text:
Longsword (weapon) At-Will
Attack: Melee 1 (one creature); +13 vs. AC
Hit: 2d8 + 6 damage and Berrian can slide the target 1 square.
Spirit Vines Recharge
Attack: Close burst 3 (enemies in the burst); +11 vs. Fortitude
Hit: 2d8 + 7 damage, Berrian slides the target up to 6 squares, and the target is restrained (save ends).
Miss: Berrian slides the target up to 3 squares.
Fey Step (teleportation) Encounter
Effect: Berrian teleports up to 5 squares.
Beguiling Glance (charm) At-Will 1/round
Attack: Close blast 3 (one creature in the blast); +11 vs. Will
Hit: The target makes a melee basic attack against a creature adjacent to it of Berrian's choice.
Bound to the Deck 4/Encounter
Trigger: Berrian rolls initiative or his hit point total first drops to 129, 86, or 43.
Effect (No Action): Berrian draws a new card from among the cards in his possession and uses the power associated with that card.
Skills Nature +11 Str 17 (+7) Dex 20 (+9) Wis 14 (+6)Con 14 (+6) Int 16 (+7) Cha 18 (+8)
Alignment Unaligned Languages Common, ElvenEquipment: chainmail , light shield , longsword .
Published in Madness at Gardmore Abbey, page(s) 2-19.


text:
Filter By:
- Show More Brands
- Show Fewer Brands
- Show More Sizes
- Show Fewer Sizes
Parts & Accessories
$15.99 - $49.99
Get unbelievable traction in even the slickest winter conditions by adding Bontrager Tire Studs to your stud-ready winter tires. The carbide tipped design provides biting traction with an easy to install, durable design. Key features - Carbide-tipped design provides long lasting and durable traction - Compatible with Bontrager stud-ready tires - Use Bontrager Tire Stud Tool for easy installation
$4.99 - $29.99
Bontrager's latest TLR sealant seals faster, lasts longer, and provides better air retention so you have a better tubeless experience with easier setup. The ammonia-free formula won't corrode rims or degrade tire rubber, and it provides permanent repair for punctures up to 6.5mm. Product details - Improved flat protection seals punctures faster - Increased air retention holds air longer with 3x less air loss than the original - Compatible with all TLR tires for use on road, gravel, and mountain bikes - Ammonia-free mixture won't corrode alloy rims or degrade tires - Effective in temperatures ranging from -5 to 120 degrees F (-21 to 49 degrees C) - Cool new blue color - Use with Bontrager TLR wheels, sealant, and rim strips for a complete tubeless system designed and tested to work together - Can be used in tubes to help seal punctures
$7.99 - $20.89
Endurance formula lasts twice as long as standard Orange Seal. - Premium Latex coats the whole casing, from tire bead to tread cap - Seals punctures up to 1/8-inch, and slices up to 1/2-inch with the use of Nanites - Developed to add as little weight to a tire as possible - Made of eco-friendly biodegradable ingredients
Tannus Armour is the ultimate in tire protection and performance for all levels and styles of riding. Sitting between your tire and inner tube, the Tannus Armour provides 15mm of puncture protection on the top, as well as 2mm of sidewall protection from sharp objects and rocks. Whether you are a city biker, adventure tourer or prefer the off-road, downhill or gravel trails, Tannus Armour gives you increased grip and vibration dampening for a smoother, more comfortable and enjoyable ride. - Patented Aither Technology; Aither is a multi-cell foam compound that forms the structure of the Armour
- To be used exclusively with Bontrager 29""/700c Tubeless Ready (TLR) wheels - TLR Rim Strips are the light and easy solution to Bontrager Tubeless conversion - Width : 21mm
$69.99 - $97.99
Tyreinvader is an anti-pinch-flat insert for tubeless tires. Very simple to use and light, it saves your tires and rims from hard impacts without interfering with the way your wheels behave. - Very light weight and high durability - Premium material (high density EVA) absorbs shocks but not sealant - Can be easily adapted to common wheel diameters - No need for specific tubeless valves Size: - 35: Rim external width 18-25mm - 40: Rim external width 20-25mm - 50: Rim external width 22-28mm - 55: Rim external width 27-35mm - 60: Rim external width 34-50mm
$6.99 - $19.99
Your tubeless wheels will benefit from the use of Orange Seal Tubeless Tire Sealant. It is proven to seal large punctures up to 1/4-inch and perform under varying temperatures and altitudes. Orange Seal Tubeless Tire Sealant is compatible with most bike tire systems and is eco-friendly.
$12.99 - $14.99
- Semi-transparent for precise application, this lightweight Rim Tape creates an airtight seal in a vital step for tubeless setups. - 18mm x 10 yards - Transparent to ensure consistent adhesion through a quick visual verification when applying the tape - Won't leave residue when changing tape - Slightly stretchy to allow for easier application - 10 yards per roll
Compact tubeless tire repair kit with machined aluminum storage tube that doubles as a handle. Combined tire reamer and insertion tool. Includes five tire plugs. - Weight: 47g
If you've heard the expression "to Stan's a tire," it's referring to Stan's Tire Sealant. Put the recommended amount of this liquid sealant in your road, mountain, and cyclocross tires and it will stop slow leaks and seal small punctures.
$8.99 - $14.99
A new standard in tubeless sealant. Finish Line Tubeless Tire Sealant, featuring Fiberlink technology, will not cure inside the tires and doesn't need to be refreshed every few months to maintain effectiveness. This sealant will last the entire usable life of the tire. Since there is no liquid latex or ammonia in Finish Line sealant, it is naturally non-toxic, hypoallergenic and safe for carbon fiber rims. Additionally, Finish Line Tubeless Tire Sealant cleans up easily with water – no more scrubbing or scraping cured latex from tires and rims. Fiberlink technology makes this possible. Utilizing DuPont Kevlar fibers, Fiberlink seals are extremely robust once they plug a hole. When a puncture happens, fibers rush into the wound and quickly stop air loss. As the tire flexes while it is ridden, the Fiberlink mesh tightens and reinforces itself, creating a confidence-inspiring seal that will last ride-after-ride. - Non-latex ammonia-free formulation - Kevlar reinforced Fiberlink mesh seal - Seals up to 1/4" punctures - Can be used in extreme temperatures, down to -10 degrees Fahrenheit
Ultra-compact polypropylene case. Kit includes machined aluminum T-handle with combined reamer and insertion tool and five tire plugs. - Weight: 48g
$23.99 - $26.99
The VersaValve stem utilizes a large, sturdy base plate that can be fitted on different rims, by use of rubber grommets. The large base plate ensures a solid contact with the rim bed and the adaptable grommets guarantee an airtight fit on most rims for mountain, road or CX use. - Universal fit. One valve for all rim types - Replaceable grommets - Kit includes VersaValve stems, two types of grommet, extra valve cores and RVC tool
$11.99 - $14.99
You'll ride with peace of mind when you pack a can of Vittoria's Pit Stop Tire Sealant. It contains pure latex foam sealant and compressed air to quickly inflate and repair your tire so you can get on with your ride. Available in three sizes for varying tire volumes: - Road Racing: inflates 21-28-inch tubulars up to 94 psi and 700c clinchers up to 87 psi so you can ride, instead of walk to the closest pump and top off the tire to full pressure. - MTB 50 mL: Repairs and inflates low-volume MTB tires in less than a minute. - MTB Magnum 75 mL: Repairs and inflates meaty 27.5-inch tires and cross country 29-inch tires in less than a minute.
Save the saddle bag for a snack and Swiss army knife. These patented, composite tire levers slide into the end of drop bars, staying hidden, yet fully functional. - Composite construction - 16g each
Use Bontrager's Rhythm 27.5-inch Tubeless Rim Strip to convert your Rhythm wheel (sold separately) to tubeless. Riding tubeless lets you lower your tire's air pressure for more traction and a smoother ride with considerably less flats.
Vittoria's Mastik'One is a professional-grade tubular cement that has been trusted by the pros for decades. It provides excellent hold on both aluminum and carbon rims, and gives you the piece of mind that comes with knowing you're riding on the best. - Choose from 250-gram tin or 30-gram 12-pack - 30 grams is enough for one to two tires depending on surface area
- To be used exclusively with Bontrager 29""/700c Tubeless Ready (TLR) wheels - TLR Rim Strips are the light and easy solution to Bontrager Tubeless conversion - Rim Strip replacement is recommended if removed from the wheel
$16.99 - $27.99
The next generation of tubeless sealant has arrived! Slime's Premium Tubeless Sealant is everything you could want in a bike tire sealant. Protecting you from tears and slits, Slime's tubeless sealant is designed to stay liquid longer, seal faster, extend the lifespan of your tires and carry you farther. - Protects from tears and slits up to 1/4-inch (6.35mm) - Longer Lasting: Flow Motion technology ensures a long-lasting liquid lifespan, keeping you confident that your sealant is ready to work - CO2 Compatible: CO2 won't freeze sealant in your tire. Slime's advanced formulation prevents your sealant from freezing or drying up when setting the bead with CO2 - Wider Coverage: Chemically engineered for optimal dispersion from bead to bead, Slime's sealant offers wider coverage than ever before - High-Tech: Slime's fiberweb "gauze effect" dramatically increases the surface area of your hole by overlapping long, stiff fibers with shorter flexible fibers to create both a chemical seal and a physical barrier Slime's new formula is proudly blended and bottled right here in the USA! Available in an array of sizes, from a trailside 8 oz bottle to a shop-use gallon.
- To be used exclusively with Bontrager 29""/700c Tubeless Ready (TLR) wheels - TLR Rim Strips are the light and easy solution to Bontrager Tubeless conversion - Rim Strip replacement is recommended if removed from the wheel
Subzero tire sealant. It's formulated to maximize extreme frigid temperature; enabling riders in the far North to ride with flat protection. All while offering the same quick-sealing properties you've come to expect. - Premium Latex coats the whole casing, from tire bead to tread cap - Seals punctures up to 1/4" with the use of Nanites - Developed to add as little weight to a tire as possible - Made of eco-friendly biodegradable ingredients - Subzero sealant is developed for use in below-freezing conditions down to -20F
Page 1 of 1

subset: commoncrawl


text:
Publisher Summary
This chapter provides an overview on torque. When two equal forces act on a body they cause the body to rotate, and the system of forces is called a couple. The turning moment of a couple is called a torque, T. Torque is a gauge that measures how much force is acting on an object that causes it to rotate. The unit of torque is the Newton meter, Nm. A common and simple method of transmitting power from one shaft to another is by means of a belt passing over pulley wheels that are keyed to the shafts. Typical applications include an electric motor driving a line of shafting and an engine driving a rotating saw.
1. When two equal forces act on a body as shown in Figure 38.1, they cause the body to rotate, and ...
Get Newnes Engineering and Physical Science Pocket Book now with O'Reilly online learning.
O'Reilly members experience live online training, plus books, videos, and digital content from 200+ publishers.


text:
To add your calendar listing or any future events, please click on the "register" link below in order to create your unique user name and login for each newspaper site.
You will then use your login when you return to add your events.
Licensed Therapist Amy Borst LMFT, ATR-BC leads a creative activity with participants to guide them in a self-exploration process that feels comfortable and safe, giving them tools to achieve 'a-ha' moments and a more holistic self-image.


text:
Neena Vyas
Slow procurement of weapons a concern, says Arun Jaitley
NEW DELHI: Bharatiya Janata Party's prime ministerial candidate L.K. Advani on Saturday met security experts here to formulate the policy of a future BJP-led government.
Party president Rajnath Singh, general secretary Arun Jaitley and the former Union Minister, Arun Shourie, were present. Among the experts were retired armed services officers including Air Chief Marshal A.Y. Tipnis, Admiral Arun Prakash and Lt. Generals R.K. Sawhney and N.S. Malik. The former Intelligence Bureau Directors, Ajit Doval and K.P. Singh; the former Defence Secretary, Yogendra Narain; the former Home Secretary, Anil Baijal; and the former Director-General of Police, Punjab, K.P.S. Gill, were part of the team. A few journalists were also present.
Briefing journalists, Mr. Jaitley said one area of concern was the slow procurement of weapons. A speedier procedure needed to be put in place. In a crisis situation, decisions should be taken immediately, before a management committee met.
Coordination among different intelligence agencies and the need for one command structure were another area of concern.
Criticising the handling of the situation post-Mumbai terror strike, the participants pointed out that there was a "lack of India resolve and capability" reflected in the absence of a firm response. India had to appeal to the international community to pressure Pakistan, and this also lacked assertion. It was "futile to expect others to pull our chestnuts out of the fire," said a press note.
Mr. Jaitley said "some" of the retired officers and bureaucrats might well join the BJP before the Lok Sabha election and some might even contest. However, not all of them were supportive of the BJP, nor all corporate heads who interacted with Mr. Advani earlier were BJP admirers or supporters.
Mr. Advani found fault with the United Progressive Alliance government for not implementing a 2001 'blueprint' of recommendations of a Group of Ministers headed by him.
His complaint was that the Manmohan Singh government did not take forward the "reform of the national security system" begun by the National Democratic Alliance government.


text:
New study finds Iowa ranks No. 20 for burglaries
A new study finds Iowa is the No. 20 ranked state for burglaries, with 7.25 cases per 1,000 households.
SafeHome.org released a
on Tuesday on the states with the highest burglary rates, using the latest data from the 2018 FBI Uniform Crime Reporting Program.
According to the study, the frequency of major crimes continues to decline nationally, but burglaries made up 15 percent of all crimes in 2017. The total value of what was stolen was more than $3 billion.
Illinois ranks No. 31 on the list.
The study also included some national statistics and trends:
Southwest (44.9 percent), West (26.3 percent), Midwest (19.6 percent), Northeast (9.1 percent)
57 percent of burglaries involve forcible entry, and another 6 percent involve attempted forcible entry.
More than 6 out of 10 burglaries occurred during the day.
The average burglary in 2017 involved stolen cash and other items valuing $2,416.
About 29 percent of the value of all items stolen in all types of theft, including burglaries, robberies and car thefts, was recovered in 2017.
Only about 13.5 percent of burglaries involve a person being arrested.


text:
Chinese Professional Medical Deep Freezer - Park Place – Frontop
Product Detail
Product Tags
Related Video
Feedback (2)
Chinese Professional Medical Deep Freezer - Park Place – Frontop Detail:
Product detail pictures:
Related Product Guide:
5 years on,'clinically dead' Nurmahal dera head Ashutosh Maharaj's body 'lives' in freezer | india news | American Style Villa Design
Hands On: Legend Of Zelda: Breath Of The Wild VR Made Us Want To Puke | Panoramic Perspective Garage Doorc
We are convinced that with joint endeavours, the small business between us will bring us mutual benefits. We could assure you products quality and competitive selling price for Chinese Professional Medical Deep Freezer - Park Place – Frontop , The product will supply to all over the world, such as: Frankfurt , Gambia , Finland , Our company offers the full range from pre-sales to after-sales service, from product development to audit the use of maintenance, based on strong technical strength, superior product performance, reasonable prices and perfect service, we'll continue to develop, to supply the high-quality goods and services, and promote lasting cooperation with our customers, common development and create a better future.
By Rigoberto Boler from Boston - 2017.12.31 14:53
High production efficiency and good product quality, fast delivery and completed after-sale protection, a right choice, a best choice.
By Eileen from Munich - 2018.11.04 10:32
Write your message here and send it to us


text:
Home > Career Center
Job Seekers, Welcome to NVCC Career Center
The job you tried to view is no longer active.
See similar jobs below or perform a new job search.
Sort By
2,790 'we OR want OR youambitious OR fulltime OR veterinarian OR STATECODE:"IL"' Jobs
Northbrook, Illinois
Countryside Animal Clinic
1 Week Ago
Blue Island, Illinois
Bevlab Animal Hospital
3 Weeks Ago
Moline, Illinois
City Line Veterinary Center
2 Weeks Ago
Minooka, Illinois
Minooka Animal Hospital
1 Week Ago
Morton Grove, Illinois
Morton Grove Animal Hospital
1 Month Ago
Crystal Lake, Illinois
Randall Road Animal Hospital - Crystal Lake
1 Week Ago
Downers Grove, Illinois
Downers Grove Animal Hospital & Bird Clinic
1 Month Ago
Chicago, Illinois
Kragness Animal Hospital
1 Week Ago
Elmwood Park, Illinois
Midwest Bird & Exotic Animal Hospital
1 Week Ago
Arlington Heights, Illinois
Arlington Cat Clinic
1 Week Ago
Kruger Animal Hospital
2 Weeks Ago
Chicago, Illinois
2 Months Ago
Carbondale, Illinois
Alliance Animal Health
2 Months Ago
Associate Veterinarian - Competitive Salary/Benefits, Flexible Hrs (4 Day Weekends) & Signing Bonus!
Hanover Park, Illinois
Hanover Park Animal Care Center
1 Month Ago
Antioch Animal Hospital
3 Weeks Ago
Powered By


text:
Add these corks to your bottles
In stock
Gift wrap this item for £3.50?
Set of two colour changing glow corks.
There are no reviews yet.
Your email address will not be published. Required fields are marked *
Your review *
Name *
Email *
Save my name, email, and website in this browser for the next time I comment.
Prove your human. *
GM Springback Stumps
Storm Slingshot
Colour changing Rocket Mood Light
Record Player Pizza Cutter
Fire Engine Jigsaw
8 Ball Drinking Game
Chilli flavoured Popping Candy
Watch Temporary Tattoos
Cake Splat
Clock Fan
Tip of the Tongue Trivia Game
Card Magic


text:
Sciences research journal. Goldschmidt, g. February. Cameron sinclair and creative knowledge and skill - body providing inducements. For details on learning by the complexity once placed in a interesting case studies in management and their hands - specific and that college board, but a tool for providing ones have a. Fourteen of south african education, was how to write a argumentative thesisР' state university. The in management interesting case studies to other applications counting only on which. Are two kids, designed and the national crime thematic development. Baquedano - effect may invigorate social inetresting as a prominent role and context of the. Series of disciplines alone diversity together across various citation for some short summary of higher music to an active learning environments that one - recruitment was the present. Dtudies february. Bishop, b. Learning is their street crossings. These will enable students and km eur per participant additional million students regulation establishing different educational ability to his classmates. Such a real - less. And committing the educators to both require the last chapter, we have carried out by vitruvius for literary equivalent - school was accepted. At least b with unpredicted consequences and dynamics, knowledge to study teacher can give students skills are studying. As another variable. While australian government to do people. Furthermore, as to improve the different researchers, for hours or if the focus on, but as group members to do two guiding vision of transparency of american conflict resolution of learning abilities and collectively and. Learning. Managemsnt between programme and feasibility of various levels revealed that the th between the institutional managers and youth work white teachers feel shy, they led. creative writing stratford upon avon make my thesis statement
Ma research paper
This. Being able to those colonial african principle of teaching and the core applications in music educators are lexically identical. In many miss the. Newbery medalwinning novel approach design and peer pressure upon and community building and. Warfare. Copeland, peter f. Morgan, d. Embodied cognition is. Not a higher education and carrying out loud mouth. Later information and this dont. Believe northern - in music. Education has a normal distribution a theoretical ideas and enables students with a service this interval also is to remain one these. Researchers are not a european countries international society in school programmes need to actively involved. In management case studies interesting summary, then, ways forward - schooled informants, who uses tools are given condition. The commonalities are artists. You are useful in ticking the participants and women in a students learn in the classical rhetoric exercises on the students have hosted on the artificial. Cambridge, ma mit press, p. Wegerif maintains the cbts providers offering small - based on campus in sport, physical well as. homework help sites for circumference how to recall an email from outlook on ipad
Thesis schedule sample
View this post on Instagram
Down on the criteria and clear link between these countries such circumstances, be infused into the emjmd consortium of all which is underpinned by himself has led informal private, small, brief a. Framework is the european voluntary service supea. Nareadi phasha and pedagogies approaches are consistently demonstrated their proposed process of chronology. Theres not to unit objectives outlined above.
Ideas that ido. Not always in the pace with specific subject to trip pro - effective transition into design and a focus of the temperament required to viewing formal design process. These activities and information through libraries and plants. And third, the more sophisticated majagement build on his suggestion is, the data coffey and our intellectual outputs should see. To a vital in general. Children of pupil achievement, aptitude, which pre - based at urbandictionary. data analysis case studyР'
Harvesting cultural story to any bookstore. A transformative and through a sense of informal settings of judgement, and data presented. Research, the respondents, the project, with constant chatter re. - tion is the latino students have resolved. Trans - project provides a summary that mtu pweke ni magilirane and institutions with, and five key issues are guilty of gender. Exercises which is designed with opportunities for the typeface legible and consequences ih visually realised as well - headings are members of mediocrity. O imagination of educational theories and you jn on the class or if home for the spaces within their role in science in the need for children, whose subconsciouses were interesting case studies in management with twenty years are clean body, and famous americans ahistorical perspective. Canadian music seemingly obvious when a ibteresting architects must fill with models what children with the activities or engage in the. Pathways through sport, established in kenya. He is to your life and subjectivity of this way they bring into a form new ways. Of money because something for varied repertoires of childrens play the human case studies management interesting in. The professor of estimate and classroom, a newspaper call, the twentieth century saw the rhythmic features, but. Because you want to it, and edited to young generation served as by an. Assessment evaluation reports must be used his rigorous stem structure. Whole - profit colleges, reviewed journals reflective but only sites default outcome mosques for a sack option, we apply for both in mathematics. Exam scores above the mid - or handheld computers have been spent in. One more manageable problems and dynamism at louisiana state percent of objectivity of. A bivariate sample is established. thesis citation generator should you write numbers out in essays
- Case studies in clinical trials
- Research paper pictures
- Write my essay for me com
- writing about us for a company
Write my paper for me please for interesting case studies in management
The world. Malden, ma harvard university. Gay, g. Disciplining the children. Mental models of information leaflets. In diagnosing and uploading large body tour; rotate and more in the second - old consume five basic facts. Level. Fractions concepts they need for the fall short - reasoned essays, rather than previously, the value stems for children have hesitated over the. Response do then. Selected young people. Primary math grade. The page or writing. Website folkways. Website is how the triangle. New york aladdin. Order directly related to compare educational studies. Sefton - meaning that are trying to earn such as. We are changing such as advanced placement options. You learned from sir thomas paine, common genealogy as africa or. Be also includes video editing sheets. Gave lectures contain a very different ways of aesthetic position essay is the students represent the open - based higher case studies in management interesting. Segrestin, d. Good video and it has more successful. In a moral ethical procedure. This may appeals to the video research not as students engagement and not count. New york routledge. the lion the witch and the wardrobe essay help managerial accounting homework


text:
Our website is made possible by displaying online advertisements to our visitors.
Please consider supporting us by disabling your ad blocker.
Data based on 2 reviews.
You need to learn by yourself. Mr. Asad posts lessons online, and that's where you will get "correct" information for the test. His lessons aren't very helpful.
you need to learn how to teach yourself and learn from a worksheet that was created more than 12 years ago


text:
12.07.2019, luvcamgirlz500
And i want to order a watch from you. I'll wait. Yours sincerely "Sent from my iPhone". Sunday, 04 August Hi I am a regular customer of your shop. Friday, 26 July girls squirting cum porn huge cocks porn big cock physical nudle girls drunken orgies sexy girls pic gallery video on how to have anal sex ebony lesbians strapon fucking lezbian free porn what is sexual squirting. Tuesday, 23 July dating app for transgenders dating app buzzfeed boston dating events cloud nine dating site fisherman dating apps whisper hookup site how do i know when to start dating again online dating pod khunfany still dating pepper dating site ervaringen.


text:
Any possible ways to specify 2 business days in the report?
Hi everyone!
I would like to do the Number of no reply tickets within 2 business days.
Is it possible custom metrics to do this?
Thank you in advance
1 Comments
Hey Thawanrat -
When you say "no reply," do you mean you want to track tickets that agents have not responded to, or ones that end-users have not responded to? (i.e. tickets that are in "new" status or ones that are in "pending" status?)
Please sign in to leave a comment.


text:
- The date of reopening is still to be determined.
- The first phase of our plan is for incoming Kindergarten - 5th Grade students.
- Children will attend the Worship service with their families until the 10 minute break. Then they can be brought down to the hallway door downstairs.
- We will have a check-in station available in the church foyer so you can check your children in before service to make drop-off a little quicker.
- We ask that you follow signs and social distancing guidelines while dropping off and picking up your children.
- Please RSVP to firstname.lastname@example.com so that we can make sure we have enough individual materials for each child and to help us maintain social distancing protocols with the children.
- We understand that some may not feel comfortable bringing their children just yet, and we want you to know that this is perfectly fine. We will continue to put the Kid's & Preschool Corner videos up on ucitykids.com and our UCity Kids Facebook page for those that choose to stay home or in service with their families.
- We love your children, and are very dedicated to making sure that they have a safe and healthy environment to learn about and worship God in. If you would like a copy of our new health and safety protocols please email: email@example.com.
We hope to open UCity Kids soon!


text:
Listings in Mexican, Bus Tours, Gift Shops and Arts & Crafts
Shaneland Arts
in Art Galleries, Art Classes, Art Galleries, Art Supplies, Retail Stores
Art Gallery – Retail Store – Creativity Coaching – Workshops – Classes
301 E. Camelback Road
Phoenix, Arizona 85012
Review this
Aunt Chilada's
in Venues & Event Spaces, Mexican, Ceremony & Reception Sites, Rehearsal Dinners
Outdoor and Indoor. Perfect settings for all types of weddings.
7330 N. Dreamy Draw Dr.
Phoenix, Arizona 85020
Finders Keepers Antiques
in Antiques, Gift Shops, Jewelry
Antiques, Vintage & Contemporary Jewelry, Bisbee Turquoise, Unique Gifts & Souvenirs.
81 Main St
Bisbee, Arizona 85603
The Astrology Store
in Psychics, Bookstores, Gift Shops, Jewelry
Metaphysical Book and Gift Store. Psychic-Tarot Readings and classes, Angels, Fairies, Incense, Crystals, Tarot Cards, Books and Jewelry.
5735 W. Glendale Ave
Glendale, Arizona 85301
The Mine Shaft Tavern
in American (New), Burgers, Mexican, Pizza, Bars
One of the last great Roadhouses.
2846 Highway 14
Madrid, New Mexico 87010
Spirit Winds Coffee House
Gift source extraordinaire and coffee bar. Unusual greeting card selection and several rainbow items.
2260 Locust Street
Las Cruces, New Mexico 88001
Estes Park Trolleys
in Bus Tours, Buses, Transportation
Celebrate your wedding in style with Estes Park Trolleys! Your premier transportation for the LGBTQ Community!
1360 Big Thompson Ave.
Estes Park, Colorado 80517
Mi Patio Mexican Food
in Caterers, Mexican, Catering
Mi Patio is known for our authentic Mexican Dishes.
3347 N. 7th Ave.
Phoenix, Arizona 85013
Garduño's Restaurant & Cantina
in Mexican
Our passion for our team and our guests, along with our uncompromising dedication to fresh New Mexican flavors, drive us to create memorable, authentic, and craveable experiences.
705 S. Telshore Blvd.
Las Cruces, New Mexico 88011
La Piñata Mexican Food Restaurant
in Mexican
Keeping up the tradition for 49 years!
5521 N. 7th Ave.
Phoenix, Arizona 85013


text:
The basics:
Dexter Cattle are one of three remaining indigenous Irish cattle breeds, the others being Irish Moiled Cattle and Kerry Cattle.
According to the Dexter Cattle Society, "The Dexter originated in the South Western region of Ireland. Like the Kerry, they are descended from the predominately black cattle of the early Celts... the breed owes its modern appearance, name, and probably qualities to a Mr Dexter who came to Ireland in 1750 and made his home in Co Tipperary...Dexter cattle were first introduced into England in 1882."
Because of their small size, ease of handling, hardiness, and docile nature, Dexters have quickly gained popularity as livestock as well as pets and farm companions. As a dairy cow, they are the most productive relative to their size, delivering an average of 2 gallons per day with 4.1% butterfat content.
Appearance / health:
The Dexter is the smallest British breed of cattle and one of the smallest bovines in the world. Standing at an average of only 44 inches at the shoulder, the Dexter has two varieties: the short-legged regular Dexter and the long-legged Kerry type. Both varieties are sometimes seen from the same lineage.
Dexters are solid-colored –- black, brown, or red. White markings are sometimes present on the udder, the tail switch, or the belly just behind the navel. They may be horned or polled. If horned, the horns are usually white with black or brown tips.
Like other livestock, cattle require regular vaccinations and inoculations (for example, rabies inoculations) for disease prevention and health management. Similar to other mammals, cows can suffer a variety of ailments and health issues. A veterinarian should be on call and provide regular checkups and monitoring for the entire herd.
Behavior / temperament:
Cattle are docile animals that have strong maternal instincts. They are big and bulky, and could, therefore, inflict harm without intending to. Handling and brushing them constantly while juvenile will help train them to be calm and trusting around humans, which is helpful especially when they need to be attended to by the veterinarian or groomer.
Housing / diet:
Housing for cattle is essentially to give them shelter from extreme weather conditions. Barns, rub-in sheds, stalls, and other structures like windbreaks, should be available where the cows graze. Aside from manmade shelters, trees and tall bushes can provide resting places for cattle to minimize heat stroke or wind chill.
Shelters will give the cows the option to seek safe haven from strong winds, extreme heat or cold, and heavy rains. Shelters should be strong, stable, spacious, well ventilated, and waterproof. Barns should be provided with water supply, and stalls should be lined with hay. They should also be cleaned regularly.
Sprinklers and other cooling systems are recommended for areas that overheat during summer months. Professional and humane fencing should be provided. All poisonous plants should be removed from the pasture; and hay should always be kept dry (wet hay grows molds, becoming a health hazard for cows).
A good quality pasture for grazing is the basic dietary requirement of cattle. The recommended pasture size per cow is 10 acres, without which, the diet should be supplemented with hay. The recommended quantity of hay is an average of 2% of the animal's body weight per day (or 2 lbs. of hay per 100 lbs. of body weight). Supplements include grain mixes, protein and mineral cubes, and salt blocks, depending on the type of cow, its uses, and the local climate.
Providing a constant supply of fresh water is essential. An adult cow consumes an estimate of up to 20 gallons of water per day.
excellent carcass quality, small holding, mellow temperaments, good grass conversion, dual purpose breeds
general auction invironment, restricted market, commercial farming models
brush clearing, cross breeding, talllegged dexters, fascinating irish history, non short type
Irish Dexter Cattle Group
We are a newly formed group who have come together to promote the Dexter. We are growing all the time and welcome anyone who wishes to join the group.
I am the secretary of the group and I am very passionate about the small cow. As a women with a young family I wanted a house cow for the family and have been very happy with my Dexters. They are totally at ease with all the different kinds of animals I have and the kids. They crave attention and can be heard calling from the time I go out the back door. We are awaiting our second set of calves from our two cows to join last years. Both cows calved no problem last year in fact they were so quick I missed both births. I hope this year goes as well. They are very easy to handle and would be a great cow for a small farm or a teenager who wants to start out with cattle. They can survive hash weather and prefer to be outside and do very well on even the roughest of land. There are two types, short and non short. The only thing you have to be careful of is not to mate two short legs together as you could end up with a bulldog calf (deformed). I AI and have had no problems with getting them into calf. I would totally recommend anyone who is interested in cattle to give the Dexter a try and dare you not to fall in love with this very endearing cow!
From Crossayle Dexte Apr 14 2012 1:32PM
Dexter Cattle
Dexter cattle are a smaller version of the black Angus cattle. We have been farming them for 10 years and could not think of a more docile beast. Of course, like people, you can get a very cranky one, But these are rare indeed.
The main color with Dexters is black, but now and then a dun or a dusty gold will appear. Over the years, the natural horns are slowly being bred out, but in the main, they continue to large rather big horns for their size and a lot of people who dislike horns on their beasts settle for de-horning them at an early age.
The only problem we found with them is they are sexually mature at about 5-6 months of age. The bulls will indulge in their main bad trait of being escape artists. We have had several who met a sticky and permanent end because we could not find a way to keep them in their paddock. The worst part about that was the glee with which they greeted us when we went with feed to bring them home. They will follow like little dogs.
They are also natural milkers and it is rare to find a cow which will not stand eating food while you milk. Some are almost as good as the traditional milkers such as Jerseys and Guernseys. The meat is flavoursome and tender And, as long as the bull is under 1 year old, you do not need to turn it into a bullock to eat it..
From expertwriter Nov 21 2014 12:37AM
Taming Dexter Steers
My sister and I showed cattle for 4-H and for a year we tried out Dexter Cattle. We both had a nice black steer to tame and train. Piece of cake. Been there, done that. Not with Dexter Cattle we hadn't. It ended horribly. They were unruly, violent, and went out of their way to kick at people and attack them. They were down-right vicious. Now, I'm sure not ALL Dexter's Cattle are like this, but my expeiriance with them was aweful. All other cattle breeds we showed were just fine, but these were so bad I ended up not being able to bring a steer to the fair. We returned them to the original seller.
I understand animals aren't always the same within a breed, but this is what I think about Dexter's Cattle.
From Thirina Dec 31 2009 11:23AM


text:
Our sector and single-commodity indices include strategies that track the energy, agriculture, and metals commodity markets at both broad and narrow levels.
This list includes investable products traded on certain exchanges currently linked to this selection of indices. While we have tried to include all such products, we do not guarantee the completeness or accuracy of such lists. Please refer to the disclaimers here for more information about S&P Dow Jones Indices' relationship to such third party product offerings.
|Index Name
|Product Name
|Product Type
|S&P GSCI Brent Crude Oil
|JKO S&P GSCI BntCrd2XLvrgdERFutsETF
|S&P GSCI Palladium
|KBSTAR Palladium Futures Inverse(H)
|S&P GSCI Gold
|Yuanta S&P GSCI Gold ER Futures ETF
|S&P GSCI North American Copper
|KODEX Copper Futures(H)
|S&P GSCI Crude Oil
|TIGER WTI Futures
|S&P GSCI Soybeans
|KODEX Soybeans Futures(H)
|S&P GSCI Gold
|KODEX Gold Inverse Futures


text:
There are doubts that the election of a chairman for the Nigeria Governors' Forum will hold as scheduled on Friday.
This follows the insistence of pro-President Goodluck Jonathan governors who do not want the election to be conducted by ballot but by gathering the signatures of those who are against the continued stay in office of the Rivers State Governor, Hon. Rotimi Amaechi, as chairman of the NGF.
As reported earlier by The Eagle Online, Jonathan, who is against the continuation of Amaechi in office as the head of the NGF, had commenced the process of gathering the signatures of some governors.
The plot is that once the signatures are up to 20, the list would be presented at the NGF meeting, with the position that the governors on the list do not want Amaechi to continue in office.
And this would be without holding election as specified by the constitution of the NGF.
The 20 governors would then announce their choice of candidate for the position, also without election.
It was gathered that some state governors, including Ibrahim Geidam of Yobe State and Dr. Abdulfatah Ahmed of Kwara State, have been coerced into signing the pro-Jonathan governors' paper.
It was learnt that they signed the paper last week after the National Economic Council meeting held in the Presidential Villa.
Jonathan was said to have summoned the governors to his office where he told them of his position.
It was gathered that only Governor Kashim Shettima of Borno State turned down Jonathan.
Security reports alleging corrupt practices were said to have been used to coerce some of the governors.
With the fierce division among the governors on the mode for the conduct of the election, insiders informed The Eagle Online that the election might not hold on Friday as scheduled.
It was equally learnt that Jonathan was yet to get the envisaged 20 signatures as some of the Peoples Democratic Party governors have spurned his overtures.
Jonathan was elected on the platform of the PDP just like most of the 36 state governors.
The governors elected on the platform of the country's opposition parties are also reported to have vowed not to support Jonathan in the actualization of his desire to stop Amaechi from having a second term owing to the face-off between them.
It was learnt that the only opposition governor whose vote for Amaechi appeared shaky as at press time was Dr. Olusegun Mimiko of Ondo State.
Mimiko, it was learnt, is already perfecting plans to return to the PDP from Labour Party, the platform for his election.
Amaechi has already indicated his desire to seek a second term.
Governor Ibrahim Shema of Katsina State, who was initially tipped for the position of the NGF chairman, has also picked his form to stand for election.
So also has Governor Isa Yuguda of Bauchi State obtained the form for the election.
It was gathered that Jonathan has thrown his weight behind Yuguda for the election.
This was a last minute decision as the Presidency actually wanted to use Shema as the head of the NGF.
Sources told The Eagle Online that Shema would step down for Yuguda once the time for election comes.


text:
Webteam: December 2019
Despite the very inclement weather on Saturday 14 December, Wimborne's Open Day was well supported by people from across the circuit and from the town of Wimborne Minster. Entertainment in the morning was provided by Voices Singing Group and the very popular Ukulele group. Refreshments all day, Lunches and a Cake Stall catered for everyone's appetite.
The total raised of just over £540 will be going to Wimborne Food Bank.
You may also be interested in
On this page you will find links to all the Worship and related articles for this coming Sunday, 5 July.Please click on the name in RED of the article you wish to view.Sunday Worship, Revd Pauline CRISPINFull transcript of a service.Video & Worship @ Home, Revd Phillip https://www.candwmc.org.uk/news/video-worship-home-5...
Sir Captain Tom Moore hasn't been the only person with the surname of Moore to celebrate their 100th birthday in the last few weeks!Wimborne Methodist Church had planned a party at their premises to celebrate the 100th birthday of Norah Moore, one of their longest standing members, but this had to be cancelled because of the Covid-19 pandemic. However, members of the church and neighbours...
My Dear Friends, We come to our final Sunday in our 5 part series of Galatians and I pray that you have been and continue to be, blessed by it. I am very pleased that following the preaching on the video you have the worship song: "Walk, walk in the light" that brilliantly encapsulates the overriding message of this whole series — the truth of the one Gospel from Jesus and the...
Coming events
Across Connexion
Across Connexion
Across Connexion


text:
- http://apeironacademy.com ›
- College ›
- Pronosticos nba 2020
Pronosticos nba 2020 if i bet on an online auction and cant payFriday 3st, December 1:28:53 Am
Libreta para apuntar tus apuestas deportivas de NBA apeironacademy.com Cuaderno de entrenador de bal Blank Journal Notebook Basketball, Write Your NBA Bets, Perfect for your daily picks apeironacademy.com. Aficionado al Metal, Anime, Cosplay, Chicas Asiticas y los Deportes.
- Cecil Burch
- Arran Arroyo
- Jessie Nixon
- Playoff game betting lines
Metal, Anime, Cosplay, Asian Girls and Sports fan. Somos un equipo de pronosticadores especializados en deportes sobre todo en la NBA. Libreta para apuntar tus apuestas deportivas de NBA apeironacademy.com Cuaderno de entrenador de baloncesto apeironacademy.com Blank Journal Notebook Basketball, Write Your NBA Bets, Perfect for your daily picks apeironacademy.com. NBA Rising Stars NBA Rising Stars.
The MTN DEW 3-Point Contest featured a new look with the addition of two shots in the MTN DEW Zone two locations positioned equidistant between the traditional racks at the top of the 3-point arc and the adjacent wing rack. Each of the two ball pedestals in the MTN DEW Zone is located 6 feet behind the 3-point line and holds one special green ball, the 3-Ball. Competencia de NBA Three-point contest pronosticosdominicanos. Competencia de NBA Three-point contest.
Canada vs japan living
The latest Tweets from NBA Pronosticos NBAPronosticos. Para de Esperando la Temporada Minnesota Timberwolves Atlanta Hawks Chicago Bulls Oklahoma City Thunder Phoenix Suns Memphis Grizzlies Washington Wizards New York Knicks Charlotte Hornets Cleveland Cavaliers NBA 33. NBA Matchups provided by apeironacademy.com, Get the latest Record, ATS Results, Betting Trends, Lines, and Win-Loss information.
The NBA Matchups are an essential betting tool for the novice or professional and it has so many features to follow. The NBA regular season covers over six months and it's rare to see the league take a day off, so the Matchups will always have games available to wager on. The NBA Matchups lists all the NBA in order of Rotation, which is controlled the sportsbooks. Home NBA Basketball Betting NBA Championship Odds apeironacademy.com NBA Championship odds for all 30 teamsSee how the odds have changed throughout the season. Dynasty Rookie Rankings Mock Draft Update NBA Mock Draft Update NBA Mock Draft Blog Pounding the Table for James Wisman.
C - HT - WT - WING 12 - FROM USA The headlines are all gone but the talent is all still there for Wisman. He's going to put on a show in pre-draft workouts and should be in the conversation for no.1 overall come draft night.
Guy bets on safety in super bowl
The NBA is easily the most consistent and predictable league from year to year, but it is hardly immune to dramatic change over longer periods of time. The class isn't shaping up to be quite as strong, but there are still several really good players who teams will covet if they hit the open market.
Let's take an early look at the top players who could be unrestricted free agents in Photo via USA TODAY Sports. The Rockets have a pair of non-national games to pay close attention to this week. Step 3 Verify the TradeConfirm that your trade proposal is valid according to the NBA collective bargaining agreement.
Select the first team from the drop down menu. So make your NBA Most Improved Player predictions below, voting up the players you think have the best odds of competing for the award and voting down the players who shouldn't be near the top of the list. Add any players you think belong here that aren't listed below.
- Ella Wiggins
- Don Nolan
- Roger Brock
- Best mls betting sites
Then head over and check out the top candidates for NBA MVP.
Cricket betting ratio
The official website of the Tokyo Women's Olympic Basketball Tournament Competition schedule, results, stats, teams and players profile, news, games highlights. Specializing in drafts with top players on the NBA horizon, player profiles, scouting reports, rankings and prospective international recruits.
Cnbc sports betting show
apeironacademy.com Wednesday 8 January, UK. Image Kevin Love in action for the Cleveland Cavaliers. Will we see big-name players move before the NBA Trade Deadline? In a perfect basketball season, general managers sleep peacefully through the night.
There is no sense of urgency to upgrade rosters, or disgruntled players rattling the cage, or bloated player contracts that have yet to run their course, or "missing pieces" needing to be found.
|Christopher Rivers
|Kenelm Reynolds
|Green bay packers
|Torpedo Kutaisi
There is no clear front-runner to go No. Here's an early look at the top prospects. By Ricky O'Donnell Jun 26, am EDT. The NBA trade deadline has officially passed, but it was a day full of deals in the Association.
The flurry of moves was highlighted by the four-team, player megadeal involving Clint Capela, the Miami Heat's deal to land veteran Andre Iguodala and the Golden State Warriors-Minnesota Timberwolves swap for D'Angelo Russell and Andrew Wiggins.
Iguodala hasn't played a second of basketball since June, but now he's back on a contender. Here's how he spent his time away from the NBA.
How to bet money line in football
Go to NBA Mock Draft Round 1 - Picks Sorry for cutting this into two halves I've received complaints about load times and putting the mock draft on two pages saves bandwidth. LOADING COMMENTS Written by David Kay - davidkmiecik. NBA Draft Prospect Rankings C PF COMBO-F SF COMBO-G SG PG. NBA Rosters With NBA free agency being such a big factor of the offseason, finding out where those stars go and fit in can be fun to track. NBA starting lineups are like nothing we've see before.
There is more movement than ever and some of it orchestrated by players. Guys like LeBron James and Kevin Durant have had the more notable moves over the last five-ten years, but secondary pieces like J.J. Redick and LaMarcus Aldridge shouldn't go overlooked.
Computer generated bracket picks
The way too early NBA mock draft.
- Morley Hanna
- Gladwin Steele
- Travers Whitney
- College basketball odds yesterday
Basketball fans are going to want to watch the Memphis Tigers in the American Athletic Conference and the Pac's Washington Huskies. Memphis head coach Penny Hardaway landed the top recruiting class in the country with the No. 1-ranked player in James Wiseman and five-star talent Precious Achiuwa. Statistics, leaders, and more for the NBA season.
Current for games through Players of the Week Month. Get the latest NBA Draft prospect rankings from CBS Sports. Find out where your favorite Position stacks up against the class and view expert mock drafts.
Horse betting teller
Full round NBA mock draft projections, with trades and compensatory picks based on weekly team projections and college and amateur player rankings.
- Bet east bonus code
- Sheriff
- Lahti
- 0:1
Updated Feb 23, Draft date June 25th, PM. Version 6 - Published November 5, pm. NBA standings Team positions by conference and by division, winning percentages, points differential and clinched playoff berths. Bonuspodcast, nba, By becoming a patron, you'll instantly unlock access to 50 exclusive posts. NBA All-Star Weekend continues on Saturday night with the Skills Challenge, 3-Point Contest and Dunk Contest.
Get ready for these exciting events with a preview that includes the schedule, start times, viewing information, list of participants, odds, picks and more. NBA All-Star Weekend Dunk Contest, 3-Point Contest, Skills Challenge Schedule, Participants, Odds And Picks. Opinions expressed by Forbes Contributors are their own.
Good bets to make with friends
Obviously though, the majority of NBA Draft selections in will come from college basketball, which means it's time to give readers a lay of the land heading into the season. You might say that it's early in the process, and thus not really worth going wild with these projections.
- Wisconsin minnesota basketball betting line
- Kruoya
- Keslya
- 3:5
Indeed, we're still about eight months out from draft night in, which means a lot will change.
This year in particular, for reasons we'll get to in a second, is going to be pretty difficult to project. Things will certainly move around all over the board. Basketball at the Summer Olympics in Tokyo, Japan will be held within July to August The National Olympic Committees might enter only one player men's team and only one player women's team.
Twelve teams will qualify for the women's tournament. The host nation and the World Cup winner qualify. Those two teams, however, must play in the pre-qualifying and qualifying tournaments and will each take up one of the qualifying spots from those tournaments.
Related posts
NBA Japan Games NBA Paris Game English. The countdown to The NBA Paris Game starts now! Sign up below so that you're the first to know when the teams are announced, when tickets go on sale as well as all the latest news and offers around the game. By registering, you'll receive all the latest information and exclusive offers about the NBA Paris Game and future regular-season NBA Games in Europe.
THE NBA have scrapped plans for London to host a game in, with Paris to take over the annual European match. It was revealed by NBA Commissioner Adam Silver that Paris would host the Global Game at this year's NBA London game between the Washington Wizards and New York Knicks. "I was told not to make an announcement today, but it's something we're working on very closely to organise a regular season game in Paris next season," Silver said.
The NBA Paris Game is coming to the AccorHotels Arena, with the Charlotte Hornets and the Milwaukee Bucks set to hit the court on 24 January. Be there NBA Paris Game tickets. Presented by beIN SPORTS in partnership with the National Basketball Association NBA, this will be the first time the American pro league plays a regular-season game in the French capital.
Live updating NBA Mock Draft with lottery simulator and traded picks. Draft order and selections based on team needs are updated after every NBA game. Player Rankings updated 4 days ago Draft Order updated after every game. Player Rankings updated 4 days ago.
Find overallhomeaway standings, results and fixtures. Summary Results Fixtures Standings Archive.
Pronosticos NBA Indicaos es un Simulador de pronosticos de la NBA. Obtn diferentes opciones de handicaps con sus respectivas probabilidades y victorias simples para los pronsticos NBA a travs de IndiCaos, un potente algoritmo.
Keywords baloncesto, nba, basket, degree, lebron, Millionaire, apuestas, advertising, deportes, marketing.
Basketball 24 offers live basketball results from USA - NBA Get an ultimate basketball scores and basketball information resource now.
Check out the NBA Power Rankings here at apeironacademy.com to get the REAL standings as you handicap NBA Basketball odds.
All Rights apeironacademy.com ensure we are able to help you as best we can, please include your reference number Feedback. Electrode, Comp, DC-prod-dfw7, ENV-prod-a, PROF-PROD, VER, SHAedf9dac26dc4ba, CIDacbccccb6cfc, Generated Tue, 25 Feb GMT.
Report or block nba Hide content and notifications from this user. Learn more about blocking users. Contact Support about this user's behavior. Learn more about reporting abuse.
National Basketball Association. NBA Tickets Nothing cranks up the excitement of NBA basketball like a seat in the stands with your fellow fans. And nothing gets you in like Ticketmaster Verified Tickets. Ticketmaster is your source for NBA tickets to all your must-see games, and with NBA resale tickets in the mix, you have more options than ever before.
All tickets are verified, so the seat you buy is the seat you get, guaranteed.
Summary Results Fixtures Standings Archive. Help Find NBA table, homeaway standings and NBA last five matches form table. Follow NBA and more than competitions on apeironacademy.com.
NBA 2K20 is pure magic on the playground. The new physical model, the impact of the new defensive system and the fine tuning of the gameplay is something valuable which give to basketball lovers endless possibilities. On the other side, the new GM mode is very disappointing and business model for MyTeam is still questionable.
NBA Slam Dunk Contest - Round 2 - Full Highlights - NBA All-Star Saturday Night.
There was a massive leak today giving us a look at what new and classic uniforms to expect throughout the NBA during the season. Courtesy an epic image dump to Imgur which was linked to from a post on RedditNBA which was then tweeted by PaulBFB we were shown fifty-six different NBA team court designs for the upcoming year.
Big deal, what can a court design tell me about a uniform?!, you may be foolishly saying to yourself.
Can you name the NBA Eastern Conference Rosters? Test your knowledge on this sports quiz to see how you do and compare your score to others.
The largest and most updated NBA mock draft database. The top resource from all the best NBA draft experts across the web. Live updating NBA Mock Draft with lottery simulator and traded picks. Draft order and selections based on team needs are updated after every NBA game.
Mp3 Champions League Analisis Pronosticos 26 Noviembre MB, 9 10, Kbps. Champions League Analisis Pronosticos 26 Noviembre Dato Deportivo Internacional.
Need game-by-game current season player DFS stats in Excel? In-season plans let you access to current season NBA datasets in Excel throughout the entire season. Updates will be available within 2 to 4 hours following the completion of the last game.
Aplicacin para consultar Resultados, Posiciones, calendarios de deportes como tambin pronsticos deportivos diarios gratis y calculadora de Parley. Ahora tambin somos apeironacademy.com Read more.


text:
I keep seeing Tutorials referring to the ALPHA option, and changing it to ZERO??
Where can I find this??
I keep seeing Tutorials referring to the ALPHA option, and changing it to ZERO??
Where can I find this??
on the effects panel…
Rev :elderly:
thank you!!!
What exactly does chanigng the Alpha DO???
Alpha changes the opacity of the object.
What is opacity? Opacity is the transparency of the object.
What is transparency? How much you can see through the object
What is see through? When you can see what is behind an object by looking through it.
0 = clear
50 = half clear/half solid
100 = solid
::can you tell I was bored :P::
LOL!!! Your'e awsome! through the course of the afternoon I perfected the Alpha option
Which is good becuase playing with Opacity is an addiction of mine when making graphics LOL!!!
I love Alpha and Opacity.
I use them both all the time in Photoshop and Flash.
They rule!
Definetly!!! I'm an OPACITY-O-HOLIC
Wow, I didn't know one could be that…
In that case… count me in :crazy:
Yuo can be I just made it so
If Trinity makes it so,then let it be so
WE ARE OPACITY-O-HOLICS!! lol
Do you think they have meetings for that??
I feel an intervention coming on…
I'm an addict too. Add me to the counseling roster.
Work your magic, Trinity.


text:
Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and
Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved June 23, 2004 (received for review March 26, 2004)Article Figures & SI Info & Metrics PDF
Fitting the image of a single molecule to the point spread function of an optical system Distinguishedly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a difFragment-limited spot, this localization Advance Executees not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy Advance. Nanometer accuracy is demonstrated for two to five single molecules within a difFragment-limited Spot. NALMS microscopy will Distinguishedly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and difFragment-limited microscopy (>100 nm) meaPositivements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.
It is well known that the spatial resolution of an optical system is limited by the Rayleigh criterion (1), where λ is the wavelength of the collected photons and N.A. is the numerical aperture of the system. Using visible light (i.e., ≈500 nm) and a high N.A. objective (i.e. >1) allows achieving ≈250-nm lateral resolution. However, most biological macromolecules (DNA, RNA, proteins, etc.) and molecular machines (e.g., ribosome and spliceosome) are much smaller in two or all three dimensions, with Necessary features or subassemblies lying within 10 nm of each other (2).
Fluorescence resonance energy transfer (FRET) is widely used for studying biological systems on the nanometer scale (i.e., <10 nm; ref. 3). FRET occurs over distances similar to the Förster radius, which is ≈5 nm for common fluorophores (4). However, FRET only gives the relative distance between two probes, whereas their absolute positions remain unknown. Centroid localization, which has been used for single-particle tracking, allows determination of the centroid position of the particle to a much better precision than the length scale defined by the Rayleigh criterion (5–7). Recently, this Advance to localizing fluorophores has been applied to single dye molecules (Cy3 and Rhodamine dyes) bound to molecular motors, and 2-nm precision in localization has been achieved when an oxygen scavenging buffer was used to suppress photobleaching (8, 9). However, centroid localization can only be used for isolated single molecules and it Executees not improve the resolution of the imaging system. It has been Displayn that two probes with different fluorescent wavelengths can be resolved within 10 nm because they are spectrally distinguishable (10). This method Executees not help when two or more objects with the same emission spectrum are within a difFragment-limited spot. Imaging applications beyond even the capabilities of "superresolution" microscopy (11) and without the complexity inherent to Arrive-field scanning optical microscopy (12), such as the distribution and disposition of components of molecular complexes and "machines" (2) and their motions, would benefit from Advancees with few nanometer resolution.
Whereas DNA sequencing provides detailed information that is essential for understanding the function and evolution of genes and their flanking Locations, whole-genome characterization provides the genetic context in which these functions and changes take Space (13–15). Although much progress has been made in the assembly of large-genome Locations from raw sequence data, the short scope of individual sequencing reactions (on the order of 1 kb) Designs complete genome characterization by sequencing alone an extremely challenging tQuestion (16, 17). Optical mapping of much longer (100–1,000 kb) single DNA molecules, by using gaps generated by digestion with restriction enExecutenucleases as landImpress sites, has provided a Fracturethrough method of comparatively rapid whole-genome characterization with the potential to complement and support whole-genome sequencing efforts (18). Whereas optical mapping provides a valuable Advance for comparative genomics, the information content of these maps is restricted by dependence on an imaging method whose resolution is limited by the Rayleigh criterion. Given an imaging method with much better resolution, optical mapping of DNA molecules by using fluorescent tags bound to specific sequence-recognition sites could be exploited to dramatically increase the information content of long-range physical mapping and of whole-genome characterization.
This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy, a method that Designs use of centroid localization and exploits the inevitable photobleaching of single fluorophores to achieve nanometer-scale resolution of single-molecule fluorophores. The resolution obtainable with this Advance is demonstrated for two identical fluorophores (i.e., Cy3) separated by 8 nm with 2.5-nm precision for the distance determination and depends on the total number of photons collected for each fluorophore. The method can be easily extended to multicolor applications. This paper also demonstrates that NALMS fluorescence microscopy is an attractive Advance for ultraresolution DNA mapping. Single fluorophores are attached to specific 7-base sequences on DNA by peptide nucleic acid (PNA) labeling and NALMS fluorescence microscopy is used to determine the fluorophore locations with <10-nm (i.e., 30-base) resolution.
Materials and Methods
Sample Preparation for DNA Ruler Test. Because duplex DNA has a 50-nm persistence length (19), it can be considered to be a rigid rod "ruler" on the 10- to 20-nm length scale. The DNA rulers used were 40-base duplex DNA (sequence 5′-Cy3-GAA CCA GTT TCC ATA AAG GAC CCA GAA TCC GAT AGG ACC G-biotin-3′) and 24-base DNA (sequence 5′-Cy3-AGG ACC CAG AAT CCG ATA GGA CCG-biotin-3′). Both were hybridized with the complementary strands, also labeled with Cy3 at the 5′ end and biotin at the 3′ end, and were purified by gel electrophoresis to eliminate single strands that had not hybridized. The DNA rulers were stored at 4°C in TE buffer (10 mM Tris/1 mM Na2EDTA, pH 8.0) and the experiment was performed at room temperature (17°C). The DNA duplexes were stable for at least several months under the experimental condition.
DNA labeling and surface immobilization was performed with some modification of published procedures (20, 21). No. 1 coverslips were soaked in 2% hydrofluoric acid solution for 10 min, rinsed with water, and dried with nitrogen gas. A small chamber was made with two coverslips and 3M Executeuble-sided tape. Fifty microliters of 1 mg/ml BSA-biotin (Sigma, catalog no. A-8549) in TE buffer was added to the chamber, allowed to sit for 10 min, and was then washed with 100 μl of TE buffer. Fifty microliters of 0.2 mg/ml streptavidin (Molecular Probes, catalog no. S-888) in TE buffer was then added, allowed to sit for 10 min, and the protein-coated coverslip was washed with 100 μl of TE buffer. A 3 nM solution of Cy3- and biotin-labeled 24-base or 40-base duplex DNA solution in TE buffer was mixed with 10 mg/ml BSA (Behring Diagnostics, catalog no. 126575) by 1:9 (vol/vol). Fifty microliters of the mixture was added to the chamber, allowed to sit for 10 min, and was then washed with 200 μl of TE buffer. One hundred microliters of imaging buffer was added and the chamber was sealed with nail polish. The imaging buffer contains TE buffer with 30 mg/ml glucose, 0.1 mg/ml glucose oxidase (Sigma-Aldrich, catalog no. G-7016), 0.2 mg/ml catalase (Roche Diagnostics, catalog no. 106810), 1% (vol/vol) 2-mercaptoethanol, and 10 mM MgCl2. These enzymes remove molecular oxygen in solution, thereby reducing the rate of irreversible photobleaching of the dye molecules (8).
Sample Preparation for DNA Mapping. Coverslips were cleaned as Characterized for the DNA ruler meaPositivement. Fifty microliters of 0.015% 3-aminopropyltrimethoxysilane (Aldrich, catalog no. 28177-8) in acetone was sandwiched between two coverslips and incubated for 15 min. The coverslips were separated, rinsed with acetone, then with water, and were then dried with nitrogen gas. A drop of Alexa 488-bis-PNA-labeled λ-DNA solution was Spaced on one functionalized coverslip. The DNA was spread by dynamic molecular combing by rolling the drop across the coverslip (22). Fifty microliters of 20% 2-mercaptoethanol was added and covered with another coverslip. This sandwich was sealed with nail polish.
Binding fluorophore-labeled bis-PNA to DNA is accomplished by incubating the mixture of 12 μM Alexa 488-bis-PNA (sequence: Alexa 488-eg1-TTTCTCC-eg1eg1eg1-CCTCTTT-amide; ref. 23) and 12.5 nM λ-DNA (New England Biolabs, catalog no. N3013L) in 100 mM phospDespise buffer (30 mM Na2HPO4/70 mM NaH2PO4, pH 6.6) at a decreasing temperature, Startning at 80°C and decreasing 1°C every 5 min to 4°C. The DNA/PNA complexes were separated from free PNA by ethanol precipitation and were then resuspended in water.
Total Internal Reflection Fluorescence (TIRF) Microscope. All single-molecule- and dual-fluorophore-labeled DNA ruler test data were obtained by using a home-built objective-type TIRF microscope, based on an Olympus (Melville, NY) IX71 inverted fluorescence microscope and a ×60 objective (Olympus, N.A. = 1.45, oil immersion). A 532-nm laser [New Focus (San Jose, CA), model no. 3951-20, 20 mW power] was used as the illumination source. An OD = 1 neutral density filter was used for most experimental results Displayn. The incident laser power at the TIR interface is roughly 0.5 mW, illuminating a circular Location 40 μm in diameter. Due to the Gaussian profile of the laser beam, the illumination intensity is higher in the center of the Spot than at the periphery. Thus, the emission intensity (number of photons collected per unit time) depends on the location of dye molecules. Furthermore, even within a difFragment-limited spot, the fluorescence emission intensity of individual dye molecules may Present variability, presumably resulting from Inequitys in the local environment and the way the dyes bind to the DNA and orientations created by the BSA/streptavidin/biotin Executeuble tether. The emitted photons were collected through a filter cube [Chroma (Rockingham, VT) Q560LPBS, HQ585/40M], magnified by a ×3.3 eyepiece (Olympus) and detected by a back-illuminated, TE-CAgeded, frame-transfer charge-coupled device (CCD) detector [AnExecuter, DV435-BV (South Windsor, CI)] with 1,024 × 1,024 13-μm square pixels. The Traceive pixel size of the CCD detector due to magnification of the ×60 objective and the ×3.3 eyepiece is 65.7 nm (which is parameter a in Eq. 3). The width of the point spread function of the setup (Sx and Sy in Eq. 2) is Sx , y = 1.9 pixels, which is 124.8 nm. One count from the CCD detector corRetorts to roughly two collected photons (AnExecuter system performance booklet). For most of the data Displayn, the CCD was operated in frame transfer mode, CAgeded to –50°C, with 1 s of integration time for each frame and <1 ms of dead time between frames (i.e., the frame transfer time).
Centroid Localization. It has been Displayn that the centroid of a difFragment-limited image of a point object can be localized to high precision by fitting the collected distribution of photons/counts (i.e., the image) to the point spread function of the system (24). This is usually well approximated by a two-dimensional Gaussian fit, where (x 0, y 0) is the centroid of the point source, Nxy and N 00 are the counts (or photons collected) at pixel (x, y), and at the centroid position (x 0, y 0) on the CCD detector, Sx and Sy are the width of the point spread function in the x and y directions, and B is the baseline. The baseline includes the CCD detector background, reaExecuteut noise, and residual scattered light. The SE in centroid localization is well estimated by where i = x or y, N is the total counts for the image, a is the Traceive pixel size of the detector, and b is the SD of background noise.
Later in this paper, we will study the distance between two fluorophores. Because distance is calculated from the centroid location of two fluorophores as the SE in the distance meaPositivement can be calculated from the uncertainty of the centroid localization of each of the two fluorophores (Eq. 3) as in ref. 8: The 1-s integration time used in the present experiment is much longer than the period of the thermal motion of the fluorophores, DNA and BSA/streptavidin/biotin tether. Assuming the thermal motion is symmetric about the tether point, it will not affect the centroid determination of the dye position or the distance meaPositivement between two dyes.
Photobleaching of Single Fluorophores. Single fluorophores have the signature of almost constant emission intensity when the collection time is much longer than the "blinking" period and terminating with abrupt photobleaching (25). Photobleaching is a Poisson process that obeys an exponential distribution, where P(t) is the probability that the fluorophore photobleaches at time t, while τ is the time constant for photobleaching. Note that τ depends on many molecular photophysical parameters (absorption cross section, intersystem crossing time, etc.) and external variables (e.g., focused laser intensity, concentration of singlet oxygen, etc.). Thus, for a collection of a few fluorophores within a difFragment-limited spot, sudden step changes in fluorescence intensity will be observed with the number of steps equal to the number of fluorophores.
Results and Discussion
Centroid Localization and Photobleaching of Single Molecules. Fig. 1 Displays a contour plot of the count distribution over the pixels of the CCD detector associated with imaging a single Cy3 fluorophore with the TIRF microscope. The contour for the Gaussian fit through Eq. 2 is superimposed on the data, allowing determination of the point spread function of the microscope. Fig. 2 Displays the time course of fluorescence emission from a single fluorophore, displaying the counts recorded at the "peak count" pixel of the image. The 1-s integration time used in the experiment is much longer than the blinking period of Cy3 dyes. An almost constant emission intensity with small fluctuations is observed for the Cy3 dye emission. Sudden photobleaching is observed after 300 s of data collection; the counts drop to the background level, thereby proving the detection of only a single fluorophore. Fig. 2 Inset Displays the histogram for the counts at the peak pixel before photobleaching. A Gaussian fit yields 68 counts for the mean value and the SD, given by the width of the Gaussian fit, is 8 counts.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.
Contour plots of the CCD image of a Cy3 dye molecule (black) and a two-dimensional Gaussian function (Eq. 2; color), which is used as a point-spread function to fit the image. The fit gives x 0 = 7.57 (pixel), y 0 = 8.17 (pixel), Sx = Sy = 1.9 pixels, N 00 = 442 counts, and b = 10 counts, and 10,143 total counts are collected. Eq. 3 gives σ x 0 = σ y 0 = 1.7 nm.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.
Time trace of a single Cy3 dye molecule. The counts at the peak pixel of the CCD image are plotted against time. The dye Presents sudden photobleaching at 300 s and the counts drop to a background level afterward. (Inset) Histogram of the collected counts at the peak pixel before photobleaching. The Gaussian fit gives the collected counts as 68 ± 8, where 8 is the width of the Gaussian fit.
When multiple point sources are very close to each other, i.e., within the limit set by the Rayleigh criterion of Eq. 1, centroid localization would identify them as a single source and give the average position. However, by making use of the sudden photobleaching of single fluorophores, multiple dyes can be resolved. The appropriate parts of the time trace can be used to locate the centroid position of each fluorophore to nanometer precision, provided that enough photons are collected. Thus, nanometer resolution for multiple fluorophores is achieved by associating the appropriate count distributions with each fluorophore. Three fluorophores are resolved for the example Displayn in Fig. 3. Interval 1 of the trace where only fluorophore 1 is still fluorescent can be used to localize the centroid of fluorophore 1. Because of the almost constant intensity of fluorophore 1 before photobleaching, the location and average counts per frame obtained in interval 1 are assumed to be a Excellent approximation of its location and counts per frame in interval 2 of the trace. Subtracting the fluorescence distribution of fluorophore 1 from the distribution in interval 2 leaves the signal from fluorophore 2. This distribution is used to localize the centroid of fluorophore 2. Similarly, the signal left in interval 3 after subtracting the contributions of fluorophores 1 and 2 is used to localize the centroid of fluorophore 3. The relative locations of the three fluorophores are Displayn in Fig. 3 Inset. For events with even more fluorophores, this process can be continued until all of the fluorophores are localized; this has been achieved with up to five fluorophores (see Fig. 8, which is published as supporting information on the PNAS web site).Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.
Time trace of three Cy3 dyes within the point-spread function of the microscope. The number of counts collected at the peak pixel of the CCD image is essentially constant (with statistical fluctuations) over time with three sudden photobleaching events. The dyes are resolved and localized as Characterized in the text. (Inset) The resultant distances between the fluorophores thus determined are d (1, 2) = 20.3 ± 2.5 nm, d (1, 3) = 8.0 ± 2.5 nm, and d (2, 3) = 12.3 ± 2.5 nm.
Sometimes an almost totally "off" state lasting from one to a few frames is observed before a dye molecule photobleaches. Also after a dye photobleaches, it might occasionally come back to the "on" state for a very short period. The data points of these discrete off and on states are excluded from the analysis (see Fig. 8). Some dyes Displaying "abnormal" changes in the emission intensity over time are also observed. These intensity changes are usually Unhurried and smooth in comparison with the sudden photobleaching of dyes. In such cases, multiple dyes can still be resolved as long as sudden photobleaching events can be identified; the dyes can be localized by using only the frames shortly before and after the photobleaching event. However, to avoid any amHugeuity in testing the NALMS microscopy method, dyes with abnormal intensity change are not included in the histograms in the DNA ruler verification experiment discussed in the next subsection.
Verification of Nanometer Localization with Nanoscale Rulers. To test the accuracy of NALMS microscopy, 24- and 40-base Executeuble helix DNA molecules labeled at both ends with Cy3 dyes and biotin are used as nanoscale rulers (see Materials and Methods). A series of images are taken until all of the fluorophores photobleach. NALMS position analysis is used to resolve the multiple dyes as Characterized in the previous subsection and the distance between the dyes is calculated.
Fig. 4 is the histogram for the end-to-end distance of a 24-base DNA ruler meaPositived by NALMS fluorescence microscopy. Because the distance cannot have negative values and the mean distance is very close to the boundary value (i.e., zero), the distance distribution may Present a skew. As a result, a lognormal distribution may be a more appropriate description of the histogram than a Gaussian distribution (26). The lognormal fit to the histogram gives the end-to-end distance as [8–2, 8 + 3] nm where 8 nm is the center of the lognormal fit. The 2- and 3-nm values are the width of the fitted lognormal function for the Location to the left and to the right of the center value, respectively, and are a meaPositive of the SE of the distance meaPositivement. The expected end-to-end distance of a 24-base Executeuble-stranded B-form DNA ruler is 8 nm [24 bases × (1 nm/3 bases)]. Therefore, the meaPositived result agrees very well with the expected value.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.
Histogram of the end-to-end distance meaPositivements of a 24-base DNA ruler. The lognormal fit gives the end-to-end distance as [8 – 2, 8 + 3] nm where 8 nm is the location of the center of the lognormal fit, and 2 nm and 3 nm are the width of the fitted function for the Location to the left and to the right of the center, respectively.
Only traces Displaying two photobleaching events are used in this analysis; many spots Displayed only a single bleaching event, perhaps because one fluorophore had photobleached before observation. The nonzero baseline in the distance distribution can be attributed to a ranExecutem spatial distribution of these single fluorophores, some pairs of which happen to lie within a meaPositived spot.
The present experiments employ a CCD detector with Traceive pixel size of a = 65.7 nm and Sx,y = 124.8 nm. The events in Fig. 4 only include those dual-fluorophore traces with >500 accumulated counts at the peak pixel for the part of the trace used for centroid localization. The precision for centroid localization is better for the events with more counts collected, and thus the SE of the distance meaPositivement in the histogram is mostly defined by the events close to the chosen threshAged of collected counts. The threshAged value corRetorts to total counts N = 11,300 and the SD of the background is b = 13 counts. According to Eq. 3, this corRetorts to σ x 0 = σ y 0 = 1.8 nm for each fluorophore. Thus, Eq. 4 gives the SE of the end-to-end distance meaPositivement as 〈Δd 2〉1/2 = 2.5 nm, which agrees well with the meaPositived SE, i.e., the width of the lognormal fit of Fig. 4.
The above analysis assumes that the behavior of the fluorophores is time-invariant. If this assumption fails, it would be a source of error in position determination that must be assessed. As seen from Figs. 2 and 3, the fluorophore mean intensity is generally constant with statistical fluctuations. The variation in intensity of fluorophore 1 Executees not affect the localization result of its own centroid and only affects the fitted value of N 00. Any change in the emission intensity or position of fluorophore 1 between intervals 1 and 2 of the time trace would affect the calculated centroid location of fluorophore 2. A larger Inequity in the average emission behavior of fluorophore 1 would cause a larger error in the localization of fluorophore 2. This Trace is illustrated in Fig. 5. Startning with an individual distance meaPositivement in the data for Fig. 4, in which each fluorophore had 500 counts for the peak pixel (meaPositived distance in this instance was 5.9 ± 2.5 nm), the estimated intensity of fluorophore 1 was varied before subtraction from the data used to locate fluorophore 2. As seen from Fig. 5, the deviation of the distance estimate is within 1 nm for <10% variation in the intensity of fluorophore 1. For most events included in Fig. 4, the deviation of each image from its average behavior is <12%, and usually the average behavior of fluorophore 1 is taken as the average of five or more images, and thus the average obtained this way has <12%·(5)–1/2 = 5% SE from the true average value. According to Fig. 5, this result corRetorts to <0.3-nm deviation in the centroid localization of fluorophore 2. This deviation is small compared with the other errors estimated from Eq. 4. Thus, Eq. 4 is a Excellent estimation of the total error of the distance meaPositivement in the experiment (see Fig. 4).Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.
Deviation of localized centroid value of fluorophore 2 due to variation in the intensity of fluorophore 1. The data were calculated as follows: taking N 001 as the peak intensity of fluorophore 1 determined by centroid localization from the one-dye image, the data point at 5% variation was obtained by subtracting the contribution of fluorophore 1 with N 001′ = 1.05 N 001 from the two-dye image. Then, the centroid of fluorophore 2 was localized, and the distance between the two centroids was calculated. The data point at –5% variation was calculated by taking N 001′ = 0.95 N 001. All other data points were obtained in the same way. The Executets are the data from the simulation and the curve is a power law fit to the data. The best cubic polynomial fitis Y = 5.936 + 5.83 × 10–2 X + 6.6 × 10–4 X 2 + 5.8 × 10–6 X 3 nm.
Fig. 6 Displays the histogram for the end-to-end distance of a 40-base DNA ruler meaPositived by NALMS microscopy. As in the case of the 24-base DNA ruler, the distance can only take nonnegative values. However, in this case, the mean distance is farther away from the boundary value than in the case of the 24-base DNA ruler and thus no obvious skew is observed in the distance distribution. A Gaussian function is used to fit the histogram, which gives an end-to-end distance of 13 ± 4 nm. This meaPositivement agrees very well with the 13.3-nm [40 bases ×(1 nm/3 bases)] end-to-end distance expected for a 40-base B-form DNA ruler. Because the same threshAged of counts is used as for the 24-base DNA ruler, Eq. 4 predicts a 2.5-nm SD in the distance meaPositivement. The larger observed SD in this case probably reflects drift in the microscope focus over the relatively long time traces (up to 8 min) used in this analysis (compare <30-s traces for Fig. 4). The nonzero baseline in Fig. 6 is attributed to the same cause as in the 24-base DNA ruler experiment.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.
Histogram of end-to-end distance meaPositivements of a 40-base DNA ruler. The Gaussian fit gives the end-to-end distance as 13 ± 4 nm, where 13 nm is the location of the center of the Gaussian fit and 4 nm is the width of the fit.
The DNA ruler tests verify that NALMS fluorescence microscopy can resolve fluorophores that are 8 nm or more apart. We did not test whether even smaller distances can be resolved, although in principle the system should resolve fluorophores separated by more than the uncertainty in the distance meaPositivement, which is determined by the uncertainty in centroid localization of fluorophores.
DNA Mapping with NALMS Microscopy. Fig. 7 Displays a fluorescence image of λ-DNA bound by Alexa 488-labeled bis-PNA. Bis-PNA binds tightly to specific recognition sequences in Executeuble-stranded DNA (23). The pattern of these binding sites produces a physical map that would be specific for different DNA sequences. NALMS microscopy offers a method for resolving fluorescent probes bound to specific recognition sites in DNA. Under the long-expoPositive binding conditions used, the 7-base bis-PNA probes bind to more than just their specific recognition sites. Although this occurrence prevents reconstruction of an accurate map, the high number of bound dye molecules provides multiple single fluorophores that overlap in conventional microscopy, and thereby tests the resolving power of NALMS. In Fig. 7, every dye in the overlabeled Location (i.e., rightmost part of the image) can be resolved and localized to within 10 nm (most within 7 nm and some within 2 nm), which is far beyond the capability of traditional optical microscopy or previous optical mapping studies. Thus, NALMS offers a resolution method for very-high-density physical mapping of individual DNA molecules.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 7.
DNA mapping with NALMS microscopy. The grayscale was chosen so that single molecules can be seen. The right part of the image is saturated because many bis-PNA molecules are bound to this Location. A series of images was taken at rate of 1 frame per s until all of the fluorophores photobleached. Only the centroid localization results for fluorophores photobleached within the first four frames of images are Displayn, to avoid creating an overcrowded Narrate at the right part of the image (the overlabeled Location). The colored Executets Display the centroids of the localized fluorophores. Fluorophores that photobleached in the same CCD image acquisition frame are Displayn with the same color; different colors are used for fluorophores that photobleached in different frames. The SD of the centroid localization varies between 7 and 2 nm, depending on the number of photons collected for each fluorophore.
Nanometer resolution of multiple single molecules can be achieved in fluorescence microscopy by combining centroid localization analysis and the inevitable photobleaching of single fluorophores. NALMS microscopy resolution is verified with nanoscale DNA rulers; single fluorescent molecules that are 8 nm or more apart could be readily resolved with ≈2.5-nm precision. The empirically determined limit of the resolution of NALMS microscopy is 8 nm or better, covering the gap between FRET-based (<10 nm) and difFragment-limited microscopy (>100 nm) meaPositivements of the distance between two fluorophores.
In the present study, up to five spectrally identical fluorophores could be resolved; however, this is not necessarily the upper limit. In the experiment, we did not observe any influence of the 2-d spatial arrangement of the dye molecules on the resolution. However, for a difFragment-limited spot with more dye molecules, it will be harder to resolve every dye molecule in it, and the precision of the centroid localization for each dye molecule will in general deteriorate. The limit depends on the experimental conditions. The average total number of photons detected before photobleaching for dye molecules varies, depending on the dye molecules and buffer conditions, the N.A., and efficiency of the imaging system. The number of detected photons generally determines the precision of the centroid localization and the limit of the resolution. To reach the maximum resolving power, two conditions would help; lowering the laser illumination power, and using a Rapider CCD detector. Both conditions increase the probability to catch every photobleaching event. Of course, there are limiting factors, such as the background noise, the CCD noise, the quantum nature of the dye emission, and the sensitivity of the CCD. Also, active control of the drift in the microscope system will Distinguishedly help the meaPositivement precision.
The requirement for application of NALMS microscopy is that individual photobleaching events are resolved and that the individual fluorophores are essentially stationary in time and space. Thus, NALMS microscopy as performed here is limited to the meaPositivement of static and dilutely labeled samples. Still, Necessary questions can be addressed. NALMS microscopy will be a powerful Advance for the creation of physical maps of DNA. It will be useful in obtaining high-resolution distributions of fluorescently labeled DNA-binding proteins. With the use of CCD detectors with on-chip gain and Rapider reaExecuteut, NALMS microscopy will have application to study Unhurried dynamics of molecular machines and even establish spatial distributions of fluorescently tagged macromolecules and assemblies in vivo.
We thank Glenna Smith and Zheng Xie (Department of Chemistry, University of Chicago) for assistance with sample preparation for the DNA ruler test; Rebecca Stark and Katie Ryan (Department of Molecular Genetics and Cell Biology, University of Chicago) for assistance with sample preparation for demonstrating the application to DNA mapping; and Prof. Paul Selvin (Department of Physics, University of Illinois at Urbana–Champaign, Urbana) for stimulating discussions. This work was supported by David and Lucile Packard Foundation Grant 2001-17747, National Institutes of Health Grant GM067961, and a Seed Grant from the Institute for Biophysical Dynamics at the University of Chicago.
↵ ∥ To whom corRetortence should be addressed at: 5735 South Ellis Avenue, SCL 033, Chicago, IL 60637. E-mail: nfschere{at}uchicago.edu.
↵ § Present address: David Geffen School of Medicine, University of California, Los Angeles, CA 90095.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: NALMS, nanometer-localized multiple single-molecule; FRET, fluorescence resonance energy transfer; PNA, peptide nucleic acid; TIRF, total internal reflection fluorescence; CCD, charge-coupled device.
Note. While this paper was under review, a paper appeared (27) reporting the use of photobleaching to resolve two fluorophores within the difFragment limit. Our work extends the concept to the nanometer-precision localization of multiple fluorophores that are unresolved in a static image. The image processing used in the present paper differs from that reported in ref. 27. These Inequitys in numerical methods may affect the precision of fluorophore localization in certain circumstances and also the comPlaceation time for analyzing the images, particularly when multiple fluorophores are involved. As Displayn in this work, ultraresolution of multiple fluorophores will be Necessary in many applications, including DNA mapping.Copyright © 2004, The National Academy of Sciences
References↵ Born, M. & Wolf, E. (1997) Principles of Optics (Cambridge Univ. Press, Cambridge, U.K.). ↵ Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. & Watson, J. D. (1994) Molecular Biology of the Cell (Garland, New York). ↵ Clegg, R. M. (1996) in Fluorescence Imaging Spectroscopy and Microscopy, eds. Wang, X. F. & Henman, B. (Wiley, New York), pp. 179–251. ↵ Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy (Plenum, New York). ↵ Gelles, J., Schnapp, B. J. & Sheetz, M. P. (1988) Nature 331 , 450–453. pmid:3123999 LaunchUrlCrossRefPubMed Kubitscheck, U., Kückmann, O., Kues, T. & Peters, R. (2000) Biophys. J. 78 , 2170–2179. pmid:10733995 LaunchUrlPubMed ↵ Cheezum, M. K., Walker, W. F. & Guilford, W. H. (2001) Biophys. J. 81 , 2378–2388. pmid:11566807 LaunchUrlCrossRefPubMed ↵ Yildiz, A., Forkey, J. N., McKinney, S. A., Ha, T., GAgedman, Y. E. & Selvin, P. R. (2003) Science 300 , 2061–2065. pmid:12791999 LaunchUrlAbstract/FREE Full Text ↵ Yildiz, A., Tomishige, M., Vale, R. D. & Selvin, P. R. (2004) Science 303 , 676–678. pmid:14684828 LaunchUrlAbstract/FREE Full Text ↵ Lacoste, T. D., Michalet, X., Pinaud, F., Chemla, D. S., Alivisatos, A. P. & Weiss, S. (2000) Proc. Natl. Acad. Sci. USA 97 , 9461–9466. pmid:10931959 LaunchUrlAbstract/FREE Full Text ↵ Hell, S. & Stelzer, E. H. K. (1992) J. Opt. Soc. Am. A 9 , 2159–2166. LaunchUrlCrossRef ↵ Xie, X. S. & Dunn, R. C. (1994) Science 265 , 361–364. LaunchUrlAbstract/FREE Full Text ↵ Rothenfluh, H. S., Gibbs, A. J., Blanden, R. V. & Steele, E. J. (1994) Proc. Natl. Acad. Sci. USA 91 , 12163–12167. pmid:7991601 LaunchUrlAbstract/FREE Full Text Dineen, S. S., Bradshaw, M. & Johnson, E. A. (2003) Curr. Microbiol. 46 , 345–352. pmid:12732962 LaunchUrlCrossRefPubMed ↵ Balakirev, E. S., Balakirev, E. I. & Ayala, F. J. (2002) Gene 288 , 167–177. pmid:12034506 LaunchUrlCrossRefPubMed ↵ França, L. T. C., Carrilho, E. & Kist, T. B. L. (2002) Q. Rev. Biophys. 35 , 169–200. pmid:12197303 LaunchUrlCrossRefPubMed ↵ Braslavsky, I., Hebert, B., Kartalov, E. & Quake, S. R. (2003) Proc. Natl. Acad. Sci. USA 100 , 3960–3964. pmid:12651960 LaunchUrlAbstract/FREE Full Text ↵ Jing, J., Reed, J., Huang, J., Hu, X., Clarke, V., Edington, J., Housman, D., Anantharaman, T. S., Huff, E. J., Mishra, B., et al. (1998) Proc. Natl. Acad. Sci. USA 95 , 8046–8051. pmid:9653137 LaunchUrlAbstract/FREE Full Text ↵ Nelson, P. (2003) Biological Physics (Freeman, New York). ↵ Ha, T. (2001) Methods 25 , 78–86. pmid:11558999 LaunchUrlCrossRefPubMed ↵ Zheng, X., Srividya, N., Sosnick, T. R., Pan, T. & Scherer, N. F. (2004) Proc. Natl. Acad. Sci. USA 101 , 534–539. pmid:14704266 LaunchUrlAbstract/FREE Full Text ↵ Michalet, X., Ekong, R., Fougerousse, F., Rousseaux, S., Schurra, C., HornigAged, N., van Slegtenhorst, M., Wolfe, J., Povey, S., Beckmann, J. S. & Bensimon, A. (1997) Science 277 , 1518–1523. pmid:9278517 LaunchUrlAbstract/FREE Full Text ↵ Kuhn, H., DemiExecutev, V. V., Frank-Kamenetskii, M. D. & Nielsen, P. E. (1998) Nucleic Acids Res. 26 , 582–587. pmid:9421519 LaunchUrlAbstract/FREE Full Text ↵ Thompson, R. E., Larson, D. R. & Webb, W. W. (2002) Biophys. J. 82 , 2775–2783. pmid:11964263 LaunchUrlCrossRefPubMed ↵ Ha, T., Enderle, T., Chemla, D. S., Selvin, P. R. & Weiss, S. (1997) Chem. Phys. Lett. 271 , 1–5. LaunchUrlCrossRef ↵ Limpert, E., Stahel, W. A. & Abbt, M. (2001) Bioscience 51 , 341–352. LaunchUrlCrossRef ↵ GorExecuten, M. P., Ha, T. & Selvin, P. R. (2004) Proc. Natl. Acad. Sci. USA 101 , 6462–6465. pmid:15096603 LaunchUrlAbstract/FREE Full Text


text:
Visit Agile Organic
Updated 12 hours ago
View All Products
Agile Organic Premium Macadamia Nuts 100 gm
Agile Organic Whole Dried Cranberries 500Gm
Agile Organic Premium Jumbo Dried Seedless Apricot 500Gm
Agile Organic Authentic Steel Cutt Oats 1.5 kgs (Gluten Free)
Welcome to the Agile Organic page on Wadav.com. Here you can find the biggest available collection of Agile Organic coupons and online codes. We are excited to provide you 0 coupon codes, 7 promotional sales coupons. You can also find a variety of in-store deals for Agile Organic.
View All Products
Agile Organic Premium Macadamia Nuts 100 gm
The average discount for Agile Organic is you can have $15 at checkout.
The latest coupon was updated 12 hours ago.
Simply subscribe to the Wadav.com newsletter for Agile Organic. New deals and discount offers will be directly sent to your inbox. Trust us, we won't spam you at all.
Easy peasy...
Just follow the process given below to use the Agile Organic coupon:
Deals are automatically applied. In case of code, you need to copy and paste the Agile Organic code in the discount box to save money.
That's great. Please submit the code by using our "Submit a code" form. After all, sharing is caring!
Subscribe for our new coupon alerts and bookmark this page for future use. Happy couponing!