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Humminbird 858c HD DI GPS/Fishfinder Combo
Key Features
7" diagonal 65K-color 16:9 display with backlight
External 50-channel precision GPS antenna
Built-in ContourXD cartography by Navionics
Down Imaging Sonar give a detailed picture
SwitchFire Sonar: two modes for the clearest image
Our Price $949.99
Sale Price $599.99
Availability: Sold Out
- Please note: This item can only be shipped to U.S. addresses.
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details & specsback to top
Product DescriptionThe Humminbird 858ci DI GPS/Fishfinder Combo has DualBeam PLUS sonar with a narrow beam (455 kHz / 16°) for a detailed view of the bottom and a wider beam (200 kHz / 25°) for finding fish and viewing the surrounding area. Down Imaging provides detailed images of what's directly beneath your boat, making it easy to decipher structures, and displays this information on a brilliant, high-definition 7" display. Pre-loaded with Contour XD cartography by Navionics with over 3,000 US lakes.
Physical/Performance Features:
• 7" diagonal 65K-color 16:9 TFT display
• 480 x 800 resolution
• CCFL backlight
• Waterproof housing
• Add more maps and memory with two SD card slots
• Includes transducer, in-dash mount, gimbal mount, and unit cover
• Power Output RMS: 500 Watts
• Power Output Peak-to-Peak: 4000 Watts
• Draw: 780 mA
• Voltage: 10V - 20V DC
Mapping Features:
• External AS GR50 50-channel WAAS precision GPS antenna
• Built-in, enhanced ContourXD cartography by Navionics features maps for major roads, continental US coastline, and over 3,000 US lakes with depth contours
• 3D chart view
• Trackplotting and chartplotting
• GPS speed
• Save up to 2,750 waypoints/favorites/locations and 45 routes directly from your device
• Save up to 50 tracks and 20,000 points per track
• Compatible with Navionics and LakeMaster cartography
Sonar Features:
• DualBeam PLUS sonar - 200 kHz / 25°, 455 kHz / 16°
• Narrow center beam focuses in detail on bottom contours and structure
• Larger beam provides a wider area for finding fish and viewing surroundings
• View beams separately, side-by-side, or blended together
• Maximum depth: 600'
• Down Imaging sonar - 455 kHz / 75°, 800 kHz / 45°
• High-frequency, ultra-thin sound waves
• Provide an incredibly detailed image of what's directly below the boat
• Complete 180° view
• Maximum depth: 250'
• SwitchFire Sonar
• Clear Mode - Filters noise in shallow or turbulent water
• Max Mode - Enhances detail, even thermoclines and currents, so you can see your bait clearly
• Selective Fish ID+ uses fish icons to indicate depth, intensity of sonar return, relative size, and which beam the fish is in
• Real Time Sonar (RTS) Window
• Sonar Echo Enhancement
• Sonar Recording
• TrueArch Technology
• Instant Image Update
• Transducer mounts on the transom or inside the hull
• Target separation: 2.5"
Other Features:
• Capable of networking with WeatherSense, CannonLink, and/or InterLink
• Trip log
• Split Screen
• Wide/narrow cone
• Independent zoom
• Bottom lock
• Screen snapshot to memory card
• Freeze Frame
• Large digits view
• Custom view selections
• Custom digital readout selection
• Selectable colors for display and background
• Temperature alarm and graph
• X-Press menu system
• One-year warranty
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Do you have questions about this product? 4 easy ways to get the answers you need:
• Live Help - Real time help for basic questions. click here
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• Answer Box - Get answers from real customers by clicking "Ask a Question" below.
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Q:can the ts3 transducer switch be used between 858cDI and minnkota trolling motors with built in trancducersAsked on 6/30/2012 by greg
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Graduate students need access to a lot more than just books, and New York University strives to offer support in all facets of the educational experience. Again, while the resources most useful to the greatest number ofmost students maywillmight be ultimately fall insidehoused in their own schools, there are several initiatives across the University that will aid students in furthering their academic career.
Beyond department-specific orientations and training for graduate instructors, students across NYU are invited to participate in the Center for Teaching Excellence, which schedules events throughout the year as part of the University's commitment to advancing standards of education. The CTE offers free individualized consulting services to graduate student teachers across NYU and, in conjunction with the Graduate Student Educational Development Program, conducts supplementary training for new and international instructors.
NYU offers a variety of services to aid in the research that informs the careers forged here. Bobst Library, the center of NYU's library system, offers an impressive number of in-house research instruments: from workshops and research seminars, to data and other bibliographical services. Here students can also meet with subject specialists dedicated to specific fields for truly individual guidance in their research projects. Bobst is also enriched by its participation in an international Interlibrary Loan system, as well as in a consortium of Manhattan libraries.
As academics is are not bound the classroom, NYU offers resources that can further advance it's the scholarly standing of its students. Throughout the year, workshops for fine-tuning CVs are offered to help all of NYU's graduate students best represent themselves. There are also grant-writing programs run through Bobst that help students search for and successfully draft proposals for internal and external grants.
Perhaps the most effective way for students to promote themselves is in person. To this end, a database of calls for papers, symposia, calls for papers, and research opportunities is in the works; for now, submit your creative works through the Tisch databases and consult the Humanities Calls for Papers database at University of Pennsylvania. The Wasserman Center hosts a number of interview preparatory workshops, among their other events, also open to graduate students.
Resources Specific to Our Graduate Schools:

subset: commoncrawl


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$129.95 Buy Now
Take on any video-editing challenge with Pinnacle Studio 18.5 Ultimate, our most complete version. Powered by a new native 64-bit architecture, Ultimate lets you create breathtaking movies with support for 4K Ultra HD video. You get all the features of Pinnacle Studio Plus and more, including NewBlue bonus effects, Blu-ray authoring, iZotope Music & Speech Cleaner, additional ScoreFitter soundtracks, and support for XAVC S for 4K and HD. Energize your videos with 2,000+ transitions, effects and templates, add the perfect soundtrack, and create a DVD in just a few steps with new Pinnacle MyDVD. Easily share online, on disc and on devices.
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Along with 64-bit speed and responsives, Ultimate offers frame-accurate precision editing on unlimited tracks. Create sophisticated picture-in-picture and multi-layer effects in HD and stereoscopic 3D.
Record content directly from your screen, then easily bring your captures to the main Timeline to add them to your movie. New in this version, record both system audio and microphone sound with ease.
Explore endless creative possibilities with over 700 presets and more than 55 plugins from industry leader NewBlue. This bonus pack includes tools for image stabilization, creative effects, exciting translations and more. (details)
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Choose the editing approach that's right for your project. For instant results, drag-and-drop your desired media elements from the library into SmartMovie to automatically make great videos—complete with music. Use Storyboard mode to easily create first drafts and visualize your movie. At any point, switch to the timeline for more traditional and precise editing.
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Experience a new and easier way to burn to DVD and AVCHD disc. Get a stylish look with 10 themed templates with menus, sub-menus and chapters. Turn your videos and photos into professional-looking multimedia disc projects, complete with image-rich menus and music.
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No movie is complete without a soundtrack. Add music with the included ScoreFitter library of royalty-free sounds and songs—they even automatically adjust to fit the length of your movies.
Get a 6-week, all-access pass to StudioBacklot.tv, featuring brand-new Pinnacle Studio 18.5 content, loads of other training and a royalty-free stock library.
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Work on projects anywhere with Pinnacle Studio for iPad2, then transfer them to Pinnacle Studio for further polishing. Try Project Packages to bundle together all of the media files related to a given movie, taking the work out of organizing and assuring that you always have all the files you need.
When you're ready to share your videos, Pinnacle Studio 18.5 Ultimate makes it easy to post your HD and 3D videos on Facebook, YouTube, and Vimeo. You can also easily create DVDs and AVCHD™ discs, as well as author Blu-ray™ discs complete with Dolby® Digital 5.1 surround sound. Export to popular formats like Adobe® Flash® FLV, MPEG-4, and DivX®, as well as popular devices like Microsoft® Xbox®, Sony® PlayStation, Nintendo® Wii™, Apple® TV and Apple® iPad®. With Pinnacle Studio Ultimate, the world is your screen.
1 Video capture hardware required
2 Pinnacle Studio for iPad app not included
|Studio Plus
|Studio Ultimate
|NewBlue bonus effects (700+ presets and 55+ plugins)
|4K Ultra HD video support
|XAVC S support
|Blu-ray™ authoring
|iZotope Music & Speech Cleaner
|Live Screen Capture
|3D MVC file format import
|Simultaneous source and master windows with drag-and-drop
|Keyframe-based effects
|Dolby 5.1 native support (import/export)
|Customizable keyboard shortcuts
|Stereoscopic 3D import, editing and export
|Stereoscopic 3D depth control
|Advanced NVIDIA 3D Vision support
|Dual-monitor 3D support, "side by side" export
|Trim editor for source clip in/out-points
|Advanced DVD Authoring
|Detach Audio
|Advanced Title Editing (Motions)
|Native 64-bit architecture
|17 Royalty-free music tracks from Triple Scoop Music
|6 weeks free training from Studiobacklot/Class on Demand (English)
|Included 2D/3D effects, transitions, titles and templates
|Video/audio tracks for multilayer editing
|6 (3/3)
|Media asset rating, advanced tagging and collections
|UI enhancements for improved ease of use
|Import and polish projects from Pinnacle Studio for iPad*
|Easy sharing to Facebook®, YouTube® and Vimeo®
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|Storyboard and timeline editing
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|Editable themed Montage effects/transitions templates
|3D auto-color adjustment for both eyes
|DVD authoring
* Pinnacle Studio for iPad app not included


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Planning a wedding, reunion, or vacation and wondering how often it rains or how hot it gets on those days? Finding out is easy!
- Search by a range of dates or the same date each year
- Search from 1960 to the present
- Download your results to a spreadsheet
- Search as many U.S. locations and as often as you wish for up to a year
Get minimum, average, and maximum measurements for the following only:
- Temperature
- Precipitation
- Wind Speed
- Snow Depth
- Visibility
- Dew Point
- Sea-Level Pressure
- Occurrences of Fog, Rain, Snow, Hail, Thunder, and Tornados
How it works:
- You log into a private website and enter your location and dates
- We pull government data reported from the weather stattions that surround your area
- You are presented with easy to read charts and graphs
Click the following links to see sample reports:
Temperature Report
Precipitation Report
Wind Report
Limitations of Use:
The data provided by our customized weather history application is intended for private use. Any means to extract data by any automated method is a violation of these terms and conditions.
Please consult our complete description of the Terms and Limitations before purchase.


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- Start It Up Episode 32: Angel Summit To Bring Investors to Chattanooga
The Two-Way
Mother Of Slain Sandy Hook Student Sits In For Obama's Weekly Address
Originally published on Sat April 13, 2013 3:16 pm
In a rare departure from tradition, Saturday's weekly presidential address was delivered not by President Obama but instead by Francine Wheeler, whose son Ben, 6, died in the Sandy Hook Elementary School shootings last December.
Flanked by her husband, David, Wheeler called for Americans to urge the Senate to pass gun control legislation that it is scheduled to begin debating in the coming week.
"We have to convince the Senate to come together and pass commonsense gun responsibility reforms that will make our communities safer and prevent more tragedies like the one we never thought would happen to us," Wheeler said (see the White House video).
In the roughly four-and-a-half minute speech, Wheeler also recalled her son for his boundless energy, his musical talents, and a desire to be like his older brother, Nate. Her address, which the White House tells the AP was written by the Wheelers themselves, comes one day before the four-month anniversary of the Newtown, Conn., murders.
"Sometimes, I close my eyes and all I can remember is that awful day waiting at the Sandy Hook Volunteer Firehouse for the boy who would never come home – the same firehouse that was home to Ben's Tiger Scout Den 6," Wheeler said, in an emotional passage that brought a brief pause. "But other times, I feel Ben's presence filling me with courage for what I have to do – for him and all the others taken from us so violently and too soon."
The Wheelers were among a group of people who lost loved ones at Sandy Hook who flew to Washington earlier this week. They told their stories to several senators, in a move that is credited with giving new momentum to a bill that would expand background checks and boost school security spending, along with other moves.
Thursday, the Senate voted by a wide margin to begin debate on the measure.


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Lewis Hamilton was relieved to take the chequered flag ahead of Nico Rosberg in Bahrain having triumphed in an "exciting" battle with his team-mate....
Lewis Hamilton was relieved to take the chequered flag ahead of Nico Rosberg in Bahrain having triumphed in an "exciting" battle with his team-mate.
With Mercedes holding a massive advantage over the chasing field, it was up to Hamilton and Rosberg to put on a show at the Sakhir circuit and that is exactly what they did.
Racing wheel to wheel at the start, Hamilton edged ahead only for Rosberg to come back him just before the first round of pit stops.
Again Hamilton held on but faced even more pressure in the final ten laps of the grand prix after the Safety Car had negated his lead.
Rosberg challenged his team-mate all the way to the finish line, even pulling ahead on occasions.
However, after 57 thrilling laps it was Hamilton who emerged victorious by just one second.
"It was exciting," said the 2008 World Champion.
"Nico drove fantastically well throughout the race. It was so fair but it was so hard to keep him behind me.
"I was on a real knife edge the whole time so it was a great relief to manage it."
As for Rosberg, the 28-year-old claims Sunday night's Bahrain GP was the "most exciting" of his racing career.
"I strongly dislike coming second to Lewis, it's not something I enjoy doing," he admitted.
"I have to say that but it was definitely the most exciting race I have ever raced in my whole career."
He added: "Today was a day for the sport, we put on a massive show as Team Silver Arrows."
Related Links
Lewis Beats Nico In Team-Mate Thriller
Gallery: Race Day In Bahrain


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Ben and JP are hard at work on a new upcoming double live album in Van Nuys, CA's legendary Sound City (home of Nirvana's "Nevermind"). The as yet untitled compilation comprised of mostly 2000 tour material will span two discs: one with full band performances, and another of solo acoustic material. "Steal My Kisses", "Sexual Healing", "Faded/Whole Lotta Love" and more are already in the can. No release date has been set.
Ben Harper & the Innocent Criminals will tour the college circuit in February and move on to Japan and Australia after that. Check out Tour Dates for the latest information.
In conjunction with San Diego radio station 91X, Toys For Tots and Yahoo! Auctions, BenHarper.net is auctioning off one pair of tickets to the upcoming "Frosty The Show, Man" on December 9th. The proceeds from the auction will benefit Toys For Tots. Click here for the auction.


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- INTERPOL Lusaka
Member since :
31 de agosto de 1966
Zambia Police Service
The preservation of peace;
- The protection of life and property;
- The prevention, detection and investigation of crime;
- Arrest and prosecution of offenders;
- Community education and liaison on issues of crime and safety.
The Zambia Police is committed to providing a high quality service to the people of Zambia by upholding and applying the law fairly and firmly. The force applies proactive methods to prevent crime, arrest those who break the law and take them to court and assist victims of crime through counselling.
The Zambia Police is committed to cultivating rapport and partnership with the community, and pledges to respect individual human rights while recognizing the community's expectations and obligations.
The service through this mission will strive to promote and supplement the overall efforts that are aimed at bringing crime levels to their minimal levels.
The Zambia Police Service is part of the Ministry of Home Affairs and is headed by an Inspector General of Police. Its headquarters are in the capital city Lusaka.
INTERPOL Lusaka
The INTERPOL National Central Bureau (NCB) for Zambia sits structurally within the Criminal Investigation Department (CID) of the Zambia Police Service.
The detective police officers at INTERPOL Lusaka work at the following NCB crime desks:
- Economic and Financial Crime;
- Cybercrime;
- Illicit Drugs;
- Intellectual Property Crime;
- Works of Art Crime;
- Fugitives and Missing Persons;
- Motor Vehicle Theft;
- Precious Stones, Minerals and Metals;
- Environmental and Wildlife Crime;
- Human Trafficking;
- Anti-Terrorism;
- Small Arms and Light Weapons;
- Stock Theft.
- To provide assistance to domestic law enforcement institutions by working with partner INTERPOL NCBs in investigating, locating, arresting and extraditing wanted criminals;
- To extend INTERPOL's criminal databases and intelligence to domestic law enforcement institutions specialized in NCB's 13 specialized areas;
- To invite domestic law enforcement institutions to participate in any regional or international events organised by INTERPOL;
- To advise, educate and raise awareness on international crime trends.
Zambia hosts INTERPOL training session against wildlife crime
Dangerous fake goods seized in INTERPOL operations across Eastern and Southern Africa
Fakes worth more than USD 1.2 million seized in INTERPOL-led operations across Southern Africa
Los traficantes de personas, drogas y armas, objetivo de varias operaciones llevadas a cabo en África Oriental y Austral
INTERPOL efectúa la mayor operación de su historia contra el tráfico de marfil a manos de organizaciones delictivas
INTERPOL colabora en un curso para frenar el tráfico de especies de flora y fauna silvestres en África Austral
El comercio ilegal de marfil y cuernos de rinoceronte objeto de una operación coordinada por INTERPOL en África austral
Las redes de tráfico de medicamentos falsificados de toda África son objeto de una serie de operaciones coordinadas por INTERPOL
Red Notices 4
LIBANGA Age today: 36 years old
MOSES Age today: 30 years old
- 17th African Regional Conference - 2003 - Lusaka
- 13th African Regional Conference - 1995 - Lusaka


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- Independent
- The Enterprise
- The Recorder
Charles County sheriff's officers went to the 8700 block of Billingsley Road in Waldorf at 12:36 a.m. May 19 after someone reported a shooting.
Police found that the victim, a 17-year-old boy, was attending a large party when someone pulled a gun and fired shots, one of which hit him in his leg. He was treated at a hospital and released.
Anyone with additional information is asked to call detective B. Buchanan at 301-609-6477.


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Taylor Swift just turned 24.
She came to Middle Tennessee from Pennsylvania at 14, and in the ensuing near-decade she has become an international superstar, and a wealthy young woman. Those are rare and laudable things, but they've been done before.
Swift, though, is unprecedented. She came to popular attention with Top 10 country hit "Tim McGraw" when she was 16, becoming the first mid-teenage singer-songwriter to do so since Janis Ian hit the Top 20 with "Society's Child" in 1967. And Swift has made the most graceful transition from teen stardom to adult music career since Brenda Lee, who recorded signature hit "I'm Sorry" at age 15 and maintained an impeccable image on her way to the country music and rock and roll halls of fame.
Yet these are not the reasons that The Tennessean recognizes Swift as the 2013 Tennessean of the Year.
Swift has become a worldwide ambassador for Tennessee's capital city, an example to millions of young (and not-so-young) people of how to turn damaged feelings into healing creativity, and a financial booster to some of the city's most important institutions. In October, the Taylor Swift Education Center opened at the greatly expanded Country Music Hall of Fame and Museum, courtesy of Swift's $4 million gift, the largest individual artist gift in the museum's history.
The center, which opened ahead of schedule in October, spans two floors and includes three classrooms, a learning lab and, coming in 2014, an interactive exhibit gallery. It gives the museum seven times more space for education.
And this month, on her birthday, Swift offered up $100,000 to the Nashville Symphony, an organization that endured severe financial uncertainty in 2013. For the second consecutive year, Swift topped DoSomething.org's list of the top 20 charitable celebrities, and much of her generosity is intended for the betterment of Nashville.
"For her to believe in us, the hometown institutions, and to be focused on Nashville speaks volumes," says Kyle Young, the director and chief executive officer of the Country Music Hall of Fame and Museum. "How often do you think she's approached to do things, all over the world? So it means so much for her to believe in us and think that investments here would help the city she clearly cares so much about."
The ambassador
In many ways, Swift carries Nashville and its music all over the world.


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Report: Washington state not the best at everything
at 4:25pm by David Kroman
Washington state is number one (at least in the hearts of Washingtonians) in so many things: marijuana, wine, apples, seafood, tech, aviation. Seattle has the highest rate of adults with a college degree. Sea-Tac set the bar with a $15 minimum wage. And Washington has more glaciers than all other contiguous states combined. It's safe to say we like being number one.With that in mind, Washington received a huge blow to its winning reputation today — our beloved state ranked as the 12th most miserable place to have Thanksgiving. That's right: According to the Estately blog, there are 38 states that are better for Thanksgiving. The ranking was based on:1. Likelihood of food poisoning (5th)2. Likelihood of relatives binge drinking (20th)3. Likelihood of political arguments at dinner (23rd)4. Dietary restrictions impacting meal quality (30th)5. Likelihood of favorite NFL team losing on Thanksgiving (3rd)6. Likelihood of guests/cooks abandoning meal for Black Friday sale (46th)Actually when you look at, we're pretty average in most categories. And we can be proud of our Black Friday ranking — no stampeding for this state. But we are exceedingly more likely to have the day ruined if the Seahawks lose (which seems unfair because 44 states have 0 football teams even playing on Thanksgiving). And apparently we all need to make sure that our raw turkey is far from our spinach salad. Use a separate cutting board please!—- D.K.


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I enjoyed the talk about antique jacks. Due to health I'm trying to sell an old Manley 2 1/2 ton floor jack.I have no idea where to sell it that it can continue to perform !!! I hope I can post this Info here as well as this contact info ! pb... email@example.com
Results 21 to 24 of 24
Thread: Antique floor jack
07-17-2008, 03:48 PM #21Junior Member
- Join Date
- Jul 2008
- Posts
- 2
07-18-2008, 01:17 PM #22Voigt Precision Welding, Inc.
Miller Dynasty 200 DX, Miller Syncrowave 250, MillerMatic 252, Hypertherm Powermax 45, Auto Arc Trailpower 8000,272+187 lb Peter Wright anvil, 120 lb Fisher-norris, and more! Buffalo drill press, Grizzly Horiz. Bandsaw, Edwards shear, Barth Shear, bantam mechanical ironworker, Hopkins fly press, Doall Bandsaw, brown and sharpe surface grinder.
2007 Silverado 2500HD (tow vehicle)
2000 Camaro SS (Race car)
07-18-2008, 02:20 PM #23Senior Member
To all who contribute to this board.
- Join Date
- Sep 2005
- Location
- S.W. Pennsylvania
- Posts
- 2,250
My sincere thanks , Pete.
Pureox OA
Westinghouse 300 amp AC stick
Miller Syncrowave 250
Hexacon 250 watt solder iron
07-21-2008, 10:49 AM #24Junior Member
- Join Date
- Jul 2008
- Posts
- 2
Sorry to get back to post a reply. I live in Cumberland Maryland. I hope somebody can put this Bear to work I'm sure it could tell some stories. It might be larger than 2.5 tons, It is about 54" long not including the handle. I'll do some more checking tonight and measure the cylander. Thanks for your interest and help ! pb...


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The National Ornamental Metal Museum in Memphis used to have classes and probably still does. Also Google "ABANA" (Artists, Blacksmiths Association of North America). They used to have lots of info.
Results 11 to 17 of 17
03-22-2009, 05:21 PM #11Junior Member
- Join Date
- Aug 2008
- Location
- Near Memphis
- Posts
- 14
03-22-2009, 05:32 PM #12Senior Member
- Join Date
- May 2007
- Posts
- 539
We were just at the Memphis National Ornamental Metal Museum a couple of weeks ago, good place to visit. Lot's of good work.
They have a working shop, casting and bending / shaping. Real friendly folks.
If you are in the area worth stopping by and taking a look.
03-22-2009, 07:09 PM #13Senior Member
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- Dec 2007
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- National City CA
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We actually have a Blacksmith shop here in escondido ca and they take volunteers They operate as part of the historical society in Grape day park Open mid week and Saturdays only. Nice shop with lot of experience running the shop. Kind of half actual blacksmith and half pre-machinist. Belt driven power hammers, etc.Miller Syncrowave 200 W/Radiator 1A & water cooled torch
Millermatic 252 on the wish list
Bridgeport Mill W/ 2 axis CNC control
South bend lathe 10LX40
K.O. Lee surface grinder 6X18
Over 20 years as a Machinist Toolmaker
A TWO CAR garage full of tools and a fridge full of beer
Auto shades are for rookies
03-23-2009, 06:44 AM #15Senior Member
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- May 2007
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- 539
vin-man, It's not that no one notices, just don't care
My Wife's Grandpa was the Blacksmith for a town in northern Illinois. Even when he retired the folks came from all around to set in the shop. Turned into the local meeting place for the old timers.
It was cool watching her face as we entered the shop, see the equipment and the wares around it.
Had to laugh, the lady that was showing us around kept saying "those bends are really easy to make". Everything is easy when you have the equipment and know how to do it. She did show us what was hard to make also. Wife and I enjoyed the visit.
03-24-2009, 08:07 PM #16Senior Member
- Join Date
- May 2007
- Posts
- 539
So, for a second ponder this, all ye faithfull.
Welding has been the last ~100 years...
Blacksmithing has been around for how long?
Course, any fool, with a couple hundred bucks can buy a welder, but to be a blacksmith, and, work with the metal, well... that can be achieved in...
For the record, and the score keepers, I am not a blacksmith or a welder, by definition.
But, I do know, what it means to be a welder, or blacksmith, and have formed a certain respect for those who have the touch, as they say
try and catch the show ' metal on modern marvels.' they really get in to the blacksmithing and when it started and were it is today.TB 325
dynasty 200sd
spoolmatic 30a/wc24
suitcase x-treme 12vs
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evolution rage 2


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Give your dog and cat an amazing food that's similar to what their ancestors ate thanks to the delicious, nutritious ingredients in Trippett formulas.
Tripett products are made from pure green tripe taken from the highest quality human grade sources and mirror what our pets' wild counterparts instinctively fed on. Green Tripe (Green simply means the tripe is pure and unbleached) contains the partially digested grasses in an animal's stomach and is rich in digestive enzymes, gastric juices, taurine, amino acids and essential fatty acids which are important to all aspects of your pets' health.
In green tripe, all of these nutrients occur naturally, making them more valuable to your pet than they would be if they were to be derived from extracted sources. Tripett contains no preservatives, no additives, no fillers or artificial ingredients. Only pure meat.
Benefits of Tripett
Tripett Offers 3 Flavors
Tripett is excellent for dogs & cats of all breeds, ages, and sizes, whether they are the family pet, or the champion of a national show! Tripett is also great for picky eaters, and pets with sensitive stomachs, colitis, skin problems, and more. When feeding Tripett, coat conditions flourish, little ones grow up healthy and strong, and seniors are rejuvenated.
Click here for reusable can lids-great for storing your pet's excess canned food in the fridge. Helps food stay fresh and last longer!
Review Rating: 4.5
Country of Origin: USA
Manufacturer: Tripett
Green Beef Tripe
Ingredients - Green Beef Tripe, Water, Garlic,Carrageenan Gum
Calories - 464.90 kcal/can
Guaranteed AnalysisCrude Protein (min) 11.0%Crude Fat (min) 7.0%Crude Fiber (max) 0.5%Moisture (max) 79.9%Ash (max) 1.0%Fatty Acids (Omega 6) (min) 0.47%Fatty Acids (Omega 3) (min) 0.20%Calcium 139 mgPhosphorus 441 mg
New Zealand Green Lamb Tripe
Ingredients - New Zealand Lamb Tripe, Water, Garlic,Carrageenan Gum
Calories - 336.60 kcal/can
Guaranteed AnalysisCrude Protein (min) 11.0%Crude Fat (min) 7.0%Crude Fiber (max) 0.5%Moisture (max) 79.8%Ash (max) 1.0%Fatty Acids (Omega 6) (min) 0.47%Fatty Acids (Omega 3) (min) 0.20%Calcium 73 mgPhosphorus 441 mg
Green Beef Tripe, Duck & Salmon
Ingredients - Green Beef Tripe, Water, Duck, Salmon, Garlic, Carrageenan Gum
Calories - 455.50 kcal/can
Guaranteed AnalysisCrude Protein (min) 10.0%Crude Fat (min) 7.0%Crude Fiber (max) 0.5%Moisture (min) 79.8%Ash (min) 1.0%Fatty Acids (Omega 6) (min) 0.49%Fatty Acids (Omega 3) (min) 0.23%Calcium 320 mgPhosphorus 405 mg
Feeding Guidelines
Tripett is intended to be supplemented with good quality dry or raw food.
The reviews below are presented for informational purposes only and are not intended as an endorsement of any product. The information is not intended to be a substitute for visits to your local veterinarian. Instead, these testimonials offer the reader information and opinions written by pet owners and/or veterinarians concerning animal health and products that they have used.
(based on 84 reviews)
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(1 of 1 customers found this review helpful)
I am going to buy this again!!
By Jenjer
from Las Vegas, NV
About Me Long-time Pet Owner
Comments about Tripett Green Tripe Canned Dog & Cat Food:
My dogs love the taste and I would most definitely buy this again. It is really good for the pickiest eaters. I love that this site sell them by the singles so that I can buy the different varieties.
Bottom Line Yes, I would recommend this to a friend
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Max (pug) and bestie Piper (pekingese)
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dogs love it
By mandy
from ohio
my 4 puglettes love this STINKY stuff, i feed as a treat 2 or 3 times a day, i give each of them a table spoon, they dance for it, it does smell bad, but they seem to disagree so i will continue to treat them with this, and it is VERY good for their digestion, so i will hold my nose for them.
(3 of 4 customers found this review helpful)
A Must Have For Older Dogs
By JJ
from Pittsburgh, PA
My 15 year old Lab/Husky was going on and off his homemade (not raw) diet more often as he aged. He has numerous food allergies and digestive issues, so finding any really yummy additions to his routine food was difficult. I purchased this to mix in and he hasn't missed a meal since. He has not had to continue FortiFlora or Gas-X since he's been on this and has had no stomach issues from it. This has also been wonderful for administering his pills. I don't even have to hide them, just sit one on top of the tripe and he doesn't even care the pill is there. I had read that it is good for their teeth as well, but at his age I didn't think I'd notice that perk. But, the plaque doesn't seem as bad on his canine teeth. It does have a distinct odor to it, somewhat bad, but I haven't been repulsed by it unless it gets really warm. It is very noticeable if it gets somewhere on the way from the can to the dog like a counter-top, dishrag, etc. But, quick clean-up and tightly bagging up any paper towels, empty cans before trashing helps. Compared to premium canned food prices, this is no more. The lack of fillers and multi-ingredients makes this a better value to me, no unwanted side effects I have to counteract with something else.
They eat it up!
By CanineMomma
from Dunnellon, Fl
About Me Professional Pet Trainer
I supplement a raw diet with the tripe and my two dogs really love it. I have given a whole can for a meal. They'd keep eating it if they could! Doesn't smell toooooo bad, I have smelled much worse in plain canned dog food. If you have never tried it, do so and your dogs will gobble it. Maybe they won't eat so much grass!
Tripett Lamb Tripe
By Maryann
from Ohio
The dogs love it. I use in in my raw mix. The cans make it conveinent.
(2 of 2 customers found this review helpful)
Tripett Green Tripe with Salmon and Duck
By kalawai2000
from Kalaheo,HI
I mix it with Only Natural Pet Raw Dehydrated Food....My girl loves it and will eat everything up....The only thing for me is the smell will knock you over but as long as my dog loves it and its good for her.....
They all love it!
By Sunny
from Florida
ALL of my 7 dogs love this food. It has no vitamins so it I mix a little of it with their dry food. Just 2-3 Tablespoons, a tiny bit of water, make a slurry then add their dry and coat it with the slurry. They devour it. This food is my go to food when any of them get loose stools or have an upset stomach. I have found it works far better for loose stools than the often recommeded white rice with a little chicken breast. It doesn't look very appetizing and the smell isn't great either. Just try not to look at it or smell it.... we may not like it but the dogs go crazy over it. When they smell me open a can of this, my Tibetan Spaniels are extra excited for their dinner that night. I've never had one turn it down and if they are picky with their dry food, add this to it, watch them gobble it up.
Saved my daisy!
By mwcgirlpapmom
from Midwest City, OK
My Daisy has CHF and takes 5 pills twice a day. AFTER TRYING EVERYTHING, my friend recommended green tripe. It only smells if you stick your nose in the can however it sure hides the taste of 5 crushed up pills!!! No more fighting to get her medicine down. I use about 1/4 cup, mix in the meds with tripe and a little water.I drizzle water over it after it has set a bit because it it very sticky. She takes a diuretic so a little soupy gets it down. She also eats kibble with the other dogs. Green beef Tripe is one of the most nutritional foods you can feed a dog. I am a grainfree mom when it comes to the babies!
Images shared by mwcgirlpapmom
Miss Daisy, Noodle and Boomer Sooner
Tags: Using Product
Dogs loved it.
By Doberman Mom
from New Orleans
Haven't tried feeding the tripe at the recommended amount yet. I am trying to gradually incorporate it into my Doberman's diet. They really go wild for it. It is too soon to say if it is doing anything for them though. Will say that if you have a queasy stomach this is not for you. Definitely has a smell you will not forget.
A "go to" food to top a good quality dry
By Deb
from Klamath, Oregon
It smells bad but the dogs love it. Good for picky eaters. Use as a topper for a good quality dry food.
© 2015 Only Natural Pet LLC - All Rights Reserved


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I am snapping a lot of pictures and lots of them I upload to Facebook. Is there any plugin out there to tag people in my catalogs?
Take the 2-minute tour ×
Photography Stack Exchange is a question and answer site for professional, enthusiast and amateur photographers. It's 100% free, no registration required.
At this moment I don't know any plugin that will do that. You can combine Picasa and Lightroom... more information on google http://lmgtfy.com/?q=lightroom+facebook+tag+people
Jeffrey Friedl produces a number of excellent Lightroom plugins and I notice he's got a new one for importing face data from Picasa:
I haven't tried it though, so I can't say if it does what you're after, but it might be worth a look?


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Action/Adventure, Suspense/Thriller
Ko will be a racy entertainer with all necessary commercial elements. Jeeva, Karthika and Ajmal in the lead. Karthika & Jeeva are journalists working for a leading newspaper. Jeeva plays a photo journalist who does an investigative story that disappoints the political mafia that rules the state. Due to this both will face various problems and how the duo emerges triumphant by successfully crossing all hurdles is the story.
Cast: Prakash Raj, Jeeva, Karthika, Ajmal
Produced by:
Directed by: KV Anand
Written by:
Running time: 2hr 35min
Opens: Apr 22, 2011 Limited
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Quick Access Biometric Rifle Gun Safe
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The all new Biometric rifle safe by Barska® is the ultimate secure quick access rifle storage system. The advanced biometric fingerprint recognition technology allows for fast secure access to your firearms with the simple scan of a fingerprint. Push the power button and hold your finger on the laser optics sensor to quickly open the safe in three seconds. The biometric safe's scanning technology is the solution to securely storing your rifles and having instant access to them in case of an emergency. No longer will you need to search for your keys or have to remember complex numerical key combinations.
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- Weight 66 lbs
Quick Access Biometric Rifle Gun Safe
Reviewed by 2 customers
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- Safe From The Grandchild
- Secure
Best Uses
- Ammo
- Handguns
- Handles All Of My Needs
- Shotguns
Comments about Quick Access Biometric Rifle Gun Safe:
I mounted it into a wall were it is out of the way.
- Was this a gift?:
- No
- Durable
- Futuristic
- Good Protection
- Narrow
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- Guns and ammo
- Storage
Comments about Quick Access Biometric Rifle Gun Safe:
Very easy to set up your fingerprints. You must bolt it down to the floor or a wall.
- Primary use:
- Personal
Quick Access Biometric Rifle Gun Safe
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Curious Britney Spears
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Reviews (142)
1 - 5 (of 142 Reviews)
I first got "lucky" wearing this so it will always have good memories for me. It's a beautiful scent that lasts a long time. One of my absolute favorites that I wear several days each week. Highly recommended!
Written by NDNgirl. on June 02, 2014Read Review details ►
in my opinion, it's her best fragrance, so light and flowery. very feminine and simple. love it.
Written by Anonymous on January 12, 2014Read Review details ►
Britney Spears Curious is by far one of the best floral perfumes I've ever had! It's not to strong or overpowering, but still has a great lasting power and projects really well. I highly recommend this scent!!!
Written by AlysonJayne on December 31, 2013Read Review details ►
Love BS Curious fragrance. BUT how can we tell the mfg. to put this in a different spray bottle. The bigger bottle requires 2 hands to spray. It's insanely awkward to spray when you're in a hurry. Terrible bottle design.
Written by Annie on December 06, 2013Read Review details ►
Curious was another scent that I tried and was hooked instantly. I had to have it, but was embarrassed to go buy a Britney Spears perfume and not being 12. Lol! I had my Mom buy it for me at first. I wore it into an Irish pub that was filled with drunk men, smoke, food and smelly construction workers and I was asked over and over again what I was wearing. The next few times I would go in the bartender would come from behind the bar just to smell me. It is a really light, intoxicating smell. I LOVE using the lotion to "freshen up" when I am out. I will also rub into my hands and then my hair.
Written by AliCat on November 10, 2013Read Review details ►
1 - 5 (of 142 Reviews)


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Start enjoying the benefits of the Club de Amigos of Fuerte Hoteles
This cosy individual or double room is fully equipped to ensure guests enjoy a delightful and relaxing holiday by the sea. The room has a balcony or a terrace with views of the golf course, the village of El Rompido or doors leading straight out into the hotel gardens.
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1st and 10Super Bowl XLV - February 6, 2011A Panoramic View in Arlington, TX - The Rob Arra Collection
Bring your wall alive with the thrilling sights of Super Bowl XLV in Cowboys Stadium, and the glorious victory of the Green Bay Packers! This incredible panoramic poster commemorates Super Bowl XLV in all its glory. Captured by the lens of legendary panoramic sports photographer Rob Arra, this print celebrates an unforgettable night in sporting history, as Aaron Rodgers and the Packers embark on another successful drive during their thrilling victory. All the details of Jerry Jones' breathtaking football palace and its magnificent Super Bowl setup are here, in perfect focus, ready to take their rightful place on the wall of any true football fan! From MVP Aaron Rodgers' electric performance, to big-time touchdowns from Greg Jennings and Jordy Nelson, to Nick Collins' key interception return, to the final moments ticking off the clock and the confetti falling from the sky, all the memories will return with each and every glance! Capped off with the official NFL and Super Bowl XLV logos, the team helmets of the competing teams, the final scoring line, and the commemorative title, this is both a stunning work of sports art and a historic collector's item, worthy of a nice frame and a prominent place on the wall of home or office. Perfect for your sports wall-of-fame, and a rare piece you likely won't find ANYWHERE else!
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|Softball » News » Schedule » Roster » Coaches » Statistics
Hardy tabbed A-Sun Pitcher of the Week
Monday, April 16, 2007
MACON, Ga. (April 16, 2007) - Freshman Marissa Hardy (Rocklin, Calif.) was named Atlantic Sun Pitcher of the Week Monday afternoon after she earned both wins for the Bucs this past weekend in Jacksonville.
Hardy improved to 3-0 on the season as she helped guide the Bucs to a pair of victories Friday night against the Dolphins. The Rocklin,
Hardy, an Interdisciplinary Studies major, becomes the first ETSU student-athlete to receive Pitcher of the Week honors since the Bucs joined the A-Sun.
The Atlantic Sun Conference hands out Player and Pitcher of the Week awards weekly during the 2007 softball campaign.
The 12-member league headquartered in
Softball Archives
2006-07 Softball


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Courtesy: Duke Today
DURHAM, N.C. - Updated: 12:45 p.m., Thursday, Jan. 17, 2013:
The updated forecast from the National Weather Service now projects snow accumulations of 2 to 4 inches in the Durham area as part of the winter storm warning between 6 p.m. today to 4 a.m. Friday.
Duke officials have been closely monitoring weather forecasts and conditions and will communicate updates as conditions warrant, but at this time plan to operate on a normal schedule tonight and tomorrow.
Crews will begin pretreating walkways, entrances and parking lots this afternoon and have plans to begin clearing any accumulation from campus roads, lots and walkways early Friday morning if necessary.
The initial focus tonight will be on clearing parking lots and walkways used for the men's basketball game. The Duke Men's Basketball game scheduled for 9 p.m. at Cameron Indoor Stadium will be held as scheduled. Crews will clear any accumulations during the game using the priority clearing plan for a basketball event. Those attending are encouraged to consult the priority clearing map to determine the best routes and parking lots to use.
Updated: 5:10 p.m., Wednesday, Jan. 16, 2013:
Durham County is under a winter storm watch for Thursday evening and night with snow accumulations of 1 to 3 inches predicted.
Duke officials are closely monitoring weather conditions and forecasts for the area. Updates will be made as conditions warrant.
Given the chance of severe weather, employees are encouraged to review Duke's severe weather and emergency conditions policy to ensure understanding of roles and responsibilities if the policy is activated.
During times of severe weather, some staff positions require on-site presence to continue operations. To ensure continuous operations of patient care, student support and research functions, managers should assign all jobs to one of three service categories:
• Essential Service: During severe weather or emergency conditions, essential service staff report or remain at work. Transportation service and childcare services may be provided for those required to work during severe weather or emergency conditions.
• Reserve Service: During severe weather or emergency conditions, these staff are assigned a status at the time of the event.
• Delayed Service: During severe weather or emergency conditions, these staff do not report to or remain at work.
More information about the policy, including a map of priorities for clearing roads, lots and sidewalks, checklists and answers to frequently asked questions, is available at the DukeALERT emergency website.


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This conversation is closed.
What is the future of "western" education? How will knowledge be conveyed to the next generation of students?
Often times, I hear from teachers and educators about how technology has revolutionized the classroom. But what has this technology really done for us?
Yes, the classroom has gotten a little streamlined, with the capability to use computers to type up essays and etc. But as a student, I wouldn't say any of this is revolutionary. It only makes learning a little more convenient. The classroom model hasn't changed for decades, if not centuries. There's a teacher, or another knowledgeable individual, who instructs a group of students, who take notes and do activities/projects under the guidance of that individual. This classroom model has remained almost unaltered despite the development of radio, television, and other things that were to have expected to "revolutionize" and change the way how we educate students.
With this being said, what do you guys all think? Will the education system and classroom model change in the future? Or will it remain the same?
May 1 2013: in reality, i haven't seen this so called revolution in effect. i've seen paper get replaced with tablets - a very expensive exercise but hardly a revolution.
there is very little use of technology to improve or make the teaching job more efficient. schools tend to be mired in tradition as much as religion.
what we will need to teach kids is how to spot propaganda (advertisers and the media have been exploiting kids' gullibility for many years now) and differentiate between opinion and information.
unfortunately, it is not the "communication revolution" that drives change in schools but the logistics of managing over-sized classes and expensive (digital) resources with severely limited budgets.
i am not seeing much in the way of real change despite all the overexcited rhetoric regarding technology..
Adelo Vant
Apr 29 2013: Maybe some of the below can provide an eyeopener for US.
- +1
USA education petition http://wh.gov/eEaI may be appropriate for US, EU ...?
Please share with US, EU and others colleagues, family, friends, and schools, FREE and OPEN online education and books. These W3 resources are affordably, and travel not required.
CourseEra - Take the World's Best Courses, Online, For Free. CourseEra is a social entrepreneurship company that partners with the top universities in the world to offer courses online for anyone to take, for free. A future where top universities educate millions of students.
There are 62 Universities partnered With Coursera. https://www.coursera.org/
edX - The Future of Online Education for anyone, anywhere, anytime. EdX is a not-for-profit enterprise of its founding partners Harvard University and the Massachusetts Institute of Technology that features learning designed specifically for interactive study via the web. A new online-learning experience with online courses.
There are 12 Universities partnered With edX. https://www.edx.org/
The Open University's mission is to be open to people, places, methods and ideas. We promote educational opportunity and social justice by providing high-quality university education to all who wish to realize their ambitions and fulfill their potential. [https://twitter.com/OpenUniversity]
United Nations - Tuition Free Online University University of the People (UoPeople) is the world's first non-profit, tuition- free online university dedicated to opening the gates to higher education for all individuals otherwise constrained.
Fritzie -
Apr 28 2013: I think there is a difference, actually, between, on the one hand,the sort of top down instruction from a live teacher lecturer or Kahn academy lecture and, on the other, the teacher as facilitator of inquiry and discussion. I think there is a difference between a model in which students receive information as individuals from someone considered an authority and in which they experiment and learn through discourse in groups.
- +1
I agree that there are common features as well- that people always have done most of their learning outside of the classroom, through pursuing and engaging in their own experiences in the course of life and work. A greater range of resources are now available for outside inquiry, with the addition of technology. This means some people focus on gathering information from others through online sources and less time experimenting than people did in the past. Some people focus more on communication-based forms of learning and on gathering prepared information rather than personal tinkering.
One thing formal education can do for the receptive learner is to provide a range of tools for lifelong learning outside the classroom, tools to add to those a learner would automatically use or be exposed to. This is one of the things that I appreciate most from my formal education. Another is to give students real time access to people who are immensely knowledgeable in the content and ways of learning in their fields, access that includes real interaction, asking and answering questions, and mentoring. That is another thing I found of enormous value in my education.Apr 28 2013: I like Khan's model for the motivated student. One of the issues is however, that not all students are motivated, but all students will be future adults. So, perhaps the idea should be broken into two parts: 1) How do we provide an opportunity for the motivated student to learn more material so as not to be bored? 2) How do we teach all the students the basic things needed to survive as adults in the future?
Motivation is one answer. The motivated student sees opportunities offered to those with a high degree of demonstrated performance and push themselves to achieve and gain access to these opportunities. The unmotivated student realizes that either do not want to compete in the academic world, are not motivated by the opportunities they see in adult life, or just do not want to do the work to learn.
Taking the last group first, I think student performing in the lower 1/3 should be taught to work as part of the learning experience in a positive way. Learning this lesson after the opportunity for education has passed you by is too late, frustrating, and creates a problem for society as unskilled, unmotivated, lazy adults hit the workforce. Somewhere, this sector seems to believe that playing the victim card is that path to survival rather than learning to work. We need to eradicate this notion.
The middle 1/3 doesn't need to be shown how to work, but needs to be shown opportunities that appeal to them and a path forward to achieving them. These opportunities need to be adult life sustaining, meaning that the need to result in careers that keep them out of poverty and sustain food, shelter, clothing, and these days...health care for their families. This might be trades or skills where a college degree is not needed to accomplish the work.
The top 1/3 knows how to work and sees opportunity. We just need to make sure the barriers such as education expenses and external influences do not inhibit them from achieving their dreams.Apr 29 2013: It has to change. Average student spends a lot time on Facebook/Youtube. So educational system (eastern/western) will change automatically. (Give it some more time plz, https://www.coursera.org/ https://www.edx.org/)Apr 29 2013: I think what you describe is common all over the world and not "Western" specific?
I agree that our current teaching model is very old and we need a new way to teach everyone because our society is getting more complex. The ongoing automation will further decrease the number of jobs that require minimum education while jobs with high requirements in IT, sciences, bio technology and similar will continue to increase.
I think we don't realize how much technology changed education (and this is just the beginning). Before the Internet era, students had to rely on their library to find answers and research material. With the Internet everyone has unprecedented access to knowledge on any topic. For example, Wikipedia is helping over a billion people around the world to understand any topic. Collaboration tools and sites allow students and teachers to collaborate. Free online courses and classes allow students with no income to learn material from top Universities like MIT etc.
We are also seeing slow but positive change in how classrooms are organized. For example, some schools are experimenting with Khan Academy and similar systems where students are more active in the learning process and teachers can focus more on individual students.
I think the future is very promising as students will have more access to wide range of online resources.
Apr 29 2013: I agree what is stated above, that the current education system is very outdated. New developments on learning styles and the progress on how we retain information will be changing our education system. I believe as a part of this change, technology will naturally flow into the new systems of education. With that said technology will not be the single key that will change the current education system. Technology will be integrated into the new education systems that develop from our new understandings.
Zdenek Smith
Apr 29 2013: Yes, society and esp. younger generation will also be part of pressure on the education system to change and adapt to new needs and views of its students.
- +1
Hopefully public education will adapt fast enough to provide its students enough skills for 21st century jobs.
Interesting times indeed. cheersApr 29 2013: definitely agreed.
technology is revolutionizing nothing in education.
education, as far as i remember, is supposed to enrich a student's memory, cognition, IQ, EQ, logic, etc. but what is really happening? with computers, now westerners are not even able to do tables of simple maths on their own. they need to Google the weight of earth and are usually confused between 300m/s and 3lac m/s being lights velocity.
i agree technology is important, but it has seriously hampered the student's development in terms of memory and cognition.
what is the use of a method which paralyses the student neck down in terms of thinking and deducing logical solutions? what do we educate students for?
now answering your question Ben:
what do you guys all think? i think technology as a point of reference is fair and fine. but if students are made dependent on technology instead of the best computer they can ever have - their brains - they are seriously nech deep in trouble with respect to their thinking abilities.
Will the education system and classroom model change in the future? it definitely will. up to a point where children will order water bottles sitting on their benches and get homework done using voice recognition programs.
Or will it remain the same? if you look at how exponentially science has developed in the past 100 years, you would agree that the education system remaining same is least probable.
cheers Ben Wang!! :)Apr 28 2013: Why do you want to limit it to the classroom?Apr 28 2013: There have been several good TED talks on this. Time will tell.


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SAR analysis of compounds that inhibit NOD1 - Set 2
The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis. ..more
BioActive Compounds: 17
Related Experiments
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH084844-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego CA
The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis.
Mutations in the NOD1 and NOD2 genes are associated with a number of human inflammatory disorders, including Crohn's disease (CD), Blau syndrome, early-onset sarcoidosis, and atopic diseases, which characteristically cause constitutive NF-kB activation. Chemical inhibitors of NOD1 and NOD2 would provide powerful research tools for elucidating the roles of these proteins in primary cultured cells from humans and in animal models.
The assay described below is a cell-based HTS assay that utilizes NF-kB-mediated luciferase reporter gene activity as a measure of NOD1 modulation. The assay uses a luminescent readout.
This dose response assay is developed and performed to confirm hits originally identified in "uHTS luminescence assay for the identification of compounds that inhibit NOD1" (AID 1578) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
This assay could be multiplexed with a HEK-293-T cytotoxicity assay, AID 2335.
1) Strober W, Murray PJ, Kitani A, Watanabe T. Nat Rev Immunol. 2006 Jan;6(1):9-20. Review. Signalling pathways and molecular interactions of NOD1 and NOD2.
2. da Silva Correia J, Miranda Y, Austin-Brown N, Hsu J, Mathison J, Xiang R, Zhou H, Li Q, Han J, Ulevitch RJ. Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1840-5. Epub 2006 Jan 30. Nod1-dependent control of tumor growth
3. Joosten LA, Heinhuis B, Abdollahi-Roodsaz S, Ferwerda G, Lebourhis L, Philpott DJ, Nahori MA, Popa C, Morre SA, van der Meer JW, Girardin SE, Netea MG, van den Berg WB. Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9017-22. Epub 2008 Jun 23. Differential function of the NACHT-LRR (NLR) members Nod1 and Nod2 in arthritis.
4. Shaw MH, Reimer T, Kim YG, Nunez G. Curr Opin Immunol. 2008 Aug;20(4):377-82. Epub 2008 Jul 2. Review. NOD-like receptors (NLRs): bona fide intracellular microbial sensors.
5.Kim YG, Park JH, Shaw MH, Franchi L, Inohara N, Nunez G. Immunity. 2008 Feb;28(2):246-57. Epub 2008 Feb 7. The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands.
6. Rescigno M, Nieuwenhuis EE. Curr Opin Gastroenterol. 2007 Jan;23(1):21-6. Review. The role of altered microbial signaling via mutant NODs in intestinal inflammation.
7. Rosenstiel P, Hellmig S, Hampe J, Ott S, Till A, Fischbach W, Sahly H, Lucius R, Folsch UR, Philpott D, Schreiber S. Cell Microbiol. 2006 Jul;8(7):1188-98. Influence of polymorphisms in the NOD1/CARD4 and NOD2/CARD15 genes on the clinical outcome of Helicobacter pylori infection.
8. McGovern DP, Hysi P, Ahmad T, van Heel DA, Moffatt MF, Carey A, Cookson WO, Jewell DP. Hum Mol Genet. 2005 May 15;14(10):1245-50. Epub 2005 Mar 24. Association between a complex insertion/deletion polymorphism in NOD1 (CARD4) and susceptibility to inflammatory bowel disease.
9. Opitz B, Puschel A, Schmeck B, Hocke AC, Rosseau S, Hammerschmidt S, Schumann RR, Suttorp N, Hippenstiel S. J Biol Chem. 2004 Aug 27;279(35):36426-32. Epub 2004 Jun 23. Nucleotide-binding oligomerization domain proteins are innate immune receptors for internalized Streptococcus pneumoniae.
10. Le Bourhis L, Benko S, Girardin SE. Biochem Soc Trans. 2007 Dec;35(Pt 6):1479-84. Review. Nod1 and Nod2 in innate immunity and human inflammatory disorders.
11. Maeda S, Hsu LC, Liu H, Bankston LA, Iimura M, Kagnoff MF, Eckmann L, Karin M. Science. 2005 Feb 4;307(5710):734-8. Erratum in: Science. 2005 Apr 29;308(5722):633. Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing
12. Li J, Moran T, Swanson E, Julian C, Harris J, Bonen DK, Hedl M, Nicolae DL, Abraham C, Cho JH. Regulation of IL-8 and IL-1beta expression in Crohn's disease associated NOD2/CARD15 mutations.
13. Hum Mol Genet. 2004 Aug 15;13(16):1715-25. Epub 2004 Jun 15. Brideau C, Gunter B, Pikounis B, Liaw A. J Biomol Screen. 2003 Dec;8(6):634-47 Improved statistical methods for hit selection in high-throughput screening.
Assay materials:
1) HEK-293-T NFKB-Luc cell line obtained from the assay provider's laboratory.
2) g-tri-DAP (Ana Spec cat #60774) obtained from assay provider's laboratory.
3) SteadyGlo (Promega)
Primary Screen and Single-concentration confirmation
Day 1 Procedure
1) Harvest HEK-293-T NFKB-Luc at 100% confluency
2) Dispense 3 uL (6000 cells)/well to every well of a 1536 TC-treated white plate (Corning # 3727).
3) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge.
4) Using a HighRes biosolution pintool equipped with V&P Scientific pins, stamp 10nl of 2mM cmpds in DMSO (col 5-48) and 10nl DMSO controls (col 1-4) to plates
5) Lid Plates. Incubate cells for 1 hour at room temp.
6) Dispense 2 uL/well of g-tri-DAP (1.875 ug/mL) in assay media containing 1.375% DMSO to columns 3-48.
7) Spin down plates for 30 sec in an Eppendorf 5810 centrifuge.
8) Lid Plates. Incubate overnight (16 hours) in 37 oC 5% CO2 incubator
Day 2 Procedure
1) Equibrate plates to room temp for 10 min.
2) Add 3 uL SteadyGlo well with Multidrop
3) Spin plates for 10 sec in a Velocity11 VSpin, shake for 30 sec.
4) Incubate plates for 20 min at toom temp.
5) Read luminescence on Perkin Elmer Viewlux.
NOD1 Dose Response assay
(This assay was multiplexed with a cytotoxicity protocol described in AID pending).
Day 1 Procedure
1) Harvest HEK-293-T NFKB-Luc at 100% confluency at 100% confluency
2) Add 1 uL/well NOD assay media with Multidrop
3) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge.
4) Serial compound dilutions: dispense 50nl 100% DMSO (columns 1-4, 47-48) or compounds (columns 5-46) using with Labcyte Echo 550 into plates from step 2.
5) Add gamma-tri-DAP to cell suspension at 0.75 ug/mL.
6) Seed 13000 cells/well in 4 uL/well to full plate HEK-293-T NFKB-Luc to Corning # 3727 white, 1536, hi-profile, TC-treated plate.
7) Spin down plates @ 500 RPM for 5 min on Eppendorf 5810 centrifuge.
8) Lid Plates. Sandwich 4 plates between 2 lidded 384 plates filled with H2O
9) Wrap plates securely in single layer of Plastic Wrap (Saran Wrap PVDC version).
10) Incubate overnight (14 hours) in 37 oC 5% CO2 incubator
Day 2 Procedure
1) Add 3 ul/well of SteadyGlo solution with Multidrop
2) Shake plates on a plate shaker for 20 min.
3) Spin plates @ 1000 RPM for 1 min using Eppendorf 5810 centrifuge.
4) Read luminescence on Perkin-Elmer Viewlux.
Compounds were tested in 1 or more ranges.
Range1 0-20 uM
Compounds are considered active if the IC50_Mean < 20 uM.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and is not applicable to this assay
2) 2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound's inhibitory potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the NOD1 inhibition assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
From ChEMBL:
Assay Type: Functional
Result Definitions
* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH084844-01
Data Table (Concise)