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Corn and Bean Salsa
Recipe by anonymous23
My mom brought this recipe home from Bunco, raving about it. I was skeptical, but she was right--it's fantastic. Now I get asked for the recipe when I make it for parties. This is best when it's made a day or two in advance so that all the flavors get a chance to mingle.
Top Review by Susiecat too
This is super-delish. The flavors wrap around each other after sitting for a few hours, and it tastes even better than when initially mixed. I used only one can of black beans instead of 2, and rice vinegar instead of red wine vinegar, but those were my only changes. Great recipe, anonymous23! Made for PAC Fall 2007.
Ingredients Nutrition
- 2 (15 ounce) cans corn, drained
- 2 (15 ounce) cans black beans, drained & rinsed
- 1 (15 ounce) can red beans, drained & rinsed
- 3⁄4 cup diced celery
- 3⁄4 cup diced red onion
- 2 medium carrots, sliced very thin (or diced)
- 1⁄3-2⁄3 cup olive oil (no substitutions)
- 1⁄3 cup chopped fresh cilantro
- 3 garlic cloves, crushed
- 3 tablespoons red wine vinegar
- 1 teaspoon cumin
- 1 tablespoon sugar
- 2 teaspoons salt
- 1 lemon, juice of
Directions See How It's Made
- In a large bowl, mix the corn, beans, celery, red onion, and carrots.
- Mix the rest of the ingredients (oil, cilantro, garlic, red wine vinegar, cumin) separately and pour over the veggies.
- Mix well.
- Serve with tortilla chips.


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energising the flow over an airfoil using synthetic jets
hello sir,
This is my final year project.. the main objective is to reduce the drag by installing synthetic jets in the quarter chord of the airfoil along spanwise direction..while testing it in windtunnel, the air flows over the airfoil and also a compressed air is blown into the synthetic jets so that it comes out of the airfoil, both the flows ie., flow over the airfoil and airflow from the synthetic jet mixes together.. this is called Mixed flow analysis..
How to solve this problem in fluent..? Is there any tutorial to solve mixed flow analysis..? please guide me sir..
you can use FLUENT to model that.
you can model realistic compressed jet or simply you can set some momentum sources on your airfoil instead if the jets flow rate are not comparable with main flow rate.
Thanks for ur reply sir,
i dont know how to interact both main flow and compressed jet flow in FLUENT..please tell some procedure to solve this problem..
Thank you..
first of all you should decide about simplifications. (as I said before you can model complete or a simplified one)
for example if you want to model complete one, you can consider some inlets over airfoil that inject high pressure air with specified angle(pressure inlet B.C.) the interactions between them are FLUENT tasks and you shouldn't care about.
Thank u very much sir,
Need ur guidance till i complete my project...is there any tutorials for this kind of problems in FLUENT.. if so please send me sir..
you can search airfoil with suction and/or injection modeling, you may find in internet. I didn't see your specific case in FLUENT tutorials.
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4 Jj - 335024 post office
Post Office name: 4 Jj
Type: Branch office (Delivery)
Pin Code: 335024
Location of 4 Jj
Taluk: Gajsinghpur
District: Ganganagar
State: Rajasthan
Contact :
4 Jj is a post office and is located in the Gajsinghpur, taluka, Ganganagar district and the pin code number is .
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View satellite images/ street maps of villages in 4 Jj, India. The Map data on this website is provided by Google Maps, a free online map service one can access and view in a web browser.
View 335024 streets list


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Human Intelligence and GeneticsBY: bluemoon silverstars | Category: Genetics | Submitted: 2010-05-28 05:38:33
Article Summary: "Human intelligence can be inherent or due to the environmental factors. Nature and nurture both are necessary for the human intelligence. Genetics plays important role in human intelligence..."
Intelligence is a God gifted thing. We can say that how quickly the organisms adapt to their environment. Some times we get so surprised when we hear about any invention of the scientists. It is their intelligence that they are able to discover and invent things. Similarly in school or colleges, teachers give lessons so efficiently and then it is the intelligence of the student that he remembers that lesson and do work on it. God has made the brain of humans in such a way that they can deal or understand new challenges of the life and they can create new ways of tackling with the problems of the life.
Intelligence can also be inherent that is the individual may get this characteristic from his parents either mother or father. Environment, characteristics and inheritance together make the foundation of the intelligence. This can be understood by this example that if the child or any other person lives all the time in a tense environment, he will never be able to think properly if he will not be able to make important decisions in his life. Similarly if a person gets proper and healthy environment, their abilities flourish and they can face the challenges of life. The intelligence of the humans is above all things. Computers and other machines can't do the things which humans can do. It is not even possible to invent such machines which can be matched with the human intelligence. Person's IQ can not match with his intelligence.
Scientists have proved that intelligence is inherent. In the recent years research has shown that intelligence in polygenic trait if there are normal circumstances for the individual. Lack of proper environment can affect the intelligence. Sometimes mutations also occur in the genes of an individual which are the major cause of the effecting the intelligence. For example, there is a genetic disease Phenylketonuria, which is caused due to the mutations in the genes. If this genetic disorder does not get the proper treatment on time, it will be possible that the individual's brain might not develop properly. As a result there can other diseases like mental illness or damage to the brain by any other means occur. This disease is caused by the mutation in the single gene. This defected gene disturbs the function of the proteins in the body and causes Phenylketonuria .
Scientists are already trying to figure out that what gene is responsible fro intelligence in humans. A team of scientists at the University f California has found a gene named HAR1F that is responsible for the intelligence in humans and this gene is not present in other animals. Scientists claim that not only the genetics but environment also affects the intelligence. If there are suitable circumstances for an individual, he will be able to live a healthy life and his intelligence will also increase. It has been discovered by some scientists that axon of the brain are responsible for human intelligence. Through the wiring of the brain or neurons, these axons move in the brain. If the movement of the axons carrying the information is fast, quickly the brain will work and send the information where it is needed. But if the brain's axons are weak, the process of sending the information will be slow and intelligence will decrease.
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- Wise-like
- Wise-like Wise"-like`, a.
Resembling that which is wise or sensible; judicious.
[1913 Webster]
The only wise-like thing I heard anybody say. --Sir W. Scott. [1913 Webster]
The Collaborative International Dictionary of English. 2000.
The only wise-like thing I heard anybody say. --Sir W. Scott. [1913 Webster]
The Collaborative International Dictionary of English. 2000.
wise-like — wiseˈ like (or (Scot) /wīsˈlīk/) adjective 1. Sensible, judicious 2. Decent 3. Fitting 4. Looking as if capable of playing one s part well • • • Main Entry: ↑wise … Useful english dictionary
WISE, STEPHEN SAMUEL — (1874–1949), U.S. rabbi and Zionist leader. Born in Budapest, Hungary, Wise was taken to the United States at the age of 17 months. From childhood he was determined to become a rabbi like his father, Rabbi Aaron Wise, who, together with alexander … Encyclopedia of Judaism
Wise Foods, Inc. — Wise Foods Inc.Wise Foods, Inc. is a private company based in Kennesaw, Georgia, that makes and sells snacks through retail food outlets in 15 eastern seaboard states as well as Vermont, Ohio, West Virginia, Kentucky, Tennessee, and Washington, D … Wikipedia
like|wise — «LYK WYZ», adverb. 1. the same; similarly: »See what I do. Now you do likewise. 2. also; moreover; too; as well: »I must go home now, and she likewise … Useful english dictionary
Wise-Rein disease — ( Lichen ruber moniliformis ) is a rare skin disease named for Fred Wise and Charles R. Rein. It is one of several diseases also known as Kaposi s disease. The disease causes numerous whitish punctiform papules and brownish macules arranged in a… … Wikipedia
Wise Blood — Infobox Book name = Wise Blood title orig = translator = image caption = First edition cover author = Flannery O Connor illustrator = cover artist = country = United States language = English series = genre = Southern Gothic novel publisher =… … Wikipedia
WISE (music) — Infobox Musical artist | | Name = Seiji Kameyama 亀山晴児 Background = solo singer Alias = WISE Born = August 10, 1979 Died = Origin = flagicon|JPN Japan Genre = Japanese Hip Hop Occupation = MC Years active = 1997 ndash;present Label = Universal,… … Wikipedia
Wise (Stetsasonic) — Infobox musical artist Name = Stetsasonic Img capt = WISE The Human Mix Machine Background = group or band Origin = Genre = Hip Hop Years active = 1981 1991 Label = Current members = Wise Daddy O Prince Paul Frukwan DBC MC Delite Bobby Simmons… … Wikipedia
Wise use — The Wise use movement in the United States is a loose knit coalition of groups promoting private property rights and use of the natural environment as a natural part of human survival. This includes use by commercial and public interests, seeking … Wikipedia
Like a Boy — Infobox Single Name = Like a Boy B side = Get Up Love You Better Artist = Ciara from Album = Released = flagicon|USA February 13 2007 flagicon|GER March 30 2007 flagicon|UK April 2 2007 Format = Digital download, CD single Recorded = Chalice… … Wikipedia


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New Delhi, Sept. 18 : Prime Minister Narendra Modi has greeted the people of Odisha on the occasion of Nuakhai.
"Nuakhai Juhar! Greetings to the people of Odisha as they celebrate the festival of Nuakhai. May this festival further happiness in society," said Prime Minister Modi.
According to the Hindu calendar it is observed on panchami tithi (the fifth day) of the lunar fortnight of the month of Bhadrapada or Bhaadra (August-September), the day after the Ganesh Chaturthi festival. This is the most important social festival of Western Odisha.(ANI)


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The Worst Property Blunders Beginning Investors Should Avoid
This post may contain affiliate links. Please read my disclosure page for more info.
As the real estate market improves, more people are investing in buy-to-let properties. It can be an excellent way to make extra money, either as a second income or as a hedge for pensioners. But before embarking on the buy-to-let adventure, educate yourself so you don't fall into these classic traps for beginning investors.
Failing to Plan
Real estate investor Andy Heller says the biggest mistake he sees investors make is not planning. Rather than finding a bargain property and then trying to decide what to do with it, Heller says to make a plan first, then look for the house to fit the plan.
If you are buying to let, decide first what area to target. Focus on neighborhoods with a lot of renters, but where there are also good employment prospects, access to public transport, and amenities — in other words, places where young adults want to live. Then spend time in the neighborhood. Find the streets that feel safest. Visit at different times of day to evaluate traffic, noise and parking situations. Don't begin your property hunt until you have a clear idea of where you want that property to be located, and be willing to wait for the right property to come along.
Failing to Anticipate Void Periods
When new landlords decide what they can afford based on anticipated rental income, they often forget to think about what happens if the property stays empty for several months. Remember that you still have to pay the mortgage, insurance and other expenses even when there is no rent coming in! Make sure you have some money set aside for this up front.
Sometimes your tenants move out during a slow rental season; other times there can be enough work to do on the property that it can't be let for a month or two. This can leave the new owner so desperate to have money coming in that they jump at the first prospective tenant. That can ultimately cause even more serious problems in the long run.
Failing to Understand Tax Requirements
Tax policies on investment properties are different from taxes on homeowners, and the new investor must be prepared to build the expenses into the monthly rental price. Income from rents is considered as taxable income, and it is important to anticipate this when calculating how much you are taking in from your investment properties.
Moreover, beginning in 2015, the Government is making significant changes in tax policy for buy-to-let properties. The website "This Is Money" states that mortgage interest relief, the 'wear and tear' allowance, and other tax relief schemes will be phased out over the next few years. Make sure you learn about these changes and plan accordingly.
Failing to Vet Tenants Thoroughly
Doing proper background research on prospective tenants is critically important, but inexperienced landlords often want to get the property occupied quickly, and hesitate at spending the money needed to verify a renter's credit and background. Not only can you end up with a disastrous deadbeat in your house, but you can also find yourself in trouble with the law if you haven't checked factors like immigration status ("Right to Rent").
Failing to Keep Up the Property
New buy-to-let owners sometimes find themselves too busy to respond to maintenance issues quickly. This can lead to unhappy tenants who move out when the letting period is up, or who file complaints against you with the local authorities. It can also lead to extensive and expensive damage to the property if the issue is a leak in a roof or inside a wall. If you don't have the time and money to handle repair and maintenance issues, investing in buy-to-let properties may not be the right move for you.
One key way to avoid many of these problems is to have a relationship with an estate agency. A good agency will not only help you find the perfect buy-to-let property for your investment goals, but will also work with you in letting the property and managing the tenants. Although working through a letting agent may reduce your profits, it can save you untold amounts of time, energy, and stress in knowing that the details are handled.
If you decide to work with a letting agent, look for one that will handle much of the routine business, such as:
–Finding and vetting tenants
–Handling move in and move out times
–Collecting rent
–Inspecting the property periodically
–Documenting and keeping up safety certifications
–Receiving and managing routine maintenance requests
–Keeping thorough documentation of inspections, repairs, and information needed for tax reporting.
An established estate agency such as StrattonCreber.co.uk has a large local operation as well as a network of nationwide service partners. This is exactly what you need on your team as a novice buy-to-let investor. With an experienced letting agent working for you, you will be able to avoid the costly learning curve that beginner investors can face on their own.
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A Comprehensive Inflation Solution. Inflationary pressure can rise without warning and can be triggered by many factors. The Multi-Asset Real Return Fund can help investors be prepared for a range of economic scenarios with a comprehensive approach that's designed to help preserve purchasing power.
Comprehensive Solution
The fund is designed to help buffer investors' portfolios from the primary sources of inflation:
- Government-induced inflation driven by low interest rates and high spending
- Imported inflation caused by weak demand for the U.S. dollar
- Commodity inflation triggered by scarce supplies or strong economic growth
Strategic Approach
The fund seeks total real return through a mix of inflation hedges, each of which offers unique performance characteristics. This approach is aimed at helping investors preserve purchasing power under a range of inflation scenarios.
Alternative Asset Classes
The Multi-Asset Real Return Fund may be a good fit for investors seeking to diversify equity and fixed income risk. Since the alternative asset classes targeted by the fund have historically had low or negative correlation to the stock and bond markets, the fund has the potential to:
- Outperform when traditional asset classes are out of favor
- Provide a buffer in market downturns
A Note About Risk
Since inflation-indexed securities trade at prevailing real, or after-inflation, interest rates, changes in these rates affect the value of such securities owned by the fund. The fund may also invest in debt securities issued by entities other than the U.S. Treasury or U.S. government and its agencies, which may increase the potential credit risk associated with the fund. In addition, the fund's commodity-related investments may be subject to greater volatility than investments in traditional securities. Generally, when real interest rates rise, the value of these securities will decline. The opposite is true when real interest rates decline. Debt securities also are subject to credit risk. Investing in foreign securities has certain unique risks that make it generally riskier than investing in U.S. securities. Investing in securities of issuers located in emerging market countries generally is riskier than investing in securities of companies located in foreign developed countries. There is no guarantee that the investment objectives will be met. Dividends and yields represent past performance and there is no guarantee that they will continue to be paid.
Fund Management
Robert Gahagan
Senior Vice President, Senior Portfolio Manager
John Lovito
Senior Vice President, Senior Portfolio Manager
Brian Howell
Vice President, Senior Portfolio Manager
Steven Brown
Senior Vice President, Senior Portfolio Manager
Peruvemba Satish, CFA
Senior Vice President and Portfolio Manager, Director of Global Analytics
Data reflects past performance for Investor Class shares, assumes reinvestment of dividends and capital gains and is no guarantee of future results. Current performance may be higher or lower than data shown. Investment return and principal value fluctuates. Redemption value may be more or less than original cost. Obtain performance data current to the most recent month end. For additional share class information, consult the prospectus. Extraordinary performance is attributable in part to unusually favorable market conditions and may not be repeated or consistently achieved in the future.
W2 If your account falls below the minimum balance, you will be notified and given 90 days to meet the minimum. If the deadline is not met, American Century Investments may redeem the shares in the account and send the proceeds to the address of record.
Expense ratios provided are the funds' total annual operating expense ratios, gross of any fee waivers or expense reimbursements. Expense ratios are as of the funds' most current prospectus.
Lipper rankings are based on average annual total returns. Portions of the mutual fund performance information contained on this page were supplied by Lipper, a Thomson Reuters Company, subject to the following: Copyright 2017 © Thomson Reuters. All rights reserved. Any copying, republication or redistribution of Lipper content, including by caching, framing or similar means, is expressly prohibited without the prior written consent of Lipper. Lipper shall not be liable for any errors or delays in the content, or for any actions taken in reliance thereon. Performance data is preliminary and subject to revision. Rankings are based on all classes available within the classification as of the date shown. Rankings are based only on the universe shown.
©2017 Morningstar, Inc. All Rights Reserved. Certain information contained herein: (1) is proprietary to Morningstar and/or its content providers; (2) may not be copied or distributed; and (3) is not warranted to be accurate, complete or timely. Neither Morningstar nor its content providers are responsible for any damages or losses arising from any use of this information.
Source: Bloomberg Index Services Ltd


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Start planning your pathway to transfer. Meet with 4-year college and university representatives here at Thomas Nelson.
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text:
- Beverley Boyd
- 2 years ago
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1 METHODOLOGICAL ASPECTS AND APPLICATIONS OF FLUORESCENT IN SITU HYBRIDIZATION TO IDENTIFY BACTERIA FROM THE GASTROINTESTINAL TRACT OF TURKEYS Agnieszka Skowrońska 1, Dariusz Mikulski 2, Marzena Mikulska 2, Piotr Zapotoczny 3, Izabela Zmysłowska 1, Piotr M. Szczypiński 4 1 Department of Environmental Microbiology, Faculty of Environmental Protection and Fisheries, Warmia and Mazury University, Prawocheńskiego 1, Olsztyn, Poland 2 Department of Poultry Science, Faculty of Animal Bioengineering, Warmia and Mazury University, Oczapowskiego 5, Olsztyn, Poland 3 Department of Agricultural Process Engineering, Faculty of Technical Sciences, Warmia and Mazury University, Heweliusza 14, Olsztyn, Poland 4 Institute of Electronics, Technical University, Wólczańska 211/215, Łódź, Poland Summary. The microflora of avian gastrointestinal tract has received increased attention as the focus of efforts to improve animal nutrition and to minimize food-borne illness in humans. This microflora is subjected to variations according to diet, environment and treatments. Conventional methods for determining the flora composition rely on the cultivation of bacteria on anaerobic selective media. This approach is time-consuming because many bacteria are difficult to culture or are uncultivable. Molecular tools introduced in microbial ecology have made it possible to study the composition of intestinal flora in a culture-independent way. Our paper describes a methodological aspects and application of FISH method with rrna-targeted fluorescent probes to identify bacteria from the gastrointestinal tract of turkey. This technique poses advantages compared with traditional culture-based methods as it does not require cultivation of the target organisms. FISH enables to address simultaneously various ecological issues: 1. to by-pass cultivation problems, 2. to identify and to accurately enumerate prokaryote sub-populations in natural systems by using sets of probes 3. to analyze their spatial distributions. Although FISH has been used widely in bacterial ecology, the method requires some adaptation to the type of samples analyzed. Applying the optimized FISH method for the analysis of turkey caecum, we were able to identify Eubacterium rectale, the genera Enterobacteriaceae, Bacteroides, Bifidobacterium, and Lactobacillus-Enterococcus groups. Keywords: bacteria, oligonucleotide probes, 16S rrna, FISH, caecum, turkey. FLUORESCENCINĖS HIBRIDIZACIJOS IN SITU (FISH) METODOLOGINIAI ASPEKTAI IR PRITAIKYMAS KALAKUTŲ VIRŠKINAMOJO TRAKTO BAKTERIJOMS IDENTIFIKUOTI Agnieszka Skowrońska 1, Dariusz Mikulski 2, Marzena Mikulska 2, Piotr Zapotoczny 3, Izabela Zmysłowska 1, Piotr M. Szczypiński 4 1 Aplinkos mikrobiologijos katedra, Aplinkosaugos ir žuvininkystės fakultetas, Varmijos-Mozūrijos universitetas, Pravochenskiego 1, Olštinas, Lenkija 2 Paukštininkystės katedra, Gyvūnų bioinžinerijos fakultetas, Varmijos-Mozūrijos universitetas, Oczapovskiego 5, Olštinas, Lenkija 3 Agrotechnikos katedra, Technikos mokslų fakultetas, Varmijos-Mozūrijos universitetas, Heveliusza 14, Olštinas, Lenkija 4 Elektronikos institutas, Technikos universitetas, Wolczanska 211/215, Lodzė, Lenkija Santrauka. Pastebėta padidėjęs domėjimasis paukščių virškinamojo trakto mikroflora norima pagerinti gyvūnų mitybą ir sumažinti maisto produktų sukeltų žmogaus ligų riziką. Mikroflora gali kisti priklausomai nuo lesalo, aplinkos ir taikomų procedūrų. Įprastiniai floros sudėties nustatymo metodai pagrįsti bakterijų auginimu atrankinėse anaerobinėse terpėse. Jie reikalauja daug laiko, nes daugumą bakterijų kultūrų sunku auginti arba jų pasėliai visai nesidaugina. Molekulinės tyrimo priemonės, įdiegtos mikroorganizmų ekologijoje, leidžia tyrinėti žarnyno floros sudėtį be bakterijų pasėlių. Straipsnyje aprašomi FISH metodo su fluorescenciniais žymekliais, orientuotais į rrna, metodologiniai aspektai ir pritaikymas identifikuoti kalakutų skrandžio bei žarnyno trakto bakterijas. Šis būdas turi privalumų palyginti su įprastais bakterijų pasėliu pagrįstais metodais, nes tiriamų organizmų nereikia dauginti. FISH leidžia vienu metu spręsti skirtingus ekologinius klausimus: 1. apeiti bakterijų dauginimo problemas; 2. panaudojant žymeklių rinkinius identifikuoti ir tiksliai apskaičiuoti prokariotinių ląstelių pogrupius natūraliose sistemose; 3. išanalizuoti jų erdvinį pasiskirstymą. Nors FISH metodas plačiai taikomas bakterinėje ekologijoje, jį reikia pritaikyti konkretiems analizuojamiems mėginiams. Pritaikę FISH metodą kalakutų aklosios žarnos analizei, mes galėjome identifikuoti Eubacterium rectale, Enterobacteriaceae, Bacteroides, Bifidobacterium gentis ir Lactobacillus-Enterococcus grupes. Raktažodžiai: bakterijos, oligonukleotidų žymekliai, 16S rrnr, FISH, akloji žarna, kalakutai. 65
2 Introduction. In modern poultry production, breeding measures which guarantee adequate development and functioning of the gastrointestinal tract are required to keep the birds in good health. Nutrition is the main factor which determines the host's resistance to bacteria colonizing the alimentary tract. Adequate and fully active gastrointestinal microflora stimulates the immune system, protects the host from intestinal infections and improves the assimilability and utilization of feed nutrients (Mead, 1997; Vispo, Karasov, 1997; van der Wielen et al., 2002). The importance of microflora of the avian gastrointestinal tract has been recognized in recent research studies to determine the effectiveness of various biostimulators (including oligosaccharides, ß-glucan, priobiotic bacteria and essential oils) with the aim of stimulating the activity of beneficial bacteria and controlling the proliferation of pathogenic bacteria (Apajalahti et al., 2001, 2004; Mitsch et al., 2004; Immerseel et al., 2005; Mikulski et al., 2008). The gastrointestinal tract of animals is colonized by a highly complex and diversified microbial community comprising several hundred types of bacteria, mostly obligate anaerobes of the domain Bacteria. Dietary nutrients provide gastrointestinal bacteria with the energy required for growth and proliferation. Their nutritional requirements vary, which is why the chemical composition and components of gastric contents largely determine the spatial distribution of different bacteria in the alimentary tract. The process of isolation and biochemical differentiation of bacteria is difficult to perform in an anaerobic environment for technical reasons. Despite numerous research efforts, adequate conditions supporting in vitro growth have not yet been developed for most bacteria (Amann et al., 1995). In the natural environment of the gastrointestinal tract, bacterial communities are highly diversified populations of interdependent bacterial strains. Conventional medical and veterinary diagnostic methods based on an analysis of phenotypic characters (morphological, biochemical, serological) are insufficient for effective identification of bacteria. The above is largely due to the phenotypic diversity of bacteria. The process of culturing a sufficient bacterial population for this type of analyses is also time-consuming. The main limitation of growing methods is posed by the fact that only 10 to 60% of bacteria isolated from the gastrointestinal tract of broiler chickens are capable of in vitro growth (Barnes et al., 1972, 1979; Salanitro et al., 1974). On the other hand, microbial populations which are successfully cultured in a laboratory do not always behave in the same way as in their natural environment where the availability of nutrients is limited and mutual metabolic interactions are observed between bacteria. Molecular biology methods (Table 1) relying on genetic material analyses were introduced to conventional microbiological practice in the late 1980s. The PCR method, PCR-rRNA cloning and sequencing generated information on the structure of microbial communities from various environments. Today, universal and specific primers enable rrna gene amplification from potentially every bacterial sample, omitting the culture stage. The amplified region can be then sequenced and compared with the sequences deposited in the GenBank (http://ncbi.mnl.nih.gov/blast/cgi). The sequencing strategy has been universally applied to compare rrna gene sequences from various microbial communities based on variable regions, and to identify the resulting affinity. The rrna sequences complementary to 16S rrna and 23 rrna genes facilitated the design of probes for identifying bacteria from both the isolated microbe as well as those known solely by the sequence. The number of deposited bacterial sequences continues to increase, and it supports the subsequent design of specific probes. Table 1. Comparison of currently applied methods for analyses of microbial communities Method Usefulness of method Limitations Growing microbial - non-representative; possibility of isolation, "ideal" cultures - time-consuming and laborious analyses - incomplete lysis of selected microbes in 16S rdna sequencing phylogenetic identification a given population; - not all rrna genes are equally amplified; - time-consuming interpretation of sequencing results DGGE/TGGE/TTGE inhibitors in PCR sample; - monitoring sudden changes in semiquantitative method; microbial populations; - identification requires cloning analyses and - fast comparative analyses access to a clone library FISH - count of microorganisms of a given type in a sample; - quantitative method; - automated image analysis; - physical association of various microorganisms - incomplete permeabilization of selected bacteria; - environment-specific probe; - information on various microbial sequences is required Fluorescent in situ hybridization (FISH) is one of the most advanced molecular biology methods which are popularly applied in both environmental and medical microbiology (Amann et al., 1995; Moter, Gobel, 2000). The FISH method is used to detect target sites in the RNA sequence of various microbes (including both cultured 66
3 microorganisms and those known solely by the sequence) with the use of fluorescent labeled probes. Based on the sequence of the 16S rrna gene, molecular probes are developed in the form of DNA fragments (rdna probes) which are capable of hybridizing specifically with rrna and rrna genes of the given bacterial type, genus species or strain. Probes may also consist of short gene fragments encoding the synthesis of toxins and hemolysins which determine the pathogenicity and antibiotic resistance of an organism (Zakrzewska-Czerwińska, 2002). The FISH method also indicates whether the sequences obtained by PCR/sequencing are represented in the investigated environment. As an additional advantage, the method enables precise enumeration of microbial populations and their physical association in diverse communities. FISH does not require DNA isolation from the studied microorganism because cells are immobilized in a solid medium. Fast-growing cells contain a sufficient number of ribosomes ( ), producing a strong signal when a labeled probe binds to the rrna of a single cell (Figure 1). A B C Figure 1. Examples of hybridization images: A Eub338 probe identifying all bacteria of the domain Bacteria, B Erec482 probe identifying Clostridium coccoides and Eubacterium rectale, C Lab158 probe identifying Lacobacillus-Enterococcus. Left side DAPI image blue-stained bacteria. Right side post-hybridization image redstained bacteria 67
4 The aim of our study was to apply the FISH method. Our paper describes a methodological aspects and application of FISH method with rrna-targeted fluorescent probes to identify bacteria from the gastrointestinal tract of turkey. Material and methods. In own study (manuscript under preparation) of the cecal digesta of 8-week-old turkeys, the authors applied the membrane filter method to determine the population size of selected bacterial groups: Bacteroides, Clostridium coccoides-eubacterium rectale, Enterobacteriaceae, Lactobacillus-Enterococcus and Bifidobacterium. Samples of cecal digesta (approximately 1.5 g) were diluted with sterile physiological saline and placed in a stomacher (Stomacher BagMixer 400 Interscience). The supernatant was collected after stomaching and fixed with paraformaldehyde for 12h at 4ºC. The fixed bacterial suspension was centrifuged ( rpm for 10 minutes), bacteria were deposited on white 0.2 µm polycarbonate filters (Millipore) by filtration with the use of sterile filtration devices (Nalgene). Filters were then stained with DAPI dye (Porter, Feig, 1980). A thick bacterial suspension with local biofilm conglomerates was observed under a fluorescent microscope. Numerous bacterial colonies prevented an effective microbial count. The suspension was further diluted (1:150) to improve microbial count effectiveness, and an adequate quantity of the bacterial suspension was deposited on white polycarbonate filters. 8.0 µm membrane filters (Sartorius) were additionally used during filtration to uniformly distribute bacteria on the filter. Filters were rinsed with sterile distilled water and excess water was removed by gentle filtration. Filters were thoroughly dried and stored in Petri dishes at -20ºC for further analyses. The hybridization procedure was conduced in accordance with the method described by Fuchs et al. (2007) and Langendijk et al. (1995). Filters were cut into sections and the buffer was prepared for hybridization (900mM NaCl, 20mM Tris/HCl [ph 7.4], 0.01%SDS, formamide, deionized water). Probes were labeled with Cy3 cyanine dye at the 5' terminal end (Thermo ElectronGmbH, Germany). The hybridization mix comprising the hybridization buffer and a probe (50 ng µl -1 ) was stored on ice. Filters were placed on slides and the buffer with the probe were applied. Filters were placed horizontally in Falcon test tubes and the remaining hybridization buffer was added. Hybridization was carried out in a darkened incubator. As suggested by reference data, formamide concentration, hybridization temperature and incubation time were modified subject to the type of the applied probe. To identify Gram(+) bacteria, lysozyme diluted in the TE- HIS buffer (100mM Tris-HCL [ph 8.0], 50mM EDTA) was applied and was incubated for 10 minutes at room temperature. Rinse buffer (900mM NaCl, 20mM Tris/HCl, 50mM EDTA [ph 8.0], 0.01%SDS, deionized water) was prepared after hybridization and filters were rinsed for minutes in a water bath. Similarly to hybridization, the temperature of the rinse buffer and rinsing time were modified subject to the type of probe. Then the filters were rinsed with sterile deionized water (80% ethanol), and dried thoroughly. Dried filters were stained with DAPI dye according to the method proposed by Porter and Feig (1980), they were moisturized with a combination of Citifluor-Vectashield oils and glued to slides. Stained bacterial cells were viewed under a fluorescent microscope (Olympus BX 51) equipped with a set of filters for DAPI (Ex nm, DM 400 nm, BA 420 nm) and CY3 (Ex nm, DM 505 nm, BA 520 nm). 20 photographs of each filter were taken from random fields for both DAPI and hybridized bacteria (Figure 1). The authors relied on the modified version of the MaZda 4.5 application (Institute of Electronics in Łódź) to count bacteria in the DAPI image, which showed all bacteria stained light blue, and in the posthybridization image, where the identified bacteria were stained red (Fig. 1). Discussion. In 1988, Stahl et al. (1988) were the first to demonstrate that 16S rrna-targeted probes can be applied in the bovine gastrointestinal tract without resorting to culture methods. They used specific oligonucleotide probes identifying the strains Fibrobacter succinogenes and Lachnospira multiparus in the alimentary tract of cattle. As regards bacteria identified in the gastric contents of birds, a typical protocol has to be changed and optimized. Sampled digesta should be thoroughly homogenized or stomached. This is very important because only a small quantity of gastric contents is to be analyzed at subsequent stages, which is why the collected sample has to be representative. Undigested residues and other particles are removed by short centrifugation (<300 g). A similar procedure is adopted regardless of the technical hybridization variant, and it comprises the following stages: (a) initial preparation of samples, including stomaching, homogenization, centrifuging and fixing (Manz et al., 1995; Langendijk et al., 1995; Barc et al., 2003), (b) for the identification of Gram(+) bacteria the use of enzymes (e.g. lysozyme, achromopeptidase, lipase) for cell wall digestion, (c) hybridization with a selected probe, (d) washing to remove the unbound probe, (e) viewing the identified bacteria under a fluorescent or confocal microscope or by flow cytometry, and documenting the results. Published data indicate that most research studies rely on the FISH method combined with fluorescence microscopy (Langendijk et al., 1995; Zoetendal et al., 2002). When combined with flow cytometry (Wallner et al., 1993; Barc et al., 2003), those methods can be deployed for multiparametric analyses and relatively rapid and sensitive quantitative analyses of bacterial populations, including those with a percentage share of less than 1%. The correct fixing of biological material is one of the prerequisites for effective hybridization in situ. Prior to hybridization, bacteria should be fixed to account for the permeabilization stage, i.e. a process which facilitates probe insertion into a cell. It preserves the integrity and morphological structure of cells and protects RNA from degradation caused by the presence of endogenous RNases. Precipitation fixatives (ethanol, methanol) or cross-linking fixatives (formalin, paraformaldehyde) may be applied. Fixing conditions may differ subject to the 68
5 type of sample, the probe and the planned method of visualizing the results. Effective fixing has a decisive impact on the visualization of bacteria and is difficult to optimize. Most authors believe 3-4% formaldehyde or paraformaldehyde is the optimal fixative because it facilitates the identification of Gram(-) bacteria. Gram(+) bacteria may be identified with the use of 50% ethanol or ethanol/formalin (9:1). An optimal hybridization environment (temperature, salt [monovalent cation] concentration in the hybridization mix, incubation time and formamide concentration) should be created to maximize reaction specificity. In most cases, formamide concentrations higher than 50% are not applied because reaction rate decreases as formamide concentration increases. Hybridization takes place in a darkened incubator at a temperature of 37ºC to 50ºC. Most 16S rrna-targeted probes applied in FISH are oligonucleotide probes containing from 15 to 25 nucleotides (Table 2). The time required for the formation of complementary hydrogen bonds between the probe and the studied rrna fragment ranges from 30 minutes to several hours. Reaction sensitivity is also determined by the type of fluorescent tracer which is covalently bonded to the probe at the 5' terminal end. The most popularly applied probe labels are fluorescein, tetramethylrhodamine, Texas Red and Cyanine-3. Their emission spectra are a measure of signal intensity emitted by every hybridized cell. Proper hybridization may be conducted with the use of membrane filters (Pernthaler et al., 2002) and gel-coated slides (Manz et al., 1992; Fuchs et al., 2007). Both methods may be successfully applied to identify gastrointestinal bacteria. Table 2. Oligonucleotide probes used for the identification of bacteria isolated from the gastrointestinal tract Probe Probe specificity Probe sequence (5' - 3') References Eub338 Bacteria without Planctomycetales GCTGCCTCCCGTAGGAGT Amann et al., 1990 Non388 Nonsense probe for identification ACTCCTACGGGAGGCAGC Wallner et al., 1993 Bif164 Bifidobacterium CATCCGGCATTACCACCC Langendijk et al., 1995 Enter1433 Enterobacteriaceae GTTTTGCAACCCACT Sghir et al., 2000 Bac1080 Bacteroides GCACTTAAGCCGACACCT Dore et al., 1998 Erec482 C.coccoides-E. rectale GCTTCTTAGTCAGGTACCG Franks et al., 1998 Lab158 Lactobacillus-Enterococcus GGTATTAGCA(C/T)CTGTTTCCA Harmsen et al., 1999 Chis150 Clostridiaceae TTATGCGGTATTAATCTYCCTTT Franks et al., 1998 Eco1531 Enterobacteriaceae spp CACCGTAGTGCCTCGTCATCA Polusen et al., 1993 Unk730 Unidentified intestinal bacteria, DF1 TAGAGCCCAGTAAACCGC Huber et al., 2004 Computer-aided image analysis is becoming an increasingly popular tool in medical microbiology. A fluorescence microscope should be equipped with efficient analyzing software, sensitive CCD cameras and at least three sets of fluorescence filters for observation at the most popular light, excitation and emission wavelengths (blue/green fluorescein, UV/blue DAPI, green/red rhodamine, cyanine). Quantitative and qualitative parameters of the investigated object can be identified in the image with the use of computer software. The image produced by the registering device is not always legible enough to identify objects with the use of simple image processing functions. As regards analyses of bacteria from turkey cecal contents, a vast number of objects which are displayed in photographs after hybridization and DAPI staining formed conglomerates that were difficult to count. A single photograph often contained around bacteria (DAPI image), some of which came into contact, overlapped or were in the process of division. Several lightness maxima were observed within the contour of a single bacterium which significantly obstructed the process of computing the binarization threshold and counting. Despite the availability of filters and transformation options, standard software does not always guarantee the achievement of the set objective. If this is the case, dedicated software should be developed. The authors relied on the modified version of the MaZda 4.5 application (Institute of Electronics in Łódź) to count bacteria. The application enables the user to change the following settings "threshold change step", "merge object step", "background intensity", "smooth object contour", "object depth" to obtain the correct segmentation threshold. The following operations support image analysis: binarization, erosion, dilation, morphological properties, median filters and histogram normalization. The program features additional options such as multiple image processing (faster count) and monitoring the correctness of the analysis by overlapping the input file with the source file. Conclusion. To conclude, the FISH method supported direct identification, visualization and location of individual bacteria in the caecum of turkeys. It is a highly sensitive analytical technique for identifying very demanding bacteria which are difficult to culture in a laboratory. The FISH method also enables to track changes in microbial structures caused by various factors. Acknowledgments. This study was supported by the Polish Ministry of Science and Higer Education (year ), as a scientific project (grant number N N ). References 1. Amann R., Ludwig W., Schleifer K-H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev Vol. 59. P Amann R.I, Binder B.J., Olson R.J., Chischolm S.W., Devereux R., Stahl D.A. Combination of 16S 69
6 rrna-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol Vol. 56. P Apajalahti J., Kettunen A., Graham H. Characteristics of the gastrointestinal microbial communities, with special reference to the chicken. World`s Poult. Sci. J Vol. 60. P Apajalahti J.H., Kettunen A., Bedford M.R., Holben W.E. Percent of G+C profiling accurately reveals diet-related differences in the gastrointestinal microbial community of broiler chickens. Appl. Environ. Microbiol Vol. 67. P Barc M.C., Bourlioux F., Rigottier-Gois L., Charrin-Sarnel C., Janoir C., Boureau H., Dore J., Collignon A. Effect of amoxicillin-clavulanic acid on human fecal flora in a gnotobiotic mouse model assessed with fluorescence hybridization using groupspecific 16S rrna probes in combination with flow cytometry. Apply. Environ. Microbiol Vol. 48: Barnes E.M., Mead G.C., Barnum D.A., Harry E.G. The intestinal flora of the chicken in the period 2-6 weeks of age, with particular reference to anaerobic bacteria. Br. Poult. Sci Vol. 13. P Barnes E.M. The intestinal microflora of poultry and game birds during life and storage. J. Appl. Bacteriol Vol. 46. P Dore J., Sghir A., Hannequart-Gramet G., Corthier G., Pochart P. Design and cultivation of 16S rrna targeted oligonucleotide probe specific detection and quantification of human fecal Bacteroides populations. Syst. Appl. Microbiol Vol. 21. P Franks A.H., Harmsen H.J.M., Raangs G.C., Jansen G.J., Schut F., Welling G.W. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group specific 16S rrna-targeted oligonucleotide probes. Appl. Environ. Microbiol Vol. 64. P Fuchs B. M., Pernthaler J., Amann R. Single cell identification by fluorescence in situ hybridization. Methods for General Molecular Microbiology Ed Harmsen H.J.M., Elfferich P., Schut F., Welling G.W. A 16S rrna targeted probe for the detection of lactobacilli and enterococci in fecal sample by fluorescent in situ hybridization. Microb. Ecol. Health. Dis Vol. 11, Huber I., Spanggard B., Appel K.F., Rossen L., Nielsen T., Gram L. Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, walbaum). J. Appl. Microbiol Vol. 96. P Immerseel F.Van, Boyen F., Gantois I., Timbermont L., Bohez L., Pasmans F., Haesebrouck F., 70 Ducatelle R. Supplementation of coated butyric acid in the feed reduces colonization and shedding of Salmonella in poultry. Poultry Sci Vol. 84. P Langendijk P.S., Schut F., Jansen G.J., Raangs G.C., Kamphuis G.R., Wilkinson M.H.F., Welling G.W. Quantitative Fluorescence in situ hybridization of Bifidobacterium spp. With genus specific 16S rrna-targeted probes and its application in fecal samples. Appl. Environ. Microbiol Vol. 61. P Manz W., Amann R., Ludwig W., Wagner M., Schleifer K.H. Phylogenetic oligonucleotide probes fort the major subclasses of Proteobacteria: Problems and solutions. Syst. Appl. Microbiol Vol. 15. P Mead G.C. Bacteria in the gastrointestinal tract of birds. Pages in Gastrointestinal Microbiology. 2. Gastrointestinal Microbes and Host Interactions. R. J. Mackie, B. A. White, and R. E. Isaacson, ed. Chapman and Hall, New York, Mikulski D., Zduńczyk Z., Jankowski J., Juśkiewicz J. Effects of organic acids or natural plant extracts added to diets for turkeys on growth performance, gastrointestinal tract metabolism and carcass characteristics. J. Anim. Feed Sci Vol. 17. P Mitsch P., Zitterl-Eglseer K., Kohler B., Gabler C., Losa R., Zimpernik I. The effect of two different blends of essential oil components on the proliferation of Clostridium perfringens in the intestines of broiler chickens. Poultry Sci Vol. 83. P Moter A., Gobel U.B. Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. J. Microbiol. Methods Vol. 41. P Pernthaler J., Glöckner F.O., Schönhuber W., Amann R. Fluorescent in situ hybridization (FISH) with oligonucleotide probes. Methods in Microbiology Vol. 30. P Porter K.G., Yvette S.F. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr Vol. 25. P Polusen L.K., Ballard G., Stahl D. Use of rrna fluorescence in situ hybridization for measuring the activity of single cells in young established biofilms. Appl. Environ. Microbiol Vol. 59. P Salanitro I.G., Fairchilds J.P., Zgornicki Y.D. Isolation, culture characteristics and identification of anaerobic bacteria from the chicken caecum. Appl. Microbiol Vol. 27. P Sghir A., Gramet G., Suau A., Rochet V., Pochart P., Dore J. Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridiza-
7 tion. Appl. Environ. Microbiol Vol. 66. P Stahl D.A. Phylogenetically based study of microbial ecosystem perturbation, in Biotechnology for crop protection, Hedin, PA., Menn JJ., and Hollingsworth RM., Eds. American Chemical Society Symposium Series 379, Van der Wielen P.W., Keuzenkamp D.A., Lipman L.J., van Knapen F., Biesterveld S. Spatial and temporal variation of the intestinal bacterial community in commercially raised broiler chickens during growth. Microb. Ecol Vol. 44. P Veldhuizen E.J.A., Tjeerdsma van Bokhoven J.M.L., Zweijtzer C., Burt S.A., Haagsman H.P. Structural requirements for the antimicrobial activity of carvacrol J. Agric. Food. Chem Vol. 54. P Vispo C., Karasov W.H. The interaction of avian gut microbes and their host: An elusive symbiosis. Pages in Gastroinestinal Microbiology. 1. Gastrointestinal Ecosystem and Fermentations. R. J. Mackie, and B. A. White, ed. Chapman and Hall, New York, Wallner G., Amann R., Beisker W. Optimizing fluorescent in situ hybridization with rrna-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry Vol. 14. P Yamamoto T., Morotomi M., Tanaka R. Speciesspecific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora. Appl. Environ. Microbiol Vol. 58. P Zakrzewska-Czerwińska J. Sondy molekularne w diagnostyce mikrobiologicznej. Biotechnologia Vol. 4. P Zoetendal E.G., Ben-Amor K., Harmsen H.J.M., Schut F., Akkermans A.D.L., Willem M. de Vos. Quantification of uncultured Ruminococcus obeumlike bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rrna-targeted probes. Appl. Environ. Microbiol Vol. 68. P Received 28 October 2008 Accepted 7 September
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47 Old Well Road #12A
Stamford, CT 06904
Sold Price: $1,088,999
Sold Date: May 11, 2017
Beds: 4
Baths: 3 | 1
Sq. Ft.: 3,400
Type: Single Family Home
Listing #99158070
Property Features
Location Information
County: Fairfield
Neighborhood Code: Springdale
Subdivision: new
Directions: Newfield Ave north to Eden Road Or Hope St north TO Eden road to Old Well Road
Interior Features
Interior: 9+ Ceilings, Central Vacuum System, Insulated Windows, Roughed-In Bath, Security System, Audio System
1/2 Baths: 1
Area Amenities: Golf Course, Health Club, Library, Medical Facility, Park, Public Transportation, Shopping/Mall
Has Fireplace: yes
Number of Fireplaces: 1
Heating: GAS, Hot Air, Hot Water, Zoned
Cooling: Cent. Air, Zoned
Appliances: Cook Top, Dishwasher, Dryer, Microwave, Range, Refrigerator, Wall Oven, Washer
Has Basement: yes
Basement Description: Bulkhead
Exterior Features
Style: Colonial
New Construction: Under Construction
Foundation: Concrete
Roof: Asphalt Shingle
Septic or Sewer: Sewer
Water: City Water
Parking Description: Attached
Has Garage: yes
Garage Spaces: 2
Lot Description: Level, Partially Fenced, Privacy Fence
Lot Size in Acres: 1
Building Total Area (Sq. Ft.): 3, 400
Waterfront Description: N/A
Has Waterfront: No
Elementary School: Per Board of Ed
Intermediate School: Per Board of Ed
Middle School: Per Board of Ed
High School: Stamford
Additional Information
Property Type: SFR
Property SubType: Single Family
Property SubType 2: Single Family
Year Built: 2017
Status: Sold
Is an REO: No
Is Aged Restricted: No
Home Owners Association: 0
Has Attic: 1
Tax Amount: 15, 000
$ per month
Year Fixed. % Interest Rate.
|Principal + Interest:
|Monthly Tax:
|Monthly Insurance:
Based on a fully amortized fixed rate loan. Ask your agent for the tax rates in your area. Insurance estimate is based on an average cost, your final premium cost will be determined by the type of coverage you select. This program only provides an estimate.
Seller's Representative:
Newbridge International Realty
Newbridge International Realty
© 2017 Greater Fairfield County CMLS, Inc.. All rights reserved.
The data relating to real estate on this web site comes in part from the Internet Data Exchange Program of the Consolidated Multiple Listing Service, Inc. Real estate listings held by brokerage firms other than William Raveis Real Estate are marked with either the IDX Detail Logo or the IDX Thumbnail Logo (a little black house surrounding the letters IDX) and detailed information about them includes the name of the listing broker.
Information Deemed Reliable but Not Guaranteed. Terms and Conditions of Use Agreement
Information Deemed Reliable but Not Guaranteed. Terms and Conditions of Use Agreement
GFC-CMLS (Fairfield) data last updated at July 26, 2017 8:05 AM ET
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text:
Q&A with Ron McNeil: The Fab Four Beatles tribute band member
Almost everyone likes the Beatles, and the band continues to be popular more than 40 years after its breakup. Unfortunately for fans, they won't have the chance to see the lads from Liverpool perform live again. This Friday, though, Beatles buffs can catch the next best thing at the Palace Theater.
The Fab Four, a Beatles tribute band, bill themselves as "the ultimate tribute." They give old fans a chance to relive their memories and allow younger fans to experience the music in a live setting. The band is known for its meticulous attention to detail, using the same instruments, sporting similar outfits and even wearing the same style of shoes as the original group.
The Daily Orange spoke with Ron McNeil, who plays the part of John Lennon, about what it takes to be in a tribute band.
The Daily Orange: How did the band start?
R.M.: I saw Ardy (Sarraf, who plays Paul McCartney) at a local Beatles convention in L.A. … And he was singing a song by Paul McCartney, and I just couldn't believe it. He sounded exactly like Paul McCartney. I just didn't know how he did it. It was like some kind of weird trick or something. So I went up to him and asked him if he would like to do it, and a couple of years later we formed a group.
The D.O.: When you all started out, how did you choose your roles?
R.M.: For me, I think I gravitate a little bit more to John as far as vocally and facially and stuff like that, and Ardy is kind of the same way, but poor Ardy, he had to learn how to play the bass left-handed. That's something I wasn't going to do. I said, "Forget it!"
The D.O.: What are your favorite songs to perform, and how does the audience react?
R.M.: We do the hits, and sometimes we'll throw a curveball, but … we cover their whole career. We do some interesting stuff, like "A Day in the Life." We perform live on stage, "Strawberry Fields," so there're a lot of songs (like) that. I think those two are the most challenging of all the songs to pull off live. It's only four guys. There're no tapes, there're no tracks, there's nothing. Everything is played on live.
The D.O.: How do you go about getting an authentic, "Beatles-esque" sound?
R.M.: I think a lot of it comes out of the instruments. We use all of the original instruments, and then, you know, just years and years of studying and just a love of the music, and trying to imitate the sounds the best we can. It's a work in progress. I mean, we're always improving, always trying to get it better.
The D.O.: Why do you think the Beatles' music still resonates with so many people today?
R.M.: I think the themes that they chose, they sing about love and peace and I think that, as long as that's still a prevalent theme, I think the Beatles' music will carry on.
Published on October 23, 2013 at 10:46 pm
Contact Zach: firstname.lastname@example.com


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Selling a car, just like buying one, is a big financial transaction. You may be familiar with CarMax as the best place to find and buy used cars, but millions of customers also count on us as an easy and convenient way to sell a car.
We're here to help answer your questions and share some tips so you can sell your car with confidence.
Here are the top 6 questions about selling a car that we've heard from customers like you.
Many owners overestimate the condition and value of their car, so it's smart to do a little research. There are quite a few online resources that can provide you with general guidance and help you estimate how much you can get for your car.
To get an offer for your car, bring your car to CarMax for an appraisal. Our trained appraisers take into account the following factors when evaluating your car and determining your offer:
"Minor imperfections have little impact on our appraisal offer, but major defects like frame or flood damage do," says Tom Marcey, CarMax's vice president of regional merchandising.
There are pros and cons to selling your car yourself. Going through Craigslist or similar websites may get you more money for your car. But for many people, the amount of time it takes and the uncertainties of dealing with strangers make it simply not worth the hassle.
Many of our customers come to CarMax for an appraisal as a starting point. "One reason our appraisal offers are good for seven days is because we know our customers may want to 'shop around' and compare other offers to ours," says Marcey. "And that's okay!"
Getting last-minute maintenance or a thorough cosmetic detailing may help your car make a positive first impression on potential private buyers. To our appraisal team, however, the long-term maintenance and care of your car is what's most important in determining our appraisal offer.
Another key factor is your car's mileage. "Keeping the miles low on any car is a great way to minimize depreciation impact over time," says Marcey.
What about after-market modifications and accessories - like a spoiler or a unique paint color? While these upgrades might make your car more distinctive, they may also limit your car's market appeal. However, certain upgrade packages that include options like leather seats and a Bluetooth connection may be beneficial when you're ready to sell.
Depending on the type of vehicle, new wear-and-tear components like brakes and tires can be a plus. High-end luxury and sports cars are often equipped with specialty high-performance tires and brakes, which can be expensive to replace.
For the average vehicle, replacing these items may not significantly affect our appraisal offer. If you have any questions about your car's appraisal, your CarMax appraiser can provide you with a detailed explanation about the factors that influenced your offer.
One key difference about CarMax is that our appraisal offer is the same whether you buy a car from us or not. That's not always the case at a dealership.
You may be familiar with "flexible" trade-in values that are standard practice for many in the industry. "If you got a generous price for your old car, it may mean you paid too much for your new car," notes Marcey.
Requirements for selling your car can vary from state to state, but there are a few key items you'll need to bring no matter where you're located.
Contact your local CarMax if you have any questions about what you need to bring with you. Once you've sold your car, don't forget to contact your insurance carrier to let them know.
When you're ready to sell your car, CarMax is a great place to start. We appraise millions of cars every year, and our goal is to make selling your car a quick and easy experience.


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Some advertesiments published by Aviaco in the Spanish printed media in the 90's:
"Con otro aire" (roughly translated, "with new, fresh air") : In the early 90's stress was made in the improvementes made in their onboard service. Until then, Aviaco had a reputation of being pretty spartan carrier.
This 1993 ad is about the size of the airline: 30 cities in their all-domestic network, 220 flights per day and more than 6 million passengers flown each year.
Las "tarifas de color" (1995) fueron la respuesta de la empresa a la creciente competencia de empresas privadas (sobre todo, Air Europa y Spanair) con precios más bajos en el mercado doméstico.
In 1995, Aviaco was promoting their "tarifas de color". They were introduced because of increading concurrency with lower fares (chiefly by Air Europa and Spanair) in the domestic market.
Dos anuncios más, de 1996 y 1997, los últimos antes de que la empresa pasara a ser una mera operadora de líneas para Iberia. El slogan de estos años era "volamos para unir" y los anuncios se centraban en las relaciones humanas que se unían con los vuelos de Aviaco.
Two further examples, from 1996 and 1997. These were their last years as and independent carrier before it started their final merge under the Iberia brand. The slogan was "volamos para unir" (roughly, "to fly, to link") and the ads were about linking feelings.


text:
- Description
A fabulous greeting card for Labrador owners and dog lovers! Featuring Sophie's illustrations of some adorable sitting and lying black Labrador dogs.
The card is part of our Labrador & Dogs collection. Any dog lover or dog owner would like to receive this card in the post!
- Part of the Greeting Cards range
- Sit! is written in Sophie's handwriting along the base of the card
- 15cm x 15cm square
- Comes with a brown envelope
- Neatly presented in a cello bag
- Blank for your own message
- Made in England
- FSC certified
- Care
- All our cards are printed onto 280gsm high quality white textured card.
- The card is a Feltmarked Board with an enhanced surface that helps to keep the printed colours bright and vibrant.
- The card is FSC Certified
- Elemental Chlorine Free - this means that it is whitened using ecological processes.
- Size
15cm x 15cm
- Delivery
UK Standard Delivery: £2.95 (3-7 working days) FREE if you spend over £50.
UK first class Delivery: £4.95 (1-2 working days).
UK Next Working Day Delivery: £7.95 (Order must be placed before midday).
Saturday delivery available if order placed by 12 noon on Friday: £12.95.
International Delivery: from £8.40 to Europe (allow 14 working days), from £10 to rest of the world (allow 14 working days). Calculated at checkout.
For more details see our Delivery information page.
- Reviews
Feefo Reviews - Average 100% (21 reviews)
Date Score Customer Comment Our Response 11-Jul-2017 Product:
Fun for a dog owning friend 06-Feb-2017 Product:
Really good quality - love it. 31-Jan-2017 Product:
a must-have for any Labrador owner. 26-Jan-2017 Product:
Excellent 21-Jan-2017 Product:
As Above 18-Jan-2017 Product:
As Above 16-Jan-2017 Product:
As Above 15-Jan-2017 Product:
As Above 14-Jan-2017 Product:
As Above 15-Sep-2016 Product:
As Above 14-Sep-2016 Product:
As Above 20-Aug-2016 Product:
Lovely good quality card. Dog lovers will adore this card. Thank you for taking the time to leave a review for the Sophie Allport Labrador Greeting Card.
02-May-2016 Product:
Excellent 17-Mar-2016 Product:
Gorgeous! more designs please! 16-Mar-2016 Product:
Lovely 02-Feb-2016 Product:
Delighted. 15-Sep-2015 Product:
Fun 01-Apr-2015 Product:
Excellent 26-Jan-2015 Product:
nice card 13-Nov-2014 Product:
Excellent Read more reviews on Feefo


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Miller, 45, who was number two to Barry Diller at USA Interactive, is expected to overhaul the online division and may cull what many see as a top-heavy management structure. He is also expected to try to win new subscribers with a more exciting blend of content and e-commerce.
AOL's advertising revenues have dropped sharply recently and its subscriber growth has slowed as US cable companies begin offering high-speed broadband internet access.
Miller's career has included the National Basketball Association's cable TV operations, Nickelodeon and the Home Shopping Network.
- More about:
- Advertising
- Broadband Technology
- Internet
- Market Research


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Subtotal: $ 0.00 USD
A week after Burt Reynolds revealed that he considers Sally Field to be the "love of his life," the actor has shared his opinion on another one of his past relationships.
This time, Reynolds is talking about his marriage to fellow actress, Loni Anderson. The pair were married in 1988 and have one son together, but divorced after just five years of marriage.
Photo Credit: AP Archives via NWI.com
Now, over 20 years later, Reynolds confesses that his whole relationship with Anderson was a huge mistake.
"I should have known that you don't marry an actress," Reynolds says in an interview with People. "That was a really dumb move on my part."
Reynolds says that it was Anderson's abuse of their finances that made her a difficult person to be in a relationship with.
"It wasn't lollipops and roses...she bought everything in triplicate, from every day dresses to jewelry to china and linens," he says.
Reynolds also says that Anderson would never wear a dress after she had been photographed in it, which was just another thing that added to her excessive spending.
"I gave her a platinum American Express card with a $45,000 credit limit. She maxed it out in half an hour," he says.
Photo Credit: Getty Images via Yahoo!
Reynolds opens up further about his life and his marriage to Anderson in his new memoir, But Enough About Me, which is set to be released on November 17.
Even though Reynolds has some regrets about his past, we hope that he can find some bits of happiness in his future. Maybe his new book will be a good start!
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Picking on General Motors long has been a national pastime for auto industry pundits. As the world's No.1 auto maker, there's always a ready supply of material. But GM is nothing if not aggressive. And lately it seems serious about putting its house in order. Not only is it churning out good-looking SUVs and trucks, and even some cars, at attractive prices, but it's also driving ahead in its pursuit of the future. For instance, the auto maker is allotting about 25% of its research ...
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