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Since, I am getting comfortable with yeast, the next thing I wanted to try baking is simple white sandwich bread. The best thing I have learned after couple of fail attempts and couple of success is that you have to be patient while working with yeast. It really takes time to shows its magic. And the outcome will always amazed you. I always considered baking bread would be a whole lot of task but within 2 1/2 hrs, I was done with everything and have put the bread in oven to bake…AMAZING!! Isn't it??
The best part when you bake your bread is your whole house is filled with freshly baked aroma which is mind-blowing. Anything 'Home-made' means you know where your food comes from and what's in it. Also, you can control the qualities of ingredients going in your food…Win-win!!
This is my favorite recipe and I loved the outcome, it is dense, hearty, crusty yet still moist and fresh. It is delicious with just some butter or jam on side. ahh..simply YUMM!! This recipe is surely a keeper in my house, I hope you guys also try this recipe and love it as much as I do.
Recipe Adapted from browneyedbaker
- 3 3/4 cup all purpose flour
- 2 1/4 teaspoon active dry yeast
- 2 tablespoon honey
- 2 tablespoon unsalted butter, melted and cooled
- 1 cup 2% milk, warm
- 1/4 cup water, warm
- 1 1/2 teaspoon salt
- In a bowl, combine milk, water, honey and microwave for 45-47 seconds or until the liquid is luke warm.
- To this fluid add the cooled butter and yeast and let it sit for 5 minutes or until the yeast gets active.
- In another bowl, mix together the salt and all-purpose flour.
- Pour the yeast mixture into bowl of dry ingredients, using a mixer with dough hook, or by hand, knead dough until smooth but not sticky.
- Coat a big bowl with the 1/2 teaspoon of oil, drop in the dough, and coat with oil all over. Seal the bowl with plastic wrap, and let it rise in a warm place until doubled, about an hour.You'll know the dough has finished rising when you poke it with your finger and it holds the depression and doesn't bounce back. At this point, punch down the dough lightly.
- Shape the dough into a rectangle about 1 inch thick, 4 inches wide, and 9 inch long. The length and width should be approximately the size of your loaf pan. Form it into a loaf by rolling the dough from the length side, pinching the crease with each rotation. In other words, shape the dough into loaves by stretching around the sides gently and forming a seam at the bottom.
- Place the loaf, seam side down, in a greased loaf pan, the ends of the loaf should touch the ends of the pan. Cover the pan with a kitchen towel and put them in a warm place to rise again for about 30 minutes or until the dough just comes lightly above the rims of the pan.
- Preheat the oven to 350 degrees with the oven rack on the middle shelf. Bake the bread for 40-45 minutes, or until the crust is well browned.
- When the bread is finished baking, remove it from the loaf pan and cool completely on a rack before slicing and enjoying the slices.
Now comes the award:
I got these awards from Deepa at Hamaree Rasoi..thanks Deepa for sharing the same with me, very kind and sweet of you dear..you are an inspiration, love your blog and recipes..Also, invite you all to visit her sites if you have not done already.
As the rule says, I have to forward these awards to 15 other bloggers to keep the ball rolling. And let the nominated bloggers know about their award. So, here's goes the name in a random order and everyone listed below, please accept the awards…Thanks!!
Aipi from US Masala, Nashira from Plateful, Ananda from A pinch of Love, KanKana from Sunshine and Smile, Sangeetha from Kothiyavunu, Shobha from Good Food , Biny from Binysrecipes, Priya from Priya's Easy and Tasty Recipes, Swathi from Zesty South Indian Kitchen , Sonia from My Creative Flavors, Sarah Naveen from @ Vazhayila, Sushma Mallya from Authentic Food Delight, Devaki from Weave a Thousand Flavors, Mary from One Perfect Bite, Joumana from Taste of Beirut


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I am going to keep my goals short an sweet this month.
July Goals:
1. My priority goal this month is to work on artwork. I am feeling the need to get back to making art, and now is the perfect time to do it. No greeting card holidays coming up for a few months now.
2. Bring 5 more of each card design to Parts and Labour. When I go back to drop off those cards, take a picture of Parts and Labour for a blog post.
3. Start research for business plan, and create a time line for writing business plan (time line to start this month).


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Dessert is pretty much synonymous with chocolate in our house. Chocolate souffles, hot fudge sundaes, and fondant au chocolat… as long as there is chocolate involved, my kids, my husband, and I just can't say no. Fruit desserts, on the other hand, can be a tough sell, but at the height of apple season, this salted caramel apple bread pudding is as much of a hit as any hot melt in your mouth chocolate concoction.
The salted caramel drizzle is what takes this dessert from good to delicious. It's a gooey blend of fall comfort food flavors. Throw a scoop of vanilla bean ice cream on top and you'll be moaning with every sweet, hot, and subtly salty bite.
Nothing about bread pudding should be confusing. Each bite should be soft and warm, simple comfort and sweetness. I cook the apples into apple sauce to keep any apple chunks from adding unnecessary crunch and texture. The apples become sweeter, and their flavor more intense, and it's a great time to infuse them with spices, adding interesting notes with Chinese five spice powder.
When the weather turns frigid and even the apples at the supermarket are dull and flavorless, we'll be able to indulge in chocolate to our heart's content. But as long as the apples are crispy and beautiful and the leaves are falling, we'll revel in this decadent bread pudding come dessert time.
- Bread Custard:
- 6 cups of leftover stale bread (1 package of sliced bread)
- 6 eggs (large)
- 1 cup of 2% milk
- 1 cup of heavy cream
- ½ cup dark brown sugar
- 2 Tablespoons of vanilla extract
- 2 teaspoons of Chinese five spice
- ¼ teaspoon of kosher salt
- Apples
- 6 granny smith apples
- ¼ cup of grade B maple syrup
- ¼ cup of dark brown sugar
- Salted Caramel
- ¼ cup of granulated sugar
- 2 tablespoons of butter
- 1 teaspoon of kosher salt
- 3 tablespoons of heavy cream (with another 2 tablespoons in case the sauce separates)
- In a large bowl, combine the eggs, milk, heavy cream, five spice powder, sugar, salt, and vanilla. Whisk until blended.
- Add bread cubes and push in until all the liquid is absorbed. Let mixture rest in the refrigerator for at least 30 minutes.
- Preheat the oven to 350 degrees Fahrenheit. Grease a bread pan with generous amounts of butter.
- Peel and dice the apples into coarse chunks. Place them in a medium saucepan with ½ cup of water, maple syrup and vanilla. Cook on medium-low heat until apples are completely soft. Using a fork, mush the apples roughly. You want to leave some texture here. This is not applesauce that needs to be fed to a baby.
- Remove the bread mixture from the fridge. Pour the apple sauce on top and swirl it a few times with a wooden spoon. You don't want to combine it too thoroughly. Leave a few spots in your batter without apple sauce and vice versa.
- Pour the mixture into the greased bread pan and bake for 50 to 60 minutes, when a knife comes out clean from the center of the bread pudding.
- To make the salted caramel glaze, pour the granulated sugar in a small frying pan. Heat on medium, let the sugar melt and turn a light copper. Take off the heat and add the butter, stirring vigorously. Once the butter is incorporated, add the heavy cream and put back on low heat for 2 to 3 minutes to bring the sauce together. If the sauce seems to be separating and chunking up, add another tablespoon or two of heavy cream. This is a glaze, you're only going to have a little bit, now is not the time to worry about being too generous with the heavy cream.
- To finish, toss in the salt and mix with a spoon until just incorporated.
- Drizzle the caramel sauce on the warm bread pudding. Watch it all meld into a delicious, fall treat. Save a little glaze to put on top of ice cream. Once you have it whipped up, you'll be adding it everywhere.
- Serve warm topped with vanilla ice cream, or after chilling.


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This truck has everything I wanted.Rides great great gas milage good power for smaller truck has more options than I will ever use but better to have and not need than to need and not haveRead more reviews at Cars.com
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TEL AVIV PRIDE 2016 – May 29th – June 6th
Day 1: Sunday, May 29th: Jerusalem
Shalom and welcome to Israel!
Upon arrival at Ben-Gurion Airport, you will be greeted by an OUTstanding Travel Representative.
Enjoy the comfort of a private transfer to your hotel in Jerusalem
Check in at your hotel in Jerusalem
Evening Welcome Dinner at one of Jerusalem's top restaurants.
Accommodations: Jerusalem
Meals included: Dinner
Day 2: Monday, May 30th: Jerusalem
Breakfast at the hotel.
Tour of the Old City of Jerusalem
After breakfast, we drive to the Mount of Olives for a panoramic view of the Old City of Jerusalem and a visit to the City of David, birthplace of Jerusalem. We shall pass through the underground tunnels through which Jerusalem was conquered and residents could slip out of the city, and experience the hidden spring where kings were crowned. Next, we walk in illuminated darkness through the waters of Hezekiah's Tunnel, where water has flowed since the time of the prophets and join the mysterious, magical journey between ancient shafts, walls and fortresses at the City of David.
On Mount Zion, visit King David's tomb, the Room of Last Supper and the Dormition Abbey and then we shall enter the Old City through Zion Gate to the Jewish Quarter. Together we tour the recently excavated and restored Roman Cardo once the ancient Roman road at the heart of the city. Our route will lead us to the Western Wall, Judaism's holiest site and the last remnant of the Second Temple. For the grand finale, we make our way to the Christian Quarter and walk along the Via Dolorosa to visit the Church of the Holy Sepulcher as we join pilgrims from around the world, venerating the Tomb of Jesus Christ.
Free evening in Jerusalem
Overnight in Jerusalem
Accommodations: Jerusalem
Meals Included: Breakfast
Day 3: Tuesday, May 31st: Jerusalem & the Dead Sea
Breakfast at the hotel
After breakfast we tour the new city of Jerusalem: A drive by the Knesset, Israel's parliament, the Supreme Court and Mount Herzl.
Visit Yad Vashem, the world's premiere Holocaust remembrance and education center, located on the Mount of Remembrance in Jerusalem.
Continue on to the Israel Museum, ranked among the world's top art and archeological museums: an experience for all the senses, and for all interests – from contemporary art, to folk art and archeology to sculpture and music. See the Model of Jerusalem as it was 2,000 years ago, as well as the Shrine of the Book where the Dead Sea Scrolls are housed.
Depart Jerusalem via the Judean Hills and visit Genesis Land, where you will experience life as it was in biblical times. Visitors to Genesis Land are greeted by Eliezer, Abraham's manservant, and by a train of camels which we will ride down to Abraham's tent of legendary hospitality.
Continue to the lowest point on earth and check in at your hotel at the Dead Sea.
Free time to swim and float on the Dead Sea
Dinner at the hotel
Overnight at the Dead Sea
Accommodations: Dead Sea
Meals Included: Breakfast, Dinner
Day 4: Wednesday, June 1st: Masada & the Galilee
Early morning wake up
Hike in the cool darkness to the very top of the ancient mountain fortress of Masada which was built in the year 30 BCE by King Herod, whose architectural feats have left their mark throughout Israel. At the beginning of the great revolt against Rome in the year 68 CE, Masada was conquered by a group of Jewish zealots, and Masada became their last stronghold. In the year 72 the Romans besieged Masada and succeeded in reaching the steep fortress after constructing a huge earthen ramp on its western side. In the year 73, the 960 Jewish zealots living at the top of Masada chose to commit suicide rather than to fall into the hands of the Romans alive. Their deeds left behind a saga of courage, heroism, and martyrdom.
On the top of Masada we will watch the day begin with a spectacular sunrise over the Moab Mountains and the Judean Desert. Then, at the top of the mountain, discover the history of one the greatest Jewish symbols of hope and pride. You will descend the mountain fortress by cable car.
Enjoy breakfast at your hotel at the Dead Sea
Free time at the Dead Sea to enjoy the SPA and the private beach
Depart the Dead Sea Resort and drive to the Galilee in Northern Israel
Visit Arbel Cliff and enjoy a breathtaking view of the Sea of Galilee and the Golan Heights.
Check in at your hotel in Tiberias
Sun and fun at a private beach on the Sea of Galilee
Dinner at the hotel
Overnight in Tiberias
Accommodations: Tiberias
Meals Included: Breakfast, Dinner
Day 5: Thursday, June 2nd: Golan/Nazareth/ Tel Aviv
Breakfast at the hotel
Enjoy a morning swim in the Sea of Galilee
Drive to the Golan Heights and enjoy a jeep tour that will take you into the depths of the beautiful and fascinating landscape of the Golan Heights, Israel's northern-most region. We will go into the heart of the hills and valleys that characterize this incredible region, with its beautiful nature, wildlife, history, and modern-day activities. Driving along dirt tracks, passing through streams, through orchards and groves, small villages, and extinct volcanic landforms.
Depart the Golan Heights and continue to Nazareth which according to the New Testament was the childhood home of Jesus. You'll visit the church of St Joseph which houses the Basilica of the Annunciation, an impressive site where the angel Gabriel informed Mary she would give birth to Jesus. Nazareth is the largest Arab city in Israel, with a distinct Middle Eastern flavor. Check out the charming local markets located in the winding cobbled streets of the Old City, as you explore the city.
Continue to Tel Aviv – a world hub of gay vibrancy and pride!
Titled the 'Mediterranean Capital of Cool' by the New York Times, this is a 24/7 city with a unique pulse, combining sandy Mediterranean beaches with a world-class nightlife, a buzzing cultural scene, incredible food, UNESCO recognized architecture, and an international outlook.
Start your Tel Aviv Pride weekend in one of the hottest parties in town!
Overnight in Tel Aviv
Accommodations: Carlton Hotel/Leonardo Art Hotel in Tel Aviv
Meals Included: Breakfast
Day 6: Friday, June 3rd: Tel Aviv Pride Parade
Breakfast at the hotel
Take part in the world-renowned Tel Aviv Pride Parade 2016
Celebrate freedom at the official Pride Beach Party
At night go wild at the official Mega Pride Party at HaOman 17 Club
Overnight in Tel Aviv
Accommodations: Tel Aviv
Meals Included: Breakfast
Day 7: Saturday, June 4th: Tel Aviv
Breakfast at the hotel
After a night of dancing and fun, enjoy some down time in Hilton Gay Beach with hot Israelis and tourists from all over the world
The party goes on! Paint the town red in another crazy gay party production.
Overnight in Tel Aviv
Accommodations: Tel Aviv
Meals Included: Breakfast
Day 8: Sunday, June 5th: The Western Galilee
Breakfast at the hotel
Morning at leisure
In the afternoon we will explore Israel's Northern Coastline: Caesarea & Haifa. Israel's Mediterranean Coast has been ruled by various empires throughout history who have created a fascinating mosaic of historic sites.
Our tour begins with a ride along the beautiful coastal highway to the ancient port of Caesarea, once an important and strategic part of the Roman Empire, today housing an impressive array of archaeological sites. Delve into history with a state of the art presentation explaining the city's colorful past. Explore lost treasures in the underwater antiquities park, go back in time with a walk inside the hippodrome and scale the impressive amphitheater, once a 15,000 seat open air auditorium which today remains largely intact and is still used for concerts and events.
From here, the beautiful city of Haifa awaits. Located on the slopes of Mount Carmel, Israel's third largest city is home to both Jews and Arabs, and is a melting pot of cultures and ideas. You'll have the chance to appreciate the beauty of Haifa with a visit to the Hanging Baha'i' Gardens – a world heritage site which is home to the Baha'i faith.
Our third and last stop on this day of touring will be Zikhron Yaakov, known for its boutique wineries. Visit one of the local wineries and taste some of Israel's finest wines.
We will drive back to Tel Aviv and enjoy a farewell dinner after the amazing 8 days in Israel.
Overnight in Tel Aviv
Accommodations: Tel Aviv
Meals Included: Breakfast, Dinner
Day 9: Monday, June 6th: Departure Day
Following breakfast, check out from the hotel and transfer to Ben Gurion International Airport for your flight back home with everlasting memories of Israel.
Goodbye & Shalom J
Tour Highlights
- Paint the town red in another crazy gay party production.
- Enjoy some down time in Hilton Gay Beach
- Golan Heights jeep tour
- Free time at the Dead Sea to enjoy the SPA
- Tour of the Old City of Jerusalem
Tour information
- Number of nights: 8
- Rates starting: 2149$
- Tour code: #p1234
Tour does not Include
- Expenses of a personal nature
- Meals throughout the entire tour other than mentioned
- Spa Treatments at the Dead Sea
- Tips to driver, guide and restaurant staff
- Any other item not mentioned above


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Tenant Shop work closely with a number of leading software providers to offer tenants and letting agents the best possible utility management service. Find out more about our integrated partners below.
VTUK specialise in providing tailor-made and bespoke property software solutions for property agents in sales, lettings and property management.
Property software is not a new concept. Many do it and do it well. We could do that, but for us, that's not enough. Of course, the functionality within our software meets our clients' needs, but we want it to offer much more than that, we want it to become an integral part of their business development and growth plans. Something that helps to drive their business, not just manage it and something that provides a genuine, identifiable return on investment. That's the basis on which we develop our range of software solutions. So whether you sell, let or manage property, at VTUK we'll have a product that meets your every need. And then some…
At VTUK our clients don't just get great software; they get software developed specifically to fit their needs. Our clients don't just get an annual call to check everything is OK; they get a dedicated account manager, regular contact and as much training as they need.
Our clients don't worry about paying for add-ons; because we don't charge them extra for access to our best-in-class third party supplier network, attending our business growth seminar programme or industry recognised round table dinner events. Our clients aren't clients, they are our partners. And when they succeed, we succeed – that's how we do business.
Blinc Tenant Referencing provides tenant referencing, rent protection warranty and property specific insurance products.
Blinc Tenant Referencing Services have designed market leading products and services for letting agents. Speed and simplicity is at the heart of what we do. We understand that this is a vital time for the agent, landlord and tenant, handling this part of the process with care, attention and a personal touch for everybody involved is absolutely essential.
Our free 'Right to Rent' verification service raises the bar in the Tenant Referencing industry. New rules and regulations have become increasingly common, our aim is to integrate these as part of our service with as minimal fuss as possible whilst also providing expert professional help and advice every step of the way.
If your goal is to offer the very best customer service for your landlords and tenants plus maximum protection, our superior range of Rent Guarantee and other insurance products offer the ultimate peace of mind for unrivalled value.
In addition to this, Blinc Tenant Referencing are proud to have taken referencing to a new level as an integrated partner with award winning Tenant Shop. No other referencing platform can offer a service which combines these essential tasks in such a seamless and efficient manner, proven to enhance the opportunity to earn commissions.
OnBoard Pro
OnBoard Pro is an innovative, cloud based pre-tenancy platform which combines the full move-in process into one efficient system.
OnBoard Pro is a young, rapidly growing and ambitious company leveraging advanced technology delivered via a Software as a Service (SaaS) solution. This software enables letting agents to deliver a significantly enhanced service to tenants and landlords. The OnBoard solution provides Agent and Tenant portals that enable credit checking, referencing, inventory management, meter reading and document storage in one easy-to-use application. The software can be branded to meet the agent or service providers' requirements and is already used by some of the best known traditional high street letting agents, digital-only operations and insurance companies.
RentPro is a comprehensive web-based property management software solution that helps landlords and letting agents manage all aspects of lettings, from rental arrears to void periods through to maintenance management.
As a web-based software solution, RentPro can be accessed from anywhere provided you have an internet connection, making remote working much easier.
RentPro is functionally rich yet intuitive to use. It is easily configured to work the way you work and uses feedback from our users to assist with its continuing evolution.
With its suite of reports and its on-screen alerts for items that need your attention, RentPro ensures you get timely information.
RentPro also allows you, should you wish, to provide landlords with their own login so that they can easily keep up-to-date with any repairs or maintenance, viewings or inspections. They can even run their own statements. What better way to demonstrate your competence and results to your landlords.
Properties can be easily marketed within RentPro at no additional cost, uploading to property portals as well as your own website and creating brochures for prospective tenants. RentPro also effortlessly matches prospective tenants with vacant and
upcoming available properties. Happy tenants equal happy landlords!
Thesaurus Technology
Thesaurus Technology produces cost effective, high quality software for estate and letting agents. They provide both desktop and internet based solutions that are suitable for all sizes of organisation.
Thesaurus Technology is an independent company that was established in 1999 and specialises in high quality software solutions (both cloud and desktop systems are available) for the estate and letting agent market. Based in Harrogate, the business provides software to help agents run their offices efficiently and gain an advantage in a competitive market.
The software is supported by a dedicated and highly skilled support team that will help you every step of the way.


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They had so many used books in great condition and most were a dollar or less. Plus they have a promotion where you buy 4 and get the 5th free. Yeah, I was in heaven! We got a few books for me and some for the kids. They were happy to have something "new" to read and look at in the car and I was happy it bought us at least a few minutes of sanity. :)
I have to say, I've been to several Savers now and really couldn't tell you about the rest of the store -- the books and I are besties. :) But it looks really clean inside so I'm sure they have more great finds. I'm thrilled that they *just* put one in down the street from me!!! I will have a fabulous library built up in no time.


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At Long Island University, we strive to prepare current and future students to lead in a fast-paced, ever-changing world and join a global network of over 265,000 alumni. As a benefactor of LIU, you will have the satisfaction of knowing your gift is having a profound impact on our students' lives.
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One and a half miles away from S. Joao da Pesqueira stands Quinta da Trovisca, 38 acres of robust vines of touriga nacional, touriga franca, sousão and tinta roriz. January is the optimal for prune, due to the local micro-climate while the vintage READ MORE
Quinta Vale d'Agodinho is located next to the Douro River which embraces 650 feet of the vineyards. Fifty luscious acres are full of the best varieties of the region. Quevedo vineyards showcase a small terroir of 50-years-old vines of strong structure, ageing potential READ MORE
Hello world!
QUEVEDO LAUNCHES THE FIRST BLOG FROM A PORT PRODUCER As of October, the Port Wine producer, QUEVEDO, has created a blog with the purpose of educating and promoting port wine worldwide, especially among first time wine consumers. Through this marketing communication initiative – READ MORE
Thank you very much for your visit. Now we wait for you in our winery! Oscar Quevedo


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Check out the 2014 Veteran Amputee Golf Classic tab for pictures from June 14th's tournament. Many thanks to our awesome volunteers and everything they did to try to get pictures of every team. If we somehow missed you or your team, we apologize and will do our best to include you in next year's pictures.
The winners of this year's tournament: Dave Bronson Jr. (VAGA Member), Dave Bronson Sr., Jon Riley, and Chris Lambert.


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Recent weeks have seen some extraordinary moves in the financial markets, as the world reacts to the spread of COVID-19. Please know that we are closely monitoring current news and economic reports, and we are committed to supporting you through this season. Feel free to contact us any time, for any reason. We are here to help.
Now that Whatcom County is in Phase 2, we will continue to hold meetings by phone or zoom, or we can now also hold meetings in person by appointment only (for up to 30 minutes and masks are required).
For information about tax filing deadlines and other COVID-19 related tax issues, access this CARES Act 2020 infosheet.
The health and safety of our clients, staff and community are our greatest concern. Thank you for your business and for your patience and understanding during this time.
Should we become unavailable to service our clients, you may call the Avantax home office at 888-438-3781 for assistance with account opening, trades, transactions and money movement.
Stay Safe with a B.O.P. At Your Back
The Long Run: Women and Retirement
When Markets React
No Cash, No Checks, No Problem
We create financial plans tailored to your goals and values.
At Steele Financial Services our mission is to provide customized financial and investment advice to empower our clients to achieve confidence and comfort in their financial lives. We manage investments holistically based on a comprehensive financial life plan. We specialize in offering sustainable and responsible investing (SRI) with environmental, social, and governmental (ESG) screens. With options such as fossil fuel free portfolios, we seek to align your investments with your values.
We can help take the mystery out of preparing for today and tomorrow. Whether you are investing to build wealth, protect your family, or preserve your assets, our personalized service focuses on your needs, wants, and long-term goals.
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Street Vision License PlateCamera Blocker and PhotoBlocker Spray 6oz
Street Vision License Plate Camera Blocker and PhotoBlocker SprayProtect your custom license plates from weather damage with this spray onprotectantSave Money Against Traffic TicketsWhy this product is legal. Traffic laws are written to state that your licenseplate must be visible at all times, and indeed this product allows your plateto be 100% visible at all times to law enforcement. There are no traffic lawswritten that state your license plate must be photogenic. This is for a 6ozbottle.
A majority of red light & speed cameras utilize strong flash to photographthe license plate on your car. Once sprayed on your license plate,PhotoBlocker's special formula produces a high-powered gloss that reflects theflash back towards the camera blinding it. This overexposes the image of yourlicense plate, rendering the picture unreadable. With PhotoBlocker, yourlicense plate is invisible to traffic cameras yet completely legible to thenaked eye.
REFLECTS traffic camera flash, helps prevent a costly ticket!FAST spray-on formula!INVISIBLE to the naked eye!EXCLUSIVE formula!BEST alternative for when you can't use an anti-camera license plate cover!ONE TIME application! One can covers FOUR license plates!Get a can before you get a ticket!
Step 1
Remove your license plate from your vehicle. Clean and drythe surface. Lay it on a dry surface.
Step 2Before spraying, be sure to lay something out to protect the surrounding area.Apply PhotoBlocker in flat even strokes until license plate is saturated. Allowyour license plate to dry until tacky, then repeat 3 -4 times until there is anice glossy shine to your license plate.
Step 3Once your license plate is dry to the touch, place it back on your vehicle andyou are done.Step 4
You are now protected. Every time you go past one of those money making trafficcameras, you have an excellent chance of avoiding a ticket.


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Who he is: Brandon Maxwell
What you need to know: Brandon Maxwell launched his eponymous collection in 2015 after a career as a successful stylist and fashion director to Lady Gaga. His collections are well-regarded for their tailored, sophisticated pieces and black and white color-palette.
On the nomination: "Almost one year ago today my life was in a completely different place. I took a leap and a chance and it was the scariest thing I think I've ever done. This nomination was such a joy and a blessing because it served as a reminder to believe in myself and give thanks for those around me who have always stood so selflessly by me."
Who they are: Monse's Laura Kim and Fernando Garcia
What you need to know: Monse launched in 2015, the brainchild of two Oscar de la Renta alums. Before her 12 years at Oscar de la Renta, Kim attended Pratt Institute and interned at Donna Karan and TSE. Garcia grew up traveling between the Dominican Republic and Spain. He studied architecture before a chance meeting with Mr. de la Renta, who encouraged him to come to NY for an internship, which led to a role as principal designer at the house.
On the nomination: "From day one, the fashion industry has been super supportive of Monse, and this nomination came as a huge surprise to us. This past year has been challenging and rewarding at the same time. One of our goals for the year ahead is to build more of a structured business within the brand."
Who she is: Ryan Roche
What you need to know: Ryan Roche launched her womenswear collection in 2011 and has created some of most luxurious knitwear in dreamy shades of peach, pink, and other classic muted colors. Her silhouettes are modern and each piece feels instantly covetable.
On the nomination: "It's such an incredible honor to be nominated, especially amongst such a talented group of designers — many of whom I count as friends! I'm so flattered every time I woman I admire wears my clothes or an editor writes about my collection. It feels like my rose-tinted mood might be contagious."
Who they are: Orley's Alex Orley, Matthew Orley, Samantha Orley
What you need to know: Orley launched with a capsule collection of Italian-made knitwear for fall 2012 and expanded into a full men's ready-to-wear collection for Spring 2014. They are known for their youthful, idiosyncratic and nostalgia driven collections. The Orleys launched a women's collection for Fall 2015 and were previously nominated for the CFDA Vogue Fashion Fund and LVMH Prize.
On their nomination: "We are really grateful to be able to do what we do. This is just the icing on the cake."
Who he is: Gypsy Sport's Rio Uribe
What you need to know: Focused on inspiring creativity, celebrating diversity, and encouraging individuality, Gypsy Sport never fails to surprise and entertain with their youthful sportswear collection rooted in street wear. Rio Uribe launched Gypsy Sport in 2012.
On the nomination: "I'm super grateful and ecstatic to be embraced by the CFDA and the fashion community as a whole."
Who he is: John Elliott
What you need to know: Elliot founded his company in spring 2012 with a focus on clean lines and tailored fits. He strives to create tomorrow's classics. He made his NYFW debut for the FW15 season.
Who she is: Brother Vellies's Aurora James
What you need to know: Aurora James launched Brother Vellies in order to share her favorite traditional African footwear with the rest of the world. Each shoe is handmade in South Africa, Kenya and Morocco.
On the nomination: "Three years ago I began down a journey that would change my life and the lives of others in a way I could have never imagined. We … worked side-by-side in South Africa, Kenya, Morocco, Burkina Fasso and Ethiopia; everything I know about accessory design I learned from people in Africa. It's like we've always said, you guys — all ships rise with the tides and this is Africa rising."
Photo by Jason Eric Hardwick.
Who he is: Paul Andrew
What you need to know: After heading up shoe and accessory design for designers like Calvin Klein and Donna Karan, Paul Andrew launched his eponymous collection in 2013.
On the nomination: "I know it sounds like a cliché, but it is really an honor to be recognized by one's peers. The fashion industry has embraced me and my brand in such a positively moving way, and I am ever so grateful for their support."
Who she is: Milliner Gigi Burris
What you need to know: Burris studied at Parsons Paris and spent time visiting the old world mercerie shops there. She was inspired to launch her own millinery for the FW 2011 season. Today, she is stocked at Colette, Matches Fashion and The Webster.
On the nomination: "It is so humbling to receive this accolade from the fashion industry. It seems a bit surreal still! I hope that this nomination will allow us a louder voice in the conversation of American fashion."


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Made from nothing more than sweetened condensed milk and very strong coffee, Southeast Asian coffee is a force to be reckoned with. Spike it with some coffee liqueur and freeze it, and you've got an adult take on frozen pops that are a great late-night treat at a cocktail party or any other time you want to get down.
What to buy: Silicone ice cube trays are key to easily unmolding the shots.
This recipe was featured as part of our Wii Gaming Menu, as well as our Drinks Around the World feature.


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Since purchasing the Eco Styler argan oil formula, I've been looking into the numerous benefits of argan oil, dubbed "liquid gold," for hair and skin. Below, I've succinctly summed up all there is to know about Moroccan argan oil. What … Continue reading
Tag Archives: scalp
Kitchen Chemistry // Household Herbs for Lush Kinks & Curls
Photo Credit: The Homemaking Cottage Product after product on the shelves of our local beauty supply stores, natural markets, neighborhood grocers, and convenience stores make promising claims of increasing shine, stimulating growth, increasing manageability, curing scalp ailments, strengthening, and adding … Continue reading
Tea & Herbs // An Organic Shampoo Alternative?
Sadly, I'm almost out of bentonite and rhassoul clay -ultra sad face- after God knows how long (seems like it took forever to get dangerously low) and as much as I love my clay wash and its fabulous results, I … Continue reading
Status Report: On the Beaux- Project Dreadlocks
Length check time! Actually not on me but for the Beaux. His hair is starting to take on a coarser texture since we've been using coconut oil/aloe vera mix on his hair and scalp. He's seen less dandruff as I've … Continue reading
The Cream of Nature Argan Oil Experience
So most of you know that I'm working towards finding my staple that works with my hair. It's a tedious task. I work outdoors and it becomes a hassle trying to retain moisture without looking like I stepped out of … Continue reading
Natural News// Tennessee Lawmaker Advocates Establishment of Natural Hair Care Schools in TN
A lawmaker in Tennessee, State Rep. Antonio Parkinson, is assisting Tennessee stylists in creating cosmetology schools centered solely around natural hair care. Brinetta Carlton, a stylist who owns her own natural hair salon, cites her experience during her days as … Continue reading


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Raleigh Child Custody Lawyers
When parents of minor children divorce, the top priority of the courts and all interested parties is protecting the children's best interests. Unfortunately, it is not always easy to determine which available environment would be best for children. Many divorce hearings therefore get bogged down in complex and emotionally-painful child custody disputes.
The Raleigh child custody attorneys of the Marshall & Taylor Law Firm, P.C., understand how important parental rights are to you. We will work hard to keep you fully informed about your rights and obligations, and present the best legal case we can build for you in court. To learn about our approach to family law, contact our offices at 919-833-1040.
We can help you settle complex child custody issues, such as:
The Basics of Child Custody
North Carolina observes the federal Uniform Child Custody Jurisdiction and Enforcement Act. The guiding principle of this act is that children should be placed with the parent, other relative, or institution that can provide the best home life for him or her. Biological parents are usually given priority unless they are shown to be negligent or abusive.
There are two basic types of child custody: physical and legal. If you are seeking full or joint custody of your child, it is important to know the differences between them.
- Physical custody: This is a parent's or other guardian's right to spend time with the child. If a person is granted primary physical custody, the child will live with him or her.
- Legal custody: This is a guardian's right to make important decisions about a child's life. Some examples include the child's education, religious instruction, and medical care. Parents commonly share legal custody, which means they have to make these choices together.
Contact Us
To discuss your circumstances with a knowledgeable Raleigh child custody lawyer, contact the Marshall & Taylor Law Firm, P.C., at 919-833-1040.


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Upon agreeing a financial settlement within the divorce process you should ensure the agreement reached is legally binding with a Consent Order. We offer a no-fuss, fast, fixed-fee service.
We can provide you with this service as long as you live within England & Wales. We will provide you with expert legal advice and a fully-drafted consent order, prepared by a specialist solicitor;with no hidden costs. For more information see also finance on divorce.
Cost of service: fixed-fee from £499 plus VAT
If you are looking for a solicitor specializing in Consent Orders, who can focus on your family legal requirements then why not contact us today.


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Be The Story
I'm always looking to work with unique people whether at home (Philadelphia Area) or when I travel for personal portraits that allow for openness and experimentation. Everyday people who are of the full spectrum of this thing we call life. If you think you'll make a unique subject, fill out the form below! I thank good people who share their time and open their lives in order for me to be able to create art.
CURRENT PROJECT| Nov. 2017 – Dec. 2019
It's Not About Disability| People Just Don't Know How To Fuck.
Open Call to anyone who has a visible disability or who utilizes mobility devices wheelchair, crutches, etc.
If this is you or you & your lover, tell me YOUR STORY! I am looking for real people who are willing to share a small part of their world with the world by embracing their passion for life, love, and sexual identity.
According to the World Health Organization, "Sexuality is an integral part of the personality of everyone: man, woman, and child; it is a basic need and aspect of being human that cannot be separated from other aspects life."
Sex and disability are often a taboo for many able-bodied people. It is rarely discussed in the same sentence; even when curiosities flood the mind. Conversations are often avoided due to the taboos that those living with physical disabilities are essentially asexual; they can't or don't have sex, can be intimate, or outright not interested in sex. Because of these mythoses, more than 50% of people living with a disability do not have any form of a "regular" sex life.
Acceptance, affection, intimacy, sex, and sexuality are all sought after, yearned for, and indulged by all humans; regardless of disability. The goal is to visually capture, document, and explore the true mythos while shattering the asexual misconceptions and stereotypes surrounding those with physical disabilities. Unveiling the vibrant lifestyles of those who live with a disability who can, want and do have sex and sexual identities while peeling away at the taboos that have made others apprehensive to engage in the conversation.
By photographing individuals and couples who have physical disabilities I hope to explore the ways that we as humans view sex, intimacy, and sexual identity as something deeper than the basic instinct. What it means to not only see one's self but to also see how our bodies can be bold and beautiful; flaws and all. In doing so, create a positive mythos that opens up the possibilities and conversations that embrace the beauty of intimacy, sex, sexual identity, and disability.
I am currently seeking individuals and couples who have some form of a physical disability to document. I'm especially looking for those comfortable with themselves enough to lay it all out. Ideally, I would like to photograph those who are interested in being a part of the story in their environments or places you may be most comfortable in.
Once completed, the images will be submitted for publication or gallery. As with any project, it may become its own life.
If nothing else, share this with someone you feel may be interested!
As an artist sometimes even we must do that which we aren't comfortable with ourselves.
Find out more about me here!


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Scripture: Joshua 12-15.
(What is God saying here?)
Joshua 13:13 (NLT)
13 But the Israelites failed to drive out the people of Geshur and Maacah, so they continue to live among the Israelites to this day.
Joshua 15:63 (NLT)
63 But the tribe of Judah could not drive out the Jebusites, who lived in the city of Jerusalem, so the Jebusites live there among the people of Judah to this day.
(How Can I apply what God is saying here to my circumstances today?)
Over the past chapters we read where Joshua and/or Moses were, with the help and direction of God, successful in overcoming a number of countries and their respective kings. I found it odd to read the two verses above where the Israelites were unsuccessful, or perhaps failed, in driving out the Jebusites and the people of Geshur and Maacah, because I know that the God they served, and of course the same God that I serve today, is very capable of overcoming all obstacles. In my further study I found where these same people that failed to fully conquer, were the same ones that caused unending difficulties for the Israelites, as recorded in the book of Judges. This sounds like there may have been personal reasons that the Israelites just didn't push hard enough to really oust these people, and it reminds me of us when we fail to seek God's power to really rid ourselves of certain sins. Often these little sins that we hold dear to our selfish little hearts are the very same things that come back to bite us and cause us extreme difficulties. I believe that God is always looking for folk that he can use who are totally sold out to Him, where we have surrendered everything to Him and cleansed ourselves of all sinful things. In fact, to substantiate my belief on this, please refer to chapter 14 vs 9 where we read that Caleb was given special favor with God because he 'wholeheartedly' followed God's commands. I believe that seeing where God gave up everything for me, yes His one and only Son, so that I might have life eternally and more abundantly, then the least I can do in return is to surrender ALL sin – yes everything that does not align with God's Word, His will and commands for me. There is no room for half-heartedness – may we all be totally sold out to Jesus!
(With God's help, what am I going to do differently today because He said it?)
There is only way that I can be the totally true, unselfish, child of God and overcome daily temptations and sin, and that is to seek Your help O God and look to You in all humility for strength and guidance to live as You would have me live – so that You can receive full benefit (ministry) in and through me. May I be a vessel usable by You – a holy and just God. May others come to know You as Lord, as you find the freedom to use this unadulterated child of Yours. Please forgive me where I have been deliberately selfish and weak and given in to sin. This is unbecoming of a child of Yours and I need Your forgiveness and strength to overcome and live a life totally sold out to You.
àWords underlined may be a hyperlink to a song that God laid on my heart while sharing this devotional this morning. Click it and be blessed by the song, like I was when I listened to it! …… I would be overjoyed to have the opportunity to pray with you about a need in your life that may, or may not have been highlighted by this Nugget today. Why not drop me a note and tell me about it? You may email me at:


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Line Drawing and Download Link
Sarracenia alata Alph. Wood -
yellow trumpets
USDA NRCS. Wetland flora: Field office illustrated guide to plant species. USDA Natural Resources Conservation Service. Provided by NRCS National Wetland Team.
Usage Requirements.
To download the original size of this image, click on the link below.
Usage Requirements.
Download a high-resolution TIFF image of Sarracenia alata (42 KB)


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March 9, 1999, Section A, Page 1Buy Reprints
A Taiwan-born scientist at Los Alamos National Laboratory was fired today for security breaches after the F.B.I. questioned him in connection with China's suspected theft of American nuclear secrets, Energy Secretary Bill Richardson said.
The scientist, Wen Ho Lee, was questioned for three days by the Federal Bureau of Investigation, starting on Friday, but ''stonewalled'' during the questioning, Mr. Richardson said.
Mr. Lee, a computer scientist working in the nuclear weapons design area at Los Alamos, N.M., has not been charged with a crime, but he has been identified by Federal officials as the prime suspect in the spying.
China denies any such theft. But Administration officials said China, working with stolen data, has made a leap in developing nuclear weapons with much smaller warheads.
The handling of the spying case brought sharp criticism from Capitol Hill. Senator Fred Thompson, an influential Republican from Tennessee, attacked the Administration for failing to notify Congress in 1997 that China had stolen nuclear secrets when the Administration certified that Beijing was no longer helping other nations build nuclear bombs.
That certification ended a 12-year ban on the sale of nuclear technology to China, an action long sought by American companies eager to bid on the estimated $60 billion Chinese market for civilian nuclear plants.
Mr. Thompson said Senators would take up a ''serious and immediate review'' of the certification issue.
The F.B.I. began to question Mr. Lee on Friday to determine whether he had given American secrets to China. The questioning continued through late Sunday, but Mr. Lee failed to cooperate, according to Mr. Richardson.
F.B.I. officials acknowledged last week that they did not have enough information then to arrest Mr. Lee, but hoped that their questioning would lead to a break.
While he agreed to talk with investigators and nuclear experts, his failure to fully cooperate has apparently still left the investigators without enough evidence to prosecute.
But Mr. Richardson dismissed Mr. Lee for ''failure to properly notify Energy Department and lab officials about contacts with people from a sensitive country, specific instances of failing to properly safeguard classified material and apparently attempting to deceive lab officials about security matters.''
Reached at home in Los Alamos this afternoon, Mr. Lee's wife, Sylvia, a former secretary at Los Alamos, refused give his whereabouts or to comment on the matter.
Mr. Lee, who officials said is in his late 50's and has been working at the lab for more than a decade, did not hire an attorney to represent him during his questioning, officials said.
Mr. Lee has been the prime suspect in a nearly three-year investigation of reports of Beijing's theft of nuclear technology, senior officials from the bureau and the Energy Department said.
Until recently, China's nuclear weapon designs were a generation behind those of the United States, largely because Beijing had been unable to produce small warheads that could be launched from a single missile at multiple targets.
But by the mid-1990's, China had built and tested such small bombs, a breakthrough that officials say was accelerated by thefts from Los Alamos.
The espionage is believed to have occurred in the 1980's, officials said, but was not detected until 1995, when American experts from Los Alamos analyzing Chinese nuclear test results found similarities to America's most advanced miniature warhead, the W-88.
By February 1996, investigators from the Energy Department and the F.B.I. searching travel records and other data at Los Alamos and other weapons labs had identified five possible suspects, officials said.
Mr. Lee quickly emerged as the prime suspect, they added.
Investigators say they now believe that Mr. Lee gave the Chinese sensitive information on nuclear detonations during a 1988 seminar, senior Administration officials said.
But after opening a formal criminal investigation in June 1996, the bureau did not at first pursue the case aggressively, according to several senior officials.
Only in the last several weeks, after prodding from Congress and the Secretary of Energy, did officials administer lie-detector tests to Mr. Lee.
After a first test in December the bureau, unsatisfied with the results, gave Mr. Lee a second test in February, and the test indicated that he was being deceptive, officials said.
The F.B.I. then decided to interview Mr. Lee on Friday.
In response to criticism of their handling of the case, bureau officials said Chinese spying is far more difficult to investigate than the more traditional espionage of the former Soviet Union.
The Chinese often take advantage of scientific exchanges and other forms of informal contacts, gathering sensitive information from such a wide range of sources that it is often difficult to pinpoint exactly how American secrets leak out.
In fact, American officials say, they have concluded that Beijing is continuing to steal secrets as major nuclear weapons laboratories in the United States have been increasingly opened to visitors from other nations.
In this case, since the reported theft of the W-88 design information apparently occurred so far in the past, the Justice Department at first lacked the grounds to obtain a secret wiretap on the suspect, making it difficult initially to build a strong criminal case.
Still the Energy Department failed to move Mr. Lee out of his sensitive position or end his security clearances for more than a year after the bureau's director, Louis J. Freeh, told Energy Department officials in September 1997 that there was no longer any investigative reason to keep him on the job.
In espionage cases, the bureau often wants suspects left alone by their employers for fear of tipping them off to the investigations.
He was finally moved out of a sensitive area several weeks ago, officials said.
Mr. Lee's wife, Sylvia, stopped working at the lab sometime in the last year or two, Energy Department officials said.
F.B.I. and Energy Department officials said her activities had raised questions while she worked at Los Alamos. In the 1980's she was invited to China to give an academic paper on high-technology computer processing even though she was only a secretary at Los Alamos.
Her husband, who was the true expert, accompanied her, according to an American official.
Energy Department officials also said co-workers questioned why she frequently invited herself to gatherings at the lab with visiting Chinese delegations.
F.B.I. officials said she was not a target of their investigation, but said it is possible that others were involved in the theft of data. They have not yet identified any other suspects.
Meanwhile, in response to criticism of the lax security at Los Alamos and other weapons labs, Secretary Richardson said today that he is ordering polygraph examinations for a few thousand Energy Department and laboratory employees during the next few years.
The order does not cover all employees with ''Q'' security clearances, which give them access to nuclear secrets, Mr. Richardson said, but it does cover those who work in the most sensitive areas.
Currently only employees of the Central Intelligence Agency are required to submit to polygraph exams on a regular basis.


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Reviews of Kulala Desert Lodge
They do not necessarily represent the views of Expert Africa.
Excellent base for exploring the Namib
We really enjoyed our stay at Kulala. It had a really nice 'feel' to it - good service and good food but not ostentatious or complicated. The staff were very good, personable and remembered our names - impressive given the visitor turnover.
Our room was very comfortable, and well laid out, with a good view. We liked the rooftop place where we could look at the stars (we weren't brave enough to try to sleep up there, but other people do). If you want to drink tea and coffee in your room, you have to ask for a thermos of hot water to take back there, but we never wanted to because there was tea and coffee always available in the main area (and cake in the afternoon).
The main area of the lodge was comfortable. All our meals here were good, and as one of us is vegetarian we particularly appreciated being given two good choices. It is good that they give waterbottles and so we could top those up at anytime.
We did two activities at Kulala - we went to Sossusvlei to climb a sand dune (we chose Big Daddy) and see Dead Vlei. This was amazing. Then in the afternoon we went to Sesriem Canyon, which was interesting but couldn't beat climbing the dune. The guide for these activities, Oscar, was great, probably the best we encountered during our stay in Namibia.
THE DUNES AND SESRIEM CANYON TRIPS WERE FANTASTICRead more about the whole safari
Kulala Desert Lodge review
This was a magical place with great facilities. It was wonderful to be able to go through the 'back door' to see the dunes.Read more about the whole safari
Kulala Desert Lodge review
Welcoming friendly staff
Hot air ballooning is a must as is early morning dune climbs.
Kulala Desert Lodge
The Kuala Lodge was also wonderful. Everyone was helpful and accommodating.
We loved being able to sleep outside on the roof watching the stars.
The excursions were fun and interesting.
The desert scenery there is spectacular.
Kulala Desert Lodge review
Very nice location. Overpriced activities but later I noticed that Wilderness Safaris are using the same price level in all their locations. The Family room was very good. Meals good but not highlight.
Activities were great and our kids fell in love with Suwama - our guide.
We have taken two sundown excursions and one morning to sand dunes. Big advantage of the Resort is the private gate which saves appr. 1 hour drive.
Excellent lodge
This is a well located lodge with friendly/helpful staff and very good guides. Excellent accommodation but insufficient veranda shade on individual lodges which restricted the ability to sit on deck after 3pm.
Was very hot - highest temp of the 2 days was 40.2 degrees. On first night only 2 other guests (one of whom worked for Wilderness and was with her sister) 2nd night - same 2 girls plus one other couple and a girl from Namibia Treks and Trails plus a Wilderness pilot. F
ood was disappointing - meat was tough (management informed and apologetic) fruit salad consisted of c. 90% apple, fresh fruit was unripe. No advice given by staff regarding food preparation eg. loin of lamb (more like very old lamb/sheep!) came with mustard sauce poured over the meat - no option advised. Little food choice - may be associated with so few guests.
No Wi-Fi but guests can use main lodge computer - good connection. Lots of activities including quad biking - a first for us - which was a fantastic experience.
Unforgettable place!
This is a stunning place. After driving the unexpectedly long (but tremendously scenic!) driveway in, we were greeted by lodge staff offering us refreshing homemade ginger "beer." We were then given a tour of the facilities (gorgeous!) and were seated on the veranda where staff members asked us about any dietary restrictions and described the activities we could partake in during our two-night stay.
At first this place seemed a bit fancy and formal compared to where we had just come from (Bullsport Guest Farm) but that feeling soon dissipated thanks to the friendly and attentive staff.
Our kulala was great. It is really more like a semi-permanent tent - the walls of the main room are canvas, though the bathroom is a type of stucco or adobe. It was furnished tastefully and appropriately for it's stunning surroundings. However you are literally at the edge of the desert, so accept that dust will be a factor here despite all the efforts to keep everything clean. The view from our little deck was amazing - the dunes are right there. We did opt to sleep on the roof one night to enjoy the stars - we highly recommend that! It definitely got chilly overnight, and while we were cozy up there, we did go back in our room at about 4 or 5am to get a few hours of solid sleep.
This lodge is run by Wilderness Safaris so you will be "indoctrinated" (in a good way) with their philosophy, particularly concerning water conservation and energy consumption.
The food is just ok. They seem to be catering to westerners and a western diet, so we didn't eat anything unusual - chicken one night and beef another. The starch and the vegetables were heavy. Same breakfast as everywhere else we went. Other than the food, the only other thing that bothered us about Kulala Desert Lodge (and it happened again at the other Wilderness Safaris place we stayed) was that there was a big group (~14 people) staying there too. Their giant table dominated the dining room and even though they weren't rowdy, being around large groups of tourists is just annoying. We went to Namibia to get away from people! Oh well.
The activities they offered us were two different sundowners (since we were staying two nights) and a morning trip to Sossusvlei. We opted for one sundowner and the Sossusvlei trip. Both were incredible. We had the same guide for both who was excellent (unfortunately we don't remember his name). We were very lucky in that we were the only ones on the sundowner, and only one other couple was with us on the Sossusvlei trip. (Perhaps that's the upside of the big group - they were all out doing group activities!) Yes you can go to Sossusvlei yourself for a lot cheaper, but having to go through the main gate at Sesriem would have meant getting up even earlier, and also you learn a lot with the guide (and you can just sit back and relax in the Land Rover!). The morning at Sossusvlei was definitely one of the highlights of our whole trip. We did drive ourselves to Sesriem Canyon which is definitely something you can do on your own.
We absolutely loved Kulala Desert Lodge. The surroundings are some of the most spectacular scenery you will ever see, and we felt very well taken care of. We wish we could have stayed longer than two nights!
Kulala Desert Lodge
Large busy lodge close to the dunes . Had an excellant guide here who made it all fun and interesting. We climbed Big Daddy ,what a sense of achievement. It was easier and even more fun coming down.
Staff very friendly and liked to chat which made you feel vey welcome and relaxed.
Kulala Desert Lodge review
very helpful staffRead more about the whole safari


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John Hampton at Wolverhampton Art Gallery
An exhibition of vibrant works by Wolverhampton artist John Hampton (1934-2012) is now showing at Wolverhampton Art Gallery till the 13th of July.
Untitled, John Hampton
Educated and trained in Bilston, Wolverhampton and Birmingham, John was a dynamic character and had an inquisitive approach to his artwork. His career spanned over five decades, during which he became Head of Art at Bilston Community College. A studio holder at Eagle Works, he was an active member of the group.
Untitled, John Hampton
This show focuses on his later works that were inspired by his garden, resulting in this colourful display of expressive and painterly compositions.
This exhibition features 26 of John Hampton's oils on canvas and mixed media works. Many of the paintings on display demonstrate his ability to use the canvas to explore light, colour and texture and use them as a metaphor to express his ideas, emotions and experiences.
The show has been co-curated by members of the Eagle Works artist group based on Great Brickkiln Street in Wolverhampton, where John Hampton was a studio holder and central figure. Joining the studios in the late 1980s, he remained actively involved with the group until shortly before his death in February 2012. Hampton acted as the Studio's Treasurer for a number of years.
Curator Marguerite Nugent said: "We're really pleased to be able to put John Hampton's work on public display. He was such an active and well-known figure within the city's artistic community, this seems a fitting tribute to his many years of dedication, both as a committed artist himself but also providing inspiration for fellow artists across Wolverhampton."
This is a rare chance to see a beautiful collection of works by a local artist, in a local setting. John's work is vibrant and enticing, capturing nature at it's peak- a perfect Summer show!


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New Research In
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Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor
Edited by Robert J. Lefkowitz, Duke University Medical Center, Durham, NC, and approved August 24, 1999 (received for review November 25, 1998)
Endocytosis of the Flaviviridae viruses, hepatitis C virus, GB virus C/hepatitis G virus, and bovine viral diarrheal virus (BVDV) was shown to be mediated by low density lipoprotein (LDL) receptors on cultured cells by several lines of evidence: by the demonstration that endocytosis of these virus correlated with LDL receptor activity, by complete inhibition of detectable endocytosis by anti-LDL receptor antibody, by inhibition with anti-apolipoprotein E and -apolipoprotein B antibodies, by chemical methods abrogating lipoprotein/LDL receptor interactions, and by inhibition with the endocytosis inhibitor phenylarsine oxide. Confirmatory evidence was provided by the lack of detectable LDL receptor on cells known to be resistant to BVDV infection. Endocytosis via the LDL receptor was shown to be mediated by complexing of the virus to very low density lipoprotein or LDL but not high density lipoprotein. Studies using LDL receptor-deficient cells or a cytolytic BVDV system indicated that the LDL receptor may be the main but not exclusive means of cell entry of these viruses. Studies on other types of viruses indicated that this mechanism may not be exclusive to Flaviviridae but may be used by viruses that associate with lipoprotein in the blood. These findings provide evidence that the family of LDL receptors may serve as viral receptors.
Hepatitis C virus (HCV), the principal viral cause of chronic hepatitis, is not readily replicated in cell culture systems, and, as yet, no information on cell receptors for the virus is available. However, several observations from studies on the role of HCV in mixed cryoglobulinemia (1–3) have provided some insights to HCV entry into cells.
Mixed cryoglobulinemia is a systemic vasculitis associated with cold-precipitable immunoglobulins in the blood. During the past 5 years, a strong association of HCV infection with mixed cryoglobulins has been established (4), and the specific concentration of HCV in type II mixed cryoglobulins that consist of polyclonal IgG and monoclonal IgM has been demonstrated (1). It also was shown that very low density lipoprotein (VLDL) is selectively associated with HCV in type II cryoglobulins (2). In studies on the cutaneous vasculitic lesions in type II cryoglobulinemia using in situ hybridization (ISH), the HCV RNA virion form (positive strand) but not the putative replicative form (negative strand) of the virus was detected in keratinocytes in lesions but not normal skin of the same patients (3). Furthermore, it was demonstrated that LDL receptors were up-regulated on keratinocytes in cutaneous vasculitis lesions compared with normal skin (3). These observations and the finding that anti-β lipoprotein precipitates HCV from infected serum (5) suggested that the low density lipoprotein (LDL) receptor also may be a receptor for HCV complexed to VLDL or LDL. This hypothesis led to this study to determine whether the LDL receptor is also a receptor for HCV and other members of the family of viruses Flaviviridae.
Materials and Methods
Reagents, Antisera Virus Stocks, and Cell Lines.
Cyclohexanedione, phenylarsine oxide (PAO), heparin sulfate, and EGTA were purchased from Sigma; 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine iodine (DiI) was purchased from Molecular Probes. Purified IgG 2a mouse monoclonal anti-LDL receptor antibody (C7 clone) was obtained from Oncogene Scientific Products (Cambridge, MA). Anti-bovine viral diarrhea virus (BVDV) envelope antibody bovine serum, α49, was provided by Marc S. Collett (Viro Pharma, Malvern, PA). Mouse monoclonal IgG 2a anti-CD-16, anti-CD-19, and anti-transferrin (CD71) were purchased from Immunotech (Hialeah, FL). Anti-μ was purchased from Jackson ImmunoResearch. Anti-apolipoprotein (αapo) E and αapo A-I were purchased from Cortex Pharmaceuticals (San Leandro, CA); αapo B was purchased from Sigma. Purified mouse monoclonal IgG αapo E (1D7), αapo A-I (3G10), and αapo B (4G3) were purchased from the University of Ottawa Heart Institute. F(ab′)2, an antibody fragment with two antigen-combining sites, preparations of mouse IgG were prepared by treating the mouse monoclonal antibodies from 30 minutes to 10 hours with 3% pepsin (Sigma) (pH 3.5) at 37°C. The F(ab′)2 fragments were isolated by column chromatography using a HR 10/30 Superose 12 column (Amersham Pharmacia). Bovine viral diarrhea virus-free donor calf serum was purchased from Boyt Veterinary Laboratory (Neosho, MO). Potassium bromide density gradient ultracentrifugation was used for preparation of VLDL, LDL, and high density lipoprotein (HDL) from normal sera, and these lipoproteins complexed to HCV from infected sera. The VLDL band [density (d) = 0.95–1.006 g/ml], the LDL band (d = 1.019–1.063), the HDL band (d = 1.063–1.21 g/ml), and HCV free of lipoproteins (d > 1.21) were isolated by aspiration and then dialyzed against Hanks' balanced salt solution (Sigma) containing 0.01% EDTA. Isolated HCV-VLDL was dissociated to HCV and VLDL by treatment with deoxycholate and fractionated by sucrose density gradient ultracentrifugation as described (6). The high density HCV fraction, free of lipoproteins, was further fractionated by column chromatography on a lecithin pretreated Superose 6 (Amersham Pharmacia). The peak of HCV present in the void volume was contaminated with small amounts of immunoglobulins that were removed by using immobilized rProtein A (Repligen). Immoblotting (dot blots) to detect small amounts of protein was performed as described (7). Sensitivity of the assay was 100 pg for IgG and IgM and 200 pg for apolipoproteins B and E. Lipoproteins were quantitated by Lowry assay using commercial kits (Sigma). Highly purified VLDL, LDL, and HDL were purchased from Cortex. Labeling of LDL with DiI was performed as described (8).
Infected human sera were used as stocks for HCV (3 × 108 genomic equivalents per millimeter [gE/ml]), GB virus C/hepatitis G virus (HGV) (2 × 109 gE/ml), and herpes simplex virus (HSV). BVDV strains NY-1 and National Animal Disease Laboratory (NADL) and vesicular stomatitis virus (VSV), Indiana strain, and respiratory syncytial virus were obtained from American Type Culture Collection (Rockville, MD). Bovine turbinate (BT) and kidney (MDBK) cell lines, Hep G2, a hepatoma cell line that is biochemically similar to hepatocytes (9), Daudi, a B cell lymphoblastoid cell line, the Molt-4 T cell line, HEp 2, a squamous carcinoma cell line, and normal fibroblasts (MRC-5) were obtained from the American Type Culture Collection. The B lymphocyte lines G4 and E11 were generated from fusion of F3B6 human-mouse heterohybridoma with peripheral B cells from patients with type II cryoglobulinemia and rheumatoid arthritis, respectively. Development of the 35G6 peripheral B cell line, cloned from a normal patient, was previously described (10). Four LDL receptor negative cell lines, GM00488C, GM02000G, GM00701C, and GMO3040B, were obtained from the National Institute of General Medical Sciences, Human Genetics Mutant Cell Repository, Coriell Institute for Medical Research (Camden, NJ). Cells resistant to infection with BVDV (CRIB) were provided by R. O. Donis (University of Nebraska, Lincoln, NE).
LDL Receptor Assays.
Cells were cultured in RPMI medium 1640 supplemented with 10% BVDV-free bovine calf serum or expression of the LDL receptor was up-regulated by culturing for 24 hours in RPMI medium 1640 supplemented with 10% lipoprotein-deficient BVDV-free medium. The cells then were washed twice with PBS (pH 7.2). Cytospin preparations were made, fixed with acetone, and blocked with 5% normal mouse serum, and the LDL receptor was visualized by incubating the slides with 5 μg/ml purified IgG 2a monoclonal anti-LDL receptor antibody followed by a 1:50 dilution of FITC-labeled goat anti-mouse [F(ab′)2] second antibody (Jackson ImmunoResearch). The demonstration of LDL receptors on adherent cells, MDBK, CRIB, fibroblasts, Hep G2, and HEp 2 was performed in the same manner except monolayers of cells were cultured and fixed on slides.
Demonstration of endocytosis of DiI-LDL by cells was performed by incubation of 2 × 105 cells for 2 hours at 37°C in 5% CO2 with 20 μg/ml DiI-LDL as described (11). The cells were washed twice with cold PBS and were fixed with 1% buffered paraformaldehyde, and cytospin preparations were made for fluorescent microscopic studies or cells in suspension were analyzed by flow cytometry. Flow cytometric analysis was performed by using the Epic XL-MCL cytometer (Coulter) using a 575 BP filter. Nonspecific binding of DiI-LDL was determined by using DiI-LDL treated with cyclohexanedione and was subtracted from the DiI-LDL binding to give specific DiI-LDL binding to cultured cells.
HCV RNA and Endocytosis Assays.
HCV RNA, reverse transcriptase (RT)–PCR, and ISH assays were performed as described (12). Specificity of the ISH method for HCV was determined by comparing monolayers of human fibroblasts inoculated with either 3 × 107 gE/ml HCV or dilutions of adenovirus or respiratory syncytial virus that produced pathologic changes in cells at 24 hours. After incubation for 24 hours at 37°C, the cultures were assayed for HCV RNA by ISH. The endocytosis assay for HCV was performed as described (12). Daudi cells (5 × 105 cells) were inoculated with 3 × 107 gE HCV or GB virus C/HGV, were incubated for 3 hours at 37°C, were washed three times, and were assayed for intracytoplasmic HCV RNA or GB virus C/HGV RNA by ISH. RT-PCR and ISH assays for GB virus C/HGV RNA were performed as described (13). The same methodology also was used for studies with HCV-lipoprotein recombinants. One hundred micrograms each of normal VLDL, LDL, HDL or cyclohexanedione-treated VLDL, or LDL were incubated with 106 gE HCV free of lipoproteins and immunoglobulins for 30 minutes at 37°C and then were added to the Daudi cells.
For cytolytic viruses, BVDV, NADL, VSV, and HSV, various dilutions of the respective viruses were incubated with monolayers of cells at 4°C for 1 hour, were washed three times with cold PBS, and were incubated with fresh medium. Virus dilutions that produced complete cytolysis at 72 hours for BVDV and VSV and 48 hours for HSV were selected. Immunofluorescent detection of intracytoplasmic BVDV was performed on acetone-fixed slides by using 1:50 dilutions of anti-BVDV serum and a FITC-labeled anti-bovine second antibody. The presence of BVDV in cells was confirmed by RT-PCR using BVDV specific primers (14).
Inhibition Studies.
Blocking of LDL receptor with various dilutions of antibodies (anti-LDL receptor, 5–20 μg/ml; control antisera, 5–20 μg/ml) or inhibitors was performed by pretreatment of cells with various concentrations of antisera or inhibitor for 15 minutes at 37°C and inoculating with virus without washing the cells. Additions of antisera during incubation period were made at 45-minute intervals. Treatment of LDL and VLDL with cyclohexanedione was performed as described (15). In experiments with cytopathic virus, cells were pretreated with 50 μg/ml of anti-LDL receptor antibody for 30 minutes at 4°C before inoculation with virus at 4°C.
Inhibition of endocytosis by PAO was assessed by pretreating cells with a range of final PAO concentration of 0.1–100 μM as described (16), and then endocytosis of LDL or HCV was evaluated by the DiI-LDL assay or HCV-ISH, respectively, as described earlier.
Endocytosis of HCV via the LDL Receptor. We previously demonstrated that endocytosis of HCV in vitro correlates with the titer of HCV in the inoculum. The percentage of cells positive for HCV RNA by ISH correlated directly with the number of gE of HCV per cell as determined by RT-PCR (12). There was also a crude correlation between intensity of ISH staining for HCV RNA and gE HCV per cell by RT-PCR. The specificity of this ISH assay for HCV is shown in Fig. 1. Cell cultures infected with viruses other than HCV were negative using this assay.
For a further investigation of endocytosis of HCV by cells in vitro, a variety of human cell cultures (see Materials and Methods) were demonstrated to have LDL receptors with the use of anti-LDL receptor antibody or DiI-LDL uptake. These cell lines then were inoculated with a high titer HCV-positive human serum. Intracellular HCV RNA then was detected by using ISH. To determine whether endocytosis of HCV correlated with the level of LDL receptor expression on cells, the well known modulatory effect of lipoproteins on the LDL receptor was used to increase the number of LDL receptors (up-regulate) on cells by culturing in lipoprotein-deficient media. Relative differences in endocytosis of LDL by various cultured cell lines could be demonstrated by the specific uptake of DiI-LDL. The specific DiI-LDL uptake of Hep G2 cells as shown in Table 1 was four times greater than of the peripheral B cell line, G4, without up-regulation.
Up-regulating these B cells produced a LDL uptake equivalent to that of the Hep G2 cells without up-regulation. These results were confirmed by immunofluorescent studies using anti-LDL receptor antibody staining and DiI-LDL uptake (Fig. 2 A and B).
Up-regulation of the LDL receptors of G4 cells resulted in 70–80% of cells staining positive for the LDL receptor, a result that was similar to the 70–80% cells positive for the LDL receptor on Hep G2 cells (Fig. 3E) and Daudi cells (Fig. 3G), also known to have higher densities of LDL receptor (11).
The percentage of cells positive for HCV by ISH was shown to correlate with the percentage of cells positive for LDL receptor by immunofluorescence using anti-LDL receptor antibody or DiI-labeled LDL. Endocytosis of HCV by peripheral B cells that showed 5–30% weakly positive cells in routine culture (Fig. 3A) when up-regulated showed a percentage of positive cells and an intensity of staining (Fig. 3B) comparable to Hep G2 cells (Fig. 3E) and Daudi cells (Fig. 3G) that were each 70–80% positive.
Direct evidence that the LDL receptor-mediated endocytosis of HCV was obtained by inhibiting endocytosis with anti-LDL receptor antibody. The endocytosis of HCV could be inhibited in a dose-dependent manner by preincubating the cells with anti-LDL receptor antibody. At sufficient concentrations of anti-LDL receptor antibody, complete inhibition of endocytosis of the virus could be demonstrated for both G4 and Hep G2 cells (Fig. 3 C–F). Similar results were obtained by using infected serum or VLDL-HCV complexes isolated from type II cryoglobulins as the inoculum in these experiments. No inhibition was observed with control mouse IgG 2a or antisera to specific cell surface antigens at a concentration up to 20 times the lowest inhibiting concentration of anti-LDL receptor: antisera to μ heavy chain, CD-19, and CD-16 surface antigens on peripheral B cells and Daudi cells and antiserum to transferrin receptor on Hep G2 cells did not inhibit the endocytosis of HCV by these cells. Moreover, treatment of Daudi cells with the endocytosis inhibitor PAO at 2 μM concentration completely inhibited the endocytosis of HCV (Fig. 3 G and H).
The role of the LDL receptor in the endocytosis of HCV was confirmed by demonstrating competitive inhibition with LDL and VLDL but not HDL, which is known not to bind to the LDL receptor. With use of hepatoma cells (Hep G2) or B cells (G4 and E11), 25–100 μg/ml of LDL or VLDL completely inhibited endocytosis of HCV whereas concentrations of HDL up to 200 μg/ml (a 5- to 20- and 10- to 40-fold molar excess over LDL and VLDL, respectively) did not inhibit. Treatment of LDL or VLDL with cyclohexanedione, which is known to alter a critical arginine residue in the LDL receptor binding site of apolipoproteins E and B (15), the main apolipoproteins found in VLDL and LDL, respectively, eliminated the inhibition by LDL or VLDL. Moreover, 25 units/ml of heparin sulfate or 2 μM EGTA inhibited endocytosis of HCV. Both are known inhibitors of LDL receptor endocytosis of lipoprotein (17).
To determine whether lipoproteins were involved in the endocytosis of HCV, we performed inhibition studies using various previously characterized antisera to apolipoproteins [αapo E, ID7 (18); αapo B, 4G3 (19); and αapo A-I, 3G10 (20)]. F(ab′)2 fragments were prepared and were used for all of the studies; inhibitory activities of the preparations were tested against DiI-labeled VLDL, LDL, and HDL isolated from a normal serum. Optimum F(ab′)2 antibody concentrations and conditions for inhibition of endocytosis were determined. Optimum conditions required addition of F(ab′)2s after pretreatment during the incubation period, and both αapo E and αapo B were required for maximal inhibition of VLDL endocytosis, whereas αapo B was sufficient for maximal inhibition of LDL endocytosis. Under these conditions, the maximum inhibition of HCV endocytosis achieved was 65%, with the remaining positive cells showing only trace staining. Pretreatment with αapo A-I gave 10% inhibition, with the remaining positive cells showing no decrease of staining compared with the control. The addition of αapo A-I during incubation did not increase inhibition. The finding that both αapo E and αapo B were required and that additional F(ab′)2s during the incubation increased inhibition was most likely attributable to the complexity of VLDL metabolism and dissociation of F(ab′)2s binding at 37°C. Hence, it could not be determined whether VLDL alone or both VLDL and LDL mediated endocytosis of HCV. Moreover, because complete inhibition could not be achieved, direct endocytosis of HCV by the LDL receptor could not be excluded.
Endocytosis experiments of isolated HCV-lipoprotein complexes and recombination experiments with HCV and lipoproteins provided more definitive data on the role of lipoproteins in endocytosis of HCV via the LDL receptor. Isolation of HCV by dissociation of HCV-VLDL complexes was unsuccessful; however, density gradient fractionation of a serum containing a high concentration of HCV produced not only HCV lipoprotein fractions but also a high density HCV fraction free of lipoprotein. Immunoglobulins contaminating the latter fraction were removed, providing a "free" HCV fraction for recombinant studies. Comparison of endocytosis of the various fractions is shown in Table 2. The HCV-VLDL and HCV-LDL, but not the HCV-HDL or high density HCV, fractions were endocytosed. Addition of VLDL or LDL but not HDL, isolated from normal serum, to the free HCV resulted in restoration of endocytosis. Cyclohexanedione treatment of the VLDL or LDL abrogated the rescue (data not shown).
Further studies were performed by using the LDL receptor-deficient fibroblast cells (21). Inoculation of these cells with HCV showed only weak endocytosis that could not be increased with preincubation of cells in lipoprotein deficient medium nor inhibited by anti-LDL receptor antibody (data not shown). Furthermore, this low level endocytosis could not be competitively inhibited with excess VLDL.
Replication of Endocytosed HCV.
Replication of HCV has been reported in Hep G2 and Daudi cell cultures. Extended cultures of Hep G2, Daudi, and G4 cells were tested serially by ISH for evidence of replication. In the Hep G2 cells, only positive-strand HCV was detected in the cells up to 1 week, but, at 3 weeks, 85% of the cells contained positive-strand HCV and 65% contained negative-strand HCV (data not shown). At 4 weeks, the cells were negative for HCV. In Daudi cells, only positive strand was detected through day 10, but on days 15 and 20, both positive- and negative-strand genome sequences were present in 80% cells. The cells died in the fourth week of culture. Only the positive strand of HCV was detected in G4 cells up to 1 week; the cells died after 1 week.
Endocytosis of Other Flaviviridae Viruses.
We investigated commercial bovine sera that were known to be contaminated with the pestivirus BVDV (22, 23). Human cell lines routinely cultured in media containing bovine serum were found to be positive for intracytoplasmic BVDV by immunofluorescence using anti-BVDV antibody. The presence of BVDV was confirmed by RT-PCR using BVDV-specific primers. Negative-strand BVDV was not detected in cells nonpermissive to infection. BVDV-positive human nonpermissive cells became negative over a 4-week culture period in noncontaminated media. Endocytosis of BVDV by nonpermissive cultured cells could be inhibited completely with anti-LDL receptor antibody but not with the control anti-transferrin receptor antibody (data not shown for this set of experiments).
With use of the cytopathic NADL strain of BVDV and permissive cells, BT or bovine kidney (MDBK) cells, anti-LDL receptor antibody but not control antiserum inhibited the cytopathic effect and positive fluorescence at 3 days (Fig. 4 A–D). Five days after infection, there was complete cytolysis of both the inhibited and control cells. Similar studies using VSV and HSV showed no inhibition of infection by anti-LDL receptor antibody for HSV (Fig. 4 E, F, and I) but some inhibition for VSV at 48 hours, which was no longer apparent at 72 hours (Fig. 4 G–I).
Additional evidence for endocytosis of BVDV by LDL receptor was obtained by using a cell line (CRIB) resistant to BVDV that was derived from a permissive bovine kidney cell line (MDBK). As illustrated in Fig. 5, the CRIB cells that do not permit entry of BVDV (24) also do not endocytose LDL. The CRIB cells were negative for LDL receptors by immunofluorescence using anti-LDL receptor antibody (data not shown). However, the absence of DiI-LDL staining (see Fig. 5) is a more sensitive indication of the absence of LDL receptor and LDL receptor endocytosis because accumulation of DiI-LDL occurs from the rapid turnover of LDL by LDL receptor in the course of cholesterol metabolism.
A third member of the Flaviviridae family, GB virus C/HGV, was reported to associate with lipoproteins in the blood (25). Evidence also was obtained for LDL receptor-mediated endocytosis of this virus, as illustrated in Fig. 6.
Several lines of evidence were presented supporting the hypothesis that HCV and other members of Flaviviridae are endocytosed via the LDL receptor. Endocytosis of HCV was shown to correlate with cell LDL receptor activity by experimental modulation of LDL receptor activity and with the striking demonstration that the CRIB cell line, resistant to infection with BVDV for unknown reasons (24), lacked detectable LDL receptor activity. Direct evidence for the hypothesis was provided by inhibiting endocytosis of HCV, BVDV, and GB virus C/HCV with anti-LDL receptor antibody. Moreover, known biochemical inhibitors of LDL endocytosis and the endocytosis inhibitor, PAO, inhibited LDL receptor-mediated endocytosis of these viruses. The reports that up-regulation of LDL receptors facilitated persistent HCV infection in vitro (26) and the induction of HCV binding with transfection of the LDL receptor gene in COS-7 cells that were unable to bind HCV (27) are consistent with these findings.
Evidence also was presented that endocytosis of HCV via the LDL receptor was mediated by the complexing of HCV to VLDL or LDL. Although, HCV also appeared to complex with HDL in the serum to the same extent as VLDL and LDL, there was no evidence of HDL-mediated endocytosis of HCV. It could not be determined from the studies performed whether the low level of endocytosis of HDL compared with those of VLDL and LDL, as shown by cytometric studies with DiI-labeled lipoproteins, was responsible for the absence of detectable levels of HDL-mediated endocytosis of HCV.
The complete inhibition by anti-LDL receptor antibody of HCV endocytosis by a variety of cells in vitro suggests that the LDL receptor may be the main mechanism for entry of HCV into cells. However, the detection of small amounts of intracellular HCV in LDL-deficient fibroblasts inoculated with HCV that could not be inhibited by anti-LDL receptor antibody suggested that receptors not related to the LDL receptor mediate viral entry in these cells. Furthermore, the finding that there was only a delaying effect of anti-LDL receptor antibody on the cytopathic effect of the NADL strain of BVDV indicates that receptors for BVDV other than LDL receptors or low affinity LDL receptors were present. This finding is consistent with the observations of Flores and Donis (24) that CRIB cells could be infected with high concentrations of BVDV. Hence, the LDL receptor may be the main but not exclusive means of entry of these viruses into these cells.
The evidence of HCV replication in Hep G2 and Daudi cells, although transient, suggests that endocytosis of HCV mediated by the LDL receptor can result in infection. Whether this route of entry can result in a productive infection in vivo and whether all members of the Flaviviridae family of viruses use this mechanism of cell entry remain to be determined. The evidence presented in this study that VSV also may be endocytosed by the LDL receptor appears to be contrary to an earlier study (28) that found interferon-α induces a soluble LDL receptor fragment that inhibits VSV infection but concluded that the LDL receptor was not a receptor for VSV. The latter conclusion was reached because VSV was found to infect LDL receptor-deficient fibroblasts. From our finding that entry of HCV into LDL receptor-deficient fibroblast was independent of the LDL receptors, it is apparent that Fischer et al. (28) did not present definitive evidence that VSV was not endocytosed by means of the LDL receptor because receptors for VSV other than LDL receptors may have been present on those cells.
LDL receptor-mediated endocytosis may not be exclusive to Flaviviridae. Rather, this mechanism may be used by all viruses that can associate with VLDL or LDL in the blood. The reported association of VSV with lipoproteins in the blood, particularly VLDL (29), is consistent with this notion.
Thus far, there only has been indirect evidence of in vivo endocytosis of HCV mediated by the LDL receptor, that is, the apparent endocytosis of HCV by keratinocytes in the inflammatory skin lesions of patients with type II cryoglobulinemia (3). In this study, evidence that BVDV contaminated media from bovine serum is endocytosed by a wide variety of human cells nonpermissive to BVDV infection suggests that this mechanism may explain the presence of positive-strand HCV that has been widely reported in human nonhepatic cells, particularly peripheral blood mononuclear cells. This mechanism must be considered before assigning HCV tropism for tissues other than the liver where clear evidence for HCV replication has been demonstrated.
The potential involvement of the LDL receptor in HCV infection has implications for the current therapy of HCV infection and also provides the rationale for a new approach to therapy—specifically, administration of anti-LDL receptor antibody or analogues of the LDL receptor binding site of apolipoproteins B and E. The effect of IFNα, the current therapy for HCV infection, may be mediated in part by the down-regulation of LDL receptors. IFNα is known to induce IL-1 receptor antagonist (30), which blocks the IL-1 receptor-mediated stimulation by IL-1. Because IL-1 is known to increase LDL receptor activity (31), IFNα would indirectly cause a down-regulation of LDL receptor activity by stimulating IL-1 receptor antagonist production, thereby decreasing IL-1 receptor-mediated stimulation by IL-1.
It has been reported that a minor group of human rhinovirus (HRV2) (32) and Rous sarcoma virus A (33) enters cells via the LDL receptor-related protein. In the latter report, evidence was presented that a viral envelope moiety binds to LDL receptor-related protein. Our study provides evidence that the family of LDL receptors may serve as viral receptors.
We are grateful to Dr. Marc S. Collett for providing BVDV reagents and advice, Dr. Ruben O. Donis for providing the CRIB cell line, Dr. Charles A. Dinarello for helpful discussions, and Polly Zorolow for editing. This study was supported in part by the Robert E. Wise, M.D., Research and Education Institute, Lahey Clinic Medical Center, Burlington, MA, and National Institutes of Health Grant 1R21AI40672.
↵‡ To whom reprint requests should be addressed at: Department of Laboratory Medicine, Lahey Clinic Medical Center, 41 Mall Road, Burlington, MA 01805. E-mail: Vincent.Agnello{at}Lahey.org.
This paper was submitted directly (Track II) to the PNAS office.
This work was presented in part at the Keystone Symposium on Molecular and Cellular Biology: Hepatitis C and Beyond, Burlington, VT, January 23–29, 1996 (abstr. 007), the Ninth Triennial International Symposium on Viral Hepatitis and Liver Disease, Rome, April 21–26, 1996 (abstr. B122), and the Fifth International Meeting on Hepatitis C Viruses Molecular Virology and Pathogenesis, Venice, Italy, June 25–28, 1998 (abstr. F3).
- αapo,
- anti-apolipoprotein;
- bovine turbinate;
- bovine viral diarrheal virus;
- cells resistant to infection with BVDV;
- d,
- density;
- DiI,
- 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine iodine;
- gE,
- genomic equivalents;
- high density lipoprotein;
- hepatitis C virus;
- hepatitis G virus;
- herpes simplex virus;
- in situ hybridization;
- low density lipoprotein;
- National Animal Disease Laboratory;
- phenylarsine oxide;
- reverse transcriptase;
- very low density lipoprotein;
- vesicular stomatitis virus
- Received November 25, 1998.
- Copyright © 1999, The National Academy of Sciences
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