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It's somewhat fascinating how quickly the glow of God's manifest glory can fade. And it's not an issue with the nature of God's glory, but a reflection of the feebleness of His people's memories.
I'm reading in Exodus 16 this morning. The nation has just come off a time of great celebration. Walking through a wall of water has a way of turning on the party lights. They have been singing to Lord. They have been dancing to the Lord. They have been rejoicing in the Lord. How come? Because they are on the other side of the Red Sea and their enemies are not. But no sooner do the lights go out then, on the morning after the night before, the singing turns to grumbling.
Because, on the other side of the Red Sea, there's a wilderness. A legitimate wilderness. Not much water. Not much food. And when you compare it to Egypt (if you can ignore the slavery, bondage, and oppression) it comes up kind of short. And despite having witnessed the power of God through ten mind-blowing plagues. Despite having seen the grace of God as the blood of the lamb on their door posts spared the lives of their firstborn. With seemingly no thought to the fact that they are hauling around the treasures of the land of their captivity. Having seemingly forgotten that they have just walked through a great sea. As the people start to interact with the desert, they murmur. As they walk in the wilderness, they complain. And they wonder about going back to Egypt.
Their wilderness experience is real. Their thirst is real. Their hunger is real. But what they fail to remember is that their God is real. And so, to remind them that He is real, He says He will feed them.
I have heard the grumbling of the people of Israel. Say to them, At twilight you shall eat meat, and in the morning you shall be filled with bread. Then you shall know that I am the LORD your God.
(Exodus 16:12 ESV)
And they wake up one morning and on the ground is God's gracious provision, "a fine, flake-like thing, fine as frost on the ground" (16:14). Not quite like anything they had seen before. Not quite like anything they had tasted before. Certainly something that had never seen served up in this manner before.
When the people of Israel saw it, they said to one another, What is it?
(Exodus 16:15a ESV)
God met their needs outside of their expectations. God provided with a solution beyond their imagination. What they felt was their greatest need on earth was met with divine provision from heaven. And they said, "What is it?" Original word? . . . Manna.
"It is the bread God has given you to eat," Moses tells them. Gather it. Pick it up. Digest it. As much as you can eat. It will be sufficient for the day. None will have too much. No one will have too little (16:15b-18). And the people said to one another, "Manna." What is it?
And I'm thinking that sometimes, when I'm wandering in the wilderness, I too think I know what I need. I know what God should do and how He should do it and when it should be done. And, when it doesn't pan out or fit into my timing, though I've known His past provision, I can find myself murmuring and complaining. Instead, I should be waiting and anticipating His provision. Even if that provision is "What is it?"
God has not directed where He will not lead. He has not sent on pilgrimage without thought of how He will provide. He doesn't lead us through great victory into a wilderness experience so that we might figure out how to go it alone. No, in fact the wilderness, often, is so that He might feed us that we might know afresh that He is the LORD our God. So let's not be surprised if God meets our needs in surprising ways. If, in the morning, we find that how He's provided is in a way we never expected.
And we'll praise Him, even as we ask, "What is it?" We'll marvel in Him, even as we partake in bread from heaven. And we'll worship Him, even as we eat the manna.
What is it . . . a reminder of God's grace. Manna . . . a reason to give God glory!


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taken from my prepaid Prestige card, Calgary Alberta. Went to withdraw a hundred but it was not there. Called Prestige and found out it went here. When I tried to take out eighty dollars that was not there.
Thank You for reading of this Complaint / Review. Complaint author: Anonymous
Country: USA
State: All USA
Phone: 8779127797
Site: www.supportsdltd.com

subset: commoncrawl


text:
Welcome to Fall, don't these galette apple mini pies look amazing! They are both so delicious, light and flaky, and the crust is awesome. My kids went apple picking this past weekend, and boy do I have apples to use up. So, I thought, why not try an apple pie galette in the Air Fryer?
I have been really busy looking for apple pie plates that will fit in the air fryer, I looked all of the obvious places, Target, Walmart and even William Sonoma, but my search ended on Amazon. I guess I should have started there.
That sounded like a plan and ordered these two mini pie plates to fit into my air fryer and they worked perfectly, I can not recommend them more.
Now, I used all the prepared pie dough to make this dessert. This is my second time making these. The first time, my family ate way too fast to take a picture of it. It was fabulous, I served it with some homemade vanilla ice cream. And, to be honest, it might be one of the most delicious desserts, I have ever eaten, even though I made it!
Next week, I will make my homemade cherry galettes but I wanted to share this with all of you, in case you were looking for an easy fall or back to school dessert.
This morning, I used my new Instant Pot, Vortex Plus, Air Fryer, which I purchased additional trays for, I found these too at Amazon and ordered them, they work perfectly, as you can see from the picture. So, if you need additional accessories for the Vortex, you can find them here:
Kitchen Items Used: (Note: Affiliate links are included in this post)
Instant Pot, Vortex Plus, Air Fryer
J.A. Henckels 6-Inch High Carbon Stainless Steel Chef's Kitchen Knife
Stainless Steel Measuring Cups, 5 Piece Set
Hudson Essentials Stainless Steel Measuring Cups Set-6 Piece Stackable Set with Spout
Anchor Glass Measuring Set–This is one of the handiest tools in the Kitchen
OXO Good Grips 12 Inch Stainless-Steel Locking Tongs
Don't Forget To Pin–
- 1 large cooking apple, I used grannysmith
- 1 teaspoon ground cinnamon
- 1 package biscuits (found in the dairy section)
- 1 cup powdered sugar
- 2 tablespoon milk or cream
Dice the apple into small pieces, then in a small bowl mix together the diced apples and cinnamon.
Make a small hole in the middle of your biscuit, which you will be filling. Be careful not to cut through to the other side.
Stuff as much filling as you can.
Grease your pan with either non-stick cooking spray or olive oil spray. Then place your stuffed biscuits on top of the tray, being careful not to crowd them, as they will expand during cooking.
Set your air fryer to 330 degrees for 5 minutes, then flip and cook for another 3 minutes.
Meanwhile, make your icing, mix together the confectionery and milk. Keep on adding milk and stirring until you get a thin coating.
Drizzle the icing over the fritters.
Plate, serve and enjoy!


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Christmas on the Farm - Shoots & Ladders - November 18th
Millie Holloman Photography
Saturday, November 18, 2017 from 8:00 AM to 4:30 PM (EST)
Event Details
Boy howdy are we gonna have fun this year!! Shoots & Ladders is heading to the Farm for our Holiday shoot. We will have stylized elements in a real farm setting. It's a twist on our typical holiday sessions as we will be outside and live animals are sure to make their way into some of the photos!
WHEN: Saturday, November 18, 2017
DETAILS: $150 session fee includes a family photo and individual images of your children during a 20 minute photo session (up to 3 kids – $25 per additional child) + 25 5×7 Christmas Cards (complete with envelopes)
This set is only ONE DAY of shooting and it's sure to fill VERY quickly (Christmas is always our busiest time for Shoots & Ladders and we sell out every single year). Both, Millie AND Amanda will be photographing families who sign up so we can accommodate everyone.
Still not sure of what to wear? We've created a Pinterest board just for this set with loads of ideas so we'll send that link your way once you sign up! We'll present the images (8-10 proofs) to you online on Monday, November 27th. To get your cards back by December 4th, we'll need you to order these right away!
Join our facebook page for updates by clicking here!
Exact location details will be emailed when session is purchased. Your session must be paid when booking. No cancellations. Payments are non-refundable. Choose your time carefully, once it's chosen it's reserved just for you. Be punctual. Showing up late gives you less time in our set, or could make you forfeit your shoot. Prints sold separately. No pets allowed.
In case of inclement weather, our rain date is November 26th. If you are unable to attend on that date, you will be offered a refund!
When & Where
Old McFaye's Farm
Castle Hayne,
Saturday, November 18, 2017 from 8:00 AM to 4:30 PM (EST)
Add to my calendar
Millie Holloman Photography
Come On In. Our studio is headquartered in the heart of historic downtown Wilmington, a charming port town on the Cape Fear River where horse-drawn carriages clickety-clack down brick paved streets. In fact, at one time, our studio housed livery stables for the downtown community. Instead of horses, these days we house scooters! Restored by architect Ligon Flynn, you enter via an open-air garden called The Atrium, a perfect outdoor space for intimate weddings. Inside, on our sprawling second floor loft, you'll discover exposed brick walls, a daylight studio, hammocks, comfy couches, and a "creepy" clown we strategically hide to surprise unsusp
Nice to Meet You. Our company has just one name, but we're an entire team of crazy, creative, passionate, professionals. Our photographers are based across North Carolina (Wilmington, Raleigh, & Charlotte), but work all over the world.
We believe that real relationships happen when people go beyond the pleasantries of a formal introduction, so we're inviting you behind the scenes into our homes, our work spaces, our hearts and our minds. Next time, let's do lunch.


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Botanical name: Nepeta catoria
Native to: Europe
Status: ACO Australian Certified Organic – Australian Grown
Actions: Sedative which helps relaxation and sleep. Aids digestion, flatulence (gas), colic, cramping and even headaches. Catnip can be used against respiratory infections, cold and flu, as well as fever, especially in children.
Description: Dried catnip can be used to aid sleeping troubles (insomnia), as well as digestive
problems. Particularly, it can sooth gastrointestinal upsets, such as indigestion, colic, cramping and flatulence (gas). Catnip is also known to increase urination, help regulate menstrual cycles and relief headaches and migraines.
Its gentle action and enjoyable taste make it a helpful remedy especially for children, with upper respiratory infections, cold and flu, as well as fever.
Catnip may also be applied directly to the skin to sooth arthritis, rheumatism, haemorrhoids and in a compress to relieve swelling.
All information on this site is for informational purposes only. We do stress that you should do your own research, or talk with your health professional if you suffer from an existing health condition, or if you are on other medication, prior to consumption of our products.
The Herb Shed offers it's products for pick up through it's showroom (by appointment) and online shop.
Our Herbs and Teas are Packed in Earth Bags to minimise portage costs


text:
Energy Drinks Market is Generating Revenue of $86.01 Billion by 2026, at CAGR 7.20% Growth Rate
Increase in demand for energy drink as it offers desirable effects and inclination of consumers toward organic energy drinks are estimated to propel the growth.PORTLAND, OR, UNITED STATES, September 16, 2020 /EINPresswire.com/ -- Energy Drinks Market by Type (General Energy Drink, Energy Shots, and Others), Form (Organic and Conventional), End User (Professional and Personal Use), and Distribution Channel (Supermarkets/Hypermarkets, Convenience Store, Retail Stores, Medical Stores, and Other Outlets): Global Opportunity Analysis and Industry Forecast, 2018–2026". According to the report, the global energy drinks market was pegged at $53.01 billion in 2018 and is projected to garner $86.01 billion by 2026, registering a CAGR of 7.20% from 2019 to 2026.
Get Instant Access to Report - Download Free PDF Sample: https://www.alliedmarketresearch.com/request-sample/5315
Increased demand for energy drinks as it offers various desirable effects such as improved memory, fast recovery and increased prominence among young generation have propelled the growth of the global energy drinks market. However, presence of alternatives including green tea and ginger root tea hinders the market growth. On the contrary, inclination of consumers toward organic energy drinks is expected to create lucrative opportunities in the near future.
The non-alcoholic energy drink segment held the largest share in 2018, accounting for more than half of the market, owing to rise in popularity among teenagers along with increasing health consciousness. Moreover, the segment is estimated to register the fastest CAGR of 6.5% during the forecast period. The report includes analysis of the alcoholic energy drink segment.
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The adult segment held the largest share in 2018, contributing more than two-fifths of the market and is projected to manifest the fastest CAGR of 6.7% during the study period. This is pertaining to rising trend among the growing adult population of corporates and athletes, who consume energy drinks for an extra dose of energy to enhance their performance.
The market across the Asia-Pacific region is anticipated to register the fastest CAGR of 7.3% during the forecast period, owing to rising demand for convenience beverage, widening media exposure and aggressive marketing by energy companies. However, the market across the North America region contributed the largest share in 2018, accounting for nearly one-third of the market. The report includes an analysis of the other regions such as Europe and LAMEA.
The report includes an in-depth analysis of the major market players such as Rockstar Inc., Red Bull GmbH, Monster Energy, PepsiCo Inc., AriZona Beverages USA LLC, Big Red, Dr. Pepper Snapple Group, National Beverage Corp., Dabur India Ltd., and Coca Cola Company.
Access Full Report Summary: https://www.alliedmarketresearch.com/energy-drink-market
Key Findings of the Energy Drinks Market :
• Region wise, Asia-Pacific is anticipated to lead the energy drinks market in 2026, growing at a CAGR of 7.30%, from 2019 to 2026.
• Based on type, the nonalcoholic segment occupied nearly 53.63% of the energy drink market share in 2018.
• By end user, the adult segment dominated the overall market in 2018, and is expected to grow at a CAGR of 6.70% during the forecast period.
• The kids segment is anticipated to grow at a significant CAGR of 6.10%.
Similar Reports:
Sports Nutrition Market size is expected to reach $44,003 million, by 20211
Cider Market is projected to reach at $16,252 million by 20232
Carotenoids Market is projected to reach $2,098 million by 20253
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Allied Market Research (AMR) is a full-service market research and business-consulting wing of Allied Analytics LLP based in Portland, Oregon. Allied Market Research provides global enterprises as well as medium and small businesses with unmatched quality of "Market Research Reports" and "Business Intelligence Solutions." AMR has a targeted view to provide business insights and consulting to assist its clients to make strategic business decisions and achieve sustainable growth in their respective market domains. AMR offers its services across 11 industry verticals including Life Sciences, Consumer Goods, Materials & Chemicals, Construction & Manufacturing, Food & Beverages, Energy & Power, Semiconductor & Electronics, Automotive & Transportation, ICT & Media, Aerospace & Defense, and BFSI.
We are in professional corporate relations with various companies and this helps us in digging out market data that helps us generate accurate research data tables and confirms utmost accuracy in our market forecasting. Each and every data presented in the reports published by us is extracted through primary interviews with top officials from leading companies of domain concerned. Our secondary data procurement methodology includes deep online and offline research and discussion with knowledgeable professionals and analysts in the industry.
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School Programs
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365 days of SL, day 34:
(Click for larger — 1920x1033 PNG, 2420 KiB)
Yesterday saw a visit to one of our favorite stores, the fabled Mushporium. We've only really been there once before, but the concept (and the name!) amused us so greatly that it's figured in our personal culture ever since.
Alas, we couldn't find the place we went to anymore. There's a small outlet in Mindulle at Route 10, which is all I still had landmarks for; we both recalled a larger store, situated at a corner somewhere, but the small outlet only had a landmark to the above place, a wide-open field that neither of us had seen before. Looking at the map (linked below), it appears that it's actually on Corsica — interesting, I didn't notice that last night. In any case, perhaps the main store just moved.
I do have a few older screenshots, at least, which I *think* are from the previous main store. So here's two bonus pictures:
(Click for larger — 1027x815 PNG, 1306 KiB. Also available uncropped; 1280x976 PNG, 1937 KiB.)
This is the old "Mushporium" logo sign; I didn't see that one around anymore now.
(Click for larger — 1280x720 PNG, 1705 KiB. Also available uncropped; 1280x976 PNG, 2318 KiB.)
And this is part of the the store itself, the area selling regular plain old fungi (as opposed to particle mushrooms and other such things). I think I actually got a free red mushroom from the place, too, although I've never had an opportunity to use it for anything.
Location (new main store): Yia Yuto (180, 175, 47)


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Increasingly, the use of pneumatic equipment compared to electrical equipment, both for performance, durability and reliability of the tool, as well as for safety, is being imposed in the manufacturing and transformation workshops of natural stone and agglomerates, avoiding the risk of electrocution in contact with water.
The quality of the supplied air directly affects the performance of the machine and therefore the final finish resulting from its use.
Although in our sector air compressors are largely intended for the supply of air in a portable pneumatic tool, we have to take into account current and future needs when choosing and sizing our compressed air installation.
Traditionally, the piston compressor has been the most chosen option by price for low and lower quality air demands. However, current high-quality pneumatic equipment such as the Alpha® pneumatic tool for which MASER is an exclusive distributor for Europe, requires a constant and dry air flow that will guarantee the correct functioning of the equipment, thus ensuring the best final finish. , the choice of a screw compressor with a built-in dryer being necessary.
But let's see before following the advantages of the screw compressor versus the piston:
Air produced: 95% of the sucked air compared to 75% of the sucked air, resulting in energy savings.
Vacuum running: in screw compressors we can regulate the idle running time (operation of the compressor without air production), compared to the start-stop system of the piston compressors, resulting in energy savings.
Sound level: 62-70 dB (A) versus 85-90 dB (A)
Duty cycle: 100% in charge versus 50-60%. The piston compressor limits the operating time due to its need to rest for cooling, compared to the continuous operation of the screw compressor thanks to its oil cooling.
Final air temperature: 20 ° C + temp. ambient versus 75 ° C + temp. environment, which helps in this case to avoid excessive water condensation.
Wear: the continuous friction of the pistons, cylinders, rings and valves causes wear causing the piston compressor to deliver less air in the long run with the same energy consumption. Screw compressors, lacking their friction blades, there is no wear and tear always producing the same air.
As important as the choice of the type of compressor is the correct installation of all its components and the correct sizing of these.
We can choose to acquire a compact group that integrates the screw, the boiler and the dryer in the same space:
Powers from 3hp to 30hp.
From 8 to 13 bar of pressure.
Up to 3,000 l / min of delivered air.
From 60 to 70 dB (A) maximum.
Preinstalled and preconfigured: plug, connect and use.
Compact: minimum space occupied.
Easy maintenance
Or we can choose to perform an installation tailored to our needs, depending on the air flow rates required at any time, the number of operators that can work simultaneously, etc.
In this case the traditional installation of compressed air will be as follows and in this order:
Compressor 8 to 13 bar pressure
Deposit from 100 to 3,000 lts
Air filter
Oil filter
Regulator, Assembly, Couplers, etc.
Although there is a tendency to dispense with the dryer to reduce investment, pneumatic equipment must work with dry air, avoiding sending water, however minimal, to the tools, which in the long run will suffer oxidation and / or corrosion problems, chalky residues and others, which will shorten the life of their equipment and cause production stoppages with the consequent economic damage.
For all this, MASER has incorporated into its product catalog the widest range of compact compressors, that is, boiler, compressor and dryer in one piece, or the possibility of acquiring each element separately to size the installation according to your requirements , or renovate and improve the existing installation
In accordance with the requirements of air flow and pressure of the range of pneumatic tool of Alpha® Professional Tools that MASER distributes exclusively, we recommend to all those professionals interested in their acquisition to verify that their current compressor meets the minimum and essential requirements for its correct operation and guarantee of finishes.
Especially for the Alpha® AIR-684 polisher and the Alpha® PSC-150 circular saw, our star products, we recommend the following models that will fully meet your technical requirements, all of them with built-in dryer:
Nuair Sirio 15hp, 10 bar, boiler of 500 liters and a flow of delivered air of 1,500 liters / min.
Nuair Star 15hp, 10 bar, boiler of 500 liters and a flow of delivered air of 1,500 liters / min.
The entire range of NUAIR compressors integrate components of the highest quality, European manufacturing and have an extensive technical service both in Spain and Portugal. Do not hesitate to contact us to request a quote or receive specialized advice from our commercial network.


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So there was an error in my code. Now I am no stranger to the odd bug(or 10), so there I was staring at the code.
I had been stepping through the code. I had put in various var_dump statements. But could I find the problem! No.
So I called over to my colleague Lee, and asked him to sit down to see if he could find the problem.
So there we were staring at the screen like two unwise monkeys, the sweat dripping off our brows as we tried to find where the problem lay.
"what you need to do" said a voice from behind us "is fail fast."
We looked round and there was Tay, another good friend, looking at the screen over our shoulders.
"eh" said Lee and I at the same time.
"well" said Tay, "If your code encounters a problem, it should fail straight away, maybe log the error as well. That way you can find the problem sooner, and fix it quicker"
Lee and I looked at each other.
"I think you might need to refactor your code mate" said Lee.
Do you write code that fails fast? I would love to hear how so why not leave a comment.


text:
Antigenic variation is an effective strategy evolved by pathogenic microbes to avoid immune destruction. Variable Ags such as the variable major protein of Borrelia hermsii, the variant surface glycoprotein of African trypanosomes, and the pilin of Neisseria gonorrhoeae include an immunodominant variable domain and one or more invariable domains that are not antigenic. Short, nonantigenic, invariable regions also may be present within the variable domain. VlsE (variable major protein-like sequence, expressed), the variable surface Ag of Borrelia burgdorferi, the Lyme disease spirochete, also contains both variable and invariable domains. In addition, interspersed within the VlsE variable domain there are six invariable regions (IR1–6) that together amount to half of this portion's primary structure. We show here that these IRs are conserved among strains and genospecies of the B. burgdorferi sensu lato complex. Surprisingly, unlike the invariable regions of variable major protein, variant surface glycoprotein, and pilin, which are not antigenic in natural infections, the most conserved of the IRs, IR6, is immunodominant in Lyme disease patients and in monkeys infected with B. burgdorferi. IR6 is exposed on the surface of VlsE, as assessed by immunoprecipitation experiments, but is inaccessible to Ab on the spirochete's outer membrane, as demonstrated by immunofluorescence and in vitro killing assays. VlsE thus significantly departs from the antigenic variation paradigm, whereby immunodominance is only manifest in variable portions. We submit that IR6 may act as a decoy epitope(s) and contribute to divert the Ab response from other, perhaps protective regions of VlsE.
To maintain chronic infection, parasitic microbes have evolved strategies to avoid or suppress the host immune response. One of the most effective evasive strategies is antigenic variation. Several pathogenic organisms, including species of protozoa and bacteria, undergo antigenic variation. The African trypanosome Trypanosoma brucei evades the immune response by successively expressing more than 100 antigenically distinct variant surface glycoprotein molecules (VSG)3 (1, 2). The VSG appears to be the only antigenic structure exposed on the surface of the organism (3). The bacterium Neisseria gonorrhoeae avoids immune attack by activating silent variable pilin genes (4). Pilin is a major component of the pilus, a predominant surface structure of N. gonorrhoeae that allows adhesion of the bacterium to various host cells (5). Relapsing spirochetemia and fever occur when the spirochete Borrelia hermsii alternates expression of at least 25 different antigenic forms of a surface-exposed and abundant lipoprotein called variable major protein (Vmp) (6, 7).
Borrelia burgdorferi, the spirochete that causes Lyme disease, expresses a surface lipoprotein, VlsE (Vmp-like sequence, expressed), that also undergoes antigenic variation (8). In the B31 strain of B. burgdorferi, this lipoprotein has a predicted molecular mass of 34 kDa and is expressed from a site (vlsE) that is located near the right telomere of the linear plasmid lp28-1 (8). The sequences of the 5′- and 3′-end segments of the vlsE-coding region, each segment encompassing less than one-third of this molecule's length, are preserved during the course of vlsE variation (9). The central portion, namely the cassette segment, which encodes the whole variable domain, recombines unidirectionally with 15 similar cassette segments that are located upstream (8, 9) from the vlsE locus. The unidirectional recombination events that result in VlsE antigenic variation only affect six regions of the VlsE variable domain (8). These six variable regions are ∼16, 22, 6, 18, 13, and 6 aa in length, respectively (9). The remainder of the VlsE variable domain, which is composed of invariable regions of about 9, 16, 7, 25, 10, and 26 aa, remains essentially unchanged during the process of antigenic variation (9).
The VSG, pilin, and Vmp each can be divided also into invariable and variable domains (10, 11, 12). As with VlsE, small invariable regions are scattered within the latter domains. Although the variable domains are highly immunogenic and serve as the major target of the host immune response (13), the small invariable regions within them as well as the larger invariable domains are not antigenic in natural infections and tend to be conserved among different isolates of the organism (10, 11). Conserved portions are probably responsible for the maintenance of a functional molecular conformation (10, 11). Abs directed to the variable domains are able to effectively kill the organisms that express the corresponding Ags. However, the organisms escape killing by successively expressing different variable domains. Pathogens thus use these variable Ags to divert the host immune response from other Ags that may be protective (13).
Nothing is known about the functional or antigenic properties of the invariable regions within the variable domain of the VlsE. As we analyzed the Ab response to a recombinant protein (P7-1), encoded by a cassette segment of the vls locus of the IP90 strain of Borrelia garinii, we made the following observation. In rhesus monkeys that had been infected with spirochetes of either the JD1 or the B31 strain of B. burgdorferi sensu stricto, Ab to P7-1 appeared within the first 3 wk postinfection (PI) and persisted thereafter, at least until wk 10 PI, the longest time point examined in this initial experiment. How could an Ag fragment so promiscuously and rapidly altered as the variable domain of VlsE (8, 14) elicit such a long-lasting Ab response to a cassette segment from another genospecies? A straightforward answer to this question was that one or more of the invariable regions within the variable domain are antigenic themselves and sufficiently conserved among Borrelia strains and genospecies to retain antigenic cross-reactivity.
To investigate this possibility, we analyzed the antigenicity of the invariable regions of the IP90 cassette segment using standard computer algorithms and determined the percent identity of their amino acid sequences with the corresponding invariable regions of published VlsE cassette segments of B. burgdorferi sensu stricto strains B31 (8) and 297 (15). One of the invariable regions (IRs), namely IR6, showed both the highest levels of antigenicity by the Hopp-Woods algorithm (16) and amino acid sequence identity among Borrelia genospecies and strains. We synthesized a peptide (C6) that encompassed the amino acid sequence of IR6 and assessed its antigenicity by ELISA in B. burgdorferi-infected mice, monkeys, and humans. We also performed antigenic competition experiments, using C6 as competitor, to determine what proportion of the mouse, monkey, and human Ab response to P7-1 was directed to IR6. Exposure of IR6 on the surface of the VlsE molecule was determined by immunoprecipitation of native VlsE of IP90 with a rabbit antiserum raised against the C6 peptide. Exposure of IR6 on the spirochetal surface was assessed by direct immunofluorescence with FITC-labeled anti-C6 Ab and by Ab-dependent, in vitro killing experiments using anti-C6 Ab and IP90 spirochetes. Here we report the results of these studies.
Materials and Methods
Animals, animal infections, and spirochetal strains
Rhesus monkeys (2–4 years old, Macaca mulatta) were infected by the bite of Ixodes scapularis nymphal ticks that were themselves infected with B. burgdorferi sensu stricto strain JD1 or B31. Mice (6–8 wk old, C3H/HeN, The Jackson Laboratory, Bar Harbor, ME) were infected with B. burgdorferi sensu stricto strain Sh-2-82 (low passage, a gift from Denee Thomas, University of Texas Health Science Center, San Antonio, TX) by s.c. needle inoculation with 1 × 108 spirochetes administered in 1 ml of BSK-H medium (Sigma, St. Louis, MO) or by the bite of B31-infected Ixodes scapularis nymphal ticks. B. garinii strain IP90 (low passage) and B. burgdorferi sensu stricto strain HB19 (high passage, lacking the lp28-1 plasmid encoding VlsE) were originally obtained from the Centers for Disease Control and Prevention (CDC; Fort Collins, CO). When required, spirochetes were cultivated in BSK-H medium as described previously (17).
Cloning, sequencing, and expression of the 7-1 cassette segment of IP90
A library of randomly sheared total DNA from B. garinii IP90 was constructed in the λZAP II bacteriophage vector (Stratagene, La Jolla, CA) following a procedure described previously (18). The library was screened with a pool of plasma collected from rhesus monkeys within the first 10 wk after tick inoculation with B. burgdorferi JD1. On immunoblots of whole cell extracts of B. garinii IP90 this plasma pool reacted strongly only with three components, namely flagellin, an unidentified 60-kDa protein, and an Ag that was the IP90 homologue of the 34-kDa VlsE of B31 (8). After several rounds of screening, 11 clones were rescued into the pBluescript phagemid (Stratagene), and the recombinant plasmids were purified and used to transform cells of the SURE strain of E. coli (Stratagene). Several transformants were selected from each original clone, the presence of the insert was confirmed, and one such transformant from each clone was grown, induced for expression, lysed, and analyzed by immunoblot analysis with the original plasma pool. One of the 11 cloned fragments (named 7-1) hybridized to all others by dot-blot hybridization. This fragment was selected for overexpression and purification on the basis of the strong reactivity of the expressed protein with the plasma Abs. Sequencing of the 7-1 insert was performed using standard procedures (19) after generating nested Bal31 deletions and subcloning the partially deleted fragments. Ab from the original plasma pool that was affinity purified using the recombinant protein (P7-1) expressed by clone 7-1 as immunoabsorbant reacted with the putative IP90 VlsE on immunoblot of B. garinii lysates. The 7-1 insert was subcloned into the pQE expression system for overexpression and purification of the polypeptide (Qiagen, Chatsworth, CA). The protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA) with BSA as a standard.
This protocol was initially used to monitor monkey Ab responses to P7-1. Ninety-six-well ELISA plates (Corning, Corning, NY) were coated with 100 μl/well of a solution of P7-1 at 0.1 μg/ml in coating buffer (0.1 M carbonate buffer, pH 9.2) at 4°C overnight. Plates were blocked with 200 μl/well of 5% FCS in PBS/T (10 mM sodium phosphate, 150 mM NaCl, and 0.1% Tween 20, pH 7.4) for 1 h at room temperature. After washing three times with PBS/T, 50 μl/well of serum samples from B. burgdorferi-infected monkeys diluted 1/100 in 5% FCS in PBS/T were incubated for 1 h at 37°C. Plates were washed and incubated with 50 μl of 1) a mixture of biotinylated goat anti-human IgG (γ-chain-specific) and IgM (μ-chain-specific) Abs at the dilution recommended by the manufacturer (Vector Laboratories, Burlingame, CA) for 1 h at 37°C, 2) avidin-HRP complex also at the dilution recommended by the manufacturer (Vector) for 30 min at 37°C, and 3) a solution containing 2 g/l ortho-phenylenediamine and 0.03% hydrogen peroxide (both from Sigma) in 0.1 M citrate-sodium-phosphate buffer, pH 5.0, for 10 min at room temperature. The reaction was stopped with 50 μl of 4 N H2SO4. The OD at 490 nm was determined. Goat anti-human IgG and IgM cross-react with the corresponding monkey Igs.
Identification of conserved sequences and prediction of antigenicity of invariable regions of the VlsE variable domain
The deduced amino acid sequence of P7-1 was compared with sequences available in the GenBank database (National Center for Biotechnology Information, Rockville, MD) using the BlastP algorithm (20). The antigenicity of the entire P7-1 polypeptide was analyzed using the Hopp-Woods scale (16), and identities of invariable regions of P7-1 with homologous cassette segments from strains B31 and 297 were calculated after aligning the cassette segments using the pam250 algorithm and MacVector 5.0 computer software (Eastman Kodak, New Haven, CT).
Peptide synthesis and conjugation to biotin
A 25-mer peptide (C6 = MKKDDQIAAAMVLRGMAKDGQFALK) and a 19-mer (C2 = DAASVNGIAKGIKGIVDAA) were prepared using the fluorenylmethoxycarbonyl synthesis protocol (21). N-terminal biotinylation was performed by the maleimide carboxylate method. The maleimide reagent was obtained from Molecular Probes (Eugene, OR), and the protocol suggested by the manufacturer was followed.
Human serum samples
A panel of 41 human serum samples was provided by the CDC. All samples were collected from Lyme disease patients who had signs and symptoms that satisfied the CDC clinical case definition (22). Four serum samples from chronic Lyme disease patients were obtained from the National Institutes of Health. Ninety-seven serum samples obtained from hospitalized patients in an area not endemic for Lyme disease were used as negative controls.
C6 peptide ELISA
Ninety-six-well ELISA plates were coated with 100 μl/well of 4 μg/ml streptavidin (Pierce, Rockford, IL) in coating buffer and incubated at 4°C overnight. The remaining steps were conducted in a rotatory shaker at room temperature. After two 3-min washes with 200 μl/well of PBS/T at 200 rpm, 200 μl of 5 μg/ml biotinylated peptide dissolved in blocking solution (PBS/T supplemented with 5% nonfat dry milk (Carnation, Nestle Food, Glendale, CA)) was applied to each well. The plate was shaken at 150 rpm for 2 h. After three washes with PBS/T as described above, 50 μl of serum (mouse, monkey, or human) diluted 1/200 with blocking solution was added to each well. The plate was incubated at 150 rpm for 1 h and then washed three times with PBS/T. Each well then received 100 μl of 0.2 μg/ml goat anti-monkey IgG (γ-chain specific; Kirkegaard & Perry Laboratories, Gaithersburg, MD), 0.5 μg/ml anti-mouse IgG (heavy and light chain specific; Sigma), or 0.1 μg/ml anti-human IgG (heavy and light chain specific; Pierce), each conjugated to HRP and dissolved in blocking solution. The plate was incubated for 1 h while shaking. After four washes with PBS/T, each for 3–6 min, the Ag-Ab reaction was probed using the TMB Microwell Peroxidase Substrate System (Kirkegaard & Perry), and color was allowed to develop for 10 min. The enzyme reaction was stopped by addition of 100 μl of 1 M H3PO4. OD was measured at 450 nm.
Competitive ELISA
Ninety-six-well ELISA plates were coated with 100 μl/well of 0.3 μg/ml of purified recombinant P7-1 dissolved in coating buffer at 4°C overnight. After blocking with blocking solution, 25 μl of this solution with 0, 1, 4, 16, 64, 256, or 1024 ng of the C6 or C2 peptides was added to each well. A 25-μl volume of a 1/100 dilution of the appropriate serum in blocking solution also was added to each well. The remaining steps were performed as related in the section describing the C6 peptide ELISA.
Preparation of rabbit anti-C6 peptide antiserum
The C6 peptide was covalently linked to keyhole limpet hemocyanin by the N-succinimidyl maleimide carboxylate method after adding a cysteine residue to the peptide's N-terminus. The maleimide reagent was obtained from Molecular Probes (Eugene, OR), and the protocol suggested by the manufacturer was followed. Six-month-old New Zealand White rabbits were given three injections at biweekly intervals of 200 μg of conjugated Ag emulsified with CFA (first injection) or IFA (remaining injections). Ten days after the last injection the Ab titer was determined by the peptide ELISA and immunoblot analysis using IP90 spirochete whole cell lysates as Ag.
Immunoprecipitation and immunoblot
Immunoprecipitation was conducted at 4°C. About 1.5 × 1010 IP90 spirochetes harvested at stationary growth phase were extracted in 4.5 ml of solubilization buffer (50 mM Tris-HCl, 1% Triton X-100, and 1 mM EDTA, pH 7.6) for 30 min. The mixture was centrifuged at 13,000 × g for 30 min, and the supernatant was collected. Each 1.5 ml of this supernatant was mixed with 30 μl of preimmune or immune rabbit serum and incubated for 30 min. Fifty microliters of drained ImmunoPure Immobilized Protein G (Pierce) pre-equilibrated in solubilization buffer was then added and allowed to incubate for an additional 30 min. After washing the gel twice with excess volumes of this buffer by centrifugation at 3000 × g for 20 min, 150 μl of nonreducing SDS-PAGE sample buffer (125 mM Tris-HCl, 3% SDS, and 20% glycerol, pH 6.8) was added. The suspension was incubated at room temperature for 30 min and then centrifuged at 16,000 × g for 30 min. Ten microliters of supernatant was loaded onto each of 10 lanes of a SDS-12% polyacrylamide minigel. Separated proteins were electrotransferred to nitrocellulose in Towbin transfer buffer. After incubating in blocking solution for 2 h, the blot was incubated for 1 h in rabbit anti-C6 serum diluted 1/2,000 with blocking solution. After three washes with PBS/T, the blot was incubated in blocking solution with 0.5 μg/ml goat anti-rabbit IgG conjugated to HRP (Pierce) for 1 h. The Ag-Ab reaction was probed in PBS/T supplemented with 0.05% 4-chloro-naphthol (Sigma), 0.015% hydrogen peroxide, and 17% methanol.
Direct immunofluorescence
Rabbit IgG was purified from preimmune or immune serum using conventional ammonium sulfate precipitation. The purity and concentration of IgG preparations were assessed using SDS-PAGE and the Bio-Rad protein assay kit, respectively. Purified IgG was conjugated to FITC according to the manufacturer's instruction (Pierce). For labeling, spirochetes were either unfixed or acetone fixed. For the fluorescent labeling of unfixed spirochetes, ∼108 spirochetes that were harvested from 1.0 ml of IP90 or HB19 culture grown to stationary phase by centrifugation at 4,000 × g for 20 min were gently resuspended in 100 μl of PBS supplemented with 2 μg of rabbit anti-C6 IgG-FITC conjugate, and incubated for 1 h. After two washes with excess volumes of PBS by centrifugation at 16,000 × g for 5 min, spirochetes were resuspended in 100 μl of PBS, applied to microscope slides, and counted under both darkfield and fluorescent microscopes. The ratio of fluorescent to total spirochetes was thus calculated. The same procedure was performed on acetone-fixed spirochetes. For this purpose, organisms harvested from 1.0 ml of culture fluid were suspended in 1.5 ml of acetone, incubated for 20 min, and then centrifuged at 16,000 × g for 5 min. After one wash with PBS, the fixed spirochetes were stained with fluorescent anti-C6 Ab as described for unfixed spirochetes. Goat anti-B. burgdorferi Ab FITC conjugate (Kirkegaard & Perry Laboratories) was used as a positive control.
In vitro Ab killing assay
As a source of complement, serum samples were collected from normal rhesus macaques, pooled, and stored in small aliquots at −70°C until used. Serum chosen for this purpose did not contain cross-reactive anti-B. burgdorferi Abs as determined by immunoblot analysis using whole cell lysates of B. burgdorferi as Ag. To perform the killing assay, spirochetes were cultured in BSK-H medium until they reached the mid-logarithmic phase (∼2 × 107 cells/ml). A total of ∼5 × 105 spirochetes in 25 μl of BSK-H medium was added to each well of a 96-well plate (Corning). A volume of 50 μl of heat-inactivated (56°C, 30 min) serum sample appropriately diluted in the same medium was already dispensed in each well. The plate was incubated at 34°C for 30 min before the addition of 25 μl of complement preparation (normal monkey serum). After 24 h of incubation at 34°C in a humidified atmosphere of 3% CO2, 5% O2, and the balance of N2, 5 μl of each sample was removed, and dead (nonmotile) and live (motile) spirochetes were counted under a darkfield microscope. Monkey anti-outer surface protein A (OspA) antiserum was used as a positive control (23).
The invariable regions of the VlsE variable domain are conserved among strains and genospecies of B. burgdorferi
As we assessed the Ab response to the recombinant P7-1 polypeptide in monkeys that had been tick inoculated with either B31 or JD1 B. burgdorferi spirochetes, we noticed that the response was detectable within the first 3 wk PI, and that it persisted at least until wk 10 PI, the longest time point measured in this initial experiment (Fig. 1⇓). We hypothesized that one or more of the invariable regions within the P7-1 variable domain were antigenic themselves and sufficiently conserved among Borrelia strains and genospecies to exhibit antigenic cross-reactivity. The deduced amino acid sequence of the B. garinii P7-1 recombinant polypeptide is shown in Fig. 2⇓. It is depicted aligned with sequences from VlsE cassette segments of B. burgdorferi strains B31 (8) and 297 (15). In B31, the cassette segment is comprised between the repeats EGAIKG (Fig. 2⇓) (8). The six variable regions of the B31 VlsE cassette segment (8) are doubly underlined. The invariable regions IR1–6 of the IP90 VlsE variable domain (Fig. 2⇓) are clearly conserved across both genospecies and strains of B. burgdorferi sensu lato. With the exception of IR1, which is 78% conserved in strain B31 and only 11% conserved in 297, all other invariable regions are between 80–90% conserved with respect to the P7-1 VlsE cassette segment of IP90 (Table I⇓).
The antigenicity of the invariable regions of P7-1 was assessed with the Hopp-Woods hydrophilicity algorithm (16). Reputedly, this method has been more successful than similar ones for identifying protein antigenic determinants (24). Overall, the invariable regions of the IP90 variable domain showed negative hydrophilicity values. Sequences with positive values were composed of fewer than six amino acids and thus were not likely to configure antigenic sites per se. In contrast, IR6 contained six or more contiguous amino acids with relatively high positive hydrophilicity values (1.7). IR6 was also slightly more conserved than other invariable regions, with a mean identity of 87% when using the IP90 sequence as the reference strain (Table I⇑).
Experimental assessment of the antigenicity of IR6.
To evaluate experimentally the antigenicity of IR6, we synthesized a peptide (C6) whose primary structure encompassed that of IR6. Serum samples from blood obtained from 10 rhesus macaques that were infected either with JD1 (six animals) or B31 spirochetes were used to examine the antigenicity of IR6 in monkeys. Anti-C6 ELISA Ab levels shown in Fig. 3⇓A were present in serum samples at 4–6 wk PI and remained as high or higher for up to 3 yr PI (data not shown).
Mice also responded vigorously to this region. Serum samples collected 4–6 wk PI from 10 mice infected with either B. burgdorferi strain Sh-2-82 (six animals) or B31 showed high levels of anti-C6 Abs (Fig. 3⇑B). This result further confirmed the antigenicity of IR6 and reaffirmed this region's antigenic conservation.
In humans, the antigenicity of IR6 was examined with the aid of a panel of 41 serum samples provided by the CDC. Ab directed to C6 was found in 35 of the 41 samples (Table II⇓). The result of this experiment also underscores both the antigenicity and the antigenic conservation of IR6, as the serum specimens in the panel stemmed from several Lyme disease endemic areas in the United States and must therefore contain Abs to multiple strains of B. burgdorferi.
Mouse and monkey sera obtained both before and after B. burgdorferi infection were also reacted with an ELISA plate coated with streptavidin alone, with no C6 peptide added. No difference between the reactivity of the samples collected before and after infection was detected (not shown), thus precluding that the anti-C6 reactivity observed was nonspecific.
IR6 may be the only immunodominant invariable region in both monkeys and humans
In addition to IR6, the VlsE variable domain contains five other invariable regions (Fig. 2⇑). To determine whether any of these regions is antigenic, a competitive ELISA was performed, using P7-1 as Ag attached to an ELISA plate, in the presence of increasing concentrations of the C6 or C2 peptides. The sequence of C2, a 19 mer, was based on that of IR2, plus three N-terminal amino acids (DAASVNGIAKGIKGIVDAA). P7-1 includes invariable regions IR1–6 (Fig. 2⇑). Ten serum samples from infected monkeys, four human serum samples randomly selected from the CDC panel, and an additional four serum samples from chronic Lyme disease patients provided by the National Institutes of Health were tested. Representative results for four human and four monkey serum samples are presented in Fig. 4⇓. Addition of C6 almost completely inhibited the binding of monkey and human serum Abs to P7-1 (Fig. 4⇓, A and B). In contrast, addition of C2 in the same range of concentrations caused no detectable inhibition (Fig. 4⇓, C and D). Thus, inhibition of Ab binding to P7-1 by the C6 peptide is specific and, more importantly, in both monkeys and humans IR6 appears to be an immunodominant invariable region within the VlsE variable domain.
When the competitive ELISA was used to analyze infected mouse serum, the maximum inhibition resulting from the addition of C6 was ∼40%, depending on the animal. Unlike with monkeys and humans, however, addition of C2 competitively inhibited binding of mouse Abs to P7-1 (up to 40%; data not shown). This indicates that other invariable regions may be antigenic in mice.
Exposure of IR6 on the surface of VlsE
We examined the exposure of IR6 on the surface of the VlsE protein by immunoprecipitation with the rabbit anti-C6 antiserum. VlsE from IP90 spirochetes was extracted in solubilization buffer and precipitated with protein G-agarose in the presence of either rabbit anti-C6 antiserum or serum obtained from the same rabbit before immunization. The presence of VlsE in the immunoprecipitates was then assessed on immunoblots reacted with the anti-C6 antiserum. The VlsE of IP90 was immunoprecipitable with the anti-C6 antiserum, but not with normal preimmune serum (Fig. 5⇓). This result indicates that IR6 is exposed on the VlsE surface.
IR6 is not accessible to Ab on the outer membrane of the spirochete
Exposure of IR6 on the spirochetal surface was assessed by immunofluorescence. While FITC-conjugated anti-C6 Ab extensively labeled all the acetone-fixed IP90 spirochetes (Fig. 6⇓B), it labeled few (<5%) of the unfixed spirochetes (Fig. 6⇓A). The unfixed spirochetes that were labeled exhibited a discontinuous fluorescent pattern, whereas fixed spirochetes fluoresced uniformly (Fig. 6⇓B). In contrast, neither unfixed nor fixed spirochetes of the B. burgdorferi sensu stricto strain HB19 high passage, which do not express VlsE, fluoresced with the FITC-conjugated anti-C6 Ab (Fig. 6⇓, C and D). Control anti-B. burgdorferi Ab-FITC conjugate labeled both fixed and unfixed spirochetes (data not shown). These results indicate that IR6 is not accessible to Ab despite the fact that VlsE is itself surface exposed (8). This is consistent with the result of the in vitro Ab killing assay; anti-C6 antiserum had no significant killing activity compared with preimmune serum, although monkey anti-OspA antiserum killed all spirochetes in the same experiment (data not shown). In vitro killing experiments were performed at least twice with the same results.
We have demonstrated that the amino acid sequences of the six invariable regions described previously in the cassette segments of VlsE (8) are conserved, at least in two Borrelia genospecies, B. garinii and B. burgdorferi sensu stricto, and we have illustrated with data from the literature that such conservation is also retained among strains of the latter genospecies. This is evident from the high level of identity between the deduced amino acid sequences of the invariable regions within the variable domain of IP90 (B. garinii), which we cloned, sequenced, and identified ourselves, and those of the B31 and 297 cassette segments (8, 14) (Fig. 2⇑ and Table I⇑).
The sequence conservation of the six invariable regions across strain and genospecies barriers indicates that these regions are important in whichever role VlsE may play in the physiology of B. burgdorferi. One would therefore expect that such sequences are not antigenic in hosts with a chronic B. burgdorferi infection or would be otherwise inaccessible to Ab, either because they are conformationally buried within the VlsE molecule or are unavailable on the spirochetal surface. The Hopp-Woods algorithm indicated that, with the exception of IR6, all the other invariable regions were either not antigenic or had lower antigenicity.
The predicted antigenicity of IR6 was confirmed in humans, monkeys, and mice (Fig. 3⇑ and Table II⇑). Sera from all these hosts reacted with the C6 peptide early and persistently in the course of infection, thus indicating that IR6 contains one or more epitopes that may be broadly antigenic regardless of host species. The antigenicity of IR6 was not only manifest independently of host species, but also regardless of whether the animals had been infected with the JD1, B31, or Sh-2-82 strains of B. burgdorferi sensu stricto. In addition, 35 of 41 human serum samples collected in the Northeast and Midwest of the U.S. from patients with acute or chronic Lyme disease also reacted with the C6 peptide. The five serum samples that had no detectable anti-C6 Ab were obtained from patients who were in the early stages of infection. Hence, the negative results may reflect the presence of very low serum Ab titers rather than the absence of cross-reactivity. The C6 peptide may thus serve as a global diagnostic probe. We are currently investigating this possibility.
No Ab reactivity was observed when the C6 peptide was not included in the assay, i.e., when plates were coated with streptavidin. Hence, the anti-C6 reactivity is specific and cannot be construed as originating from polyclonal B cell stimulation during B. burgdorferi infection (25, 26).
The absence of or relatively low antigenicity of IR1–5 was underscored by the results of the competition experiments (Fig. 4⇑). The fact that the binding of essentially all the human and monkey Ab that reacted with P7-1 could be inhibited simply by adding an excess of the C6 peptide indicates that in these hosts IR6 is possibly the only immunodominant invariable region within the VlsE variable domain. In mice the results were different, as we were unable to fully inhibit the P7-1 reactivity of mouse anti-Sh-2-82 or anti-B31 Abs with an added excess of the C6 (or C6 and C2) peptide(s). This result entails that some of the other IRs may be antigenic in mice. Indeed, when synthetic peptides based on the five remaining IRs listed in Fig. 2⇑ were used as ELISA Ags, Ab responses to IR2 and IR4 were evident in infected mice, but not in monkeys or humans (34). The immunodominance of IR6 was further underscored by the long term persistence of anti-C6 Abs in infected monkeys and in patients with chronic Lyme disease (not shown).
The Hopp-Woods algorithm also predicted, as expected, that some variable regions of the cassette segment are strongly antigenic (data not shown). Thus, at least theoretically, the VlsE variable regions could satisfy the antigenic variation paradigm that variable domains are immunodominant. In the case of VlsE, however, variable region immunodominance of individual VlsE molecules may be difficult to achieve, for the recombination process that underlies the mechanism of VlsE variation occurs very rapidly (14). Despite strong antigenicity, no variable region might at any time be represented on a large enough number of bacterial cells or otherwise be expressed long enough to become immunodominant. Indeed, within the first 4 days PI, as many as 11 predicted amino acid changes per VlsE variant can occur during a B. burgdorferi B31 infection in C3H/HeN mice (14), and unlike with T. brucei, N. gonorrhoeae, and B. hermsii, VlsE serotypes of B. burgdorferi have never been found. In fact, it has been estimated that amino acid substitutions within the variable domain of VlsE could result in as many as 1030 different combinations (9), a number much larger than the diversity of the Ab repertoire of vertebrate host species (109–1011). Even though some of the combinations may share antigenic specificities, it seems unrealistic that VlsE serotypes may emerge during infection.
Our finding that IR6 is exposed on the VlsE surface (Fig. 5⇑) but not on the spirochetal surface (Fig. 6⇑) is in agreement with the antigenicity and immunodominance of this region. Features of protein domains, such as surface accessibility, hydrophilicity, flexibility, and proximity to a site recognized by Th cells, are all important in positively determining domain antigenicity (27). Except for the possibility that solubilization with Triton X-100 may have partially denatured VlsE and thus artificially exposed IR6, the result of the immune precipitation experiment indicates that IR6 is accessible on the surface of native VlsE. It is also hydrophilic, as assessed by the Hopps-Wood algorithm. The molecular surface exposure of IR6 was indicated also by the fact that a small proportion of unfixed spirochetes fluoresced when incubated with FITC-labeled anti-C6 Ab (Fig. 6⇑A).
Most of the unfixed spirochetes failed to label with the FITC-conjugated anti-C6 Ab (Fig. 6⇑A). This indicates that IR6 is not exposed on the spirochetal surface. The small proportion of unfixed spirochetes that bound anti-C6 Ab probably had some degree of membrane damage. Although our procedure for labeling of unfixed spirochetes entailed very gentle manipulations, it is possible that portions of the VlsE molecule not normally exposed on the spirochete surface were nonetheless uncovered in a fraction of the spirochetes. The fragility of the outer membrane of B. burgdorferi has been noted by other investigators (28). Loss of the lp28-1 plasmid, which encodes VlsE, during in vitro cultivation (8) also could explain a negative labeling result. However, the fact that nearly 100% of acetone-fixed spirochetes from the same culture were labeled allows us to rule out this possibility (Fig. 6⇑B). Acetone fixation probably exposed regions of the VlsE not accessible to Ab on an intact spirochete. As expected, the HB19 spirochetes not expressing VlsE did not bind anti-C6 Ab regardless of whether the organisms were fixed or unfixed (Fig. 6⇑, C and D). Hence, the anti-C6 reactivity is singularly specific.
The result of the in vitro Ab killing experiment was entirely consistent with our interpretation of the immunofluorescent observations, as absence of killing is most likely due to failure of the anti-C6 Ab to bind to the spirochetal surface. Complement-dependent killing of B. burgdorferi is facilitated by anti-surface Ab binding and does not depend on the complement-activating properties of the Ab. B. burgdorferi spirochetes are able to activate complement through an Ab-independent mechanism (29). Taken together, our immunofluorescence and in vitro killing results indicate that IR6 is inaccessible to Ab on the spirochetal surface. Because VlsE is surface exposed and is a lipoprotein (8), it is probably anchored to the spirochetal membrane only by its N-terminal lipid moiety and is not otherwise embedded in the membrane. It follows that VlsE is probably densely packed on the spirochetal surface, interacting with either neighboring VlsEs or other vicinal surface-exposed molecules.
In the variable Ags that have been thoroughly investigated to date, such as the VSG, Vmp, or pilin, the function attributed to variable portions, because of their immunodominance, has been to act both as target of the immune response and as decoy with respect to invariable portions of the same molecule or other molecules. On the other hand, the role ascribed to these Ags' invariable portions, which are characteristically nonantigenic, has been to contribute to preserving a functional conformation (10, 11). What, then, is the role of the antigenicity and immunodominance of IR6? It has been hypothesized that chronic host exposure to immunodominant Ags or epitopes diverts the immune system from responding to less antigenic but functionally important Ags or epitopes, thus serving as a protective strategy for persistent pathogens (30). Recently, it was demonstrated that when the immunodominant V3 loop epitope of gp120 of HIV-1 is masked through site-directed targeting of N-linked glycosylation, the dominant, type-specific, neutralizing Ab response is shifted away from V3 to epitopes in the first variable domain (V1) of gp120 (31). Ab responses to conserved domains of gp120 also were observed (31).
We submit that IR6 may act as a decoy epitope and contribute to subvert the Ab response to B. burgdorferi. As mentioned before, it is conceivable that the promiscuity of VlsE antigenic variation is such that variable regions of VlsE do not, or do not always, become immunodominant. The variation may serve to inhibit the formation of high avidity Abs to the variable VlsE regions, but not to divert the response away from invariable VlsE regions and invariable domains or other less antigenic, but functionally neutralizing, B. burgdorferi Ag. It should be considered that only about half the length of the mature VlsE protein is variable, compared with more than two-thirds of proteins such as VSG, pilin, and Vmp. Moreover, more than half the variable domain of VlsE is encompassed by invariable regions, including one immunodominant IR6 identified in this study. Conserved regions of VlsE other than IR6 thus may be exposed, per force, on the spirochete surface. IR6 would serve as the decoy epitope for such domains by suppressing protective immune responses through mechanisms such as clonal restriction and/or idiotypic dysregulation, as has been invoked for HIV-1's V3 (32, 33).
Secretarial help from Christie Trew and the photographic skill of Murphy Dowouis are gratefully acknowledged.
↵1 This work was supported by Grants AI35027 and RR00164 from the National Institutes of Health and by a grant from SmithKline Beecham Biologicals.
↵2 Address correspondence and reprint requests to Dr. Mario T. Philipp, Tulane Regional Primate Research Center, Tulane University Medical Center, 18703 Three Rivers Road, Covington, LA 70433. E-mail address: philipp{at}tpc.tulane.edu
↵3 Abbreviations used in this paper: VSG, variant surface glycoprotein; Vmp, variable major protein; VlsE, Vmp-like sequence, expressed; PI, postinfection; CDC, Centers for Disease Control and Prevention; Ospa, outer surface protein A.
- Received May 27, 1999.
- Accepted September 8, 1999.
- Copyright © 1999 by The American Association of Immunologists


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The nitrite preservatives in processed meats such as bologna, bacon, ham, and hot dogs form carcinogenic nitrosamines, but also reduce the growth of botulism bacteria—forcing regulators to strike a balance between consumers risking cancer, or a deadly form of food poisoning.
Bacon & Botulism
Nota del Doctor
This reminds me of the cooked meat carcinogen issue. If we undercook meat, then we can get food poisoning (see for example Fecal Bacteria Survey; Chicken Out of UTIs; U.S. Meat Supply Flying at Half Staph). But if we make sure meat is well-done, we risk exposure to carcinogens produced when muscle flesh is exposed to high temperatures (see Fast Food Tested For Carcinogens; Muscle Tremors & Diet; Carcinogens in Roasted Chicken?). See Are Nitrates Pollutants or Nutrients? for why nitrites from meat can be harmful, while nitrites from nitrates in vegetables can be helpful.
For more context, also check out my associated blog posts: Using Greens to Improve Athletic Performance; Top 10 Most Popular Videos of the Year; Strawberries Can Reverse Precancerous Progression; and How Chemically Contaminated Are We?
If you haven't yet, you can subscribe to my videos for free by clicking here.
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Shutters are a crucial part of the decoration and protection of your house. Their usage is embedded in history. However, there are new ideas you need to embrace in the installation and care of exterior louvered shutters.
Interior Vs Exterior Installation
The installation of shutters was traditionally outside the window. This was meant to protect your window panes from damage by the storm or projectiles that may be thrown towards your house. This is sufficient justification for the installation of shutters outside the house.
The exterior installation has faced a number of criticisms. It alters the design and appearance of your house. This may affect its aesthetic value and also cause a reduction in price once it is placed in the market. This is why homeowners are moving away from installing shutters outdoors if there is a better option.
Interior installation is preferred if your area is not prone to storms or strong wind. This allows you to maintain the architectural design that was envisaged for the house. It also allows you to stick to architectural instructions provided for your estate.
Interior installation helps you achieve the other benefits of installing shutters. They include protecting your interior from spying eyes, direct sun, and wind. It will also protect interior properties from damage in case a storm hits your place through the window panes will still be damaged.
Modern Materials for Manufacture of Shutters
Shutters were traditionally made of wood. It was preferred because of its ease to fabricate, strength and durability. Wood was also easy to maintain because it was a natural part of the environment. It is still used but it comes with the challenge of high cost. Wood also requires regular painting for maintenance purposes.
Iron, steel, and aluminum shutters have also gained popularity over the years. They are factory manufactured and have to be ordered based on the size of your window or door. The challenge with these metals is their cost and maintenance. It is expensive to get shutters made of metals compared to other options like synthetic.
Metallic Louvered Shutters come with a high maintenance bill. You have to clean the shutters and paint them on a regular basis in order to maintain their good appearance. This can prove to be an expensive venture. However, metal is the best material when you are searching for shutters to protect your house from storm damage.
Vinyl is a new revolution when ordering and installing shutters. The shutters are per-ordered based on window and door sizes. Vinyl comes with several advantages that homeowners should consider when making an order.
Vinyl is cheaper than all other materials. This is because of the use of fabrication in manufacture. It allows you to get protection from direct sunlight and stormy weather without spending a fortune.
Vinyl shutters are durable. Vinyl is resistant to weather damage and will not require a lot of maintenance attention. As a result, it takes years before any damage or need for any replacement. This reduces the overall cost of installing shutters on your house.
Vinyl is easy to maintain- vinyl shutters do not require a lot of maintenance attention. Once installed, they are never repainted. Vinyl is also resilient in the wake of tough weather. As a result, you will never bother about the shutters for the years they will be on your wall.
The choice of material depends on personal preference and budget. Work with experts to help you explore the best options in the market and choose one that delivers value for money.
Shutter Style
Shutters come in different styles and designs. The shutters may be solid or louvered. The design or style will affect the appearance of your house and also air supply into the house.
Solid shutters are used for providing protection against storms. Because they are not serrated, they can withstand strong winds and even flying debris. However, solid shutters will prevent fresh air and light from getting into the house. As a result, it becomes uncomfortable or will demand the installation of AC systems that are expensive to maintain.
Exterior louvered shutters are perfect when you need fresh air and light into the house. Depending on the material used, they will protect your interiors from damage by wind, storms or flying projectiles, among other elements. You do not need to use artificial lighting and air conditioning that are expensive. They can be regarded as the environmentally friendly shutter option available in the market.
Cost of Installing Shutters
Homeowners and developers are always cautioned about the cost of shutter installation. Shutters can be expensive considering that you have to install them on every window and door. However, they should not be too expensive.
The cost of shutters should be balanced with ensuring that you get shutters that meet your expectations. Buying exterior louvered shutters cheap might appear advantageous. However, you need to consider the quality of material, its appearance, and long term maintenance needs.
Synthetic material like vinyl has proven to be the most attractive option when you need the benefits of vinyl without spending a fortune. Vinyl comes in different colors and textures. It, therefore, allows you to give the house your desired appearance without spending too much.
Installation should also be done by a professional even with the cost in mind. Professional installation ensures that you consider the surface on which you are installing and therefore make the right choices. Without professional installation, the shutters will be weak, haphazard and even fail to meet your expectations.
Shutters and Personal Preference
There is a great desire by homeowners to maintain a personalized appearance on properties. This will only be possible when you install shutters that reflect your personal aspirations in terms of design, and sizes. There are numerous choices that you can rely on in the market. With these choices, you can make your home as personal as you desire.
Professional installation of Louvered Shutters is important to maintain their integrity. Work with professionals who understand the designs and installation of shutters. Be ready to invest in quality shutters to keep your house in perfect shape during storms and beautiful all other seasons.


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Five major poultry employers sign up to new training scheme
A host of UK poultry companies have already signed up to the new Poultry Meat Training Initiative, which aims to standardise training across the sector.
Vion UK, Cargill Meats Europe, Cranberry Foods, Faccenda, and PD Hook/2sisters have all committed to the scheme, equating to more than 2500 Poultry Passports being issued over the few months to employees.
The scheme is designed to standardise the training carried out across the poultrymeat sector with each job role linked to a set of minimum training requirements.
Central to the Poultry Passports is an online database holding employees' training records managed by its administrator, Poultec Training. Employers and employees have access to their training records via a secure log-in which is renewable on a yearly basis.
The cost is set at £20 a person a year and to support its producers, Assured Chicken Production has provided a 25% subsidy for the first £4000 worth of passports purchased, with further reductions available for bulk purchases.
The initiative is a collaborative project between the British Poultry Council, National Farmers Union, Lantra and Poultec. For more information about the scheme, contact Nathan Raines at Poultec Training on 01362 850 983.


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But it was always my notion, that children should not dispute their parents' authority.
But, if it be to ease her heart, and not to dispute my will, you may hear her out.
The beauty of the Turkish women is still a matter of dispute.
I could not dispute the evidence of the bit of shivered glass.
Now, where there is an et cetera in an agreement, there is always an opening for dispute.
And then there was our dispute at Albany--in the Patroon's mansion, you will recall.
There had been some dispute about payment, and the Britisher had slapped the dragoman's face.
Baxter says of him, complainingly, "he would not dispute with me at all."
Provided we agree about the thing, it is needless to dispute about the terms.
I dispute the claim, said the Englishman, restraining himself by a powerful effort.


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Who Won the 8th Season of Dancing with the Stars?
Who won the 8th season of Dancing with the Stars?
Olympic Gold medalist gymnast Shawn Johnson, who thrilled the world with her balance beam performance in the Beijing Olympics, can now hang a new piece of hardware on her wall. Shawn Johnson and partner Mark Ballas won the 8th season of ABC's hit Dancing With the Stars by one of the narrowest margins of victory in the show's history.
ABC television's Dancing with the Stars 8th ceason gleefully celebrated a new winner Tuesday night. This season, which premiered on March 9, 2009, saw 13 couples vying for the number one spot. The competition must have been fierce, as ABC television reports the outcome of the competition had its narrowest margin ever. In a finale that was declared as the most competitive in the show's history, Olympic gold medalist Shawn Johnson took home the mirror ball trophy.
Shawn Johnson won with less than a one percent lead in the final vote, narrowly beating out actor Gilles Marini. The finale was declared as the most competitive ever. Shawn Johnson's professional partner in the 2009 dance competition was Mark Ballas. Actor Gilles Marini, with dance partner Cheryl Burke, took second place in the competition. Melissa Rycroft, who became famous after an appearance on the Bachelor, took third place with her dance partner Tony Dovolani.
Shawn Johnson was born in Des Moines, Iowa. She is the daughter of Doug and Teri Johnson, who were smart enough to enroll their daughter in a gymnastics class at the tender age of three after they noticed her climbing up cabinets and jumping down from tables. Wanting to channel this particularly dangerous behavior into something healthy, they figured gymnastics was the route to go. Boy, dd they have good timing — at the age of six, Shawn was among the first students of Liang Chow, who opened a gymnastics school in Des Moines. Chow's training really paid off.
Unlike many top notch athletes even at her age, Johnson attends public school — specifically, Valley High School in Des Moines. She enteried the 2008–09 school year as a junior. Most elite gymnasts train around 40 hours a week and choose to work with private tutors rather than attend school. Shawn Johnson trains instead for just 20-25 hours a week and maintains a "normal life" in high school — where she is currently on the A Honor Roll and takes part in the regular extra-curricular activities of the average teenager. In this way, Johnson is a great role model for young athletes, who often get sucked into the world of elite sports and miss out on a "regular life".
Anyone who's watched the Dancing with the Stars series knows how much work the competitors must put into the show. The consistent practice and workouts and dance routines can be extremely taxing on the human body. Many contestants have left the show with injuries, and on several occasions injuries related to dancing have been highlighted on the show or made much of in the media.
In fact, Dancing with the Stars has a whole team of doctors and chiropractors working behind the scenes to keep the dancers in top condition. It was in season three, back in September of 2006, when show coordinators behind the Dancing with the Stars reality series decided they were in desperate need of a chiropractor's help. They hired a local chiropractic service, and DWTS contestants have had access to onset chiropractic care at the filming location in Los Angeles ever since.
In that same season, Emmitt Smith (former NFL running back) won the Dancing with the Stars competition. This is a bit ironic, as Emmitt Smith has long been a vocal celebrity advocate for chiropractic care, famously advising fellow NFL player LaDainian Tomlinson of the San Diego Chargers to find a good chiropractor. Smith was even shown on TV during training for Dancing with the Stars receiving an 'adjustment" from his chiropractor, reportedly to spread the health benefits of chiropractic practice and take away any "stigma" associated with chiropractic care.
Congratulations to Shawn Johnson and Mark Ballas. Not only is Shawn Johnson a national hero for her performance at the most recent Olympics, she is now enjoying her fifteen minutes of Dancing with the Stars fame. No one was surprised to hear that Dancing with the Stars is set for another season, the show's ninth, on ABC. The show is incredibly popular, and is a wholesome option for family viewing at a time when television is crammed full of murder, sex, and other questionable behavior.


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For people.
To receive expert guidance during their graduate school search.
And to recruit students to their graduate programs...by offering expert guidance to prospects.
An Emory professor and his grad student first launched in early 2013. We made it better with v2 in 2015, and with v3 in 2018.
Successful recruiting is mentoring, not marketing. Friendly outreach and helpful advice generates good will. Just be yourself.
Choosing the right graduate program to launch your career is a complex decision. We help you gather and synthesize all the information you need to make it a smart one.
TJ has spent his entire adult life in college, first as an undergrad, then as a PhD student, then a fellowship, followed by joining the faculty! That lifetime of experience grounds what we do. Simplicity, utility, and effectiveness is what matters. For TJ something is worth doing if it helps people find the right graduate program, while teaching them how to get into it.
Brian focuses a lot on business strategy, which mostly involves the member experience, along with more boring stuff like business processes, yada, yada, yada. He has keen interests in designing and delivering technology-based consumer-focused solutions. Brian is a busy man, recently completing his MBA at the Fuqua School of Business at Duke University.
We can tell you that Jared focuses on product development. But the simple truth is that Gradschoolmatch is Jared's baby. Jared joined us after we asked him for help in fixing a few bugs in our beta platform during a holiday school break! After he graduated from Emory U, Jared shook off offers from IBM and others to build out Gradschoolmatch.
Camila drives our communications while sharing her strong voice internally to help shape the strategic direction of Gradschoolmatch. Shortly after graduating with a dual MS, MBA from the Ross School at the University of Michigan she heard our story. As an ex-graduate student, she gets the importance and wants everyone to know of it. She's on the team!
We're an Atlanta startup made with soul. Helping you is who we are. That's all that matters to us.
We write stuff. Our content on the graduate education space is for you, whether you're looking for programs or for students.
151 Ted Turner Dr NW
Atlanta GA, 30303


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Get your free Connect the Dots companion workbook
The goal of this book is to bring the fun and ease back into networking; to give you the tools you need to network successfully, and meet some new, interesting people. Too many people hate networking because they were never taught how to do it. But we management consultants have been schooled in networking and naturally do it without blinking an eye. I'm going to share all of these tips and tricks with you, so that you have the right tools to be successful.
This companion workbook will help you put things into perspective.
What is Connect the Dots?
Somewhere in the recent past, networking became an almost
meaningless mechanical exchange of handshakes, business cards,
and no authentic value.
This book shows you the techniques that will help you bring the
ease and pleasure back into networking. After all, networking is
about making meaningful relationships and building a genuine
support network. And, can't we all use a little help?
The goal of the book is to bring the fun and ease back into
Networking and to meet interesting people. The book teaches
people some techniques to build the natural ease of networking.
Too many people hate networking and yet it's a necessity of life.
It's a necessary life skill as no one gets ahead in life without a
little help.
You can make more friends in two months by becoming
interested in other people than you can in two years by trying
to get other people interested in you.
– Dale Carnegie
Connect the Dots Features
Networking is the start of building mutually beneficial relationships; whether that's to make new friends, new connections, find new opportunities, or new adventures. Who doesn't want to meet interesting people to be inspired. There is no shortage of a reason to put ourselves out there.
This book provides you with the tools, and the process, for networking in person – and on social media. How to introduce yourself that makes you more intriguing, how to follow-up, easy & fun ice-breaker, the etiquette of networking, and ultimately how to start building relationships that will create more opportunities and have fun while doing it!
- Techniques to meet more interesting people
- How to craft a great intro that generates intrigue in you
- Ice-breaker for all different aspects of your life (that are not cheesy)
- Remove the stress of small talk
- Discover different networking approaches & tactics
- Engage others in a fun way that is authentically you
- Build mutually beneficial relationships
- Be seen as a value creator
- Develop network road map, including leveraging social media
- And so much more…


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Lead System Analyst
- Foster, CA 96027, USA
- Full-time
Company Description
As the world's leader in digital payments technology, Visa's mission is to connect the world through the most creative, reliable and secure payment network - enabling individuals, businesses, and economies to thrive. Our advanced global processing network, VisaNet, provides secure and reliable payments around the world, and is capable of handling more than 65,000 transaction messages a second. The company's dedication to innovation drives the rapid growth of connected commerce on any device, and fuels the dream of a cashless future for everyone, everywhere. As the world moves from analog to digital, Visa is applying our brand, products, people, network and scale to reshape the future of commerce.
At Visa, your individuality fits right in. Working here gives you an opportunity to impact the world, invest in your career growth, and be part of an inclusive and diverse workplace. We are a global team of disruptors, trailblazers, innovators and risk-takers who are helping drive economic growth in even the most remote parts of the world, creatively moving the industry forward, and doing meaningful work that brings financial literacy and digital commerce to millions of unbanked and underserved consumers.
You're an Individual. We're the team for you. Together, let's transform the way the world pays.
Job Description
Open VisaNet (OVN), is the Next Generation VisaNet. OVN will use best-in-class technology components to push the envelope as a highly scalable, secure, cost-effective and distributed application on commodity hardware. Open VisaNet (OVN) is one of Visa's Key initiatives which will help Visa support in-country and global processing solutions while providing Visa an opportunity to move away from proprietary hardware systems. Realizing the vision of OVN requires the collaboration of an exceptional group of software engineers, security specialists, DevOps engineers, payment experts, infrastructure architects and business analysts. To support our development roadmap, we are creating a function to ramp up the creation and delivery of business requirements which articulate the current VisaNet business logic, both functional and non-functional. Reporting to the Sr. Director of Technical Requirements and Business Analysis, this role in Foster City will be accountable for generation and publication of business requirements for more than 330 discrete services and functions.
Essential Functions
- Define and document detailed functional and non-functional (performance and operational) requirements for OVN.
- Work with Architecture and development teams to refine and maintain templates for the requirements artifacts that will be produced by the team.
- Work with vendors to support the Knowledge Extraction process (reverse engineering of existing business logic), define artifact templates and use those artifacts efficiently in the documentation of requirements
- Work with Architects and development teams to ensure detailed requirements are accurate, complete and can be understood by a diverse group of users.
- Collaborate with the quality assurance team on creation of testing artifacts.
- Manage the lifecycle of requirement artifacts to elaborate requirements of OVN as needed.
- Coach development teams on the specific processing requirements of Visa switching, clearing and settlement in relationship to OVN requirements.
- Participate as needed in presentations to and discussions with key clients and stakeholders to share details about OVN and its value to the payment processing environment.
- Consult on a regular basis with leadership to align expectations and prioritize deliverables.
- In addition to the responsibilities for requirements elaboration, the candidate will perform the role of a Product Owner on an assigned sprint team as needed.
- Acquire a good understanding of the Scaled Agile Framework (SAFe) concepts and play an active role in SAFe activities as required for the Product Owner role.
- Participate in Team Demos and Retrospectives
Basic Qualifications
- 10 years of work experience with a Bachelor's Degree or at least 8 years of work experience with an Advanced Degree (e.g. Masters/MBA/JD/MD) or at least 3 years of work experience with a PhD
Preferred Qualifications
- 12-15 years of work experience with a Bachelor's Degree or 8-10 years of experience with an Advanced Degree (e.g. Masters, MBA, JD, MD) or 6+ years of work experience with a PhD
- 12 years of experience in information technology with a Bachelor's degree or equivalent
- Extensive experience with producing high quality requirements, documentation and user stories.
- Team player with broad experience collaborating with Subject Matter Experts, system architects and senior software engineers.
- Ability to work with multicultural teams across the globe.
- Ability to work across time zones as required.
- Strong knowledge of MS-Office products for Windows including Word, Excel, Visio, and SharePoint and Wiki.
- Experience with advanced authoring software tooling such as Adobe FrameMaker, Acrobat, etc., or the aptitude to quickly adapt to using such tools as required.
- Demonstrated ability to handle multiple priorities with minimal direction from management.
- Strong customer service, analytical, research, interpersonal and problem-solving skills
- Capable of forming and advocating for an independent viewpoint.
- Demonstrated skills in influencing, negotiating and communicating.
- Must demonstrate longer-term strategic thinking and staying ahead of state-of-the-art curve in order to keep abreast with what's possible technically, operationally and professionally.
- 10+ year track record of relevant work experience and an undergraduate degree in Information Systems, English, Journalism, Communications, or a related technology focused field. Master's degree is a plus.
- Knowledgeable about Visa systems or applications.
- Significant knowledge of the payment business and products and services provided by VisaNet.
- Professional experience in ideation and incubation, which has led to shipping and supporting products in Production is a plus.
Additional Information
Work Hours This position requires the incumbent to be available during core business hours.
Travel Requirements This position requires the incumbent to travel for work 2-5% of the time.
Mental/Physical Requirements This position will be performed in an office setting. The position will require the incumbent to sit and stand at a desk, communicate in person and by telephone, frequently operate standard office equipment, such as telephones and computers, and reach with hands and arms.
EEO Statement Visa is an EEO Employer. Qualified applicants will receive consideration for employment without regard to race, color, religion, sex, national origin, sexual orientation, gender identity, disability or protected veteran status. Visa will also consider for employment qualified applicants with criminal histories in a manner consistent with EEOC guidelines and applicable local law.