen Park area has several unnamed roads, and a number of parks with little or no detail.
• The extent and connections of the cycle lanes along San Jose Ave could be improved.
• Mount Olympus (near 17th and Clayton) needs better street data (perhaps too far away).
Personal tools
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
6405.0 - Export Price Index, Australia, Jan 1997
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 18/03/1997
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• About this Release
Measures f.o.b. port-of-export price movements for Australian merchandise exports. Index numbers are published for 'All Groups'; 10 groups defined in terms of Sections of the AHECC; 'Industry of Origin' groups based on Divisions and Subdivisions of ASIC, and 6 selected Sections of SITC.
This publication has been converted from older electronic formats and does not necessarily have the same appearance and functionality as later releases.
© Commonwealth of Australia 2013
Unless otherwise noted, content on this website is licensed under a Creative Commons Attribution 2.5 Australia Licence together with any terms, conditions and exclusions as set out in the website Copyright notice. For permission to do anything beyond the scope of this licence and copyright terms contact us.
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cccc_CC-MAIN-2013-20
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
5657.0.40.001 - Common Funds, Australia, Jun 2000
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 31/08/2000
© Commonwealth of Australia 2013
Unless otherwise noted, content on this website is licensed under a Creative Commons Attribution 2.5 Australia Licence together with any terms, conditions and exclusions as set out in the website Copyright notice. For permission to do anything beyond the scope of this licence and copyright terms contact us.
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Research article
General and species-specific transcriptional responses to downy mildew infection in a susceptible (Vitis vinifera) and a resistant (V. riparia) grapevine species
Marianna Polesani1, Luisa Bortesi1, Alberto Ferrarini1, Anita Zamboni1, Marianna Fasoli1, Claudia Zadra2, Arianna Lovato1, Mario Pezzotti1, Massimo Delledonne1 and Annalisa Polverari1*
Author Affiliations
1 Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy
2 Department of Agricultural and Environmental Science, University of Perugia, B.go XX Giugno 72, 06121 Perugia, Italy
For all author emails, please log on.
BMC Genomics 2010, 11:117 doi:10.1186/1471-2164-11-117
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2164/11/117
Received:12 November 2009
Accepted:18 February 2010
Published:18 February 2010
© 2010 Polesani et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
Downy mildew is a destructive grapevine disease caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni, which can only be controlled by intensive fungicide treatments. Natural sources of resistance from wild grapevine (Vitis) species are used in conventional breeding approaches, but the signals and effectors involved in resistance in this important crop species are not well understood.
Results
Early transcriptional changes associated with P. viticola infection in susceptible V. vinifera and resistant V. riparia plants were analyzed using the Combimatrix microarray platform. Transcript levels were measured 12 and 24 h post-inoculation, reflecting the time points immediately preceding the onset of resistance in V. riparia, as determined by microscopic analysis. Our data indicate that resistance in V. riparia is induced after infection, and is not based on differences in basal gene expression between the two species. The strong and rapid transcriptional reprogramming involves the induction of pathogenesis-related proteins and enzymes required for the synthesis of phenylpropanoid-derived compounds, many of which are also induced, albeit to a lesser extent, in V. vinifera. More interestingly, resistance in V. riparia also involves the specific modulation of numerous transcripts encoding components of signal transduction cascades, hypersensitive reaction markers and genes involved in jasmonate biosynthesis. The limited transcriptional modulation in V. vinifera represents a weak attempted defense response rather than the activation of compatibility-specific pathways.
Conclusions
Several candidate resistance genes were identified that could be exploited in future biotechnological approaches to increase disease resistance in susceptible grapevine species. Measurements of jasmonic acid and methyl jasmonate in infected leaves suggest that this hormone may also be involved in V. riparia resistance to P. viticola.
Background
Plasmopara viticola (Berk. and Curt.) Berl. and de Toni is an oomycete pathogen that causes downy mildew in grapevine. This devastating disease causes partial or total crop losses and has a severe secondary environmental impact due to the repeated fungicide applications required as a control measure. P. viticola is an obligate pathogen that obtains nutrients from infected plant cells through specialized structures known as haustoria, which also allow the exchange of signals involved in the establishment of compatibility [1]. In susceptible grapevine genotypes, compatibility is probably achieved through a lack of recognition. Some oomycetes can secrete effectors that suppress host cell defense responses but such effectors have yet to be described in P. viticola [2,3].
Although European V. vinifera cultivars are highly susceptible to P. viticola, Muscadinia species and several American and Asian Vitis species exhibit varying levels of resistance, allowing quantitative trait loci (QTLs) and major resistance genes to be mapped [4-9]. Efforts to introgress these traits into cultivated V. vinifera genotypes by conventional breeding have produced some resistant interspecific hybrids, but further work is needed to couple strong resistance with high quality wine production [10]. This process will be greatly accelerated by the availability of the grapevine genome sequence [11,12] and high density genetic maps [13,14].
Detailed resistance mechanisms have been described in a few model species [15], and these often involve a signal transduction cascade triggered by infection which induces the resistance response. Plants can recognize general elicitors (or pathogen-associated molecular patterns, PAMPs) and specific elicitors encoded by pathogen Avr genes, as well as byproducts of pathogen activity (damage-associated molecular patterns, DAMPs), through a wide repertoire of receptors, with intriguing similarity to the innate immune system in animals [16,17]. Defense responses include strengthening the cell walls [18], the synthesis of pathogenesis-related (PR) proteins and antimicrobial compounds such as phytoalexins [19], and the hypersensitive response (HR), in which cells undergo programmed cell death in the infected region to block further spreading of the pathogen [20].
Wild American grapevine species may enjoy a higher level of constitutive resistance to P. viticola because of the higher basal level of certain antimicrobial compounds [21-25]. Post-infection resistance mechanisms have also been described in wild Vitis species, including the accumulation of reactive oxygen species, PR proteins, antimicrobial compounds, peroxidases and HR activation [26-31]. Although V. vinifera is susceptible to P. viticola, it can defend itself against other pathogens indicating the defense components are in place but are not activated in response to this pathogen [28]. The early signaling events underlying defense responses in grapevine have only recently been described [32-37] but a systematic survey of the V. vinifera genome has identified more than 200 resistance gene analogs, many localized in genomic regions associated with P. viticola resistance in wild Vitis spp. [12,38], as well as orthologs of Arabidopsis genes that regulate defense pathways [39,40].
In this paper we describe the early transcriptional changes associated with P. viticola infection in both susceptible Vitis vinifera and resistant Vitis riparia plants, performed on a Combimatrix Grapevine Microarray, the broadest transcriptomics resource available for Vitis species http://www.combimatrix.com/tech_microarrays.htm webcite. Transcriptomic approaches have been used to analyze plant-pathogen interactions in model species. Although several grapevine diseases have been investigated using Affymetrix [23,36,37] or Operon grapevine chips [33], P. viticola is not among them. Our study therefore provides the first broad overview of the molecular events underlying the early response to P. viticola infection in susceptible and resistant grapevine species and will provide valuable candidate genes that could be used to develop mildew-resistant commercial grapevine plants.
Results
P. viticola developmental stages
After inoculating plants with P. viticola, we followed the progress of the infection by looking at the developmental time-course of the pathogen. On that basis we chose which RNA samples were most suitable for microarray analysis. Leaf samples were collected at 12, 24, 48 and 96 hours post-inoculation (hpi) and stained with aniline blue for microscopy (Figure 1). Zoospores were localized over stomata by 12 hpi in both species, and germ tubes, primary hyphae and the first haustoria could be identified. By 24 hpi, further mycelium development appeared to be delayed in V. riparia. By 48 hpi, a mycelium network with many haustoria was observed in V. vinifera, whereas branched hyphae with only a few haustoria were observed in V. riparia. At 96 hpi, V. vinifera tissues were completely invaded by mycelia and heavy sporulation followed, whereas only small patches of mycelium were visible in V. riparia, and sporulation was severely impaired or absent. This established that the resistance response in V. riparia probably began within the first 24 hpi, and we therefore chose 12 and 24 hpi as the relevant time-points for microarray analysis.
Figure 1. Analysis of P. viticola infection steps. Infected leaf disks from V. vinifera (left panels) and V. riparia (right panels) were collected at 12, 24, 48 and 96 hpi, stained with 0.05% aniline blue and observed under an epifluorescence microscope. Panels A, B, C, D and F: magnification 200×; panels E, G and H: magnification 100×. Arrows indicate primary hyphae, arrowheads haustoria. Bars = 80 μm.
Reliability of hybridization data
Both phylogenetic analysis [41,42] and previous cross-species microarray analysis using Vitis species [22] suggested that a V. vinifera microarray should reliably detect transcriptional changes in V. riparia. However, a certain level of sequence divergence between the two species could increase the random noise in the hybridization data and possibly result in a lower correlation between V. riparia replicates compared to V. vinifera replicates. We tested healthy samples collected at 12 and 24 hpi, which served as controls in the infection experiments, and found no evidence for differences in the correlation between replicates for each species. The average Spearman's rank correlation coefficient (r) between V. vinifera replicates was 0.9678 (range 0.9536-0.9782), which was comparable to the V. riparia replicates (r = 0.9349; range 0.9017-0.9680).
We also checked the intensity distribution of log2-transformed data, the overall hybridization intensity and the number of absent calls (i.e. transcripts with a fluorescence signal below a calculated threshold, see Materials and Methods) for the two species. The intensity distributions of data derived from uninfected samples of each species were normal-like and similar. The average log2-transformed abundance values were 8.57 ± 2.07 and 7.40 ± 2.74 (across-replicate average ± SD) in V. riparia and V. vinifera, respectively. The number of probe sets assigned an absent call was 7,712 in V. riparia, and 7,306 in V. vinifera. These observations confirmed the reliability and comparability of the microarray results in the two grapevine species.
Interspecies differences in basal gene expression
Differences in basal gene expression between the two grapevine species were determined by comparing matched uninfected control samples for the steady-state levels of all 24,571 transcripts represented on the microarray. However, because it has been suggested that resistance in V. riparia could in part reflect constitutive physical or chemical barriers, we also focused on defense-related transcripts (i.e. those functionally associated with disease resistance, stress, the cell wall and secondary metabolism). Because the 12 hpi samples were harvested in darkness and the 24 hpi samples in daylight, data from the different time-points were normalized and compared separately to avoid the detection of genes regulated by light. We identified 5550 and 6379 transcripts with statistically significant differential expression at 12 and 24 hpi, respectively (Additional files 1 and 2). At both time points, ~ 48% of the differentially expressed transcripts were more abundant in V. riparia and ~ 52% were more abundant in V. vinifera. Broadly similar results were obtained when restricting the analysis to defense-related transcripts. Here ~ 45% of the differentially expressed transcripts were more abundant in V. riparia and ~ 55% were more abundant in V. vinifera (Additional files 1, 2 and 3).
Additional file 1. Differences in basal gene expression levels between the two species at 12 h after mock-inoculation with distilled water. The file contains a list of transcripts showing statistically significant differential expression, with a False Discovery Rate (FDR) ≤5%. The fold change of V. vinifera vs. V. riparia expression levels (Fold Change Vv/Vr) is reported, along with the q-value (%) indicating the FDR. A separate list reports the subset of defense-related genes, functionally categorized as 'resistance', 'stress', 'cell wall' and 'secondary metabolism' considered in Additional file 3.
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Additional file 2. Differences in basal gene expression levels between the two species at 24 h after mock-inoculation with distilled water. The file contains a list of transcripts showing statistically significant differential expression, with a False Discovery Rate (FDR) ≤5%. The fold change of V. vinifera vs. V. riparia expression levels (Fold Change Vv/Vr) is reported, along with the q-value (%) indicating the FDR. A separate list reports the subset of defense-related genes, functionally categorized as 'resistance', 'stress', 'cell wall' and 'secondary metabolism' considered in Additional file 3.
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Additional file 3. Comparison between defense-related genes in V. vinifera and V. riparia at 12 and 24 h after mock-inoculation with distilled water. Defense-related genes considered for the comparison are those functionally categorized as 'resistance', 'stress', 'cell wall' and 'secondary metabolism' and are shown in the 'defense-related' lists in Additional files 1 and 2. The tables on the left show the total numbers of genes whose basal expression is higher in V. riparia (overexpressed in Vr) or V. vinifera (overexpressed in Vv) within each category. The tables on the right report mean logarithmic fluorescence values of transcripts within each category (mean Vr and mean Vv), the ratio of the means calculated for each genotype and the resulting fold change. Microarray fluorescence data from the two time-points were normalized and analyzed separately to avoid detecting basal differences based on the response to illumination.
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To exclude genes regulated by light in only one of the species, we also retrieved the subset of 2176 transcripts present at both time points (Additional file 4). In this group, many transcripts were more abundant in one species at one time point but more abundant in the other species at the other time point, and there was a trend showing that 74-78% of such transcripts were more abundant in V. vinifera and 22-26% were more abundant in V. riparia, depending on which time point was examined. When restricting the analysis to defense-related transcripts, the results were almost identical (74-76% vs. 24-26%) (Additional files 4 and 5). Overall, these data indicated that resistance in V. riparia does not reflect differences in the basal expression of defense-related genes.
Additional file 4. Subset of transcripts showing a difference in basal expression level between V. vinifera and V. riparia at both the 12 and 24 h time points after mock-inoculation with distilled water. The file contains a list of transcripts showing statistically significant differential expression, with a False Discovery Rate (FDR) ≤5%. The fold change of V. vinifera vs. V. riparia expression levels (Fold Change Vv/Vr) is reported, along with the q-value (%) indicating the FDR. A separate list reports the subset of defense-related genes, functionally categorized as 'resistance', 'stress', 'cell wall' and 'secondary metabolism' considered in Additional file 5.
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Additional file 5. Comparison between defense-related genes in the subset of transcripts differentially expressed in the two species both at 12 and 24 h after mock-inoculation with distilled water. Defense-related genes considered for the comparison are those functionally categorized as 'resistance', 'stress', 'cell wall' and 'secondary metabolism' and are shown in the 'defense-related' list in Additional file 4. The tables on the left show the total numbers of genes whose basal expression is higher in V. riparia (overexpressed in Vr) or V. vinifera (overexpressed in Vv) within each category. The tables on the right report mean logarithmic fluorescence values of transcripts within each category (mean Vr and mean Vv), the ratio of the means calculated for each genotype and the resulting fold change.
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Transcriptional changes in V. vinifera and V. riparia in response to P. viticola infection
Figure 2 shows the total number of transcripts that are differentially expressed (fold change ≥2) in the two species at 12 and 24 hpi (full list provided in Additional file 6). In both species, the majority of modulated transcripts were upregulated.
Figure 2. Transcriptional changes associated with P. viticola infection. Piled histograms represent the number of genes induced (gray bars) or repressed (black bars) in V. vinifera (Vv) and V. riparia (Vr), at 12 and 24 hpi with P. viticola.
Additional file 6. Differential gene expression in V. riparia and V. vinifera following infection with P. viticola. The file lists transcripts showing a statistically significant differential expression (fold change ≥2, FDR ≤5%) in P. viticola infected samples of V. riparia (Vr) and V. vinifera (Vv), in comparison to their respective mock-inoculated controls, at 12 and 24 hpi. Species-specific and 'common' transcriptional changes associated with infection are also reported in separate lists for easier access.
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V. riparia responded very quickly to infection, with 733 transcripts modulated at 12 hpi (707 induced, 26 repressed) whereas only 124 were modulated in V. vinifera (all induced) at the same time-point. At 24 hpi, 339 transcripts were modulated in V. riparia (283 induced, 56 repressed) whereas 135 were modulated in V. vinifera (129 induced, 6 repressed). The transcripts were assigned to functional categories on the basis of literature evaluation (Figure 3). Although the 'unknown function' category was predominant in both species, there were important differences in other categories. In V. riparia, signal transduction components accounted for 18% of the modulated transcripts at 12 hpi (almost invariably induced by infection) falling to 9% at 24 dpi, metabolic functions accounted for 18% of the modulated transcripts at 12 hpi increasing to 27% at 24 hpi, and defense-related functions accounted for 8% of the modulated transcripts at 12 hpi increasing slightly to 11% at 24 hpi. In V. vinifera, defense-related functions accounted for 22% of the modulated transcripts at 12 hpi increasing to 24% at 24 hpi, whereas metabolism and signal transduction accounted for 10-15% of modulated transcripts at both time-points. Other functional categories each accounted for up to 6% of modulated transcripts in both species at both time-points. Considering that a significant proportion of the differentially expressed genes are modulated at both time points, there were 870 differentially expressed transcripts in V. riparia and 187 in V. vinifera, with many modulated at both time points. Transcripts showing the greatest induction in response to infection (30-80-fold) tended to be induced in both species, albeit to different levels. They were predominantly defense-related transcripts and are discussed in more detail below.
Figure 3. Functional categories of transcripts modulated in V riparia and V. vinifera following infection with P. viticola. Transcripts modulated in V. riparia (A) and V. vinifera (B) after infection with P. viticola at 12 hpi (left panels) and 24 hpi (right panels) were manually grouped in functional categories on the basis of literature evaluation. Induced genes are represented in light gray, while repressed ones are in black. The total percentage of modulated transcripts within each category is shown next to each bar. The complete list of genes is available in Additional file 6.
Common transcriptional changes in response to infection
Figure 4 shows the proportion of genes whose induction/repression in response to infection was observed in both species or was restricted to one or the other. This can be represented by a repartition of modulated transcripts by species, either at each time point (Figure 4A) or collectively (Figure 4B). We consider the second approach more useful because it defines modulations occurring in both species as common transcriptional changes, even though they may not occur at the same time. However, the first approach shows how specificity evolves over time, in some cases with different profiles in different functional categories.
Figure 4. Specificity of transcriptional changes in infected V. vinifera and V. riparia within selected functional categories. A. Proportion of transcripts modulated in V. vinifera (Vv) or V. riparia (Vr) or in both species at either 12 (upper panel) or 24 hpi (lower panel). B. Proportion of transcripts modulated in V. vinifera (Vv) or V. riparia (Vr) or in both species considering either time point collectively.
The data show clearly that most of the transcriptional modulation observed in V. riparia had no parallel in V. vinifera, indicating that many of the changes in all functional categories were restricted to V. riparia. In contrast, most of the transcriptional modulation observed in V. vinifera also occured in V. riparia (Figure 4A). However, when each species was considered separately at each time-point, it was clear that the number of transcripts uniquely modulated in V. vinifera increased from 12 to 24 hpi, possibly reflecting the establishment of a compatible interaction. Interestingly, the strength of modulation among the common genes was invariably much higher in V. riparia, at both 12 hpi (Figure 5A) and 24 hpi (data not shown).
Figure 5. Common transcriptional changes in V. vinifera and V. riparia following infection with P. viticola. A. Intensity of the transcriptional changes of 'common' genes in V. riparia and V. vinifera at 12 hpi. Each functional category is shown in a different color. B. Distribution of the 147 'common' genes, modulated in both species at one or both time points, into functional categories.
When we considered as 'common' any gene that is modulated in both species irrespective of the time-point, we detected 147 common transcripts, always modulated in the same direction in both species (Figure 5B and Additional file 6). Moreover, because the timing of the response is also relevant, it is notable that 30% of the common transcripts were modulated earlier in V. riparia (12 hpi) and later in V. vinifera (24 hpi), although there is no qualitative difference associated with this delayed response (Additional file 6).
After discounting transcripts with no assigned function, the largest proportion of common transcripts were related to disease resistance (22%, Figure 5B). Within this category, about half of the transcripts modulated in V. riparia were also modulated in V. vinifera, including several encoding stilbene synthases and PR proteins such as chitinases, β-1,3-glucanases and PR-10. The difference in expression between the species was especially notable for these genes (Figure 5A). After resistance, the next largest group of common transcripts was related to signal transduction (15%, Figure 5B). This group included many transcripts encoding WRKY transcription factors, all strongly induced by infection at both time points, but again much more strongly induced in V. riparia (6-22-fold in V. riparia; 2-5-fold in V. vinifera). Approximately 12% of the common transcripts had metabolic functions, including a cell wall apoplastic invertase and an alternative oxidase, both of which were induced to a greater extent in V. riparia. Only a few genes related to photosynthesis were modulated in both species, and these were downregulated by infection (Additional file 7).
Additional file 7. Representative V. riparia and V. vinifera transcripts modulated after infection with P. viticola. A selection of representative transcripts modulated in both species ('common') or specifically in V. riparia (Vr) or V. vinifera (Vv) after infection with P. viticola. Target descriptions are provided, corresponding to gene annotations in the source databases, along with the corresponding functional category and the microarray fold change (FC) value for each time point.
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Specific transcriptional changes in response to infection
Many transcriptional changes occurred solely in V. riparia, and the most prevalent functional categories among the modulated transcripts were general metabolism and signal transduction, the latter especially at 12 h. In the general metabolism category (22%; Figure 4B) most transcripts showed 2-3-fold induction, although a few were induced strongly, such as those encoding major enzymes in phenylalanine biosynthesis (up to 40-fold induction). Genes encoding enzymes in the Calvin cycle were repressed, in agreement with the decline in photosynthesis-related transcripts, whereas those involved in glycolysis and the pentose phosphate pathway were induced. Protein metabolism also appeared to be strongly influenced by infection, as shown by the large number of modulated transcripts related to ubiquitinylation, particularly those encoding different RING-H2 finger proteins, which are involved in proteolytic degradation (induced up to 14-fold). Transcriptional changes involving lipid metabolism included the upregulation of genes encoding biosynthetic and catabolic enzymes, and enzymes involved in jasmonic acid synthesis (e.g. allene oxide synthase and cyclase, omega-3 fatty acid desaturase). Several signal transduction pathways were affected including calcium signaling, ethylene signaling, MAP kinases, phosphatases, receptor-like proteins and numerous transcription factors. Overall, 68% of the signal transduction genes induced in V. riparia were never modulated in V. vinifera, and the vast majority were induced by 12 hpi (Figure 4; Additional file 6). Particularly strong modulation was observed for certain zinc-finger proteins (up to 16-fold induction) and WRKY genes (transient 3-4-fold induction) (Additional file 7).
We found that many resistance-related genes were induced to a greater or lesser extent in both species but those involved in the hypersensitive response were mostly restricted to V. riparia. These included several Avr9/Cf-9 rapidly elicited proteins [43] and a homolog of the tobacco Hin1 gene (12-fold induction) which is considered a HR marker [44]. Another HR marker, a homolog of the tomato hsr203J gene [45,46], was induced 40-fold in V. riparia and only 5-fold in V. vinifera at 12 hpi (Additional file 7).
There were few genes specifically induced in V. vinifera at 12 hpi, but the number increased substantially by 24 hpi. These genes represented several different functional categories and were not particularly informative with regard to the establishment of compatible interactions (Figure 4). Resistance and stress-related genes were well represented but on the whole it appeared that V. vinifera mounts a much less specific response to infection, which may be considered as an unsuccessful attempt to establish resistance.
Validation of microarray analysis by real-time RT-PCR
The microarray data for 10 differentially expressed transcripts, whose induction index varied from 0.1-fold to 34-fold at either 12 or 24 hpi, were validated by real-time RT-PCR analysis. As shown in Additional file 8, the magnitude of change determined by the more sensitive real-time RT-PCR technique was in accordance with the microarray data and in some cases revealed even greater differential expression, suggesting that the microarray results underestimated actual changes in gene expression.
Additional file 8. Real-Time RT-PCR analysis of selected genes. The figure reports the comparison of transcriptional changes of selected genes as determined by microarray (white bars) and Real-Time RT-PCR analysis (black bars). The black bars indicate the average fold change obtained for the three independent biological replicates, and the error bars indicate the standard deviations. Individual fold change values and standard errors for each Real-Time experiment are available in Additional file 9.
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Determination of jasmonate levels in infected leaves
The microarray data indicated that genes encoding enzymes involved in biosynthesis of jasmonic acid were strongly induced in V. riparia shortly after infection. We therefore measured the amount of jasmonic acid (JA) and methyl jasmonate (MeJA) in the leaves of both species before infection and at the four post-infection time-points discussed above. The basal levels of MeJA were higher in V. riparia than in V. vinifera. There was a sharp increase in the levels of both jasmonic acid and MeJA in V. riparia leaves 48 hpi, which was followed by rapid decline to below pre-infection levels (Figure 6). In V. vinifera, there was no change in the basal level of jasmonic acid after infection and only a limited increase in MeJA levels at 24 and 48 hpi.
Figure 6. Endogenous levels of jasmonic acid and MeJA in V. riparia (Vr) and V. vinifera (Vv). Measurements were taken using leaf samples collected at 12, 24, 28 and 96 hpi with P. viticola (Pv) or on the mock-inoculated control samples (w) at the corresponding time-points. Values are the average of three measurements, with standard errors.
Discussion
Analysis of P. viticola developmental stages
Infected tissues were examined under a microscope at 12, 24, 48 and 96 hpi, to determine the most suitable time-points for microarray analysis and to observe sporulation. The localization of zoospores over stomata at 12 hpi in both V. riparia and V. vinifera confirmed previous reports that zoospores can locate stomata with equal efficiency in susceptible and resistant species [27,30]. Restriction of pathogen growth in V. riparia is a post-infection phenomenon that begins when the first haustoria enter mesophyll cells, resulting in the thickening of cell walls, necrosis of guard cells, the accumulation of phenolics and peroxidases, and in some cases a hypersensitive reaction depending on environmental conditions [9,30,47]. This correlates well with the specific induction of genes related to hypersensitivity and phenylpropanoid synthesis. Pathogen spread was severely impaired between 24 and 48 hpi in comparison to V. vinifera, suggesting that the resistance mechanism is already in effect before this time point, consistent with the strong transcriptional reprogramming observed at 12 hpi, when the first haustoria form.
Reliability of hybridization data
Because we used a V. vinifera microarray to assess differential gene expression in V. vinifera and V. riparia we performed experiments to confirm the reliability of cross-species hybridization. The successful outcome was not unexpected because V. vinifera arrays have previously been hybridized with RNA from other Vitis species [22,23,48]. Indeed, cross-species microarray hybridization is widely used in animals and plants [49-52], and although the data must be interpreted with caution, it remains a valid approach when dealing with groups of closely related species where sequence information is only available for one member [53]. The average signal intensity and the number of absent calls in the hybridization data were similar in V. riparia and V. vinifera, and comparison of replicates within each species suggested a similar level of variation. This probably indicates that polymorphisms within each species provide nearly as much sequence variation as the differences between species, as previously shown by singlenucleotide polymorphism analysis [54]. Moreover, the only direct comparison between V. vinifera and V. riparia was performed to assess differences in basal gene expression, while most of the comparisons were made between sampling time points in the same species, preventing such misinterpretation of hybridization results.
Interspecies differences in basal gene expression
The comparison of basal gene expression in healthy V. vinifera and V. riparia plants 12 and 24 h after a mock infection procedure revealed substantial variation in the expression of thousands of genes, but no overall bias towards either species.
V. riparia is a major source of resistance against P. viticola [4,6,13,55,56] and although major resistance genes have been identified [8] it has been suggested that some resistance may be conferred by constitutive differences in defense-related gene expression. We therefore focused on defense-related transcripts (resistance, stress, cell wall and secondary metabolism categories) to see if there were any broad trends. Although the levels of individual transcripts varied widely, overall levels were similar in the two species (Additional file 3).
The 'cell wall' category contained more transcripts expressed preferentially in V. riparia and the average signal intensity was also higher, but the differential expression of various cell wall enzymes did not explain how the modified cell wall might help to prevent pathogen spread. The 'resistance' and 'stress' categories, in contrast, included more transcripts preferentially expressed in V. vinifera. Many grapevine species accumulate stilbene derivatives, such as resveratrols and viniferines, in response to pathogens [57,58] and we found that one stilbene synthase was preferentially expressed in V. riparia at 12 hpi, two were more abundant in V. riparia at 24 hpi, whereas five were more abundant in V. vinifera. Several PR protein genes were also more strongly expressed in V. vinifera, which is perhaps surprising because the genes are strongly induced by infection in V. riparia but not in V. vinifera. These data confirm that the response to P. viticola infection in V. riparia is not mediated by higher constitutive expression of defense genes and is essentially a post-infection process [26,28,30].
The absence of any significant differential expression of 'secondary metabolism' transcripts in pre-infection samples supports this conclusion, given that secondary metabolism, especially the phenypropanoid pathway, is often considered an important component of plant resistance [59]. In a previous microarray-based comparison of a susceptible and a resistant V. vinifera cultivars, Figueiredo and co-workers [21] identified only 12 genes preferentially expressed in the uninfected resistant cultivar, one of which encoded phenylalanine ammonia lyase, whereas 17 genes were preferentially expressed in the susceptible cultivar. Other authors have reported that stilbene synthase and phenylalanine ammonia lyase mRNA are not detected in healthy leaves but are induced by infection or abiotic stresses, proportionally to the resistance phenotype observed and are therefore considered elicitor-induced responses [24,25].
In the subset of transcripts showing differential basal expression at both time points, about 75% were more strongly expressed in V. vinifera and about 25% were more strongly expressed in V. riparia. When the analysis was restricted to defense-related transcripts the same broad trend was observed. Taken together, these findings suggest there is a stronger diurnal fluctuation in basal gene expression in V. riparia compared to V. vinifera, but provide no evidence that the resistance phenotype in V. riparia is caused by the constitutive expression of resistance genes maintaining a constant state of readiness.
Broad transcriptional changes associated with P. viticola infection
The infection of both species with P. viticola results in the rapid induction of many genes, although their number and the magnitude of induction are much greater in V. riparia (Figure 3). Transcript profiling in other grapevine diseases [23,33,36,37] has focused on compatible interactions, for which large transcriptional changes are observed. The only incompatible interaction studied in this manner is that between V. aestivalis and the powdery mildew agent Erysiphe necator [23]. This is another biotrophic, haustoria-forming grapevine pathogen, which might be expected to adopt strategies similar to P. viticola with similar consequences. In V. aestivalis only three genes were shown to be modulated by infection by E. necator. The same authors also investigated the compatible interaction with V. vinifera, which responded with a broad remodeling of the transcriptome. Our data show that both V. vinifera and V. riparia respond to downy mildew infection with a massive transcriptional change, which is much more pronounced in the resistant species as suggested by several large scale analyses of incompatible interactions in other species [60-63]. Many similarities can be identified between the responses against powdery and downy mildew in V. vinifera based on the annotation of probes on the chips, although a complete and detailed comparison cannot be carried out because different array platforms were used in each case.
Overlapping transcriptional responses to infection in V. vinifera and V. riparia
As expected, there were overlaps in the transcriptional changes in each species in response to infection, with most of the genes induced in V. vinifera constituting a weak subset of those induced in V. riparia at the same time-points (Figures 4 and 5). The limited response in V. vinifera appears to reflect an abortive attempt to achieve resistance, since most of the common modulated transcripts fall into the 'resistance' category (Figure 5). The activation of genes encoding PR proteins and enzymes in the phenylpropanoid pathway was anticipated based on data from model species [19,59]. Interestingly, many of the common modulated transcripts are not only expressed at higher levels in V. riparia than V. vinifera, but also at higher levels than the genes in the same family that are uniquely expressed in V. riparia, e.g. PR-10, stilbene synthases and WRKY transcription factors. For example, the six WRKY genes whose induction is common to both species (TC59548, TC66456, TC71038, TC57604, TC53734, TC68615) are induced 6-22-fold in V. riparia, whereas those solely expressed in V. riparia are induced 2-5-fold (TC60897, TC51831, TC51732, TC53072, TC55553, TC64282). It therefore appears that V. vinifera can only weakly execute those responses that are strongly induced in V. riparia.
It is interesting to highlight the induction of an apoplastic invertase (TC56057), a sink-specific enzyme that catalyzes the irreversible cleavage of sucrose into hexoses, both in V. vinifera and V. riparia (2-3-fold and 7-9-fold, respectively). The rapid induction of invertase activity has also been observed in tomato roots resistant to the necrotrophic fungal pathogen Fusarium oxysporum [64]. Likewise, in barley challenged with powdery mildew, an apoplastic invertase was induced more strongly and rapidly in a resistant cultivar [65]. Hexoses produced by the invertase could be seen as a nutrient source for pathogens, but also as a supply of extra energy required for the activation of defense responses [66,67] whose accumulation might suppress photosynthesis in line with our data on photosynthetic genes. Most importantly, sugar can also be used to trigger defense gene expression [68,69] hence the suggestion to consider apoplastic invertase as a true PR protein [66].
All the common genes were modulated in the same direction by both species, indicating they probably fulfill the same functions in defense. Inverse regulation of the same gene in genotypes with different infection outcomes could be interpreted as part of a pathogen defense suppression strategy [70]. Indeed, susceptibility to P. viticola is associated with broad downregulation of gene expression at later time-points [71] but our data show that such downregulation does not occur early in the infection.
Quantitative and kinetic differences between compatible and incompatible interactions have been elegantly described in Arabidopsis [61]. The incompatible interactions produced a more robust and intense transcriptional response and the proposed quantitative model suggested that a high level input signal is generated in resistant plants in response to infection, determining the robustness of the system.
The specific transcriptional response in V. riparia
Although both species responded to infection with broad changes in gene expression, the response was strongest and fastest in V. riparia, with a peak of gene induction at 12 hpi. This response had transient and permanent components, since the expression of about half the genes fell back by 24 hpi (Figure 2). The strong transcriptional response of V. riparia together with its histological reactions to the pathogen is reminiscent of R-gene dependent resistance in other species [16], although the molecular determinants are unknown in this case.
When transcripts with unknown functions are excluded, the genes induced specifically in V. riparia fall into a number of functional categories whose expression appears to be coordinated. At 12 hpi, many genes encoding signal transduction components are induced, and this is followed by a wave of metabolic genes that are induced 24 hpi. This may indicate that an initial burst of signaling activity reprograms metabolism to provide a 'defense mode'. Among the different signaling pathways affected, calcium is known to be an important second messenger in resistance [72] as shown by the induction specifically in infected V. riparia, of calmodulins and calmodulin-binding proteins, calcium transporting ATPases, and proteins with similarity to calreticulin and calcineurin B-like proteins, all known to contribute to calcium homeostasis in the cell and to the definition of specific calcium signatures [73]. Several different ethylene response factors are also strongly induced solely in V. riparia at 12 hpi, and this hormone has also been implicated in resistance [74]. The possible involvement of ethylene in P. viticola resistance is further supported by the very strong induction of the ACC oxidase gene TC64623 (20-fold in V. riparia compared to only 3-fold in V. vinifera) and the 5-fold induction of an ACC synthase gene (TC60326) specifically in V. riparia.
Several genes with homology to known receptor-like protein kinases and leucine-rich repeat receptor-like proteins are specifically induced in V. riparia, especially at 12 hpi. These genes are known to mediate pathogen recognition and trigger defense responses in many species [75]. Although the ligands for these receptors are unknown, hundreds of genes encoding receptor-like proteins have been identified in V. vinifera [12,13], some of which map in linkage groups associated with resistance. Two MAP kinase kinase genes (TC62930, TC53469) were induced specifically in V. riparia at 12 hpi, consistent with the upregulation of three MAP kinases, two specifically in V. riparia at 12 hpi (TC66292, TC56256) and one also induced in V. vinifera at 24 hpi (TC61436). Interestingly, the TC66292 and TC56256 genes are related to Arabidopsis MAP kinase 3 (MPK3), the ortholog of tobacco wound-induced protein kinase (WIPK), which acts together with salicylic acid-induced protein kinase (SIPK) in resistance responses [76]. The absence of a SIPK homolog among our induced genes is consistent with its predominantly post-translational mode of regulation [77].
Several families of transcription factors are also specifically upregulated in V. riparia, especially WRKY factors and other zinc-finger proteins. WRKY factors are regulated by interaction with MAP kinase in other species [78,79] which provides a link in the signaling network we have outlined above. WRKY factors bind to DNA motifs known as W-boxes which are often found in defense genes, so they are regarded as important regulators of resistance [80].
It is well established that primary metabolic reprogramming underlies defense in biotrophic interactions and many genes in this category are specifically induced in V. riparia. Further analysis of our data suggests that specific pathways are involved: gycolysis (GADPH, enolase), the pentose phosphate pathway (glucose 6-phosphate dehydrogenase) and the Krebs cycle (pyruvate dehydrogenase, citrate synthases, succinyl-CoA ligase) are all induced, and could supply both energy and precursors for the biosynthesis of aromatic amino acids. Indeed, we observed the strong and specific induction of a group of genes controlling all the key steps in phenylalanine biosynthesis, including genes with homology to 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases (6-30 fold at 12 hpi), chorismate synthase and mutase, and prephenate dehydratase, correlating with the induction of PAL (GSVIVT00013936001) and other genes involved in the hydroxycinnamic acid biosynthesis. Enzymes involved in lipid metabolism are also induced specifically in V. riparia. These include enzymes involved in lipid synthesis (e.g. acetyl-CoA carboxylase, β-ketoacyl-CoA synthase) and degradation (e.g. 13-lipoxygenase, acyl-CoA oxidase, acetoacetyl-CoA thiolase), and enzymes involved in the synthesis of jasmonates (omega-3 fatty acid desaturase, allene oxide cyclase, allene oxide synthase).
Genes encoding anti-oxidant enzymes and genes involved in protein degradation are also strongly and specifically induced in V. riparia, e.g. many RING-H2 domain proteins involved in ubiquitinylation are induced at 12 hpi. Interestingly, a rice RING-H2 protein associated with incompatible (but not compatible) interactions with Magnaporthe grisea is induced following treatment with different resistance-inducing chemicals, and transgenic plants constitutively expressing this gene are resistant to several pathogens, as well as drought and oxidative stress [81]. This demonstrates how modulated transcripts identified in our experiments provide promising candidates for biotechnology-based disease resistance programs.
Surprisingly, 'resistance' as a functional category, is relatively poorly represented among genes expressed specifically in V. riparia, many of them instead being common to both species. However, as already stated, many of the common resistance genes are more strongly modulated in V. riparia, and the V. riparia-specific group does include a number of genes strictly related to hypersensitivity, such as those encoding rapidly elicited Avr9/Cf-9 proteins (e.g. TC63609, TC61603) [43], two hypersensitive-induced response proteins (TC63023, TC63883) and two homologs of known HR markers in other species - tobacco Hin1 [44] and tomato hsr203J [45,46] - both of which are specifically or preferentially induced in V. riparia at 12 hpi. The HR has previously been implicated in resistance response to downy mildew in V. riparia [27]. Several additional defense genes are strongly induced in V. riparia, including those encoding PR proteins (such PR-4 and PR-10) and enzymes involved in the synthesis of antimicrobial compounds, as already reported in grapevine infected with powdery and downy mildew [23,28].
The specific transcriptional response in V. vinifera
Although most modulated transcripts in V. vinifera are also modulated in V. riparia, there is a small collection of genes induced specifically in V. vinifera. The genes involved in this specific response do not suggest any coordinated and explicit mechanism related to the establishment of compatibility in V. vinifera. It is possible that the analysis of early transcriptional changes provides more information on resistance than susceptibility (the former involving a pro-active transcriptional response by the plant) and transcriptional changes associated with compatibility are established later [71].
Jasmonate levels in healthy and infected plants
Resistance to biotrophic pathogens is often dependent on salicylic acid-mediated defense responses [82]. Jasmonates were originally associated with defense against herbivores and necrotrophic pathogens [83] but have more recently been implicated in resistance against biotrophes, such as powdery and downy mildews in Arabidopsis and in grapevine [84-87] and in resistance induced by BABA and by β-1,3-glucan sulfate against P. viticola [88,89]. Jasmonates interact with other danger signals such as salicylic acid and ethylene to determine the ultimate outcome of an infection, in a manner dependent on the specific plant-microbe interaction. Our data support a role for jasmonates in establishing or maintaining V. riparia resistance against P. viticola, given the significant increase in the levels of both jasmonic acid and MeJA at 48 hpi only in this species, concomitant with the effective arrest of pathogen growth, although much later in comparison to the transcriptional reprogramming described above. More experiments are needed to determine the precise timing of this accumulation in relation to pathogen arrest and to reveal how much of the response to P. viticola can be considered jasmonate-dependent in grapevine.
Conclusions
We compared two grapevine species, V. riparia and V. vinifera, the former resistant to the pathogen P. viticola and the latter susceptible to infection. Comparative transcriptome analysis of healthy leaves and leaves representing two early infection stages allowed us to characterize the molecular events involved in the establishment of resistance in Vitis riparia.
Our data strongly support the view that resistance in Vitis riparia is a post-infection phenomenon, characterized by a rapid wave of signal transduction (12 hpi) followed by a shift in primary and secondary metabolism (24 hpi) to implement a defense mode. In contrast, early transcriptional changes in V. vinifera indicate a weak and abortive defense response and do not provide information about the possible downregulation of resistance mechanisms by pathogen effectors, which might occur later on. Basal levels of defense gene expression in the two species do not seem to be responsible for the different infection outcomes.
The upregulation of genes involved in jasmonic acid biosynthesis and the increase in jasmonate levels indicate that this hormone may play a role in V. riparia resistance against P. viticola, although signal transduction-related genes are already upregulated before a detectable increase of jasmonate accumulation. Our broad comparative characterization of resistant and susceptible phenotypes has provided several candidate genes that could be used for additional functional analysis and for the development of disease-resistant commercial grapevine varieties in the future.
Methods
Plant material and P. viticola infections
Vitis vinifera cv. Pinot Noir and Vitis riparia cv. Gloire de Montpellier plants were grown in vitro at 27°C with a 16-h photoperiod (50 μE/m2/s) as described by Blaich [90]. The P. viticola isolate was harvested in experimental fields in 2007 and propagated axenically on surface-sterilized detached Pinot Noir leaves maintained in Petri dishes. Five days after inoculation, sporangia were collected from freshly-sporulating leaves using a microtip equipped with a nylon filter and connected to a vacuum pump. In order to obtain uncontaminated sporangia, the inoculum was repeatedly propagated under axenic conditions on plants growing in vitro.
Fully expanded leaves of 8-10-week-old in vitro plants were infected by applying 50-μl drops containing 50,000 sporangia per ml on the adaxial leaf surface (or distilled water as a control). The concentration of sporangia was determined using a hemocytometer. For microscopy, leaf disks were collected 12, 24, 48 and 96 hpi, stained with 0.05% (w/v) aniline blue in 0.1% (w/v) Na2CO3 (pH 10), and observed under an epifluorescence microscope (Leica DM/RB, excitation filter BP 340-380 nm; dichroic mirror 400 nm; suppression filter LP > 430 nm). For microarray analysis, leaf disks were collected 12 and 24 hpi, immediately frozen in liquid nitrogen and stored at -80°C. Three independent biological replicates of the artificial infection were performed.
Combimatrix array conception
The analysis was performed on a Combimatrix Vitis vinifera chip produced by the Plant Functional Genomics Center at the University of Verona. The chip contained 24,571 non-redundant probes in triplicate, composed of 35-40-mer oligos. Probes were designed using the program oligoarray 2.1 [91] and were based on tentative consensus sequences (TCs) derived from the TIGR Vitis vinifera Gene Index release 5.0 (19062 probes), singletons with a 3' poly(A) tail (1904 probes), expressed sequence tags (55 probes) and on genomic sequences produced by the International Grape Genome Project [11] that were not already represented by the TCs (3490 probes). TC annotations were derived from the TIGR Gene Index, release 5.0 and EST annotations were obtained by aligning sequences against UniProtKB/Swiss-Prot database with BLASTX. Nine bacterial oligonucleotide sequences provided by CombiMatrix, 40 probes designed on seven Ambion spikes and 11 additional negative probes based on Bacillus anthracis, Haemophilus ducreyi and Alteromonas phage sequences were used as negative controls. Three or four replicates of each probe were distributed randomly across the array. Two technical and three biological replicates were used for each hybridization experiment.
RNA preparation, hybridization and microarray analysis
RNA was isolated according to Reid et al. (2006) and quantified by spectrophotometry (ATI Unicam) and using an Agilent 2100 Bioanalyzer. Total RNA (1 μg) was amplified using the SuperScript Indirect RNA Amplification System (Invitrogen, USA), to incorporate amino-allyl UTP molecules (aRNA) and a fluorescent label (Alexa Fluor 647). The purified, labeled aRNA was quantified by spectrophotometry and 4 μg was hybridized to the Combimatrix array according to the manufacturer's directions. Pre-hybridization, hybridization, washing and imaging were performed according to the manufacturer's protocols http://www.combimatrix.com/support_docs.htm webcite. The array was scanned with a ScanArray 4000XL (Perkin-Elmer, USA) and TIF images were exported to Microarray Imager 5.8 (CombiMatrix, USA) for densitometric analysis. Probe signals higher than negative control values plus twice the standard deviation were considered as 'present'. Data were normalized by quantile normalization and differentially expressed genes were identified using the Two Class Unpaired Statistical Analysis of Microarrays method [92] with a False Discovery Rate (FDR) < 5%. Expression data are available from the National Center for Biotechnology Information (NCBI) [GenBank: Gene Expression Omnibus accession number GSE18596].
Real-Time RT-PCR
Real-Time RT-PCR experiments were carried out in biological triplicates with the same RNA samples taken for microarray analysis, using the SYBR® Green PCR master mix (Applied Biosystems, Foster City, CA, USA) and the Mx3000P Real-Time PCR System (Stratagene, La Jolla, CA, USA). Complementary DNA was synthesized from DNase-treated total RNA using the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). Gene-specific primers were designed for the 10 target genes as well as the actin transcript TC81781 (see Additional file 9). Each 25-μl reaction comprised 300 nM each primer and cDNA synthesized from 40 ng of total RNA (three replicates for each reaction) and began with a 50°C hold for 2 min and a 95°C hold for 10 min followed by 40 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 20 s. Non-specific PCR products were identified by analyzing dissociation curves. The amplification efficiency was calculated from raw data using LinRegPCR software [93]. The relative expression ratio value was calculated for treated samples relative to the corresponding untreated sample at the same time-point according to the Pfaffl equation [94]. SE values were calculated according to Pfaffl et al. [95].
Additional file 9. Details of the Real-Time RT-PCR analysis. The file contains: the sequence ID of each gene analyzed by Real-Time RT-PCR; the corresponding primer pairs used for the amplification (FOR = forward primer, REV = reverse primer); an indication of the region amplified by each primer pair (3' UTR = 3'untranslated region; CDS = coding sequence; CDS-probe = region of the coding sequence covered by the microarray probe; CDS-3' UTR = region between the coding sequence and the 3'untranslated region); the time point after treatment at which the leaves were sampled; the Real-Time RT-PCR results, reported as fold change (FC) relative to the untreated control sample and with the standard error (SE), for each species (Vv = V. vinifera Vr = V. riparia) and divided in biological replicates (1, 2 and 3); the mean values of the FC for the three replicates for each genotype. The corresponding microarray results for the same transcripts are also reported as fold change at the end of the list.
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Analysis of endogenous jasmonic acid and methyl jasmonate levels
Frozen plant material (500 mg fresh weight) was pulverized under liquid nitrogen, mixed with 4 ml methanol and filtered into a vial. After repeating this procedure twice, the extract was divided into two aliquots and the solvent evaporated under nitrogen at room temperature. To estimate the jasmonic acid content, 2 ml of ethereal trimethylsilyldiazomethane (2M in diethyl ether, Sigma-Aldrich) was added to the dried sample and incubated for 30 min before stopping the reaction under a gentle stream of nitrogen. The dried sample was mixed with 1 ml 30% NaCl and methylated jasmonic acid was extracted by solid phase micro-extraction (PDMS 100 μm film thickness, Supelco) while stirring at 60°C for 30 min. Blank analyses were carried using saline. Preliminary recovery studies were performed by adding known amounts of jasmonic acid (5, 10, 50, 100 and 200 ng) to grapevine leaf tissue prior to extraction, with recovery in the range 85-93%.
To estimate the levels of endogenous MeJA, plant material was extracted by solid phase micro-extraction without the derivatization step. The amount of endogenous MeJA was then subtracted from the total methylated jasmonic acid level to calculate the concentration of JA in the samples [96]. The limit of detection for jasmonic acid as MeJA was 2 ng/g.
GC-analysis was performed with a Varian CP-3800 (Varian Inc., Palo Alto, CA, USA) equipped with a 1177 split/splitless injector, a Factor-Four 5 capillary column (Varian 30 m, ID 0.25 mm, F.t. 0.25 μm), a FID detector and a Galaxie Workstation software (Varian Inc.) [96]. GC-MS analyses were also used to confirm the efficacy of the methylation procedure with a Varian Saturn 2100 GC-MS operating in the electron impact mode (EI), equipped with a multiple-ion detector and a Factor-Four 5 capillary column (Varian 30 m, ID 0.25 mm, F.t. 0.25 μm) as described [96].
Authors' contributions
MP performed grapevine infections, microscopic examinations, RNA extractions and microarray hybridizations. LB helped writing the manuscript and prepared all Figures and Additional files. AF collaborated in designing the Combimatrix grapevine gene chip, in fluorescent data extraction, and performed microarray statistical analysis. AZ collaborated in statistical analysis of microarray and real time RT-PCR data. MF performed real time RT-PCR experiments. CZ performed jasmonic acid and methyl jasmonate measurements. AL was responsible for growing in vitro plants and for P. viticola maintenance and collaborated to infection experiments. MPz and MD hold the responsibility of the Plant Functional Genomic Centre that produced the Combimatrix grapevine gene chip and collaborated to experimental design. AP conceived the study, participated in all steps of the analysis and wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We are grateful to Dr. Andreas Kortekamp for helpful suggestion on in vitro grapevine cultivation and for critical reading of the manuscript. This work was supported by the project 'Programma quadriennale per il completamento e l'attività del Centro di Genomica Funzionale Vegetale dell'Università degli Studi di Verona' granted by the Cariverona Bank Foundation and by the project 'Structural and functional characterization of the grapevine genome (VIGNA)' granted by the Italian Ministry of Agricultural and Forestry Policies (MIPAF).
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Research article
Analysis of the melon (Cucumis melo) small RNAome by high-throughput pyrosequencing
Daniel Gonzalez-Ibeas1, José Blanca2, Livia Donaire3, Montserrat Saladié4, Albert Mascarell-Creus5, Ana Cano-Delgado5, Jordi Garcia-Mas4, Cesar Llave3 and Miguel A Aranda1*
Author affiliations
1 Departamento de Biología del Estrés y Patología Vegetal, Centro de Edafología y Biología Aplicada del Segura (CEBAS) - CSIC, Apdo. correos 164, 30100 Espinardo (Murcia), Spain
2 Departamento de Biotecnología, Instituto de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV) - UPV, Camino de Vera s/n, 46022 Valencia, Spain
3 Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas (CIB) - CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
4 IRTA, Center for Research in Agricultural Genomics CSIC-IRTA-UAB, Campus UAB, Edifici CRAG, Bellaterra (Cerdanyola del Vallès), 08193 (Barcelona), Spain
5 Molecular Genetics Department, Center for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB, Campus UAB, Edifici CRAG, Bellaterra (Cerdanyola del Vallès), 08193 (Barcelona), Spain
For all author emails, please log on.
Citation and License
BMC Genomics 2011, 12:393 doi:10.1186/1471-2164-12-393
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2164/12/393
Received:12 April 2011
Accepted:3 August 2011
Published:3 August 2011
© 2011 Gonzalez-Ibeas et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21-24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon.
Results
We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analysed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs.
Conclusion
We have discovered and analysed a large number of conserved and melon-specific sRNAs, including miRNAs and their potential target genes. This provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melon-virus interactions.
Background
Melon (Cucumis melo L., family Cucurbitaceae) is an important horticultural species cultivated in temperate, subtropical and tropical regions worldwide, with Spain being the largest producer in Europe and fifth in the world [1]. The melon genome has 12 chromosomes and is thought to contain 450-500 Mb of DNA, which is 3-4 times more than Arabidopsis [2]. Melon is a useful model for the analysis of fruit traits because of the vast morphological, physiological and biochemical diversity within the species, which can be exploited to dissect the biological processes controlling color, flavor and texture and how these properties arise during fruit development [3,4].
Despite the importance of melon, not much was available in the way of genomic sequence information prior to the establishment of a functional genomics consortium in 2004, which developed a range of tools and accumulated more than 33,000 expressed sequence tags (ESTs) and ~17,000 tentative consensus sequences (unigenes) [5]. This EST collection has been expanded recently with the addition of 94,000 new ESTs from full-length enriched cDNA and standard cDNA libraries from various melon tissues and cultivars in the framework of the International Cucurbit Genome Initiative [6]. These ESTs as well as other resources are now accessible in a public database [7]. The unigene sequences have also been used to construct an oligonucleotide microarray, which has been applied in the analysis of fruit quality traits, ovary development and pathogen resistance [8]. In addition, a melon sequencing consortium has recently produced a high-quality draft of the melon genome (unpublished data). Although these resources provided significant advances in the analysis of melon gene expression, the small RNA (sRNAs) component of the melon transcriptome has not been studied in detail. These important molecules have been studied in other crop species and have been shown to fulfill a number of critical regulatory roles [9-12].
sRNAs are short, non-coding RNAs 21-24 nucleotides (nt) in length which are found in protists, fungi, plants and animals [13]. In plants, their roles include maintenance of genome stability, initiation of heterochromatin assembly, post-transcriptional regulation of gene expression and protection against viruses using an RNA-based immune system. The most abundant and best-characterised sRNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are widely studied because of their regulatory activity, particularly in development, pathogen resistance and stress responses [13]. miRNAs are cleaved from stem-loop precursor molecules that derive from single stranded non-coding transcripts. miRNAs regulate protein-coding genes post-transcriptionally by mediating RNA cleavage or translational repression. Unlike miRNAs, siRNAs are generated from double-stranded RNA precursors and function on cognate RNA or DNA molecules by instigating degradation or promoting RNA-directed DNA methylation, respectively. cis-acting siRNAs (ca-siRNAs) arise from and target endogenous loci such as transposons and DNA repeats to direct cytosine methylation and chromatin modifications [14]. Natural antisense-transcript siRNAs (nat-siRNAs), which derive from pairs of natural-antisense transcripts, guide the cleavage of one of the two parent transcripts, leading to the production of a series of secondary 21-nt siRNAs of unclear function [15,16]. Finally, trans-acting siRNAs (ta-siRNAs) derived from TAS genes, which transcribe long primary non-coding RNAs as precursors for ta-siRNA biogenesis. TAS primary RNAs are cleaved by specific miRNAs and are sequentially processed into 21-nt ta-siRNAs starting from the miRNA-cleaved end, to generate clusters of phased siRNAs [17,18]. In addition to endogenous sRNAs, exogenous siRNAs from virus genomes can be detected in virus-infected plants as a part of the RNA-based immune system [19].
RNA viruses that infect melon are responsible for significant yield losses as well as poor fruit quality [20,21], particularly the widespread Watermelon mosaic virus (WMV, genus Potyvirus, family Potyviridae) [22,23]. Recently, a collection of accessions representing cultivated melon and its wild relatives was screened to identify sources of resistance to mosaic-inducing viruses [24]. TGR-1551 was identified as a resistant accession based on the lower WMV titer compared to susceptible genotypes (e.g. melon cv. Tendral) and the absence or mildness of the mosaic symptoms normally observed in systemically infected leaves [25]. Melon necrotic spot virus (MNSV, genus Carmovirus, family Tombusviridae), although less economically important, may also cause yield losses, and epidemic outbreaks have been reported worldwide [26,23]. In melon, resistance to MNSV is controlled by the single recessive gene nsv, which encodes eukaryotic translation initiation factor 4E (Cm-eIF4E) [27]. This resistance is effective against all MNSV strains (e.g. MNSV-Malfa5) except MNSV-264 [28]. Studies of chimeric viruses have shown that the MNSV 3' untranslated region (3'-UTR) contains the resistance-breaking determinant of MNSV-264, and that it functions as a cap-independent translational enhancer [29,30].
We constructed 10 sRNA libraries from a range of healthy and virus-infected melon tissues, and we sequenced a set of endogenous and exogenous sRNAs using the pyrosequencing-based 454 technology from Roche [31]. To gain insights into the role of sRNAs on key aspects of fruit development, maturation and pathogen defense, samples from two stages of the developing ovary, fruits 15 and 45 days after pollination, and photosynthetic cotyledons from resistant and susceptible melon accessions infected with WMV and MNSV were analysed. In a previous study, we reported the profile of virus-derived sRNAs (viRNAs) from cotyledon samples [32]. Here we report a catalog of endogenous melon sRNAs, including miRNAs from known families and new candidate miRNAs potentially unique to melon, focusing on the number of sequence reads as a reflection of their expression profiles. Potential targets for these miRNAs in the melon transcriptome were identified.
Results
cDNA libraries and sequencing of small RNAs
We used high throughput sequencing data to analyze the composition of the small RNA transcriptome (sRNAome) of melon and compare the results to data in publicly-available RNA and genomic databases. Ten sRNA libraries were constructed from total RNA extracted from fruits, ovaries and healthy and virus-infected melon cotyledons (Table 1). PCR amplification products corresponding to each library were pooled in equal amounts and sequences were obtained by multiplexed high-throughput pyrosequencing (Roche 454). This produced 447,180 raw sequences, each ~100 bases in length, 432,743 of which had a complete 3' adaptor in the correct position. Based on these data, we estimated a sequencing error rate of 3.7%. After removing reads where one or the two adaptors could not be identified, 398,450 useful sequences with 3' and 5' adaptors were selected. Only 44 sequences comprising ligated adaptors without an insert were identified. Although we pooled similar amounts of PCR products from each library, different numbers of sequences were obtained according to the 5' adaptor sequence barcode (Table 1). For instance, the fruit and ovary libraries (15d, 45d, c1 and c5) were poorly represented providing a collection of fewer than one third of the number of sequences from the other six libraries. A set of 186,698 non-redundant sRNA sequences was generated for downstream analysis. The representation of sequences with different lengths in the redundant and non-redundant sRNAs datasets is shown in Figure 1. The most abundant sequences were 21, 24, 20 and 22 nts. A few sequences shorter than 20 nt were also retrieved, and these probably represent cloning artifacts and/or degradation products. Sequences > 30 nt in length in our dataset predominantly represented combinations of other melon sRNAs identified in our work. Detailed data are provided in Additional file 1.
Table 1. Description of small RNA libraries from different melon tissues
Figure 1. Representation of sequences with different lengths in the melon sRNA data set. (a) All sequences. (b) Non-redundant sequences. The number of sequences is expressed as a percentage of the total number of sequences.
Additional file 1. Length distribution of the small RNA data set for each library. Length distribution of melon sRNAs for each library (listed in Table 1). Sequence numbers are shown as a percentage of the total number of sequences obtained from every library. Data are given for total (with redundancy) and unique (no redundancy) sequences.
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Identification of known miRNAs
In order to identify known miRNAs, the melon sRNA data set was used as a BLAST query against the Arabidopsis small RNA database (ASRP) [33] and the microRNA database (miRbase) [34]. We identified 46 melon unique sequences corresponding to 26 miRNA families. Thirty nine sequences were identical to known miRNAs from other plant species, while 7 additional species were sequence variants highly conserved (up to two mismatches allowed). In order to clearly identify each melon sequence, melon miRNAs were named according to the homologous reference miRNA from each database (Table 2). For each reference miRNA, we found that ~3% of the corresponding melon sequences differed at one or two sites with mismatches distributed randomly along the sequence, so these were considered sequencing errors. Only specific sequence variants that represented more than 3% of the total population for each reference miRNA were considered biologically relevant. We identified only two non-conserved miRNAs, corresponding to ath-miR2111a from Arabidopsis and peu-miR2910 from Populus euphratica, respectively (Table 2). The largest diversity of miRNA species was found in ovary samples and the lowest in fruit samples.
Table 2. Known plant miRNAs identified in melon
The abundance distribution of different miRNAs in each library was estimated based on sequencing frequencies as shown in Figure 2. We used sequencing data for quantitative profiling of small RNAs, though estimation of abundance based on sequencing frequencies could be misleading due to limited sequencing depth. Many miRNAs differed in abundance according to the source library. Nevertheless, most of the redundancy reflected the accumulation of miR159a, which accounted for more than 14,000 sequences in total. Figure 2A, B compares the accumulation of miRNAs in healthy versus WMV-infected melon tissues from genotypes Tendral and TGR-1551. Melon miRNA species with similarity to Arabidopsis miR156abcdef, miR160abc and miR168ab, which target mRNAs encoding squamosa promoter binding proteins, auxin response factors (ARFs) and argonaute-like proteins (AGO), respectively, showed different trends in the genotypes tested. For example, miR168ab is more abundant in healthy Tendral tissues compared to infected tissues whereas it is more abundant in WMV-infected TGR-1551 tissues than in healthy tissues. Other known miRNAs in our sequenced set were generally more abundant in healthy tissues irrespective of the melon variety tested. For example, miRNAs with similarity to Arabidopsis miR159a and miR167d, which target MYB transcription factors and ARFs, respectively, followed this trend in both genotypes albeit with differences in magnitude. Comparison of the two libraries from ovary and fruit samples (Figure 2C, D) revealed that miRNAs were particularly abundant and diverse in ovaries compared to fruits. Several miRNAs appeared to be temporally regulated during ovary development (e.g. members of the miR160, miR164, miR167, miR169, miR319 and miR390 families) whereas others were equally abundant at both ovary stages (miR156, miR167 and miR171 families). Fruits contained far fewer miRNAs than ovaries, and only miRNAs similar in sequence to Arabidopsis miR159a, miR164ab and miR397a showed significant differences in accumulation (with trends opposite to those seen in ovaries). These findings indicated that miRNAs in melon were expressed in specific tissues and in response to particular physiological conditions. In Arabidopsis, most of these miRNAs target mRNAs encoding transcription factors with roles in development, such as hormone signal transduction and organ identity. Figure 2E, F compares the accumulation of miRNAs in healthy and MNSV-infected tissues. Similar accumulation profiles were observed in both samples for most of the miRNAs identified. Exceptionally, miRNAs similar to Arabidopsis miR396a, miR396b and miR162a, which regulate transcripts encoding GRF transcription factors and DCL proteins, respectively, showed opposite accumulation patterns.
Figure 2. Relative accumulation of conserved miRNAs in melon samples used for sRNA library construction. Total reads for each miRNA in each library were normalised relative to the total number of reads from the library, and expressed per 10,000 reads. (a) Cotyledons from melon cv. Tendral inoculated with WMV-M116 compared to mock inoculated cotyledons of the same cultivar. (b) Cotyledons from the melon accession TGR-1551 inoculated with WMV-M116 compared to mock inoculated cotyledons of the same accession. (c) Stage C1 and C5 ovaries from melon cv. Piel de Sapo. (d) Fruit from melon cv. Piel de Sapo 15 days after pollination (15d) compared to fruit from the same cultivar 45 days after pollination (45d). (e) Cotyledons from melon cv. Tendral inoculated with MNSV-alfa5 compared to mock-inoculated cotyledons of the same cultivar. (f) Cotyledons from melon cv. Tendral inoculated with MNSV (chimeric virus) compared to mock inoculated cotyledons of the same cultivar.
Identification of miRNA/miRNA* duplexes
DCL-mediated cleavage of miRNA precursors having the characteristic stem-loop structure gives rise to miRNA duplexes where one of the two strands is the guide miRNA (the functional molecule) while the near-perfect complement sequence is known as the passenger miRNA, or miRNA*. The miRNA* is rapidly degraded but transient species can be cloned and therefore sequenced. We identified 16 miRNA* sequences complementary to some of the 46 miRNAs in our dataset (Table 2), nine of which had the predicted sequence based on the fold-back structure of their presumptive precursors with internal mismatches and two additional terminal nucleotides forming a 3' tail (Figure 3A), whereas the other six had a different number of protruding nucleotides and were considered non-typical (Figure 3B).
Figure 3. Duplexes of mature miRNA and passenger (miRNA*) sequences identified in the melon sRNA collections. (a) Typical duplex structure. (b) Non-typical duplex structure (number of protruding nucleotides ≠ 2).
The number of sequenced miRNA*s was generally much lower than the number of mature sequences but there were some remarkable exceptions. For example, for miR396a we counted 134 miRNA and 84 miRNA* sequences, as opposed to miR159a for which 14,651 miRNA sequences but no corresponding miRNA* sequences were retrieved in the sequenced collections (Table 2). The most extreme example was miR157d, for which we recovered the same numbers of miRNA and miRNA* sequences.
Identification of putative melon-specific miRNAs
After identification of known miRNA sequences and other sRNA sequences (see below), 108,454 unique melon sRNAs remained unclassified, from which the most abundant (28.6%) were 24-nt species. Initial analysis confirmed that 36,783 (33.9%) of these sequences had a perfect match in the melon genome. The frequency distribution was highly skewed: 33,621 sequences had fewer than 25 hits (24,488 originated from a single locus), and only 659 sequences had more than 100 hits.
Sequences that were 21, 22 or 24 nt in length with a maximum of six hits in the genome were selected as potential novel miRNAs, and flanking genomic regions were analysed according to three consecutive criteria. First, we used miRanda software to detect sequences complementary to the potential miRNA inside the flanking regions. Second, potential miRNAs with precursors less than 70 nt in length were discarded. Finally, the MFEI index, which is used to distinguish miRNA precursors from other coding and non-coding RNAs and is based on free energy estimates and nucleotide composition [35], was calculated for each precursor and the results were sorted accordingly (the more negative the index, the better the precursor).
Predicted miRNA precursors and their genomic flanking regions that were found to be similar in sequence to previously described transposons were discarded. Other predicted miRNA precursors with intramolecular folding potential showed no similarity to known transposon sequences although their secondary structures were similar to those of known foldback transposons; these were characterised by strong negative MFEI indexes and high miRanda scores, both features consequence of high sequence complementarity in the pairing stem sequence. For some of these precursors, several uncharacterised melon sRNAs mapped on them in both the sense and antisense orientations (e.g. a11_62726 in Figure 4), up to 85 in some cases. Therefore, these were also considered unsuitable miRNA candidates. Three other potential miRNAs were shown to be the miRNA* sequences of known miRNAs that had not been picked up in our initial screen.
Figure 4. Secondary structures of putative novel melon-specific miRNA precursors. Some of the sRNAs identified as potentially novel and melon-specific miRNAs (listed in Table 3) were selected based on quality criteria and are shown as examples. Red lines represent regions where miRNA/miRNA* duplexes are located. Identifier a11_72726 represents an example of unsuitable miRNA candidate.
After manually inspecting the remaining secondary structures, 77 loci that fulfilled the structural criteria for annotation of plant miRNAs [36,37] were selected as plausible miRNA precursors; we also added to this list 7 other loci that had an asymmetric bulge involving 3 bases inside the putative miRNA duplex. From them, 43, 20 and 21 corresponded to sequenced sRNAs of 21, 22 and 24 nt in length, respectively (Table 3). Six selected sequences are shown as examples in Figure 4. By checking the pairing sequence on the stem of the predicted precursors, miRNA*s for seven candidate miRNAs were found in the sequenced set. Therefore these miRNAs were regarded as authentic miRNAs that conformed to the biogenesis and expression criteria for confident miRNA annotation [37]. The remaining sequences, not supported by the complementary passenger strands, were classified as candidate miRNAs. Most of the potential novel miRNA were represented by a small number of sequences, a single sequence in more than half of the cases, but six exceptional candidates were represented by more than 10 sequences (Table 3). As occurred for conserved miRNAs, sequence variants were identified for some novel miRNAs (Table 3) which mapped on the genomic melon sequence with slight variations in length and position relative to the most abundant sequence. In the absence of a reference sequence from any database, these variants were counted. Sequencing errors of differential cleavage of potential miRNA precursors possibly explain these length and positional polymorphisms.
Table 3. Potential novel melon specific miRNAs
Prediction of potential miRNA targets in the melon transcriptome
To identify potential targets of miRNAs, we screened melon unigenes in the publicly-available database [7]. Two independent searches were performed using miRanda [38] and TargetFinder [39], and the results were compared. Each program scores potential targets based on sequence complementarity, with high scores better in miRanda, and low scores better in TargetFinder. Both algorithms identified a common set of presumptive targets albeit with different scores, and the few discrepancies involved targets with low confidence scores.
Targets in Arabidopsis defined by miRanda generally have a score ≥170, and using this value as cutoff we found 150 melon unigenes as potential miRNA targets, the best of which are summarized in Table 4 (a complete list is provided in Additional file 2). The potential miRNA targets in Table 4 generally had similar annotations to their Arabidopsis counterparts, although there are some exceptions. For example, melon unigene cHS_39-F10-M13R_c is a predicted target of miR159a but it is annotated as positive regulator of brassinosteroid signaling rather than a MYB or TCP transcription factor, which is sensitive to miR159 regulation in Arabidopsis. Many of the melon unigenes identified as potential targets were not annotated, and some had previously been identified as potential miRNA precursors [5].
Table 4. Best quality miRNA targets identified in melon unigenes
Additional file 2. Known miRNA targets identified in melon unigenes. Complete set of all known miRNA targets identified in melon unigenes.
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Interestingly, the highest miRanda scores (> 300) were achieved for transcripts with two separate miRNA targets on the same molecule. For example, unigene c15d_05-D02-M13R_c had two target sites for miR390ab separated by ~200 nt. When this region was used as a BLAST query against the melon sRNA dataset, a group of 257 sequences (more than 92% of them being 21 nt long) was identified with nearby clusters of related 21-nt sequences in both the sense and antisense orientations, which is reminiscent of the ta-siRNAs biogenesis mechanism [18] (Figure 5). Both sites (complementary to miR390 family members in unigene c15d_05-D02-M13R_c) had similar miRanda scores, they did not contain mismatches or G:U wobbles involving nucleotides 9-11 and were phased 21 nt one of each other. The number of sRNA copies was different in each cluster and were more abundant in sense orientation compared to antisense orientation. Two registers of phased 21-nt siRNAs were observed. One of them was phased with the miR390 complementary sites but the other one was not. A representative sequence from each cluster was selected and used to search for potential targets in melon unigenes, identifying > 100 transcripts with a miRanda score > 170. Several of these transcripts were annotated as ARFs and ubiquitin related gene products (Table 5).
Figure 5. Identification of potential ta-siRNA transcripts. Four unigenes were found to have two miRNA target sites each. For each unigene, the region delimited by the two miRNA suites was used as a BLAST query against the sRNA data set and only c15d_05-D02-M13R_c generated hits. Unigenes are represented by black horizontal lines. The miRNA site boundaries are represented by vertical arrows. Potential ta-siRNAs are represented in the inset by colored short horizontal lines mapped onto the unigene.
Table 5. miRNA targets identified in melon transcripts for potential ta-siRNAs derived from unigene c15d_05-D02-M13R_c
The remaining unigenes with two predicted miRNA target sites listed in Table 4 were annotated as protein-coding transcripts and no sRNAs were identified with similarity to the region flanked by the two target sites (Figure 5), suggesting that they did not account for authentic ta-siRNA-producing loci. Targets were also sought in the reverse-complement sequences of melon unigenes, because a small proportion of the ESTs could be incorrectly oriented as an artifact of the cloning procedure [5]. Twenty-eight unigenes were identified as potential miRNA targets using the same criteria described above, most of which were found to be non-annotated (Table 6). In this new set of data, unigenes with two targets were used again as a BLAST query against the melon sRNA dataset but no hits were obtained, so these unigenes were no longer considered as potential ta-siRNAs.
Table 6. Best quality miRNA targets identified in reverse-complement sequences of melon unigenes
With some exceptions, several miRNA targets with miRanda scores ≥170 (see Additional File 3) were identified for each of the potential novel melon-specific miRNAs listed in the previous section.
Additional file 3. Novel melon-specific miRNA targets identified in melon unigenes. Complete set of all novel melon-specific miRNA targets identified in melon unigenes.
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Characterization of other melon sRNAs
Next, we blasted our sRNA sequences against RNA and genomic databases to search for other sRNA species by sequence similarity (Figure 6). sRNAs similar to transfer RNA (tRNA), trans-acting siRNA, small nucleolar RNA (snoRNA) and transposons were the least abundant, whereas ribosomal RNAs (rRNA) were largely the most abundant non-coding sRNA species (Figure 6A). Intriguingly, exogenous virus-derived sRNAs were as abundant as other endogenous plant sRNAs, at least in the case of MNSV. Most of the sRNAs identified had complete sequence similarity with the reference RNA from each database (Figure 6A), even if up to two mismatches were allowed in BLAST comparisons. The only exception were sequences similar to ta-siRNAs, for which 14 melon sRNAs with similarity to Arabidopsis TAS3a|D7(+) and TAS3a|D8(+) were identified, 2 containing 1 mismatch, and 12 containing 2 mismatches. All of them mapped very close in the melon genome and in a different region than c15d_05-D02-M13R_c unigene (the other potential source of ta-siRNAs, see above). To determine if they were authentic melon ta-siRNAs, we selected a 600 bp window sequence upstream and downstream from the genomic location determined in the melon genome for each candidate; then, a BLAST query against the melon sRNA dataset was performed, revealing that at least 126 sequences (95 of them being 21-nt in length) mapped in this region and were arranged according to a near 21-nt phase spacing (data not shown).
Figure 6. Types and frequencies of known melon sRNAs. (a) Number of sRNAs with similarity to known sequences in public databases: tRNA = transfer RNA; snoRNA = small nucleolar RNA; LSUrRNA = large subunit of ribosomal RNA; SSUrRNA = small subunit of ribosomal RNA; taRNA = trans-acting-si-RNAs; transposon = transposon sequences; chloroplast = melon chloroplast genome; mitochondrion = melon mitochondrial genome; MNSV = Melon necrotic spot virus genome; WMV = Watermelon mosaic virus genome; miRNA = microRNA. (b) Mapping of small RNAs onto the melon chloroplast genome. X-axis represents genome nucleotide positions. Y-axis represents the number of sRNAs mapped at each position. The chloroplast genome is represented by a horizontal black line (156,018 nt in length) at y = 0, and the 5' ends of sRNAs are represented by points (blue: sense sRNAs, pink: antisense sRNAs). The frequencies of antisense sRNAs are shown as negative numbers. (c) Number of melon sRNAs with similarity to the melon chloroplast genome.
Many sRNA sequences also generated hits in the plastid genomes (30,239 sRNAs corresponding to 4,254 unique plastid sequences). When these sRNAs were mapped onto the melon chloroplast genome (unpublished data) (Figure 6B), two clusters of sequences resolved in regions presumably annotated as chloroplast rRNA. These regions lie within two inverted genomic repeats, and sRNAs were accordingly identified in both the sense and antisense orientations. Some of the chloroplast sRNAs had previously been cloned in other species [40]. For example, melon sRNA a33_398374 (sequence AGT TAC TAA TTC ATG ATC TGG C) was the most abundant melon plastid sRNA (18,054 counts), and a matching sequence is present in more than 900 chloroplast genomes. It is located in an intergenic region and may target a methyltrasferase transcript, although there is no direct evidence that it has silencing functions. Melon sRNA a14_392967_ (sequence GGT AGT TCG ATC GTG GAA TTT) was less abundant (166 counts), it is present in 10 different chloroplast genomes, and it may target a transcript encoding an electron carrier protein. Interestingly, different numbers of plastid sequences were obtained from each library (Figure 6C). For example, in the virus-resistant melon accession TGR-1551 there was no difference in the number of sRNAs with hits to melon chloroplast genome between healthy and virus inoculated samples, but in the virus-susceptible accession Tendral, more sRNAs were counted in inoculated samples (Figure 6C).
Unlike chloroplast sRNAs, only 7,854 sRNAs (corresponding to 2,384 unique sequences) matched the melon mitochondrial genome (unpublished data). These sRNAs were mapped on the mitochondrial genome sequence and formed three clusters, again corresponding to the sites of rRNA genes (data not shown).
Discussion
In this report, we describe the first screen for melon sRNAs by deep sequencing. In total, 398,450 high-quality sequences were generated, representing 90% of the total raw reads. RNA species 21, 24, 20 and 22 nt in length dominated the sRNA transcriptome in melon with the 21-nt class being the most abundant in our libraries. Molecules of 24-nt processed by DCL3 are often the most abundant endogenous plant sRNAs [13], but this may vary according to species. For example, 24-nt sRNAs are more abundant in Arabidopsis, rice and tomato [41,42,9], whereas 21-nt sRNAs are more abundant in grapevine, wheat and conifers [12,43,44]. It is also possible that the composition of the sRNA population of a given plant species varies according to tissue and physiological conditions, as seems to be the case of melon (see Additional file I). Perhaps the higher proportion of 24-nt sRNAs found in melon ovaries compared to the other tissues reflects the predominance of developmental processes based on epigenetic events in the ovary.
Recent studies have shown that cis-acting siRNAs arising from heterochromatin, transposons and other repeat elements account for the greatest proportion of endogenous sRNA populations in plants [13,45-48]. In melon, only ~7,000 sRNAs matched known transposon sequences, in contrast to ~60,000 sRNA sequences matching ribosomal RNA, which may simply reflect the paucity of melon transposon sequence information in databases, as only 1.5% of the melon genome has been annotated for transposable elements [49]. Transposon sequences in different species show more divergence than rRNA sequences, so the representation of transposon-related sRNAs could increase when a more accurate and complete annotation of the melon genome becomes available. We also identified two sets of ta-siRNAs in our data, which mapped to different loci in the melon genome thus revealing the presence of at least two potential TAS genes. One locus was not represented in the melon unigene database, most likely because of its incomplete coverage. The sequence of the other locus was similar to that of a non-annotated melon transcript, and contained two registers of sRNAs in a 21-nt phase bounded by two target sites to miR390ab, reminiscent to TAS3 genes. Non-coding transcripts containing two miR390 complementary sites that give rise to phased siRNAs have been described in other organisms. In the moss Physcomitrella patens, both 3' and 5' target sites are cleaved. In Arabidopsis, the 5' miR390 complementary site contains a mismatch and two G:U wobbles involving positions 9-11 and, despite it is not cleaved, it binds the silencing complex and is required for full AtTAS3 function in vivo [50]. In melon, both 3' and 5' miR390 had perfect complementarity at positions 9-11, suggesting that both could be cleaved, as opposed to Arabidopsis, to specify a phased register for ta-siRNA biogenesis. Interestingly, an additional siRNA register that is likely independent of miR390-directed cleavage of the putative melon TAS transcript was observed. This alternative register might be determined by the processing activity of TAS transcripts by one of the most abundant melon primary ta-siRNAs during generation of secondary ta-siRNAs (Figure 5), as proposed for alternatively phased TAS3 ta-siRNAs in Arabidopsis [18,50]. Since there are additional TAS loci in other plant genomes, it is reasonable that other melon TAS loci remain to be discovered.
More than 30,000 of our sRNA sequences matched the plastid genome, suggesting intense sRNA activity in this organelle. Mitochondrion-specific sRNAs were less abundant in comparison. The abundance of plastid sRNAs varied by source, with fewer sequences obtained from the ovary and fruit libraries compared to the cotyledon libraries, perhaps reflecting a relationship between chloroplast sRNA activity and photosynthesis. Interestingly, there was no significant difference in sRNA accumulation when comparing infected and healthy TGR-1551 cotyledons (resistant to WMV) whereas more sRNA accumulated in healthy Tendral (susceptible to WMV and MNSV) cotyledons than in infected ones. Whether or not this is related with the resistance phenotype is a matter of speculation.
More than 28,000 melon sRNAs in our sequenced collections matched known miRNAs in other plants, and 46 distinct melon sRNA species could be assigned to 26 known miRNA families. Although we generated a relatively low number of sequence reads, our data nevertheless were in good harmony with previous studies of miRNA profiling based on exhaustive sequencing of sRNA populations (e.g. in grapevine, 24 million reads, 26 known miRNA families and 26 non-conserved miRNA families; in tomato, 721,874 reads, 30 known miRNA families; and in orange, 13,106,573 reads, 42 highly-conserved miRNA families) [12,11,9]. This probably reflects the generally-accepted high level of expression reported for conserved miRNAs.
In addition to known miRNAs, 84 sRNA sequences derived from genomic loci with intramolecular folding capacities and not previously described as miRNAs in other plant species were predicted as potential melon-specific miRNAs. In most cases, only one sequence was counted from each of these miRNAs, which is consistent with reports suggesting that species-specific miRNAs are usually expressed at low level and in a tissue-specific manner [41]. The candidates listed in Table 2 include a number of special cases, i.e. miRNAs with miRanda scores ≥195 and very strong secondary structures including an internal loop, resembling type III foldback transposons [51,52,9]. Although these sequences do not match known melon transposons, they were not considered as miRNA candidates because accurate homology-based transposon annotation and prediction occasionally needs to be complemented with ab initio approaches based on structural features [53,49]. However, even not considering this particular group, our data indicate that most of the precursors we identified are candidates to encode melon-specific miRNAs.
The accumulation of miRNAs was estimated by census sequencing and this showed that there is more miRNA diversity and that miRNAs are more abundant in ovaries than fruits. Although miRNAs are involved in many processes, 60-70% of known plant miRNAs control the expression of transcription factors that regulate critical developmental processes, such as proper specification of floral organ identity or leaf polarity, and over-expression or knockout of MIRNA genes led to severe developmental defects [48,54,13]. It is likely that the greater abundance of miRNAs in the early ovary stages compared to fruit reflects the more significant developmental activity in ovaries, and confirms that meristems and other developmentally active tissues are good resources for miRNA screening.
The comparison of healthy and virus-infected melon tissues showed that generally miRNAs were less abundant in infected tissues. Viruses interfere with and exploit endogenous RNA-silencing pathways using diverse strategies [55,19]. For example, the potyvirus silencing suppressor HC-Pro has been shown to suppress the miRNA pathway by inhibiting miRNA assembly into AGO1-containing silencing complexes and unwinding of miRNA/miRNA* duplexes, causing accumulation of stable duplexes [56]. Several studies have shown that virus infection can regulate the accumulation of mRNAs targeted by miRNAs without affecting the abundance of the miRNAs themselves, or even by promoting a slight accumulation [57-59]. In contrast, we found that miRNA accumulation was generally depressed in infected plants compared to controls subjected to mock inoculations. A notable exception was miR168ab, which was upregulated in the resistant genotype but downregulated in the susceptible one. This miRNA has previously been shown to be involved in controlling the expression of ARGONAUTE1 (AGO1), the catalytic subunit of the RNA-induced silencing complex responsible for slicing of target mRNAs [60]. Recent work has described the enhanced expression of miR168 and AGO1 mRNA in virus-infected plants specifically and independently of other miRNAs [61-63]. The contrasting miRNA profiles observed in the TGR-1551 and Tendral varieties suggests that silencing may underly the resistance of TGR-1551 to WMV, although this is a hypothesis that will require further research.
We have identified more than 150 melon unigenes as potential targets for the known and novel miRNA sequences discovered in this investigation. Many animal transcripts are targets for more than one miRNA but this phenomenon is uncommon in plants [64]. Accordingly, most of melon unigenes identified as potential targets featured only a single miRNA site. miRNAs that are conserved across species tend to have conserved targets too, and our data confirm this is the case in melon. However, several unigenes predicted with high confidence as targets for conserved miRNAs had different annotations to the corresponding target genes in Arabidopsis, although these may represent false positives that would fail additional validation. Furthermore, the non-conserved targets of conserved miRNAs can be cleaved at a lower frequency than conserved targets [12]. For these two reasons, the selection of targets for individual validation experiments can be challenging.
An interesting alternative for miRNA target discovery in a genome-scale is the analysis of the small RNA degradome [65], as this avoids the a priori selection of potential targets. High-throughput gene expression profiling techniques such as microarray hybridization can also help to predict miRNA targets because some times a negative correlation between the abundance of miRNAs and their target mRNAs can be identified [66,67]. We have used microarrays to monitor gene expression profiles in healthy TGR-1551 and Tendral plants and plants infected with WMV. When compared with our miRNA data, we were able to identify two unigenes encoding AGO proteins that were differentially expressed and showed contrasting expression profiles in susceptible and resistant genotypes (Gonzalez-Ibeas and Aranda, unpublished data). The same profile was observed for miR168 accumulation, suggesting that miR168 may be involved in virus resistance and providing the basis for future experiments.
Conclusion
We have analysed and catalogued a collection of melon endogenous sRNA obtained through massive cDNA sequencing and have identified known miRNAs and ta-siRNAs (conserved in other species) as well as potential melon-specific miRNAs with no database matches. We have also identified potential targets for these miRNAs in the melon transcriptome. Census sequencing (i.e. counting the number of sequence reads for each sRNA) was used to profile their expression in different tissues, and in healthy vs. virus-infected cotyledons. By comparing the predicted targets and the differential expression profiles we were able to provide insights into the role of miRNAs in the regulation of fruit development and plant-virus interactions.
Methods
Plant material
Small RNA libraries were prepared using material from three melon accessions: 1) the Tendral cultivar (Semillas Fitó, Barcelona, Spain), which is susceptible to MNSV and WMV, 2) the breeding line T-111 of the cultivar Piel de Sapo (Semillas Fitó, Barcelona, Spain), and 3) the genotype TGR-1551 (germplasm collection of "Estación Experimental La Mayora" (EELM-CSIC), Málaga, Spain), which is resistant to WMV.
Melon plants were grown under greenhouse conditions (~25/20°C, 16-h photoperiod, ~70% relative humidity) in 0.5-L pots with substrate (Tendral and TGR-1551) or in soil bags with the capacity for four plants (Piel de Sapo). Fruits of 15 and 45 days after pollination (DAP) were collected and mesocarp tissues were recovered and used for RNA extractions. Virus infected samples were obtained from completely expanded cotyledons rubbed with carborundum (ø = 0.037 mm) and the corresponding viral inoculum. MNSV-infected melon cotyledons exhibiting lesions and marrow leafs systemically infected with WMV were ground in cold inoculation buffer (0.2 M phosphate buffer pH = 8.0, 0.1% (v/v) beta-mercaptoethanol, 0.03 g/ml activated charcoal) for inoculum preparation. Mock-inoculated control cotyledons were rubbed with inoculation buffer and carborundum alone.
Cotyledons were harvested at 1, 3, 5 and 7 days post-inoculation (dpi) and pooled for RNA extraction. Fruit samples were prepared as previously described [8]. Ovaries were collected at stages C1 and C5 (Mascarell-Creus et al., unpublished). The C1 stage corresponds to flower emergence from the inflorescence bud, when the outermost perianth organs commence development and no floral whorls are visible. The C5 stage corresponds to anthesis, when the flower is ready to be fertilized and all floral organs are fully formed, including the yellow petals that attract pollinators. Under normal growth conditions, C1 to C5 development takes approximately 5 days.
Small RNA library construction
Total RNA was extracted using Trizol-Reagent (Sigma Chemical Co., St. Louis, MO, USA) and 300 μg were used to construct sRNAs libraries as described [57,32]. The 3' adaptor was replaced with a pre-activated 5'-adenylated oligonucleotide (5'-rAppCT GTA GGC ACC ATC AAT 3ddC-3') (Integrated DNA Technologies, Coralville, Iowa, USA) to avoid sRNA circularisation.
Ten chimeric RNA/DNA oligonucleotide 5' adaptor variants were generated by modifying the four-nucleotide identifier (barcode): 1-1, ATC GTA GGC ACC UGA UA; 1-2, ATC GTA GGC CAC UGA UA; 1-3, ATC GTA GGC UGC UGA UA; 1-4, ATC GTA GGC GUC UGA UA; 2-1, ATC GTA GCG ACC UGA UA; 2-2, ATC GTA GCG CAC UGA UA; 2-3, ATC GTA GCG UGC UGA UA; 2-4, ATC GTA GCG GUC UGA UA; 3-1, ATC GTA GAC GCC UGA UA; 3-2, ATC GTA GAC CGC UGA UA. After each ligation step, sRNA was purified by 17% denaturing polyacrylamide gel electrophoresis. The purified, ligated sRNA was reverse transcribed with SuperScript® III reverse transcriptase (Invitrogen BV/Novex, Groningen, Netherlands) and the cDNA was amplified with AmpliTaq Gold® DNA Polymerase (Applied Biosystems, Foster City, CA, USA) using 3' PCR FusionB and 5' PCR FusionA primers [57]. The PCR primers contained the "A" and "B" tag sequences compatible with 454 technology [31].
DNA amplicons were gel-purified using 4% Metaphor Agarose and isolated using the QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany). The quantity and quality of the DNA amplicons were determined using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA) and an Experion Automated Electrophoresis System (Bio-Rad, Hercules, California, USA). The same quantity of DNA from each library was pooled and sequenced using the 454 Life Science Technology platform (Lifesequencing S.L., Paterna, Valencia, Spain). Sequence data in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE28653 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE28653 webcite.
Bioinformatics
The sRNA sequences were parsed from FASTA-formatted files containing 447,180 reads from two independent 454 sequencing runs and assigned to specific libraries by identifying the sRNA/adaptor boundaries and barcode analysis. Sequences were analysed with standard Python scripts [68] and the BioPython library [69]. Only sequences with the 3' and 5' adaptors in the correct position were considered. Known sRNAs were identified by searching public databases using BLAST version 2.2.19 [70] and allowing up to two mismatches. The following databases and sequences were searched: Transfer RNA Database (version 2009) [71], Plant Small Nucleolar RNA Database (v1.2) [72], SILVA (ribosomal RNA database, v100) [73], The Arabidopsis Small RNA Project (ASRP) Database [33], Rfam Database 10.0 [74], miRBase (release 16) [34], The Plant Repeat Database [75], Cucumis melo chloroplast genome (unpublished data), Cucumis melo mitochondrial genome (unpublished data), MNSV genome (GenBank accession AY122286.1), WMV genome (GenBank accession AY437609.1). In the case of miRNAs, melon sequences were named with the reference miRNA from each database in order to distinguish miRNA species of each family. miRNA targets were identified using miRanda v3.0 [38] and TargetFinder Perl script 1.5 [39]. Putative novel melon-specific miRNA genes were identified by using the candidate miRNA as a BLAST query against the melon genome (unpublished data). For each hit, 600 bp of sequence upstream and downstream of the alignment was used to search for a near-perfect reverse complement (miRNA*) sequence with the miRanda algorithm. Regions lacking a corresponding miRNA* sequence were discarded. Minimum genomic regions (> 70 nt) containing miRNA and miRNA* sequences were selected as potential precursors. Those corresponding to protein-coding genes were identified by BLAST searches against the Arabidopsis protein database (TAIR) and were discarded, whereas non-coding potential precursors were manually inspected and used to predict the RNA secondary structure with Mfold [76] and for calculation of the MFEI index [35]. Precursors that met structural miRNA criteria were selected for further evaluation [36,37].
Authors' contributions
DGI prepared RNA from infected and mock-inoculated samples, constructed all the sRNA libraries, carried out the trimming and analysis of the sRNA sequences and wrote the manuscript. JB provided bioinformatics analysis support. LD and CL provided guidance for the preparation of the sRNA libraries and additional technical support. ACD, AMC, MS and JGM prepared RNA from fruit and ovary samples. MAA supervised DGI including writing of the manuscript, and conceived this study together with DGI, JGM and CL. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by grants AGL2009-07552/AGR, BIO2006-13107 (Ministerio de Ciencia e Innovación, Spain) and MELONOMICS (Fundación Genoma España, Spain). We thank Mari Carmen Montesinos and Blanca Gosalvez for their technical assistance and Richard M. Twyman richard@writescience.com for editorial assistance.
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Research article
Early life factors, childhood cognition and postal questionnaire response rate in middle age: the Aberdeen Children of the 1950s study
Yuji Nishiwaki1, Heather Clark2, Susan M Morton3 and David A Leon1*
Author Affiliations
1 Department of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, London, UK
2 Dugald Baird Centre, University of Aberdeen, Aberdeen, UK
3 Liggins Institute and School of Population Health, University of Auckland, Auckland, New Zealand
For all author emails, please log on.
BMC Medical Research Methodology 2005, 5:16 doi:10.1186/1471-2288-5-16
The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2288/5/16
Received:7 January 2005
Accepted:5 May 2005
Published:5 May 2005
© 2005 Nishiwaki et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
Little is known about the relationship between early life factors and survey response in epidemiological studies of adults.
Methods
The Children of the 1950s cohort is composed of 12,150 children (boys 51.7%) born in Aberdeen 1950–56 and in primary schools in the city in 1962. Information on birth weight, gestational age, growth, behaviour and socio-economic position at birth and in childhood were obtained from contemporaneous records. Cognitive test scores at ages 7,9 and 11 years were also available from school records. The outcome was response to a postal questionnaire sent (2001–2003) to surviving cohort members in middle age.
Results
Of 11,282 potentially mailed subjects, 7,183 (63.7%) returned questionnaires. Response rates were highest among females, and those whose parents were married at birth, were in a non-manual social class at birth or in childhood, had fewer siblings, were taller and heavier in childhood for their age and had lower Rutter B behavioural scores. Childhood cognitive test scores at every age were strongly and positively related to the response rate to a postal questionnaire independently of other early life factors monotonically across the entire range of test scores. Those in the bottom fifth at age 11 had a response rate of 49% while those in the top fifth 75%.
Conclusion
The strength and consistency of the association of childhood cognition with questionnaire response rate in middle age is surprisingly large. It suggests that childhood cognition across the entire normal range is a powerful influence on the complex set of later behaviours that comprise questionnaire response. The extent of possible response bias in epidemiological studies of the associations between childhood characteristics (particularly those related to cognition) and later health is probably larger than is generally realised, at least in situations where the survey instrument is a postal questionnaire.
Background
The potential bias introduced by non-response or non-participation is an issue that affects much of observational epidemiology. Extending our understanding of the factors that influence non-response is thus an important area of research in its own right [1,2]. It is well known that many characteristics measured in adulthood such as gender, age, socio-economic position, education, health status and smoking habits are associated with response rate [2-10]. However, very little is known about the association of characteristics in infancy and childhood with response or recruitment rates in adulthood. This is partly because there are relatively few contexts in which unbiased data on early life characteristics are available for the total population irrespective of later response behaviour. The increasing interest in life-course epidemiology [11,12], in which adult disease is related to factors across an individual's life course, makes an investigation of these early-life influences on response rate particularly pertinent at this time.
In this paper we look at the influence of a range of infant and childhood characteristics on adult questionnaire response rates in a large cohort study. We pay particular attention to childhood cognition as we are also interested in the broader question of why adult health is related to childhood cognition [13-15]. A priori one might expect childhood cognition to be related to response rates, partly through its link with educational attainment, that is known to be strongly associated with response rates. However, to date there have been very few studies of the association of factors in early life, including cognition, and questionnaire or survey response in adult life. In an analysis of the UK 1946 national birth cohort, Wadsworth et al. reported that lower cognitive score at 8 years of age was associated with previous refusal or failure to contact by the study nurse at age 53 years [16]. However, the findings from this paper may not be readily generalizable, as the study is unusual in that it involved repeated contact with participants over their entire life time. More typically, life-course studies have information from infancy and childhood from historic sources, and then attempt a first survey follow-up well into adult life.
We have used the Aberdeen Children of the 1950s historical cohort study to investigate the association between early life factors, including childhood cognition, and response to a postal questionnaire conducted in middle age that represented the first attempt to make direct contact with the study subjects since childhood.
Methods
The Aberdeen Children of the 1950s study
The background to this historical cohort study has been previously described [17]. Briefly, this cohort is a large subset of the Aberdeen Child Development Study (ACDS) [18]. This was conducted in the early 1960s to estimate the population prevalence of mental subnormality in children and to investigate its etiology. Aberdeen was chosen as both obstetric and educational records were of a high standard and the population was relatively stable.
The ACDS was comprised of all 15 thousand children born 1950–56 who were in primary school in Aberdeen, Scotland in December 1962. In order for the ACDS to achieve its main objectives, particular care was taken by the original investigators to include all children of primary school age in Aberdeen regardless of their cognitive function. Thus children in schools for individuals with moderate or severe learning difficulties were included as well as those in ordinary primary schools. However, an unknown but small number of children with very severe learning difficulties who did not attend any school will not have been included in the original cohort.
In December 1962 these children (aged 7–12 years) were administered a range of age-appropriate reading tests and were requested to provide their own and parental demographic information. In March 1964 they were resurveyed and behavioral information was collected by class teachers using a detailed behavioural inventory for each child. Information was also obtained retrospectively from school test records (cognition at 7 and 9 years of age) and school medical records such as height and weight. Cognition scores at 11 years of age were obtained prospectively for most children who were aged 8 years or more in December 1962.
The Children of the 1950s cohort is comprised of the 12,150 individuals (males 51.7%) in the ACDS who were born in Aberdeen and had been successfully linked to information from obstetric records held in the Aberdeen Maternity and Neonatal Databank. The process of revitalising this cohort was begun in 1998. This included tracing of the vital status and area of residence and address of the study participants and attempting to mail a questionnaire to those subjects believed to be currently resident in the UK. This 21 page questionnaire covered a range of topics including living conditions, occupation, education, income, height and weight, health, health-related behaviours, and parental vital status. A copy of the questionnaire is available from the authors on request. This was the first time that a direct attempt had been made to contact any of the cohort members since childhood, with the exception of a small subgroup (less than 1 in 5) who had been followed up in various investigations of respiratory function since the mid-1980s, known collectively as the WHEASE study [19,20].
Tracing of cohort members
The tracing of the current vital status and area of residence of the cohort members was carried out by the National Health Service Central Register (NHSCR) for Scotland, England and Wales. Using name, date of birth and the fact that the subjects were known to have been resident in Aberdeen in 1962, the NHSCR was able to identify all but 136 (1.1%) of subjects in their records.
Questionnaire mailing
The mailing of questionnaires to cohort members was begun in May 2001. For confidentiality reasons, the primary mailing to subjects believed to be resident in Scotland (n = 9,261) was carried out on our behalf by the Information and Statistics Division (ISD) of the National Health Service in Scotland who had access to current addresses. Those not responding to this initial mailing were re-mailed by ISD in August 2001. For those resident in England and Wales (n = 1,062), local health authorities undertook the mailing exercise (February – August 2002). A small proportion of subjects (n = 328) were given the questionnaires as a part of the WHEASE study. Those not returning a questionnaire after a mailing from ISD or health authorities and those who were not mailed through these channels for other reasons (mainly that a precise address could not be determined by ISD) were sent to the Driver and Vehicle Licensing Agency (DVLA) (n = 4,355). They agreed to send out questionnaires to those people who they could identify in their database (October 2002). However, in order to protect confidentiality DVLA were not able to tell us which subjects they were successful in identifying and sending a questionnaire to. Finally, between December 2002 and February 2003 a small number of non-responders (n = 556) were sent questionnaires via their siblings who had already responded earlier on. However, we were unable to ascertain how many of these questionnaires were actually delivered to their intended recipients.
Figure 1 summarizes the final outcomes of the questionnaire survey responses. We attempted to contact all subjects regardless of their early life characteristics including cognition. However, of our original 12,150 subjects we did not mail those who were dead (n = 479), were known to have emigrated from the UK (n = 291) or were members of the armed forces (or family of those in the armed forces) or those who were in prison or in a long stay psychiatric hospital (n = 62). Of those we tried to mail through the national health service for a small number (n = 27) the Health Authority or Health Board or General Practitioner refused to forward the questionnaire to the subjects' address. Finally we did not attempt to mail the very small number subjects (n = 9) who were residents of the Western Isles of Scotland or North Ireland.
Figure 1. Profile of mailing questionnaire in the Aberdeen Children of the 1950s study. Potential receiver includes subjects who were not traced through National Health Service Central Registry or cancelled General Practice registration as well as surviving cohort members.
Cognition and other factors in early life
The Moray House Picture Intelligence Test, Schonell and Adams Essential Intelligence Test and Moray House Test were used as cognitive tests at 7, 9 and 11 years respectively. The Moray House Test at 11 consisted of two ability tests (verbal reasoning 1 & 2) and two attainment tests (Arithmetic, English). The scores from these tests were combined together with a teacher's estimate to yield a "total transfer score"[21] that played a crucial role in determining the type of secondary school the child went onto. All these cognitive test scores were standardised to give means of 100 and standard deviations (SDs) of 15 using national norms for cognition at 7 and 9 and local norms for cognition at 11.
The following additional factors were also considered in this study. Sex and year of birth of children were obtained from the survey in 1962. Birth weight was measured in pounds and categorized into 4 groups (<6.5, 6.5–7.5, 7.5–8.5 and ≥8.5). Gestational age was categorized into 5 groups (≤38, 39, 40, 41 and ≥42 completed weeks of gestation). Height for age was derived from height and age at first medical examination, which was at around 5 years of age. A sex-specific z-score was calculated for each child within 3 month age bands. Using this z-score, subjects were classified into 4 groups (≤-1, -1 to 0, 0 to 1, ≥1 SD). Weight for age z-score categories were derived using the same approach. As a behavioural factor, Rutter B score was assessed by the children's class teachers based on 26 questions. A score of 9 or over was defined as indicating psychological disturbance [22,23]. In this analysis, scores were divided into four (0, 1–2, 3–8, and ≥9). Father's occupational class at birth and in 1962, number of siblings in 1962 and mother's marital status at birth were also obtained.
Ethical approval
The revitalisation of the Children of the 1950s cohort including the questionnaire survey was approved by the Scottish Multi-Centre Research Ethics Committee, the Scottish Privacy Advisory Committee and various local ethics committees in Scotland and England and Wales.
Statistical analysis
Absolute response rates (or more properly response proportions) were examined in cross-tabulations according to explanatory factors of interest. The associations between response proportions and explanatory factor were quantified in terms of odds ratios. Subjects who returned a questionnaire were considered as responders even if the questionnaire included some degree of item non-response. However, subjects who returned a questionnaire that was completely blank were classified as non-responders. In the original data, children who were considered not capable of being meaningfully tested using the standard instruments because of their degree of cognitive impairment (n = 8) were arbitrarily allocated a score of 50. As exclusion of these subjects did not change results, we included them in the analyses.
Logistic regression was used to model the association of explanatory variables with response coded as a binary variable. The trend in odds ratios were assessed by the score test. As cognitive test scores for females were slightly higher than those for males, sex-specific quintiles of cognitive test scores were used for categorization. Sex-adjusted odds ratios for a one SD increase in cognition scores at 7, 9 and 11 years were compared with those additionally adjusted for other early life factors. In addition, sex-adjusted odds ratios for cognition scores were compared with those adjusted for cognition scores at different ages. Interactions among early life factors were tested using the likelihood ratio test. All analyses were conducted using STATA 8 [24].
Results
The average age (range) of the subjects at the start of the questionnaire survey in May 2001 was 48.1 years (45.1 to 51.3). Of 11,282 (5,742 males, 5,540 females) potentially mailed subjects, 7,183 (3,432 males, 3,751 females) returned questionnaires. This constituted an overall response rate of 63.7%. A total of 3,831 (34.0%) did not respond, 245 (2.2%) refused to participate and 23 (0.2%) were unable to complete or returned blank questionnaires (Figure 1). Females had an appreciably higher response rate (67.7%) than males (59.8%). Table 1 summarises the association of response rate with key explanatory variables for males and females separately. In males and females response rates were higher for subjects whose mothers were married and whose fathers were in a non-manual social class when they were born. Response rates were also higher among those who were had fewer siblings, were taller and heavier for their age in childhood and did not exhibit symptoms of behavioural disorders (indicated by a low Rutter B score). Tests for interaction of each explanatory variable with sex were all non-significant with the exception of mother's marital status at birth where the married category was associated with a greater response rate in females compared to males. There was no association of response rate with year of birth or birth weight or gestational age (not shown).
Table 1. Questionnaire response rates by early life factors
The associations of response rate with cognitive score at ages 7, 9 and 11 years for both sexes combined are shown in Figure 2. The almost monotonic increase in questionnaire response rate with each 5 point increase in score at each age is very striking. Response rates by quintile of cognitive score are summarised separately by sex in Table 2. Response rates increased progressively at all ages from the bottom to the top fifth of cognitive test score in both sexes. Each of the four components of cognition measured at 11 years (verbal reasoning × 2, English and arithmetic) showed similar associations with response and were adequately summarised by association with the total score. There was no evidence of a statistically significant interaction between sex and cognitive scores at any age, and thus in the remaining analyses we present results for both sexes combined.
Table 2. Questionnaire response rates by sex-specific cognition score quintiles
Figure 2. Response rates by cognition test scores at 7, 9 and 11 for males and females combined. Five point bands were used for cognition scores.
Sex-adjusted odds ratios for one SD increase in cognition scores at 7, 9 and 11 were slightly attenuated (less than 6%) by adjustment for socio-economic position, height and weight for age and Rutter B score (Table 3) but nevertheless remained substantial. Cognitive test scores at ages 7, 9 and 11 years are highly correlated (Pearson correlation coefficients: 0.74 to 0.88). How far the associations of response rate with cognitive score at each age are independent is also examined in Table 3. Nearly all of the effects at ages 7 and 9 were removed by adjustment for cognitive test score at age 11 years. In contrast, the effect of cognition at 11 was only slightly attenuated by adjustment for cognitive test score at 7 or 9.
Table 3. Odds ratios for questionnaire response by cognition scores with adjustment for other early life factors and cognition at different ages
Discussion
We have found that a wide range of characteristics in infancy and childhood were associated with questionnaire response rate. Cognitive test score in childhood was particularly strongly related to the probability of responding to the questionnaire survey independently of other factors, with an almost monotonic increase in response rate, with no evidence of a threshold effect. At age 11 response rates among those in the top fifth of the cognitive test score distribution had a response rate that in absolute terms was 25% higher than in the bottom fifth.
Although the possibility of obtaining biased samples in postal questionnaires with incomplete response rates has been well documented in the survey and epidemiological literature [3,5,7,25,26], the emphasis has been mainly upon the determinants of response related to characteristics of subjects at the time they receive the survey instrument. Ours is the first study that has looked at early life influences on response rate to a postal questionnaire in middle age in a cohort not previously contacted in adult life. A similar analysis by Wadsworth and colleagues [16] of childhood influences on later continued participation in the British 1946 birth cohort, in which subjects had been repeatedly contacted throughout life, found that continued participation was least likely among those who had been in the most disadvantaged socio-economic circumstances in childhood and those with lowest cognitive test scores at 8 years of age. The sex-adjusted odds ratio for avoidable loss to follow-up at age 53 years in the top compared to the bottom quarter of childhood cognition was 0.52 (95% Confidence Interval; 0.42 to 0.64). However, these analyses did not involve any multi-variable adjustments, so it is unclear how far this association may be explained by confounding with socio-economic and other factors.
Finding an association between childhood cognition and questionnaire response rate in adult life was expected. However, the strength and consistency of this association, with almost monotonic increases across the entire range of test scores, are surprising. At least two explanations for this powerful effect should be considered. The first one would hypothesise a pathway via education. High cognitive test scores in childhood are highly predictive of higher levels of education. Subjects with higher level of education may have higher response rate because they may be more health conscious, more interested in research and not feel intimidated by a relatively substantial questionnaire. The second possibility is a more direct pathway, where cognitive ability such as a problem solving (akin to completing a 21 page questionnaire) played an important role. Our study was not able to definitively distinguish between these two pathways. However, it should be noted that the effect of cognition at 11 was independent of cognition at 7 and 9 years, whereas the effect of cognition at 7 was attenuated substantially by adjusting for cognition at 11. The test at age 11 explicitly included components that measured educational attainment (mathematics and English), and the overall score was used as one of the key criteria for determining secondary school. Thus cognitive score at 11 is more likely to reflect educational progress to that age than the score at 7 years, suggesting that our data are most consistent with the link between childhood cognition and response rate being via attained educational level.
Some limitations of this study should be noted. Firstly, bias by undelivered mail might be possible. We do not know which subjects actually received a questionnaire. If cognitive test score was positively related to the probability of actually receiving the questionnaire this would generate, or at least contribute to, the observed gradient. However, this seems unlikely. As we have shown elsewhere [17], the lower a person's cognitive test score the less likely it was that they moved away from the Aberdeen area, and thus the lower the probability that the NHS had an incorrect or out-of-date address for them. Secondly, our data set included an appreciable proportion of subjects who did not have a cognitive test score at 11 (n = 2,339, 20.7% of 11,282). However, 80% (n = 1,873) of those without score at 11 were missing it simply because they had not reached this age during the survey period. We repeated the multivariate analyses after excluding cognition at 11 and confirmed that odds ratios for cognition at 7 and 9 were essentially unchanged. Therefore, we do not believe that selection bias due to this missing data could explain the observed associations. Finally, it should be noted that our findings may not automatically apply to other survey methods such as telephone and face to face interview.
Any postal questionnaire survey should be designed and undertaken to achieve the maximum possible response rate. However, in many contexts there will remain major concerns about non-response bias. In these situations the powerful links that exist between early life characteristics and response needs to be taken into account when interpreting the data. This can be done in part by employing sensitivity analyses. Moreover, in some situations multiple imputation methods may also be useful[27,28].
Conclusion
Our results indicate that the interpretation of associations between childhood and later life factors in life-course studies using postal questionnaires need to take account of the fact that factors in childhood can be strongly related to response rate. Particular caution is needed if the outcomes are related to childhood cognitive function. Sensitivity analyses that explore the extent of such biases are strongly recommended.
Quite apart from these methodological conclusions, the study finding shows that this simple and routinely collected information on cognition is extraordinarily predictive of the complex collection of later-life behaviours and circumstances that jointly generate the likelihood of a completed questionnaire being returned.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DAL, SMM and HC designed the questionnaire survey. HC supervised the data collection. YN analyzed, interpreted the data and drafted the paper in consultation with the other authors. All authors read and approved the final manuscript.
Acknowledgements
We are very grateful to Raymond Illsley for providing us with the data from the Aberdeen Child Development Survey and for his advice about the study. Graeme Ford played a crucial role in identifying individual cohort members and in helping us initiate the process of revitalisation. Sally Macintyre, Doris Campbell, George Davey Smith, Marion Hall, Bianca de Stavola, Susan Morton, David Batty, David Godden, Di Kuh, Debbie Lawlor, Glyn Lewis and Viveca Östberg collaborated with David Leon to revitalise the cohort. Heather Clark managed the study at the Dugald Baird Centre, Aberdeen with the assistance of Margaret Beveridge. We would also like to thank staff at the ISD (Edinburgh), GRO (Scotland) and NHSCR (Southport) for their substantial contributions, John Lemon who undertook the linkage to the Aberdeen Maternity and Neonatal Databank and Valerie Mccormack for the statistical advice. The Aberdeen Children of the 1950s Study was funded as a component project (G0828205) of a Medical Research Council Co-operative Group Life-course and trans-generational influences on disease risk (G9819083). A project on cognition and adult health in the cohort has been funded by the Chief Scientists Office, Scottish Executive Health Department, which currently funds HC. YN was funded by the Uehara Memorial Foundation.
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Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2288/5/16/prepub
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Tuesday, June 19, 2012
The Intertsection of Book Burning and Blogging Your Lunch
There's not much book burning and blogging your lunch have in common. One is both evil and vile, while the other is considered the ultimate waste of space on the Internet. Turns out, their both excellent ways to make change happen.
Check these stories out: Leo Burnett Detroit Saves Local Library by Threatening Book Burning and Nine-Year-Old's Cafeteria Food Blog Goes Viral.
Both are stories of small taking on, and winning against big. Brilliant, all around.
You don't even have to read the book burning story, you can catch it all on this 3 minute video right here:
And who would have ever thought a 9 year could have raised $134,000 in charity?
Sometimes the Internet is truly awesome.
LinkWithin
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<D <M <Y
Y> M> D>
: Is Marriage to Unix the Answer to Enterprise Linux? You don't ask that kind of question unless you already have an answer handy.
: Moeh.
: The standard model: Gotta catch 'em all!
: Does anyone know of a hosting service which would sell me a disk allotment of x megabytes, and allow me to host arbitrarily many domains within that quota? I forsee a future in which I have a million tiny domains, and I don't want to have an account for each domain.
: He's an intriguing bow-shock. She's a poorly understood gas cloud. Can they get along in the suburbs?
[Main]
Unless otherwise noted, all content licensed by Leonard Richardson
under a Creative Commons License.
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You are here: Home / Data and maps / Maps and graphs / EU-15 and EU-27 greenhouse gas emissions from energy supply and use (excluding transport) compared with energy demand
EU-15 and EU-27 greenhouse gas emissions from energy supply and use (excluding transport) compared with energy demand
Created : Nov 12, 2009 Published : Dec 04, 2007 Last modified : Nov 29, 2012 11:38 AM
For France and Italy, sectoral projections ´with additional measures´had to be gap-filled to calculate EU15 greenhouse gas projections
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Since sectoral emission projections for Bulgaria and Cyprus were not available, greenhouse gas projections for the EU27 are calculated on the basis of projections reported by 25 Member States. The 2005–2010 percent variation for the EU25 was applied to Cyprus and Bulgaria to obtain an EU27 projection for 2010. No additional measures were reported for Belgium, Denmark, Luxembourg, Netherlands, Spain, Sweden, United Kingdom, Hungary, Latvia, Lithuania, Malta and Poland. For these Member States, the ´with existing measures´ projections were used for the calculation of the EU27 ´additional measures´ projections.
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Hubbert, Marion King
Hubbert, Marion King
This article has been reviewed by the following Topic Editor: Ida Kubiszewski PhD
Marion King Hubbert (1903-1989), an American geophysicist best known for his accurate prediction of the peak in oil production in the lower 48 United States. In 1956, he published what is now known as the “Hubbert Curve”, a simple mathematical model of oil supply; he used this to predict that the peak of crude-oil production in the United States would occur between 1966 and 1971. It actually occurred in 1970. Hubbert's estimate of future supplies were much lower than those accepted by many American petroleum companies and leaders of the U.S. Geological Survey (USGS), producing a long-running and public debate about the future of oil supplies that continues to this day. Hubbert also made numerous fundamental contributions to geophysics. He demonstrated that fluids can become entrapped under circumstances previously not thought possible, leading to a major reassessment of techniques to locate oil and natural gas deposits. Hubbert also explained the puzzling displacement of enormous blocks of material, known to geologists as overthrust faults, as a consequence of fluid pressure between such blocks and underlying materials.
Some of his best known works include Energy from Fossil Fuels and Nuclear Energy and the Fossil Fuels.
Further Reading
Marion King Hubbert (Handbook of Texas Online)
Citation
Cutler J. Cleveland (Lead Author);Ida Kubiszewski PhD (Topic Editor) "Hubbert, Marion King". In: Encyclopedia of Earth. Eds. Cutler J. Cleveland (Washington, D.C.: Environmental Information Coalition, National Council for Science and the Environment). [First published in the Encyclopedia of Earth February 10, 2007; Last revised Date February 10, 2007; Retrieved May 18, 2013 <http://www.eoearth.org/article/Hubbert,_Marion_King>
The Author
Cutler J. Cleveland is Professor of Earth and Environment at Boston University, where he also is on the faculty of the Center for Energy and Environmental Studies. Professor Cleveland is Editor-in-Chief of the Encyclopedia of Energy (Elsevier, 2004), winner of an American Library Association award, the Dictionary of Energy (Elsevier, 2005), Handbook of Energy (Elsevier, forthcoming), and is the Founding Editor-in-Chief of the Encyclopedia of Earth. He is the recipient of the Adelma ... (Full Bio)
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Upcoming events
From Forensics Wiki
Revision as of 07:02, 2 June 2008 by Frnzxguy (Talk | contribs)
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PLEASE READ BEFORE YOU EDIT THE LISTS BELOW
Events should be posted in the correct section, and in date order. An event should NEVER be listed in more than one section (i.e. Ongoing/Continuous events should not be listed in Scheduled Training). When events begin the same day, events of a longer length should be listed first. New postings of events with the same date(s) as other events should be added after events already in the list. If a provider offers the same event at several locations simultaneously, the listing should have a single (ONE) entry in the list with the date(s) and ALL locations for the event. Please use three-letter month abbreviations (i.e. Sep, NOT Sept. or September), use two digit dates (i.e. Jan 01 NOT Jan 1), and use date ranges rather than listing every date during an event(i.e. Jan 02-05, NOT Jan 02, 03, 04, 05).
Some conferences or training opportunities may be limited to Law Enforcement Only or to a specific audience. Such restrictions should be noted when known.
This is a BY DATE listing of upcoming conferences and training events relevant to digital forensics. It is not an all inclusive list, but includes most well-known activities. Some events may duplicate events on the generic conferences page, but entries in this list have specific dates and locations for the upcoming event.
This listing is divided into four sections (described as follows):
1. Calls For Papers - Calls for papers for either Journals or for Conferences, relevant to Digital Forensics (Name, Closing Date, URL)
2. Conferences - Conferences relevant for Digital Forensics (Name, Date, Location, URL)
3. On-Going / Continuous Training - Training opportunities that are either always available online/distance learning format or that are offered the same time every month (Name, date-if applicable, URL)
4. Scheduled Training Courses - Training Classes/Courses that are scheduled for specific dates/locations. This would include online (or distance learning format) courses which begin on specific dates, instead of the "start anytime" courses listed in the previous section. (Name, Date(s), Location(s), URL) (note: this has been moved to its own page.)
The Conference and Training List is provided by the American Academy of Forensic Sciences (AAFS) Digital and Multimedia Sciences Section Listserv. (Subscribe by sending an email to listserv@lists.mitre.org with message body containing SUBSCRIBE AAFS-DIGITAL-MULTIMEDIA-LIST) Requests for additions, deletions or corrections to this list may be sent by email to David Baker (bakerd AT mitre.org).
Contents
Calls For Papers
Title Due Date Website
Anti-Phishing Working Group eCrime Researchers Summit Jun 05, 2008 http://www.ecrimeresearch.org/2008/cfp.html
Economic and High Tech Crime Summit Jun 06, 2008 http://summit.nw3c.org/speakers/call_for_speakers.cfm
1st Workshop on Open Source Software for Computer and Network Forensics Jun 07, 2008 http://conferenze.dei.polimi.it/ossconf/call_for_papers.php
Call for Chapter: Handbook of Research on Computational Forensics, Digital Crime and Investigation: Methods and Solutions Jun 30, 2008 http://www.dcs.warwick.ac.uk/~ctli/Call_For_Chapters_2.html
2009 DOD Cyber Crime Conference Jul 01, 2008 http://www.dodcybercrime.com/9CC/call_for_papers.asp
ANZFSS - 19th International Symposium on the Forensic Sciences Jul 06, 2008 http://www.anzfss2008.org.au/content/view/56/63/
DeepSec 2008 Jul 15, 2008 https://deepsec.net/cfp/
American Academy of Forensic Sciences Annual Meeting Aug 01, 2008 http://www.aafs.org/default.asp?section_id=meetings&page_id=aafs_annual_meeting
5th Annual IFIP WG 11.9 International Conference on Digital Forensics Oct 15, 2008 http://www.ifip119.org/Conferences/WG11-9-CFP-2009.pdf
Conferences
Title Date/Location Website
Fourth Annual Cyber Security and Information Intelligence Research Workshop (CSIIRW-08) May 12-14, Oak Ridge, TN http://www.ioc.ornl.gov/csiirw
Ohio HTCIA Spring Training Conference May 12-14, Lakeland Community College, OH http://www.ohiohtcia.org/conference.html
LayerOne 2008 Information Technology Conference May 17-18, Los Angeles, CA http://layerone.info
EuSecWest Security Conference 2008 May 21-22, London, England http://eusecwest.com/
3rd International Workshop on Systematic Approaches to Digital Forensic Engineering May 22, Oakland, CA http://conf.ncku.edu.tw/sadfe/sadfe08/
4th GFIRST National Conference Jun 01-06, Orlando, FL http://www.us-cert.gov/GFIRST/index.html
Techno-Security 2008 Jun 01-04, Myrtle Beach, SC http://www.techsec.com/html/Techno2008.html
Gartner IT Security Summit Jun 02-04, Washington, DC http://www.gartner.com/it/page.jsp?id=507478&tab=overview
6th International Conference on Applied Cryptography and Network Security Jun 03-06, Columbia University, New York City, NY http://acns2008.cs.columbia.edu/
RECON 2008 Jun 13-15, Montreal, Quebec, Canada http://recon.cx/2008/
Usenix Annual Technical Conference Jun 22-27, Boston, MA http://www.usenix.com/events/usenix08/
International Association of Forensic Sciences Annual Meeting Jul 21-26, New Orleans, LA http://www.iafs2008.com/
17th USENIX Security Symposium Jul 28-Aug 01, San Jose, CA http://www.usenix.org/events/sec08/
Blackhat USA 2008 Briefings & Training Aug 02-07, Las Vegas, NV http://www.blackhat.com/html/bh-link/briefings.html
2nd International Workshop on Computational Forensics Aug 07-08, Washington, DC http://iwcf08.arsforensica.org
Defcon 16 Aug 08-10, Las Vegas, NV http://www.defcon.org
GMU 2008 International Training Symposium Aug 11-15, Fairfax, VA http://rcfg.org/
Digital Forensic Research Workshop Aug 11-13, Baltimore, MD http://www.dfrws.org
International Workshop on Digital Crime and Forensics in conjunction w/4th International Conference on Intelligent Information Hiding and Multimedia Signal Processing Aug 15-17, Harbin, China http://www.dcs.warwick.ac.uk/~ctli/CFP_IWDCF2008.html
1st Workshop on Open Source Software for Computer and Network Forensics Sep 07-10, Milan Italy http://conferenze.dei.polimi.it/ossconf/index.php
11th International Symposium on Recent Advances in Intrusion Detection Sep 15-17, Cambridge, MA http://www.ll.mit.edu/IST/RAID2008/
4th International Conference on IT Incident Management & IT Forensics Sep 23-25, Mannheim, Germany http://www.imf-conference.org/
VB2008 anti-malware conference Oct 01-03, Ottawa, Canada http://www.virusbtn.com/conference/vb2008/
ANZFSS - 19th International Symposium on the Forensic Sciences Oct 06-09, Melbourne, Australia http://www.anzfss2008.org.au/
13th European Symposium on Research in Computer Security Oct 06-08, Malaga, Spain http://www.isac.uma.es/esorics08/
Economic and High Tech Crime Summit 2008 Oct 07-08, Memphis, TN http://summit.nw3c.org/
3nd International Annual Workshop on Digital Forensics & Incident Analysis Oct 09, Malaga, Spain http://www.icsd.aegean.gr/wdfia08/
Anti-Phishing Working Group eCrime Researchers Summit Oct 15-16, Atlanta, GA http://www.ecrimeresearch.org/
2008 HTCIA International Training Conference Oct 22-28, Atlantic City, NJ http://www.htcia.org/conference.shtml
DeepSec 2008 Nov 11-14, Vienna, Austria https://deepsec.net/
2009 DoD Cyber Crime Conference Jan 24-30, St. Louis, MO http://www.dodcybercrime.com/
5th Annual IFIP WG 11.9 International Conference on Digital Forensics Jan 25-28, Orlando, FL http://www.ifip119.org/Conferences/
American Academy of Forensic Sciences Annual Meeting Feb 16-21, Denver, CO http://www.aafs.org/default.asp?section_id=meetings&page_id=aafs_annual_meeting
On-going / Continuous Training
Title Date/Location or Venue Website
Basic Computer Examiner Course - Computer Forensic Training Online Distance Learning Format http://www.cftco.com
Linux Data Forensics Training Distance Learning Format http://www.crazytrain.com/training.html
SANS On-Demand Training Distance Learning Format http://www.sans.org/ondemand/?portal=69456f95660ade45be29c00b0c14aea1
MaresWare Suite Training First full week every month, Atlanta, GA http://www.maresware.com/maresware/training/maresware.htm
Evidence Recovery for Windows Vista™ First full week every month, Brunswick, GA http://www.internetcrimes.net
Evidence Recovery for Windows Server® 2003 R2 Second full week every month, Brunswick, GA http://www.internetcrimes.net
Evidence Recovery for the Windows XP™ operating system Third full week every month, Brunswick, GA http://www.internetcrimes.net
Computer Forensics Training and CCE™ Testing for Litigation Support Professionals Third weekend of every month (Fri-Mon), Dallas, TX http://www.md5group.com
Scheduled Training Courses
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About this Journal Submit a Manuscript Table of Contents
Journal of Robotics
Volume 2010 (2010), Article ID 860790, 9 pages
doi:10.1155/2010/860790
Research Article
Haptic Perception with Self-Organizing ANNs and an Anthropomorphic Robot Hand
Lund University Cognitive Science, Kungshuset, Lundagård, SE-222 22 LUND, Sweden
Received 18 August 2009; Accepted 30 December 2009
Academic Editor: Noriyasu Homma
Copyright © 2010 Magnus Johnsson and Christian Balkenius. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
We have implemented and compared four biologically motivated self-organizing haptic systems based on proprioception. All systems employ a 12-d.o.f. anthropomorphic robot hand, the LUCS Haptic Hand 3. The four systems differ in the kind of self-organizing neural network used for clustering. For the mapping of the explored objects, one system uses a Self-Organizing Map (SOM), one uses a Growing Cell Structure (GCS), one uses a Growing Cell Structure with Deletion of Neurons (GCS-DN), and one uses a Growing Grid (GG). The systems were trained and tested with 10 different objects of different sizes from two different shape categories. The generalization abilities of the systems were tested with 6 new objects. The systems showed good performance with the objects from the training set as well as in the generalization experiments. Thus the systems could discriminate individual objects, and they clustered the activities into small cylinders, large cylinders, small blocks, and large blocks. Moreover, the self-organizing ANNs were also organized according to size. The GCS-DN system also evolved disconnected networks representing the different clusters in the input space (small cylinders, large cylinders, small blocks, large blocks), and the generalization samples activated neurons in a proper subnetwork in all but one case.
1. Introduction
Haptic perception, that is, active tactile perception, is of outmost importance in the field of robotics since a well-performing robot must be able to interact with objects in its environments. However, haptic perception is also important in supporting and sometimes also in substituting the visual modality during the recognition of objects. Like humans, robots should be able to perceive shape and size as well as to discriminate between individual objects by haptic exploration.
The modelling of haptic perception as well as the implementation of haptic perception in robots have been neglected areas of research. Robot hand research has mainly focused on grasping and object manipulation [14], and many models of hand control have focused on the motor aspect rather than on haptic perception [5, 6], although there are some exceptions [717].
Previously we have designed and implemented haptic size perception systems [1821], haptic shape perception systems [2225], and haptic texture/hardness perception systems [26, 27].
The haptic size perception systems used a simple three-fingered robot hand, the LUCS Haptic Hand I, with the thumb as the only movable part. The LUCS Haptic Hand I was equipped with 9 piezo-electric tactile sensors. This system used Self-Organizing Maps, SOMs, [28] and a neural network with leaky integrators and it successfully learned to categorize a test set of spheres and cubes according to size.
The haptic shape perception systems used a three-fingered 8 d.o.f robot hand, the LUCS Haptic Hand II, equipped with a wrist for horizontal rotation and a mechanism for vertical repositioning. This robot hand was equipped with 45 piezo-electric tactile sensors. This system used active explorations of the objects by several grasps with the robot hand to gather tactile information. The LUCS Haptic Hand II was not equipped with any proprioceptive sensors, that is, sensors that register joint angles, but the system used the positioning commands to the actuators as a substitute. Depending on the version of the system, either tensor product (outer product) operations or a novel neural network, the Tensor Multiple Peak SOM, T-MPSOM [2325], was used to code the tactile information in a useful way while a SOM was used for the categorization. The system successfully learned to discriminate between different shapes as well as between different objects within a shape category when tested with a set of spheres, blocks, or cylinders.
The haptic texture/hardness perception systems employed a microphone-based texture sensor and a hardness sensor that measures the displacement of a stick pressed at the object with a constant force. With these sensors, we implemented systems that automatically evolved monomodal as well as bimodal representations of texture and hardness [26], and also a system that evolved monomodal representations of texture and hardness while at the same time learning to associate these representations. The latter was done by using a variant of the SOM called Associative Self-Organizing Map (A-SOM) [27].
This paper explores a somewhat different approach to shape and size perception which is based solely on proprioception. Using the position of each joint as the only input, we have designed an anthropomorphic robot hand and self-organizing systems that can discriminate objects and categorize them according to shape and size [2931].
When designing a neural network based on self-organizing perception system a natural question comes up, namely, what kinds of neural network architectures are most suitable to use. A common choice is the self-organizing map (SOM) that we have used in previous work. This is often a very good choice but it suffers from some limitations, for example, the topological structure is fixed and the number of neurons in the neural network has to be preset by the system designer. Other limitations are that parameters like the learning rate, the initial neighbourhood size, and the decreasing rate of the neighbourhood size also have to be set manually by the designer.
To address these limitations we have, in addition to a SOM-based system, explored and compared three other haptic systems based on the same robotic hand. These systems are based on alternative neural network architectures that avoid some or all of the limitations with a SOM-based system. The four systems differ in one respect, namely, in the kind of self-organizing neural network employed to cluster the input. The first system uses the SOM, the second uses the Growing Cell Structures (GCS), the third uses the Growing Cell Structures with Deletion of Neurons GCS-DN [32, 33], and the fourth uses the Growing Grid (GG) [34].
2. LUCS Haptic Hand III
The LUCS Haptic Hand III is a five-fingered 12-d.o.f anthropomorphic robot hand equipped with 11 proprioceptive sensors (Figure 1). The robot hand has a thumb consisting of two phalanges, whereas the other fingers have three phalanges. The thumb can be separately flexed/extended in both the proximal and the distal joints and adducted/abducted. The other fingers can be separately flexed/extended in their proximal joints, whereas the middle and the distal joints are flexed/extended together. All this is similar to the human hand. The wrist can also be flexed/extended as the wrist of a human hand. The phalanges are made of plastic pipe segments and the force transmission from the actuators, which are located in the forearm, are handled by tendons inside the phalanges in a similar way to the tendons of a human hand. All fingers, except the thumb, are mounted directly on the palm. The thumb is mounted on an RC servo, which enables the adduction/abduction. The RC servo is mounted on the proximal part of the palm, similar to the site of the thumb muscles in a human hand. The actuators of the fingers and the wrist are located in the forearm. This is also similar to the muscles that actuate the fingers of a human hand. The hand is actuated by in total 12 RC servos, and to get proprioceptive sensors, the internal potentiometers in the RC servos, except the RC servo that actuates the wrist, have been included in the sensory circuit (Figure 2). The resistances of these potentiometers are proportional to the angle of the different joints.
Figure 1: The LUCS Haptic Hand III while holding a screw driver, in open position seen in a front view and in a side view. Some of the actuators in the forearm can also be seen in the side view. The 12-d.o.f robot hand has five fingers, is of the same size as a human hand, and all its parts have approximately the same proportions as their counterparts in a human hand. Each finger can be separately flexed/extended in the proximal joint, whereas the medial and distal joints are flexed/extended together as real human fingers. As a human hand, the thumb has only a proximal and a distal phalang. These can also be separately flexed/extended. In addition the thumb can also be adducted/abducted in a way similar to the human thumb. The wrist is capable of flexion/extension. The actuators of the LUCS Haptic Hand III are controlled via an SSC-32 (Lynxmotion Inc.). The proprioceptive sensors are scanned with a MAX396CPI multiplexor chip and digitalized using an NiDaq 6008 (National Instruments). The NiDaq 6008 converts multiple analog input signals to digital signals, which are conveyed to the computer via a USB-port. The robot hand is equipped with two multiplexor chips, which means it is prepared for 21 additional sensors.
Figure 2: The circuits involved in the proprioceptive part of the LUCS Haptic Hand III. The NiDaq 6008 converts multiple analog input signals to digital signals that are conveyed to the computer via an, USB-port. The MAX396 chip is a multiplexor circuit for selection of sensor channels.
The software for the LUCS haptic hand III is developed in C++ and Java, and much of it runs within the Ikaros system [35, 36]. Ikaros provides an infrastructure for computer simulations of the brain and for robot control.
3. Self-Organizing ANNs
3.1. Self-Organizing Map
The SOM consists of an grid of neurons with a fixed number of neurons and a fixed topology. Each neuron is associated with a weight vector . During adaptation, the weight vectors for the neurons are adjusted to a degree which is determined by a neighbourhood function with a size that decreases with time. The adaptation strength also decreases with time. The SOM variant used in our experiments is a dot product SOM with Gaussian neighbourhood. The adaptation algorithm works as follows.
At time , each neuron receives an input vector . The neuron associated with the weight vector most similar to the input vector is selected,
The weight vectors of the neurons are adapted according to:
where is the adaptation strength with when and the neighbourhood function is a Gaussian function the width of which decreases with time.
3.2. Growing Cell Structures
The GCS has a variable number of neurons and a -dimensional topology, where can be arbitrarily chosen. The adaptation of a weight vector in the GCS is done in a similar way as in the SOM, but the adaptation strength is constant over time and only the best matching unit and its direct topological neighbours are adapted. The GCS estimates the probability density function of the input space by the aid of local signal counters that keep track of the relative frequencies of input signals gathered by each neuron. These estimates are used to indicate proper locations to insert new neurons. The insertion of new neurons by this method will result in a smoothing out of the relative frequencies between different neurons. The advantages of this approach are that the topology of the network will self-organize to fit the input space, the proper number of neurons for the network will be automatically determined and the learning rate and neighbourhood size parameters are constant over time. The basic building block and also the initial configuration of the GCS are a -dimensional simplex. Such a simplex is for a triangle. The variant of the GCS algorithm used in our experiments works as follows.
The network is initialized to contain neurons with weight vectors randomly chosen. The neurons are connected so that a -dimensional simplex is formed.
At time step , an input vector activates a winner neuron for which the following is valid
where is the Euclidean distance, and the squared distance between the input vector and the weight vector of the winner neuron is added to a local error variable :
The weight vectors are updated by fractions and , respectively, according to:
where is the set of direct topological neighbours of .
A neuron is inserted if the number of input vectors that have been generated so far is an integer multiple of a parameter . This is done by finding the neuron with the largest accumulated error and the neuron among its direct topological neighbours which has the weight vector with the longest distance from the weight vector of the neuron , insert the new neuron in between, remove the earlier connection , and connect with and and with all direct topological neighbours that are common for and . The weight vector for is interpolated from the weight vectors for and :
The local error counters for all neighbours to are decreased by a fraction that depends on the number of neighbours of :
The error variable for is set to the average of its neighbours:
and then the error variables of all neurons are decreased:
In GCS-DN, a neuron (or several if that is necessary to keep a consistent topological structure of -dimensional simplices) is deleted, provided that the network has reached its maximum size; at the same occasions new neurons are inserted. Thereafter, new neurons are inserted again according to the algorithm described above until the network has reached its maximum size again. This process is repeated a preset number of times, in our experiments 250 times.
3.3. Growing Grid
The GG can be seen as an incremental variant of the SOM. It consists of an grid of neurons with a fixed topology but with and increasing with time as new rows and columns are inserted. In addition to a weight vector , each neuron also has a local counter variable to estimate where to insert new rows or columns of neurons in the grid. The self-organizing process of a GG is divided into two phases: a growth phase and a fine-tuning phase. During the growth phase, the grid grows by insertion of new rows and columns until the wanted size of the network has been achieved. During the fine-tuning phase, the network size does not change and a decreasing adaptation strength is used. The size of the neighbourhood is not decreasing with time. Instead the network is growing with a constant neighbourhood size and therefore the fraction of all neurons that are adapted decreases over time. The variant of the GG algorithm used in our experiments is described below.
Growth Phase
Initialize the network to contain neurons with weight vectors randomly chosen. At time , an input vector is generated and received by each neuron in the grid.
The neuron associated with the weight vector most similar to the input vector is selected:
Increment the local counter variable for : The weight vectors of the neurons are adapted according to: where is the adaptation strength and the neighbourhood function is a Gaussian function. Notice that and are not functions of though.
A new row or column is inserted if the number of input vectors that have been generated so far is an integer multiple of the current number of neurons in the grid. This is done by finding the neuron with the largest value of the local counter variable and the neuron among its direct topological neighbours which has the weight vector with the longest distance from the weight vector of the neuron . Depending on the relative positions of and , a new row or a new column is inserted.
If and are in the same row, then a new column is inserted between the columns of and . The weight vectors for the new neurons are interpolated from their direct neighbours in the same row.
If and are in the same column, then a new row is inserted between the rows of and . The weight vectors for the new neurons are interpolated from their direct neighbours in the same column.
Adjust or to reflect the new numbers of rows and columns in the grid. Reset all local counter values:
If the desired network size has not been reached, then go to step 2, that is, generate a new input vector.
Fine-Tuning Phase
This phase is similar to the growth phase but the adaptation strength is now decreasing with time and no insertions of new rows or columns are done. This phase stops after a preset number of iterations.
4. Proprioception-Based Systems
All the four systems (Figure 3) consist of the LUCS Haptic Hand III, sensory and motor drivers, a commander module that executes the grasping movements, and a Self-Organizing ANN (SO-ANN). The kind of SO-ANN employed is the only thing that distinguishes one system from another. The sensory driver scans the proprioceptive sensors when requested to do so by the commander module, while the motor driver translates high-level motor commands from the commander module to positioning commands for the robot hands servo controller board. When the commander executes a grasp, and the robot hand is fully closed around the object, the sensory driver scans the 11 proprioceptive sensors and outputs an eleven-element vector to the SO-ANN, which is adapted.
Figure 3: Schematic depiction of the systems. The commander module executes the grasps by sending high-level motor commands to the motor driver, which translates and conveys the information to the servo controller board of the robot hand. When the robot hand has become fully closed around the object, the commander module requests a scanning of the 11 proprioceptive sensors of the robot hand. The sensory information is conveyed as a vector to a Self-Organizing ANN (SO-ANN). The SO-ANN is a Self-Organizing-Map, a Growing Cell Structures, a Growing Cell Structures with Deletion of Neurons, or a Growing Grid.
The SOM-based system uses a 225 neurons dot product SOM with plane topology, which uses softmax activation with the softmax exponent equal to 10 [37]. It is trained by 2000 iterations.
The GCS-based system grows, by inserting a new neuron every 19th iteration, until a size of 225 neurons has been reached.
The GCS-DN based system grows until a size of 225 neurons has been reached, also by inserting a new neuron every 19th iteration, then the deletion/insertion process described in Section 3.2 is repeated 250 times. Finally this yields a number of disconnected networks with altogether 225 neurons.
The GG-based system grows by inserting a new row or column each time the number of time steps since the previous insertion equals a multiple of the current grid size, that is, until with . The growth phase lasts until a minimum grid size of 225 neurons has been reached, then the model runs in fine tuning mode for 1000 iterations.
We have trained the systems with 10 objects (see Table 1 objects a–j). These objects are either cylinder shaped or block shaped. There are five objects of each shape category. All objects are sufficiently high to be of a nonvariable shape in those parts grasped by the robot hand, for example, a bottle is grasped on the part of equal diameter below the bottle neck.
Table 1: The 16 objects used in the experiments with the four systems. The objects a–j were used both for training and testing, whereas the objects 1–6 were used in the generalization tests.
During the grasping tests, the test objects were placed on a table with the open robot hand around them. If the objects were block shaped, we always placed the widest side against the palmar side of the robot hand.
To simplify the testing procedure, each object was grasped 5 times by the robot hand, that is, in total 50 grasps were carried out, and the sensory information was written to a file. Then the SO-ANN were trained and tested with this set of 50 samples. The training phase for the SOM system lasted for 2000 iterations. The GCS system was trained until a network size of 225 neurons was reached. The GCS-DN system was trained until a network size of 225 neurons was reached and then the insertion/deletion process described in Section 3.2 was repeated 250 times. The GG system was trained with a growth phase which lasted until the minimal network size reached 225 neurons, and then for 1000 iterations in fine tuning mode.
Each fully trained system was tested with the original training set and in addition with three new block-shaped and three new cylinder-shaped objects of variable sizes (see Table 1, objects 1–6) as described in the next section.
5. Generalization Tests
We have also tested if the systems were able to generalize their knowledge to new objects, that is, to objects not included in the training set. To this end we used 6 new objects, Table 1. 1–6, 3 cylinder shaped objects and 3 block shaped objects. The new objects were of variable sizes. The fully trained systems were fed by input from grasps of the new objects under the same conditions as the objects in the training set. Each object in the new set was grasped once and the activity in the SO-ANN for each system was recorded.
6. Results
The results are depicted in Figure 4. Figure 4(a) shows the centres of activation in the SOM in the fully trained SOM-based system when tested with the training set and the test set. The SOM seems to be organized according to shape. Four groups of objects can be distinguished in the map, large block shapes, small block shapes, large cylindrical shapes, and small cylindrical shapes. The SOM also seems to be organized in a clockwise manner according to size. The result of the generalization experiment shows that all test objects are mapped so that they are ordered according to size in the same way as the objects in the training set, and that they are also correctly mapped according to shape. The activations in the SOM also indicate that it is possible to discriminate individual object of the training set to a large extent and this is also true for the test objects, since each of the test objects is also mapped so that it can be identified as the most similar object of the training set. The results with the SOM-based system are thoroughly described in [29].
Figure 4: The test results of the four systems. (a) The SOM-based system is organized according to shape and size. Groups of large blocks, small blocks, large cylinders, and small cylinders can be distinguished. The activations tend to be located according to size of the objects in a clockwise manner. Individual objects can be discriminated to a large extent. (b) The GCS-based system produces similar results as the SOM-based system, that is, it is organized according to shape and size. The GCS-based system separates large blocks, small blocks, large cylinders, and small cylinders, and the objects are represented according to size with the smallest objects uppermost and the largest lowermost in the GCS. Also individual objects can be discriminated to a large extent. (c) The GCS-DN based system self-organized into subnetworks, where one or more subnetworks represent the categories large blocks, small blocks, large cylinders, and small cylinders. (d) The GG-based system separates large blocks, small blocks, large cylinders, and small cylinders, and the grid is organized according to size. Individual objects can be discriminated to a large extent. The 6 test objects (indicated with the numbers 1–6) not included in the training set activated neurons at proper locations perfectly in all systems but the GCS-DN based system. In that system, object 1 triggered activation in the wrong subnetwork. (see Table 1 for the meaning of the labels).
Figure 4(b) shows the centres of activation in the GCS in the fully trained GCS-based system. Only the part of the GCS which is activated by some object is shown in the figure. This system produces similar results as the SOM-based system, that is, the organization of the GCS separates large block shapes, small block shapes, large cylinder shapes, and small cylinder shapes. The GCS is also organized according to size with the smallest objects represented uppermost in the GCS and the largest in the lowermost part. The ability for discrimination of individual objects is approximately similar as that for the SOM-based system. Also this system activates neurons at proper locations when fed with the objects of the generalization test set.
Figure 4(c) shows the final network structure of the fully trained GCS-DN based system. As can be seen, this network structure consists of several disconnected subnetworks. This is due to the removal of neurons that represent parts of the input space with a low value of the probability density function. As a result, such a network tends to self-organize into subnetworks that represent different clusters in the input space. This is also what happened in our experiments. As indicated in the figure, one or more subnetworks can be seen as representing one of the categories large block shapes, small block shapes, large cylinder shapes, and small cylinder shapes. The objects of the generalization test set activate neurons in the proper subnetworks except in one case, namely, the test object 1 is a large block but is identified as a large cylinder.
Figure 4(d) shows the centres of activation in the GG in the fully trained GG-based system. This system produces similar results as the SOM-based system and the GCS-based system, that is, the organization of the GG separates large block shapes, small block shapes, large cylinder shapes, and small cylinder shapes. As indicated in the figure, the GG is also organized according to size. The ability for discrimination of individual objects is approximately similar as that for the SOM-based system. All 6 objects of the generalization test set are mapped so that they can be associated with the correct shape category and identified with the most similar object of the training set.
7. Discussion
We have experimented with four self-organizing systems for clustering of proprioceptive data collected by our anthropomorphic robot hand, the LUCS Haptic Hand III. All four systems were able to cluster the sensory information according to shape, and all four of them resulted in networks which preserve the size ordering of the training objects. The systems could also discriminate individual objects, more or less. The systems have proven to have an excellent generalization capacity. This is clearly illustrated in the categorization of the 6 new objects that offered different characteristics of shape and size.
The results are interesting because they reveal that the proprioceptive information encompasses information about both the shape and the size of the grasped objects, and in addition information that enables discrimination of the individual objects to some extent.
In comparison with our earlier systems for haptic shape perception [2225], the current systems have turned out to be much more able to correctly categorize objects according to shape in a much wider size range, and this is done with a less computationally expensive model. The current systems were also able to map the sizes of the objects in an ordered fashion, and to discriminate between objects as long as they were not too similar. A human would probably have a similar problem if she, like our systems, was not able to detect the material properties of the objects or expressed differently, if all object were of exactly the same material and weight.
The SOM-based, the GCS-based, and the GG-based systems performed at approximately a similar level. This could be an argument for using the alternative neural network architectures GCS and GG instead of the SOM, because that reduces the number of parameters that have to be set. According to Fritzke [38], the performance of the GCS is actually slightly better than the performance of the SOM in complex and realistic problems. The results of our experiments in [39] also point in that direction.
The GCS and the GCS-DN also have the virtue to get organized into networks whose topology reflect the probability density function of the input space. The GCS-DN is especially interesting since it has the property to automatically form disconnected subnetworks that represent clusters in the input space. It should be possible to implement an online version of the GCS-DN algorithm that never stops and that should result in a set of networks, that reflects the probability density function of the input space, which changes if the probability density function happens to be nonstationary. In other words, if the probability density function of the input space changed, then the set of subnetworks would change by the deletion of some subnetworks and the split, followed by growth of others.
It should be mentioned that the graphical presentation of GCS and GCS-DN could be improved. Fritzke [32] suggests a method on how to embed these kinds of networks in the plane for better visualizations. In this method, a physical model is maintained where the neurons are considered as discs influenced by attractive and repulsive forces.
The success with the GCS-based, the GCS-DN based, and the GG-based systems suggests an increased focus on our part on these kinds of self-organizing neural networks. The advantage of getting rid of several parameter settings like network size, learning rate, and neighbourhood settings can be important to succeed with more complex cognitive models with several coupled neural networks at multiple levels. To be forced to set all the parameters in a good way for all included neural networks with complex dependencies in such a model could prove to be overwhelming.
It would be interesting to compare our systems to self-organizing systems developed by others. Heidemann and Schöpfer [11] describe a haptic system, which consists of a plate with a touch sensitive array mounted on a robot arm. The system explores an object by sequences of contacts and feeds a self-organizing neural architecture with input. The system was able to learn to recognize 7 different objects when tested.
Natale and Torres-Jara [14] describe a system consisting of an upper body humanoid robot with a hand equipped with dome-like tactile sensors, which are sensitive to pressure from all directions, as well as position sensors (proprioception). The system also includes a camera together with a visual system for coarse localization of the object. The information gathered by the system was used as input to a SOM. When evaluated with 4 different objects, a bottle, a box, and two cups, these objects were mapped differently. However, the cups could not be distinguished from each other.
When compared with the two systems described above our current systems stand out in that they are able to categorize the objects according to shape, order them according to size, as well as recognize individual objects to a large extent.
In the future, we plan to increase the use of neural networks like GCS and GG as an alternative to the SOM in our haptic systems. By doing so, we will reduce the number of parameters that have to be set explicitly and this should yield more robust systems.
Because of the successful approach with using proprioceptive information as a base for haptic shape perception as well as size perception, we will in the nearest future continue our research in haptic perception with the following task: try to bring the proprioceptive systems to their absolute limits, for example, by exploiting the possibility of the LUCS Haptic Hand III to carry out a more active exploration than simply grasping the objects in only one way. This can be done by adducting/abducting the thumb and by flexing/extending the wrist differently in different grasps
At a later stage, we will study the interaction between haptics and vision. This would be interesting because these modalities interact to a considerable extent [40].
Acknowledgment
The authors want to acknowledge the financial support from Stiftelsen Landshövding Per Westlings Minnesfond to the LUCS Haptic Hand III.
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About this Journal Submit a Manuscript Table of Contents
Mediators of Inflammation
Volume 2013 (2013), Article ID 367245, 11 pages
http://dx.doi.org/10.1155/2013/367245
Review Article
Role of Mitogen-Activated Protein Kinase Pathways in Multifactorial Adverse Cardiac Remodeling Associated with Metabolic Syndrome
1Division of Cardiology, Geneva University Hospital, Faculty of Medicine, Foundation for Medical Researches, 64 Avenue de la Roseraie, 1211 Geneva, Switzerland
2Department of Internal Medicine, University of Genoa, V.le Benedetto XV 6, 16132 Genoa, Italy
3First Clinic of Internal Medicine, Department of Internal Medicine, University of Genoa, V.le Benedetto XV 6, 16132 Genoa, Italy
Received 5 November 2012; Revised 21 December 2012; Accepted 21 December 2012
Academic Editor: Massimo Collino
Copyright © 2013 Mohamed Asrih et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Metabolic syndrome has been widely associated with an increased risk for acute cardiovascular events. Emerging evidence supports metabolic syndrome as a condition favoring an adverse cardiac remodeling, which might evolve towards heart dysfunction and failure. This pathological remodeling has been described to result from the cardiac adaptive response to clinical mechanical conditions (such as hypertension, dyslipidemia, and hyperglycemia), soluble inflammatory molecules (such as cytokines and chemokines), as well as hormones (such as insulin), characterizing the pathophysiology of metabolic syndrome. Moreover, these cardiac processes (resulting in cardiac hypertrophy and fibrosis) are also associated with the modulation of intracellular signalling pathways within cardiomyocytes. Amongst the different intracellular kinases, mitogen-activated protein kinases (MAPKs) were shown to be involved in heart damage in metabolic syndrome. However, their role remains controversial. In this paper, we will discuss and update evidence on MAPK-mediated mechanisms underlying cardiac adverse remodeling associated with metabolic syndrome.
1. Introduction
The prevalence of metabolic syndrome is rapidly increasing in the western world [1]. Metabolic syndrome has been defined as a cluster of multiple disorders including insulin resistance, abdominal obesity, dyslipidemia, increased blood pressure, hypercholesterolemia, and proinflammatory state [2]. Several definitions have been historically proposed during the last decades, also including oxidative stress, leptin resistances and endothelial dysfunction as key pathophysiological mechanisms contributing to the increase of cardiovascular risk that affect metabolic syndrome patients [2, 3]. Considering these paradigms, it is clear that the metabolic syndrome is a fully heterogeneous construction, raising important scientific limitations for meta-analyses of clinical investigations. Although some common components (such as dyslipidemia, hypertension, and hyperglycemia) are recurrent in the different definitions [4], these other disorders might represent an important variable in the analysis of different cohorts. Several conditions included in the metabolic syndrome have been shown as strongly associated with an acceleration of atherogenesis and an increased incidence of acute ischemic events [1]. In addition, some processes (mainly systemic insulin resistance and inflammation) have been proposed to contribute to physiological organ remodelling and pathological damage in metabolic syndrome. In particular, different chronic adverse heart remodeling and the development of liver steatosis have been widely described [5, 6]. In this paper, we will focus on the pathophysiology of heart remodelling and damage during metabolic syndrome. Metabolism syndrome patients affected by diabetes are associated with altered myocardial substrate metabolism, which has emerged as an important contributor to the development of cardiomyopathy [7]. In diabetes and concomitant metabolic syndrome, an increased cardiac fatty acid metabolism and reduced glucose metabolism have been reported [7]. Although initially profitable, the rate of fatty acid uptake reaches a point where it exceeds the rate of fatty acid oxidation, thereby promoting the accumulation of lipids, resulting in lipotoxicity and associated cardiac dysfunction [8]. This leads to some complications, such as cardiac hypertrophy, which is identified as a cardiac pathological remodeling. However, cardiac remodeling does not necessarily refer to pathological adaptation of the myocardium. Indeed, short-term compensatory mechanisms are beneficial for the heart because it adapts cardiac output to physiological or pathological loading conditions such as exercise, hypertension, or aortic stenosis. In contrast, sustained overload leads to maladaptive and detrimental remodeling, as reported in detail by Buckberg and coworkers [9]. Altogether, these studies suggest that cardiac remodeling in metabolic syndrome depends on the different component disorders and might not be a disease per se but rather an adaptive response.
Several intracellular signaling pathways, continuously sensing the extracellular stimuli and modulating the different intracellular responses, have been investigated to characterize their role in cardiomyocyte modifications and potential injury associated with metabolic syndrome. Mitogen-activated protein kinases (MAPKs) are cytosolic signaling proteins that become activated after specific phosphorylation [10]. In response to wide extracellular stimuli, MAPKs have been shown to modulate various cellular processes, such as cell growth and cell size regulation [11]. Although not specifically performed in models of metabolic syndrome, in vitro studies using isolated cardiomyocytes have shown that MAPKs might be involved in cardiac hypertrophy via three traditional phases: (i) the activation of specific transmembrane proteins; (ii) intracellular signal transduction; (iii) the activation of cytosolic and nuclear events [12]. Since cardiac hypertrophy is characterized by increased cell size, it has been suggested that MAPKs might play a critical role in cardiac remodeling in hypertensive patients with metabolic syndrome. This appears to be achieved by modulating the activity of numerous transcription factors that target specific genes involved in structural response of the myocardium. In this paper, we will provide an overview on the role of MAPKs in the adverse cardiac remodeling that is associated with metabolic syndrome.
2. Different Structural Adverse Cardiac Remodeling in Metabolic Syndrome
Diagnostic criteria of metabolic syndrome (as indicated by ATP III classification [24]) have been reported to independently predict the development of diastolic dysfunction and cardiac hypertrophy [13, 19, 20] (Table 1). Cardiac hypertrophy is commonly defined as an increase in heart size or more particularly as an increase in ventricular size with or without increased wall thickness relative to body size [25]. This cardiac modification was shown to be a common alteration in subjects with different stages of obesity, which is one of the central features in metabolic syndrome [15, 16, 2628]. These studies have used noninvasive methods such as echocardiography and magnetic resonance imaging (MRI) to assess cardiac adaptations in obese patients. The results indicate that obesity is associated with a high prevalence of cardiac hypertrophy, characterized by an enhancement of left ventricular cavity size as well as wall thickness. Moreover, it has been observed that wall thickness was increased to greater extent than left ventricular cavity size, revealing a concentric instead of eccentric cardiac hypertrophy. Indeed, computed tomography and MRI demonstrated that fat tissue deposits are well detectable within the heart of obese subjects and commonly accumulate anterior to the right ventricle [2931]. Although noninvasive methods are useful to characterize cardiac size, structure, and function, they have limitations; in that they do not allow analyzing the biochemical composition of the hypertrophic heart in metabolic syndrome. Interestingly, postmortem studies confirmed the presence of cardiac hypertrophy and cardiac fat tissue deposits in obese patients [17, 18]. The amount of epicardial fat has been reported to be correlated with both visceral fat and the severity of ventricular hypertrophy [14]. Several studies have been conducted in animal models to better understand the cardiac adaptation in diabetes [32, 33]. Diabetes was associated with an increase in left ventricular internal dimension during diastole (LVIDD) and systole (LVIDS) in rats [34]. This adverse cardiac remodeling was independent of hypertension [34]. These results are consistent with a previous study, demonstrating that diabetic rats develop an eccentric left ventricular hypertrophy associated with a decreased cardiac systolic function, and related to impaired collagen turnover [35].
Table 1: Different disorders in metabolic syndrome are associated with cardiac structural and functional changes.
The Prospective Cardiovascular Münster (PROCAM) study, recruiting a cohort of 2754 males aged 40–65 years over a four-year period, showed that patients, who had either hypertension or diabetes, had a 2.5-fold increased risk of cardiovascular morbidity. However, when developing both, patients had an eightfold increase in cardiovascular risk. This multiplicative relevance was confirmed by a twentyfold increase of cardiovascular risk in patients with concomitant diabetes mellitus, hypertension, and abnormal lipid profile [36]. Since the metabolic syndrome might include all these disorders by definition, it is easy to understand that the alterations of an atherosclerotic targeted organ, such as the myocardium, might result from both acute and chronic ischemia. However, also in the absence of traditional atherosclerotic complications, the measurement of diastolic function worsened progressively during metabolic syndrome [13], indicating an adverse cardiac remodeling independent of cardiac necrosis and potentially related to the soluble mediators increased during the disease. In line with these results, different studies have confirmed the development of left ventricular diastolic dysfunction in subjects presenting metabolic syndrome [20, 21, 37, 38].
Interestingly, hypertensive patients with concomitant metabolic syndrome have been shown to present increased left ventricular mass and wall thickness as compared to patients exclusively affected by hypertension [39]. Furthermore, the authors showed that metabolic syndrome might induce an adverse cardiac remodeling via different pathophysiological mechanisms with a multiplicative effect. Metabolic syndrome patients had not only abnormal diastolic left ventricular relaxation, but also increased cardiac hypertrophy [40]. Cardiac hypertrophy predisposes individuals to cardiac arrhythmias, congestive heart failure, and diastolic dysfunction [41]. Consistently, several evidences have revealed a positive association between the metabolic syndrome and the severity of left ventricular hypertrophy [39, 40, 42]. Indeed, metabolic syndrome induces abnormal loading, which may favor the left ventricular hypertrophy. Since pathological cardiac hypertrophy has been associated with sudden cardiac death, heart failure, and stroke [43], it was proposed that this cardiac alteration might further increase cardiovascular risk in metabolic syndrome [44].
Increased myocardial fibrosis and stiffness have been also observed in animal models of obesity and metabolic syndrome [45]. Since collagen and fibrosis determine tissue compliance, cardiac deposition of this protein might promote left ventricular diastolic dysfunction and negatively affect diastolic function [46, 47]. Several other molecular mechanisms (i.e., insulin resistance aggravating asymptomatic myocardial inflammation and association between visceral obesity and myocardial adiposity) have also been suggested to potentially induce such cardiac structural alterations [48, 49].
Most importantly, the renin-angiotensin-aldosterone (RAA) system might also be involved cardiac fibroblast proliferation and collagen synthesis, thereby increasing cardiac fibrosis [50]. The active role for RAA system was confirmed by the finding that pharmacologic inhibition of this pathway ameliorates heart failure [51, 52], also in metabolic syndrome patients [53]. Thus, these studies suggest that RAA system could be a critical player underlying cardiac modifications in hypertensive metabolic syndrome patients.
3. Pathophysiological Mediators of Adverse Cardiac Remodeling in Metabolic Syndrome
Cardiac structural remodeling in metabolic syndrome might be due to different pathological triggers. Hypertrophic growth accompanies heterogeneous metabolic syndrome disorders, including not only diabetes and hypertension, but also coronary heart disease and ischemic cardiac remodeling. Mechanical alterations have been classically described as major causes inducing an adverse cardiac remodelling [54]. However, metabolic syndrome has been associated with left ventricular hypertrophy [40] independently of ischemic cardiac remodeling. Given the upregulation of several hormones and cytokines in metabolic syndrome patients [55], a potential role on cardiac remodeling has been proposed for these molecules [56]. In particular, a hormonal and inflammatory dysregulation might contribute to the development of cardiac hypertrophy and fibrosis [55]. For instance, increased aldosterone plasma levels in patients with metabolic syndrome [57, 58] might be directly associated with the development of left ventricular hypertrophy [59] or cardiac fibrosis [60]. Although clinical studies have reported that aldosterone induces left ventricular hypertrophy [61], the mechanism by which aldosterone promotes cardiac hypertrophy remains unclear. Okoshi and coworkers showed that aldosterone directly induced cardiac hypertrophy and atrial natriuretic peptide (ANP) mRNA expression (a molecular marker of cardiac hypertrophy) in neonatal rat ventricular myocytes [62]. These results were accompanied by enhanced activation of ERK1/2- and JNK-mediated pathways. This critical role of the ERK pathway in the development of cardiac hypertrophy (in response to endothelin-1) was also confirmed by the in vitro abrogation of cardiomyocyte hypertrophy in the presence of the pharmacological inhibitor of ERKs [63, 64].
On the other hand, aldosterone has been shown as a potent inducer of cardiac fibrosis [60, 65, 66]. Therefore, emerging evidence indicates a crucial role for aldosterone in maladaptive cardiac remodeling in metabolic syndrome [67, 68].
Considering the hypothesized inflammatory etiology of the metabolic syndrome [69], it was proposed that elevated circulating levels of cytokines, adipocytokines, and chemokines might actively regulate cardiac remodeling and via the activation of inflammatory signaling pathways [7072]. Amongst several mediators, tumor necrosis factor (TNF) and interleukin-6 (IL-6) were shown to promote both insulin resistance [73] and cardiac hypertrophy [74], suggesting that inflammation in metabolic syndrome might be a central feature in atherogenesis as well as in cardiac hypertrophy. In particular, TNF expression, which was shown to be increased in response to pressure overload in the adult heart [75], might be one of the most important mediators of cardiac hypertrophy in metabolic syndrome with hypertension [76]. The molecular pathways potentially involved in metabolic syndrome-induced cardiac hypertrophy have been only partially investigated. To summarize, mechanical stress remains the main responsible of adverse cardiac remodelling in metabolic syndrome, such as hypertension. However, considering the potential dysregulation of inflammatory and hormonal systems, the cardiac pathophysiology in metabolic syndrome might be partially influenced also by these soluble molecules. In the following sections, we will review the pathophysiological role of MAPKs in cardiac remodeling in a general context and also particularly in metabolic syndrome.
4. Role of MAPK in Cardiac Remodeling
4.1. Extracellular Signal Regulated Kinases (ERKs)
One of the most studied MAPK pathways is the Ras/Raf/ERKs pathway. Extracellular stimuli such as stress or hormones activate diverse receptors at the cell surface, driving intracellular recruitment and activation of the guanosine triphosphate (GTP) small G-protein (Ras). This, in turn, induces Raf-1 kinase translocation to the plasma membrane and Raf-1-mediated phosphorylation of MEK proteins (MEK1 and 2). Thereafter, ERKs are activated by MEKs and regulate a large number of nuclear and cytosolic proteins [77] that directly modulate numerous intracellular processes.
For instance, pressure overload was shown to influence ERK-mediated intracellular signaling as well as extracellular matrix deposition within the heart [78]. In response to chronic pressure overload, cardiomyocytes start to grow leading to heart enlargement and hypertrophy. Pressure overload induced by transverse aortic constriction (TAC) in rodents was shown to mediate hypertrophic effect through ERK activation [79]. In addition, Esposito and coworkers showed that TAC procedure is associated with the activation of all three major MAPKs (ERK1/2, p38 MAPK, and JNK) in mice [80]. Consistent with the animal studies, clinical researches reported increased cardiac activation of ERK1/2, JNK, and p38 MAPK in failing human hypertrophic hearts [81]. In line with these results, ERK1/2 activation has been shown as a key element directly promoting cardiac hypertrophy [26, 82].
It was proposed that ERK1/2 induces the activation of various transcription factors by modulating their phosphorylation level, hence leading to hypertrophy. Indeed, using a model of phenylephrine-induced cardiac myocyte hypertrophy, Babu and coworkers revealed that ERK1/2 pathway is involved in Elk-1 upregulation in a model of phenylephrine-mediated hypertrophy [83]. Therefore, these studies suggest a central role for ERK in the pathophysiological development of cardiac hypertrophy. The molecular mechanisms downstream of this pathway remains to be clearly defined.
4.2. Janus Kinases (JNKs)
The hypertrophic effects of JNKs are still controversial. Wang and coworkers showed that specific activation of the JNK pathway was associated with hypertrophy in neonatal cardiomyocytes overexpressing MEK7 [84]. In line with these results, transfection of ventricular myocytes with MEK1 (a MAPK-activating JNK) leads to cardiac hypertrophy via JNK activation [85]. By contrast, transgenic mice selectively overexpressing MEK7 in the cardiac tissue were not shown to develop cardiac hypertrophy despite JNK1 and JNK2 upregulation [86]. Although these mice died from congestive heart failure, they had normal cardiomyocytes size and they did not develop ventricular hypertrophy. Importantly, mice overexpressing MEK7 also presented diastolic dysfunction and paradoxically high levels of ANF mRNA, which is considered a marker for cardiac hypertrophy [86]. The reduction in connexin 43 and gap junctions between ventricular cardiomyocytes might explain the absence of hypertrophy in the presence of increased ANF expression [86]. Also in the case of JNK, the exact mechanisms through which it regulates cardiac hypertrophy remains poorly determined.
4.3. p38 MAPK
In addition to JNK and ERK1/2, p38 MAPK was also intensively investigated. There are at least four isoforms of p38 MAPK that have been involved in cardiac remodeling and inflammation [87]. This signaling pathway is predominantly involved in the inflammatory response, and it can be activated by proinflammatory cytokines, chemokines, and hormones [8890]. MEK 3, 4, and MEK 6 are the upstream kinases that directly activate p38MAPK [82]. Several downstream transcription factors have been identified as potential p38MAPK substrates, including activating transcription factor-1 (ATF-1), ATF-2, Elk-1, serum response factor (SRF), growth arrest, and myocyte enhance factor 2C (MEF 2C) [9193]. Since a large amount of studies has been performed in vitro in neonatal rat cardiomyocytes, the role of p38 MAPK in cardiac clinical modifications remains to be confirmed. Nevertheless, p38 MAPK has been implicated in the regulation of both cardiac growth and hypertrophy [94]. Indeed, the inhibition of the p38 MAPK pathway via pharmacological inhibitors or adenovirus blunted the hypertrophic effect associated with p38 MAPK activation [9496]. These hypertrophic effects of p38 MAPK phosphorylation in cardiomyocytes are also supported by another study where specific activation via adenovirus in ventricular muscle cells induced cardiac hypertrophy [84].
By contrast, Choukroun and coworkers have shown that p38 MAPK is not required for agonist-induced hypertrophy in cardiomyocytes [98]. To determine the role of the p38 MAPK pathway in the response to endothelin-1 (ET-1, a hypertrophic agent), neonatal rat cardiomyocytes were concomitantly treated with ET-1 and the selective p38 MAPK inhibitor, SB203580. The results revealed that this inhibitor had no effect on ET-1-induced hypertrophy, hence suggesting that p38 MAPK activation may not be required during cardiac cell hypertrophy [98]. Since these preliminary studies present some limitations (due to the use of the cardiomyocyte model [immature neonatal cells] and the modest specificity of pharmacological inhibitor, that can inhibit also other intracellular pathways), a genetic approach was also performed. Transgenic mice specifically overexpressing MEK 3 and MEK 6 (upstream activators of p38 MAPK) in the heart do not exhibit cardiac hypertrophy, despite the development of ventricular wall thinning and premature death with signs of congestive heart failure [99].
Taken together, these basic research studies suggest that p38 MAPK, ERKs, and JNKs may all be involved in promoting cardiac hypertrophy (Figure 1). In particular, p38 MAPK activation might also contribute to cardiac fibrosis, while ERK and JNK activation might promote cardiomyocyte growth and defects in gap junctions, respectively. The activation of a single MAPK-mediated cascade might not be sufficient to determine a clinically relevant adverse cardiac remodeling.
Figure 1: Intracellular pathways mediating hypertrophic cardiac remodelling in metabolic syndrome. Intracellular pathways mediated by phosphatidylinositol 3-kinase (PI3-K) activation play a pivotal role in insulin-mediated glucose transport in cardiomyocytes. Metabolic syndrome is associated with impaired intracellular signaling that could be activated by various stimuli, including inflammatory cytokines. These pathways, mainly dependent on MAPK activation (composed of extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK], and p38 MAPK), could be also triggered by chirurgical manipulation (such as transverse aortic constriction [TAC]). MAPK activation might be considered as a critical mechanism aggravating cardiac hypertrophy as well as cardiomyocyte insulin resistance in metabolic syndrome.
5. Potential Role of MAPK in Metabolic Syndrome-Related Cardiac Adverse Remodeling
As suggested above, the alterations in cardiac MAPK activation might be induced in metabolic syndrome by insulin resistance and abnormal inflammation. The association between these molecular dynamics was so relevant that an altered MAPK activation pattern might also be a potential cause of hyperinsulinemia [100]. This pathophysiological role of MAPK in metabolic syndrome-induced heart remodeling was confirmed for JNK activation that was associated with both developments of insulin resistance and cardiac hypertrophy in metabolic syndrome [82, 101]. Assessing the role of ERKs in metabolic syndrome cardiac remodeling is much more complicated since ERK2-knockout mice are not viable [102, 103]. On the other hand, ERK1 knockout mice are viable and fertile. Thus, ERK1-deficient mice were investigated, and they were shown to be protected from diet-induced obesity and insulin resistance [104]. Nonetheless, mice lacking the ERK1 negative regulator p62 presented altered metabolism with increased adipogenesis, reduced energy expenditure, and reduced insulin sensitivity [105]. However, these animals were not investigated on concomitant cardiac remodeling and might be considered a good model to assess metabolic syndrome adverse heart remodeling.
Inflammatory mediators such as TNF are elevated in metabolic syndrome [106]. Condorelli and coworkers showed that Akt and the JNK MAPK mediate TNF-induced hypertrophy in cultured cardiomyocytes [107]. TNF-mediated eccentric cardiac hypertrophy in response to intermittent hypoxia was shown to be mediated by ERK and STAT3 activation in adult rat myocardium [97]. On the other hand, IL-6 levels might also be involved in the determinism of cardiac hypertrophy [22]. Consistent with this hypothesis, Hirota and coworkers demonstrated a concomitant overexpression of both IL-6 and IL-6 receptor in a mouse model of cardiac hypertrophy [108]. Although these observations suggest a potential role for IL-6 in metabolic syndrome, the potential activation of intracellular signaling pathways by this cytokine remains unclear. Cardiotrophin-1 (CT-1), a newly discovered member of the IL-6 family and a key regulator of metabolism were also reported to be upregulated in metabolic syndrome [109, 110]. This cytokine was shown to favor cardiac hypertrophy and fibrosis in mice [23]. Thus, CT-1 might also underlie adverse remodeling in metabolic syndrome and thus represent an attractive pathophysiological target.
Although evidence for the role of p38 MAPK in metabolic syndrome is still lacking (except for some animal studies in diabetic fibrotic cardiomyopathy) [111], enhanced MAPK signaling was shown to increase both insulin sensitivity and promote hypertrophic cardiac remodeling (Table 2). Thus, these proteins might represent a promising target to improve metabolic, inflammatory, and cardiac pathophysiology in metabolic syndrome.
Table 2: Role of MAPK activation in metabolic syndrome-associated cardiac hypertrophy.
6. Conclusions
In the past decades, relevant progresses have been made in the attempt to define the role of MAPKs in metabolic syndrome and in its clinical manifestations, including heart adverse pathophysiological remodelling. The heart structure has been described to be directly influenced by the mechanical stress, characterizing certain components of the metabolic syndrome (such as hypertension). Importantly, this adverse cardiac remodelling might be partially regulated by elevated hormones and inflammatory cytokines. MAPKs might represent the final pathways commonly activated within cardiomyocytes by both mechanical and soluble determinants. Despite some limitations due to animal and in vitro models, MAPK (mainly JNK and ERK) activation in the peripheral organs (including the heart) in metabolic syndrome was shown to induce insulin resistance and to increase inflammation. On the other hand, although the effect of p38 MAPK and JNK phosphorylation remains controversial, mounting evidence indicates ERK1/2 as responsible for promoting cardiomyocyte growth. We believe that MAPKs might be considered as potential therapeutic targets for drugs aimed to improve metabolic and cardiac dysfunctions in metabolic syndrome. Indeed several inhibitors of MAPK signaling proteins are currently available. Some of them are already being tested in clinical trials for oncological disorders and may theoretically find application also for the treatment or prevention of heart dysfunction in metabolic syndrome. Alternatively, strategies targeting downstream effectors/transcription factors in these cascades could also be a viable therapeutic option.
Acknowledgments
This paper was funded by EU FP7, Grant no. 201668, AtheroRemo to F. Mach. This work was also supported by the Swiss National Science Foundation Grants to F. Mach (no. 310030-118245) and F. Montecucco (no. 32003B-134963/1). This work was supported by a grant from Novartis Foundation to F. Montecucco.
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85. M. A. Bogoyevitch, J. Gillespie-Brown, A. J. Ketterman et al., “Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart: p38/RK mitogen-activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/reperfusion,” Circulation Research, vol. 79, no. 2, pp. 162–173, 1996. View at Scopus
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91. A. Besnard, B. Galan-Rodriguez, P. Vanhoutte, and J. Caboche, “Elk-1 a transcription factor with multiple facets in the brain,” Frontiers in Neuroscience, vol. 5, article 35, 2011.
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96. D. Zechner, D. J. Thuerauf, D. S. Hanford, P. M. McDonough, and C. C. Glembotski, “A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression,” Journal of Cell Biology, vol. 139, no. 1, pp. 115–127, 1997. View at Publisher · View at Google Scholar · View at Scopus
97. L. M. Chen, W. W. Kuo, J. J. Yang et al., “Eccentric cardiac hypertrophy was induced by long-term intermittent hypoxia in rats,” Experimental Physiology, vol. 92, no. 2, pp. 409–416, 2007. View at Publisher · View at Google Scholar · View at Scopus
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Max Afford - Chronological Bibliography
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Pseudonym. See: Malcolm Afford (or view all titles by this pseudonym here)
Copyright (c) 1995-2011 Al von Ruff.
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Bibliography: Author's Introduction (Swords and Deviltry
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Title: Author's Introduction (Swords and Deviltry
Author: Fritz Leiber
Year: 1989
Type: ESSAY
ISFDB Record Number: 1094446
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USA Reverse Phone Lookup – Instantly Trace The Identity Of Any Unknown Phone Number Owner In US
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Frustrated of receiving frequent late night prank calls, harassing telemarketing calls or blank calls from unknown phone number?
Los Angeles, CA, USA, January 19, 2013 - (PressReleasePoint) - Most have come to take for granted the fact that can now look down at telephone and see who is calling before even answer the phone. But, sometimes there are numbers that just don't recognize -- and that could mean trouble. USA Reverse Phone Lookup makes it easy to look up numbers online and determine exactly who is behind that number.
Of course, people don't use reverse phone lookup services solely to see who is calling them! There is the added convenience of being able to see who the children are calling, who the spouse, boyfriend, or girlfriend is calling, or who is calling them. Especially in today's world, it is important to keep an eye on children and their electronic interactions and USA Reverse Phone Lookup can do just that.
There was a time when could actually go down to the library and look in a giant reverse phone directory, but obviously those days are over. It is not unusual for a person to have dozens of different phone numbers during their life now, so a website like USA Reverse Phone Lookup can really help. Whether searching for the name behind a number have written down, or trying to see who is pestering in the middle of the night, they will help find out.
In order to use the service, will need is the area code and phone number and the website will do the rest. If they don't have a name behind the number, which usually only happens when the number is extremely new, then they won't charge for looking it up. However, if they have the information, then can choose to pay for a one time use or buy a subscription that will enable to look up even more numbers in the future.
In addition to just getting the owner's name of a phone number will also get information such as where the number is physically located, whether or not it is a cell phone or a land line, if it is a business line or a personal line, and even some more personal information as to who owns the line.
Would all like to trust those who call and those who live with, but sometimes just need to know a little more information to ease mind. If loved one is making or receiving calls that just don't recognize, or are interested in finding out who is calling all the time, then a service like USA Reverse Phone Lookup can help. They service that they provide is completely anonymous - so no one will know that their number is being investigated - and will have the information that need in a matter of seconds.
For more information about reverse phone lookup or to instantly trace an unknown phone number visit USA-ReversePhoneLookup.Com
About USA-ReversePhoneLookup.Com
USA Reverse Phone Lookup - Instantly Reverse Lookup Any Phone Number powered by Phone Detective is a one stop resource to reverse lookup any phone number within United States and Canada. With USA Reverse Phone Lookup users will get access to 400 million cell phone, landline, business and unlisted phone numbers operating in US. Users can get all information they need to know about reverse phone lookup service in one place, they can also reverse lookup an unknown phone number and instantly trace the caller for legal purpose.
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SEC Football and IP
by Stephan Kinsella on August 17, 2009
in Intellectual Property
The SEC Would Prefer That You Not Mention SEC Games To Anyone
America’s fastest conference is developing a new “media policy” that severely restricts how much audio, video and “blogs,” reporters can dish out during live games. (Hint: Not much.) Oh, and fans in the seats are subject to the policy too.
We all know that you can’t disseminate anything without the express written consent of Major League Baseball, but the Southeastern Conference’s new fan policy seems to suggest that any attempt by people inside the stadium to let people outside the stadium know what’s going on would be against the rules. No Twittering. No Facebook photos. No YouTubes. Nothing. If your friend wants to know the score, he can read the paper the next day.
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Place:Sydney, New South Wales, Australia
Watchers
NameSydney
Alt namesPort Jacksonsource: Rand McNally Atlas (1989) p 352
Sidneysource: Van Marle, Pittura Italiana (1932)
TypeCity
Coordinates33.917°S 151.167°E
Located inNew South Wales, Australia (1788 - )
source: Getty Thesaurus of Geographic Names
source: Family History Library Catalog
the text in this section is copied from an article in Wikipedia
Sydney is the state capital of New South Wales and the most populous city in Australia. It is located on Australia's south-east coast of the Tasman Sea. As of June 2010, the greater metropolitan area had an approximate population of 4.6 million people.[1] Inhabitants of Sydney are called Sydneysiders, comprising a cosmopolitan and international population.
The site of the first British colony in Australia, Sydney was established in 1788 at Sydney Cove by Arthur Phillip, commodore of the First Fleet, as a penal colony.[2] The city is built on hills surrounding Port Jackson which is commonly known as Sydney Harbour, where the iconic Sydney Opera House and the Harbour Bridge feature prominently. The hinterland of the metropolitan area is surrounded by national parks, and the coastal regions feature many bays, rivers, inlets and beaches including the famous Bondi Beach and Manly Beach. Within the city are many notable parks, including Hyde Park and the Royal Botanic Gardens.
Sydney is a high ranking world city and has hosted multiple major international sporting events, including the 1938 British Empire Games (now known as the Commonwealth Games) and the 2000 Summer Olympics. The main airport serving Sydney is Sydney Airport and its main port is Port Botany.
History
the text in this section is copied from an article in Wikipedia
Radio carbon dating suggests that the Sydney region has been inhabited by indigenous Australians for at least 30,000 years.
The traditional indigenous inhabitants of Sydney Cove are the Cadigal people, whose land once stretched from south of Port Jackson to Petersham. While estimates of the population numbers before the arrival of the First Fleet in 1788 remain contentious, approximately 4,000–8,000 Aboriginal people lived in the Sydney region before contact with British settlers. The British called the indigenous people the "Eora", because being asked where they came from, these people would answer: "Eora", meaning "here", or "from this place" in their language.[3] There were three language groups in the Sydney region, which were divided into dialects spoken by smaller clans. The principal languages were Darug (the Cadigal, original inhabitants of the City of Sydney, spoke a coastal dialect of Darug), Dharawal and Guringai. Each clan had a territory, the location of each territory determined the resources available. Although urbanisation has destroyed much evidence of these settlements such as shell middens, a number of Sydney rock engravings, carvings and rock art remain visible in the Hawkesbury sandstone of the Sydney basin.
In 1770, British sea captain Lieutenant James Cook landed at Botany Bay on the Kurnell Peninsula. It is here that Cook made first contact with an Aboriginal community known as the Gweagal. Under instruction from the British government, a convict settlement was founded by Arthur Phillip, who arrived at Botany Bay with a fleet of 11 ships on 18 January 1788. This site was soon determined to be unsuitable for habitation, owing to poor soil and a lack of reliable fresh water. Phillip subsequently founded the colony one inlet further north along the coast, at Sydney Cove on Port Jackson on 26 January 1788. The official proclamation of the founding and naming of Sydney took place nearly two weeks later on 7 February 1788. The original name was intended to be Albion, but Phillip named the settlement after the British Home Secretary, Thomas Townshend, Lord Sydney, in recognition of Sydney's role in issuing the charter authorising Phillip to establish the colony.
In April 1789, a catastrophic epidemic disease now thought to be chickenpox spread through the Eora people and surrounding groups, with the result that local Aborigines died in their thousands, and bodies could often be seen bobbing in the water in Sydney Harbour. The cause of the epidemic has always been a matter of speculation and controversy, but if it was chickenpox, an outbreak of shingles among the convicts being among the most likely explanations. In any event, the results were catastrophic for the Eora people and their kin and by the early 1800s the Aboriginal population of the Sydney basin "had been reduced to only 10 percent of the 1788 estimate", or an estimated 500 to 1000 Aboriginal people between Broken Bay and Botany Bay.[4]
There was some violent resistance to British settlement, notably by the warrior Pemulwuy in the area around Botany Bay, and conflicts were common in the area surrounding the Hawkesbury River. By 1820 there were only a few hundred Aborigines and Governor Macquarie had begun initiatives to 'civilise, Christianise and educate' the Aborigines by removing them from their clans.[4] Macquarie's tenure as Governor of New South Wales was a period when Sydney was improved from its basic beginnings. Roads, bridges, wharves and public buildings were constructed by British and Irish convicts, and by 1822 the town had banks, markets, well-established thoroughfares and an organised constabulary.
The 1830s and 1840s were periods of urban development including the development of the first suburbs, as the town grew rapidly when ships began arriving from Britain and Ireland with immigrants looking to start a new life in a new country. On 20 July 1842 the municipal council of Sydney was incorporated and the town was declared the first city in Australia, with John Hosking the first elected mayor. The first of several Australian gold rushes started in 1851, and the port of Sydney has since seen many waves of people arriving from around the world.
Rapid suburban development began in the last quarter of the 19th century with the advent of steam-powered tramways and railways. With industrialisation Sydney expanded rapidly and, by the early 20th century, it had a population of more than a million. In 1929, the novelist Arthur Henry Adams called it the "Siren City of the South" and the "Athens of Australia". The Great Depression hit Sydney badly in comparison to other Australian cities. One of the highlights of the Depression era, however, was the completion of the Sydney Harbour Bridge in 1932. There has traditionally been a rivalry between Sydney and Melbourne since the gold rushes of the 1850s made the capital of Victoria Australia's largest and richest city. Sydney overtook Melbourne in population in the early years of the 20th century, and continues to be the largest city in Australia. During the 1970s and 1980s, Sydney's central business district (CBD), with a great number of financial institutions including the headquarters of the Reserve Bank, surpassed Melbourne as the nation's financial capital. Throughout the 20th century, especially in the decades immediately following World War II, Sydney continued to expand as large numbers of European and later Asian immigrants took up residence in the metropolitan area.
Research Tips
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Australian Bureau of Statistics
Celebrating the International Year of Statistics 2013
ABS Home > Statistics > By Release Date
3307.0 - Social Statistics, Australia: Divorce, Mar 1969
Previous ISSUE Released at 11:30 AM (CANBERRA TIME) 15/08/1969
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• About this Release
These related publications have been grouped together under the later title Divorces, Australia (Cat no 3307.0) although the title has changed over the period of publication.
Publication details
Title: Social Statistics, Australia: Divorce
Period covered: 1960-1969
Frequency: Annual/Quarterly
Related Links: Continued by: Divorce
This publication has been scanned from the paper version using character recognition software. This provides a full-text searching capability once downloaded.
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Tell me more ×
Answers OnStartups is a question and answer site for entrepreneurs looking to start or run a new business. It's 100% free, no registration required.
I am wondering what my next step should be in implementing my idea.
Should I design a smaller step of the website (I mean script and the actual design)? I can't use SEO to get traffic. I was thinking of advertising/sponsoring with people who already have a larger following and fit my target audience (GEN Y, Z or people who are up to date with technology).
Should I approach Youtube partners and give them money or sponsor them? Tell me how you would approach this (getting numbers wise).
My main idea to encourage signups and referrals is to have a referral contest and give my users a taste of what's to come. People who refer a lot of other friends will win items like iPads, Gift cards, Video games, etc. Do you think this is a good idea? I am trying to show potential investors the value of my business.
My idea is giving people what they want when they want. It's a recession. Not a lot of teens can get a job and pay for things they want. These things could be games, clothes, electronics, anything. On my site they can do what they normally do, earn points for it, and win those items. One example of "doing what they normally do" would be watching videos.
I know that this might look like a scam but that's why I want to do this referral contest - to show people, before they invest a lot of their time, we are not scammers.
share|improve this question
4 Answers
up vote 1 down vote accepted
Take a look at a website called Lockerz. They are doing exactly what you are planning on doing, and they have over 17 million members. I am not saying this to discourage you, rather I am saying that "your idea" is definitely viable yet based on the amount of work already done to develop Lockerz awesome and proprietary platform - your barrier of entry is now very high.
Your question is somewhat disorganized and really lacks a lot of clarity. It seems to me that you have this an idea with a lot of little details scattered around. I would encourage you to really downsize your idea and launch it on a really small scale (maybe friends and family) to test it. Your tech only needs to be functional at this point. From their, take their feedback and gradually build and refine your platform based on your user's feedback. Rather than pay for promos that may or may not result in new members, make an incentive for your members to invite their friends.
share|improve this answer
Most of the text books designed to work with special websites that explain material in videos and interactive games, however, I doubt that these websites get as much traction as some "cute kittens" videos on YouTube.
Primarily, the school sites are recommended by teachers, therefor -- not cool. Links to "cute Kittens" is distributed by peers -- cool factor.
If you can figure out how to tap into psychology of that behavior, then you can spend some money to develop a beta -- until then, I'm afraid it'll be a waste of your resources (including time).
Best of luck.
share|improve this answer
I have heard of better ideas, and seen them fail. I have also seen worse ideas (TWITTER) do amazing. The point is, you really dont know until you build ... Try to build a beta as suggested by slatecaster and do it on a shoestring budget. I think advertisers would pay if people were forced to view their messages. For example, Mercedes benz might pay you 1 cent for every video viewed. I dont expect that the revenues will be too high. Best of luck
share|improve this answer
I know for a fact there are a few of these sites around, the downside to them is that people soon realise that you have to answer a lot of surveys, sign up to a lot of partner programs and refer your friends and yet you still don't earn that many points after you've done it all.
If you can somehow make your site work in regards to points, make it easy for users to get points by rewarding them properly for doing tasks you would consider time consuming, then you have a winner on your hands. Most sites skimp on points because they don't really want to give away cool prizes without turning over a massive profit doing so.
Points websites are considered spammy and somewhat legally fraudulent (legal but bordering fraudulent) in that they make exorbitant amounts of money from partnership programs, referrals and affiliate commissions because of their users, but it's a small minority who get cool prizes and mostly benefit off of the hard work of the other site users.
Whilst it's possible for you to change the perception of how points based websites work, it's not going to be easy. A referral program that rewards top referrers with iPad's and other goodies sounds great though, I would roll with that. I've had great success with Facebook Advertisements in regards to those types of things.
Good luck.
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{
"content_type": "text/html",
"provenance": "cccc-CC-MAIN-2013-20-0000.json.gz:17092",
"uncompressed_offset": 16473366,
"url": "arthritis-research.com/content/14/2/R61/abstract",
"warc_date": "2013-11-22T14:53:14.000Z",
"warc_filename": "<urn:uuid:5c06adec-9a3e-47b2-a495-b907d02d6ff1>",
"warc_url": "http://arthritis-research.com/content/14/2/R61/abstract"
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Research article
Value of self-performed joint counts in rheumatoid arthritis patients near remission
Helga Radner*, Johannes Grisar, Josef S Smolen, Tanja Stamm and Daniel Aletaha
Author Affiliations
Department of Internal Medicine III, Division of Rheumatology, Medical University of Vienna, Waehringerguertel 18-20, Vienna, 1090, Austria
For all author emails, please log on.
Arthritis Res Ther 2012, 14:R61 doi:10.1186/ar3777
Published: 14 March 2012
Abstract
Introduction
To determine the validity and reliability of patients' self-performed joint counts compared to joint counts by professional assessors in rheumatoid arthritis (RA) patients in different disease activity states.
Methods
In patients with established RA we determined the inter-rater reliability of joint counts performed by an independent evaluator and the patient using intraclass correlation (ICC), and agreement on activity in individual joints by kappa statistics. We also performed longitudinal analyses to assess consistency of assessments over time. Finally, we investigated the concordance of joint counts of different assessors in patients with different levels of disease activity.
Results
The reliability of patient self-performed joint counts was high when compared to independent objective assessment (ICC; 95%confidence interval (CI)) for the assessment of swelling (0.32; 0.15 to 0.46) and tenderness (0.75; 0.66 to 0.81), with higher agreement for larger joints (kappa: 0.57 and 0.45, respectively) compared to smaller joints (metacarpo-phalangeal joint (MCPs): 0.31 and 0.45; and proximal interphalangeal joint (PIPs): 0.22 and 0.47, for swelling and tenderness, respectively).
Patients in remission according to the Simplified Disease Activity Index (SDAI ≤ 3.3) showed better concordance of the joint counts (swollen joint count (SJC) ties 25/37, tender joint count (TJC) ties 26/37) compared to moderate/high disease activity states (SDAI > 11; MDA/HDA: SJC ties 9/72, TJC ties 21/72). Positive and negative predictive values regarding the presence of SDAI remission were reasonably good (0.86 and 0.95, respectively). A separate training session for patients did not improve the reliability of joint assessment. The results were consistent in the longitudinal analyses.
Conclusions
Self-performed joint counts are particularly useful for monitoring in patients having attained remission, as these patients seem able to detect state of remission.
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{
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{
"content_type": "text/html",
"provenance": "cccc-CC-MAIN-2013-20-0000.json.gz:17101",
"uncompressed_offset": 31862995,
"url": "blog.mozilla.org/theden/2012/07/23/search-any-site-from-the-firefox-awesome-bar/",
"warc_date": "2013-11-22T14:53:14.000Z",
"warc_filename": "<urn:uuid:5c06adec-9a3e-47b2-a495-b907d02d6ff1>",
"warc_url": "http://blog.mozilla.org/theden/2012/07/23/search-any-site-from-the-firefox-awesome-bar/"
}
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Search any site from the Firefox Awesome Bar
We all spend a lot of time searching for things on the Internet. Wouldn’t it be great if we could cut down on the searching and get to the finding? You can do just that with Firefox!
Let’s say that you love to shop on Amazon. Head over to Amazon.com, find the search field and right-click on it. From there, select “Add a Keyword For This Search…” from the dropdown menu. That will open up the Add Bookmark box, where you’ll enter a name for the bookmark (in this case, we’ll call it “Amazon.com”) and a keyword for the search (let’s use “Amazon”).
Congratulations! You’ve set up a smart keyword search for Amazon.
Now, let’s imagine you’re surfing the Web and you come across a review for a gadget that you simply must have. It’s called the Super Gadget and you just can’t wait to buy it. Without even leaving the site you’re on, just head up to the URL bar (the Awesome Bar) and type in “Amazon Super Gadget” (keyword followed what you’re searching for) — then hit return. Voila! Welcome to the Super Gadget page on Amazon.com.
And this works on any site with a search bar – Wikipedia, IMDB and any others you can think of.
There’s a few seconds back in your day. You’re welcome.
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{
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"uncompressed_offset": 46174059,
"url": "ccsenet.org/journal/index.php/jmsr/article/view/21219/0",
"warc_date": "2013-11-22T14:53:14.000Z",
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"warc_url": "http://ccsenet.org/journal/index.php/jmsr/article/view/21219/0"
}
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Effects of Annealing Temperature on Raman Scattering and Electrical Properties of Te-Doped Nanostructured Black Silicon
Yuanjie Su, Ting Zhang, Peng Zhang, Jing Jiang, Zhanfei Xiao, Shibin Li, Zhiming Wu, Yadong Jiang
Abstract
In this paper, the influence of annealing temperature on Raman scattering and transport properties of Te-doped nanostructured black silicon has been studied. We prepared the black silicon samples by wet etching, i.e. alkaline etching and metal assisted etching. The nanopores on the surface of black silicon were produced by metal assisted etching. The black silicon samples were annealed at different temperature of 600oC, 700oC, and 800oC According to the Raman scattering results, the peak intensity of Si increases with the increase of annealing temperature. However, the full width at half maximum (FWHM) is inversely proportion to the annealing temperature. Thermal annealing removes the Raman peak of amorphous Si at 480 cm-1. In term of Te dopant atoms, the peak intensities of annealed samples are much lower than that of unannealed one. Subsequent Hall Effect measurement shows that annealing treatment improves electronic transport properties of Te-doped nanostructured black silicon.
Full Text: PDF DOI: 10.5539/jmsr.v2n1p34
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Journal of Materials Science Research ISSN 1927-0585 (Print) ISSN 1927-0593 (Online)
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"provenance": "cccc-CC-MAIN-2013-20-0000.json.gz:17127",
"uncompressed_offset": 65641458,
"url": "ddowiki.com/page/Dungeons_%26_Dragons",
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| Edit | | History |
From DDO wiki
(Redirected from Dungeons & Dragons)
Jump to: navigation, search
D&D or DnD generally refers to the traditional pen and paper (PnP) fantasy role-playing game, Dungeons & Dragons. Where as DDO refers to the game covered by this wiki - Dungeons & Dragons Online.
Dungeons & Dragons Online is based on 3.5 edition rule set of the pen and paper game and the Eberron world settings.
[edit] External links
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{
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"provenance": "cccc-CC-MAIN-2013-20-0000.json.gz:17134",
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"url": "dungeons.wikia.com/wiki/SRD:Medium_Water_Elemental",
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Wikia
SRD:Medium Water Elemental
Talk0
9,503pages on
this wiki
This material is published under the OGL
MEDIUM WATER ELEMENTALEdit
Water Elemental, Medium
Size/Type: Medium Elemental (Water, Extraplanar)
Hit Dice: 4d8+12 (30 hp)
Initiative: +1
Speed: 20 ft. (4 squares), swim 90 ft.
Armor Class: 19 (+1 Dex, +8 natural),, touch 11, flat-footed 18
Base Attack/Grapple: +3/+6
Attack: Slam +6 melee (1d8+4)
Full Attack: Slam +6 melee (1d8+4)
Space/Reach: 5 ft./5 ft.
Special Attacks: Water mastery, drench, vortex
Special Qualities: Darkvision 60 ft., elemental traits
Saves: Fort +7, Ref +2, Will +1
Abilities: Str 16, Dex 12, Con 17, Int 4, Wis 11, Cha 11
Skills: Listen +3, Spot +4
Feats: Cleave, Power Attack
Environment: Elemental Plane of Water
Organization: Solitary
Challenge Rating: 3
Treasure: None
Alignment: Usually neutral
Advancement: 5–7 HD (Medium)
Level Adjustment:
A water elemental can’t venture more than 180 feet from the body of water from which it was conjured.
Water elementals speak Aquan but rarely choose to do so.
COMBATEdit
A water elemental prefers to fight in a large body of water where it can disappear beneath the waves and suddenly swell up behind its opponents.
Water Mastery (Ex): A water elemental gains a +1 bonus on attack and damage rolls if both it and its opponent are touching water. If the opponent or the elemental is touching the ground, the elemental takes a –4 penalty on attack and damage rolls. (These modifiers are not included in the statistics block.)
A water elemental can be a serious threat to a ship that crosses its path. An elemental can easily overturn small craft (5 feet of length per Hit Die of the elemental) and stop larger vessels (10 feet long per HD). Even large ships (20 feet long per HD) can be slowed to half speed.
Drench (Ex): The elemental’s touch puts out torches, campfires, exposed lanterns, and other open flames of nonmagical origin if these are of Large size or smaller. The creature can dispel magical fire it touches as dispel magic (caster level equals elemental’s HD).
Vortex (Su): The elemental can transform itself into a whirlpool once every 10 minutes, provided it is underwater, and remain in that form for up to 1 round for every 2 HD it has. In vortex form, the elemental can move through the water or along the bottom at its swim speed. The vortex is 5 feet wide at the base, up to 30 feet wide at the top, and 10 feet or more tall, depending on the elemental’s size. The elemental controls the exact height, but it must be at least 10 feet.
The elemental’s movement while in vortex form does not provoke attacks of opportunity, even if the elemental enters the space another creature occupies. Another creature might be caught in the vortex if it touches or enters the vortex, or if the elemental moves into or through the creature’s space.
Creatures one or more size categories smaller than the elemental might take damage when caught in the vortex (see the table below for details) and may be swept up by it. An affected creature must succeed on a Reflex save when it comes into contact with the vortex or take the indicated damage. It must also succeed on a second Reflex save or be picked up bodily and held suspended in the powerful currents, automatically taking damage each round. An affected creature is allowed a Reflex save each round to escape the vortex. The creature still takes damage, but can leave if the save is successful. The DC for saves against the vortex’s effects varies with the elemental’s size. The save DC is Strength-based.
Creatures trapped in the vortex cannot move except to go where the elemental carries them or to escape the whirlwind. Creatures caught in the whirlwind can otherwise act normally, but must make a Concentration check (DC 10 + spell level) to cast a spell. Creatures caught in the whirlwind take a –4 penalty to Dexterity and a –2 penalty on attack rolls. The elemental can have only as many creatures trapped inside the vortex at one time as will fit inside the vortex’s volume.
The elemental can eject any carried creatures whenever it wishes, depositing them wherever the vortex happens to be. A summoned elemental always ejects trapped creatures before returning to its home plane.
If the vortex’s base touches the bottom, it creates a swirling cloud of debris. This cloud is centered on the elemental and has a diameter equal to half the vortex’s height. The cloud obscures all vision, including darkvision, beyond 5 feet. Creatures 5 feet away have concealment, while those farther away have total concealment.
Those caught in the cloud must make a Concentration check (DC 15 + spell level) to cast a spell.
An elemental in vortex form cannot make slam attacks and does not threaten the area around it.
Skills: A water elemental has a +8 racial bonus on any Swim check to perform some special action or avoid a hazard. It can always choose to take 10 on a Swim check, even if distracted or endangered. It can use the run action while swimming, provided it swims in a straight line.
Water Elemental Sizes
––—––—— Vortex ——––––—
Elemental Height Weight Save DC Damage Height
Medium8 ft.280 lb.151d610–30 ft.
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[maemo-developers] OS 2007 / 770 hacker edition with sound and video
From: Daniel Stone daniel.stone at nokia.com
Date: Fri Mar 2 21:54:33 EET 2007
On Fri, Mar 02, 2007 at 01:51:46PM -0600, ext Levi Bard wrote:
> >The main news is that sound is now working including media and system
> >sounds.
> >Video works to some extent, though not the sample clip that comes with the
> >image which is intended for the N800.
>
> Does this mean the accelerated Xsp pixel doubling works?
I haven't tested this hypothesis, but it should. (FWIW, it's also fixed
in the next firmware release for the N800, but using it for video is
pointless anyway.)
Cheers,
Daniel
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28 January 2008
Welsh Death-Wish
As someone with a Welsh first name, I have always taken an interest in the Welsh language and efforts to promote it and keep it in the land of the living. Alas, this ain't one of them:
Scores of writers are refusing to let their works be scanned for an online archive at the National Library of Wales because they are not being paid.
A year after a near-£1m project was awarded to digitise modern Welsh writing, a dispute between authors and the library has not been resolved.
The library is putting some 3.5m words from 20th Century English and Welsh periodicals and magazines on the web.
But literature promotion agency Academi wants writers to be paid a share.
Academi chief executive Peter Finch said: "It's an extremely exciting programme: what's wrong with it is there is no small sliver in there for paying the writers.
Hello??? The "small sliver" is that your words live on and people can read the bleddy things. Refusing to allow works written in Welsh to be digitised (which costs money) is a sure way to ensure that the language languishes and becomes even more marginal in the digital age. (Via paidContent UK.)
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User:AgiStachowiak
From OpenWetWare
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(Contact Information)
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Teaching Lab: 617.452.2886 (Room 56-322)<br>
Teaching Lab: 617.452.2886 (Room 56-322)<br>
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<font color = 990OCC>astachow AT mit DOT edu</font color>
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<font color = 990OCC>astachow AT mit DOT edu<br>
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20109 DOT talk AT gmail DOT com</font color>
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Revision as of 16:26, 21 September 2011
Contents
Contact Information
Technical Instructor
Department of Biological Engineering
Massachusetts Institute of Technology
Office: 617.324.1940 (Room 16-319)
Teaching Lab: 617.452.2886 (Room 56-322)
astachow AT mit DOT edu
20109 DOT talk AT gmail DOT com
Background and Education
Prior to joining the teaching faculty here in Biological Engineering, I was myself educated at MIT for ten years (this is sometimes derisively referred to as being a "lifer"). I first acquired an S.B. in Chemical Engineering (2001), then a Ph.D. in Polymer Science and Engineering under the auspices of the Department of Materials Science and Engineering (2007). My interest in the interface between biology and materials came about late in my undergraduate career, and I was fortunate to pursue this interest at the graduate level in Darrell Irvine's lab. In addition to a challenging and engaging research experience, graduate school offered me a chance to hone my teaching (assistant) chops, first in 3.012 (thermodynamics), then in 20.361 (biotechnology). I have a passion for the written word, whether reading literature or enabling effective and elegant science communication. My greatest interest now is in fostering a science-literate citizenry by multiple tacks: at the college instructor level, this means developing the abilities of students to navigate a large body of information, to understand the logic of experimental design, and to communicate their findings to myriad audiences.
Core Teaching Values: Goals and Expectations
Lessons from year three
Lessons from year two
Lessons from year one
• Transparency - I would like to convey to you why you are learning what you are learning at every stage, and also how you will be held accountable for this knowledge. In turn, I expect you to keep me abreast of any bugs or features of the course.
• Responsibility - I hope to take the basics (keeping up with classwork, being considerate of your labmates) for granted. Rather, I aspire for us to engage in issues both intrinsic and seemingly peripheral to the course, such as the ethical implications of biotechnology.
• Adaptability - No matter what your next career may be, adaptation to unexpected outcomes and the ability to redefine your strategy and goals will be vital. In my own case, flexibility and experimentation with pedagogical methods is a priority. Please do not hesitate to give me feedback.
Teaching History
Classes
Fall 2007 - Laboratory Fundamentals of Biological Engineering, subject 20.109 - instructor
Spring 2008 - subject 20.109 - instructor and lecturer
Fall 2008 - subject 20.109 - instructor
Spring 2009 - subject 20.109 - instructor and lecturer
Fall 2009 - subject 20.109 - instructor
Fall 2009 - Thermodynamics of Biomolecular Systems, subject 20.110 - recitation instructor
Spring 2010 - subject 20.109 (OpenCourseWare version here) - instructor and lecturer
Fall 2010 - baby-raising!
Spring 2011 - subject 20.109 - instructor and lecturer
Other projects
Summer 2008 — primary author of departmental guidelines for undergraduate education best practices
Summer 2009, 2010 — co-organizer of departmental TA training
Research Interests
1. Biomaterials - especially natural and synthetic polymers.
2. Immunology - particularly T cell motility and lymphoid chemokines.
Publications
Current Recommended Reads
• 2010 One day in my free time I will review pregnancy and infancy books...
• 2009 Spring/Summer here
• 2008 Year in Review here
• Winter 2008
• How Doctors Think, by Jerome Groopman (review)
• Woman: An Intimate Geography, by Natalie Angier (review)
• Summer 2007
• Popular science - Endless Forms Most Beautiful, by Sean Carroll
• Fiction - Gilead, by Marilynne Robinson
Personal tools
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