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Chinese Company ZTE to come up Android Nougat OS 7.0 on all ZTE Axon 7 smartphone in January.
Recently LG has announced android nougat OS 7.0 for LG G5 smartphones while Samsung for all Samsung Galaxy S7 and One Plus for all OnePlus 3 phones.
ZTE Axon 7 smartphone has a 5.5 inch quad-HD screen and a resolution of 1440×2560 pixels. It contains fingerprint scanner and powered by the flagship-grade, Snapdragon 820 SoC, with the Adreno 530 GPU.
It sports 4GB RAM, and comes with a 64 GB built-in storage space. The storage space can further be expanded up to 256 GB. The ZTE Axon 7 includes a 3250mAh battery, and supports the Qualcomm Quick Charge 3.0 technology, which allows the device to charge at very rapid speeds. The smartphones also provide a 20MP primary camera on the back and an 8MP front camera.
The company has not finalized that what kind of update the smartphone will contain. It can be a beta upgrade, or a full-fledged final version of the Nougat OS.
Also Read: OnePlus Unveiled Bullets (V2) Headphones in India at Rs 1199
Also Read: Micromax unveiled Canvas Spark 4G in India at Rs 4,999
Also Read: Lava Unveils 'X80' Tablet at Rs 6,299
Also Read: Chinese Company Huwaei unveils Mate 9, Mate 9 Porsche Design smartphones
Also Read: Vivo to unveil V5 Smartphone on November 15 in India


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Parents Educating Children is a network of home school families in Palm Beach County. They share their experience and resources among members to enrich their children's education with a wide range of interesting opportunities. Today, these bright shiny faces came from Jupiter and Palm Beach Gardens to tour the Dreamweaver Studios.
Friday, March 5, 2010
Parents Educating Children
Posted by admin at 11:25 AM
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- Home
- Diffusers
- Oils
- Accessories
- Health
- About
All of the products on this website have a purpose - to help you lead a more natural life, to avoid chemicals and to support your body from the inside out.
We believe that the body is designed to repair itself naturally, but is prevented by the toxic load placed on it from the chemicals in our environment, in our food and in our water supply. Even worse, the body's automatic inflammatory and adrenal response to threats such as toxins, stress and leaky gut syndrome creates its own problems and illnesses over prolonged periods of time. To heal from the inside out, the body needs help to detoxify, to reduce unnecessary inflammation and to restore its natural balance – both physically and emotionally.
Our Products
The products we offer are designed to help your body do this, from the detoxifying dōTERRA therapeutic-grade Zendocrine blend, to the anti-inflammatory properties of our Turmeric Chai Tea and the Traditional Chinese Medicinal benefits of Sunny Pills. Everything is designed to restore balance. Our soaps and face creams are hand-made from organic ingredients, heavily laced with dōTERRA essential oils and without a chemical in sight - making them suitable for even the most sensitive skins. The MONQ range of personal aromatherapy inhalers is brand new to Australia and an exciting addition to our product offering. The design is funky, the oils are organic and the terpenes from the blends are nature's blessing. We love them. While the doTERRA oils are superb, the quality is second to none, and I have no hesitation whatsoever in recommending them, you might not see the whole doTERRA product range on these pages - we've included all of the oils and the products we agree with and encourage you to research the ingredients of those we have left out. That way you can be assured we are only offering you the best.
Finally, our range of diffusers are not just attractive, they have the added bonus of offering important health benefits too, such as the aromatherapy benefits of essential oils and purifying and cleansing your home environment. I have mine misting away on my desk as I write this, with Bergamot for creativity and Frankincense for concentration. We will continue to add beneficial products to our range as we research them, but our aim is to offer a niche selection of hand-picked products rather than a confusing supermarket array. If you are interested in wholesaling any of the items listed you can enquire here. Just let us know what you're interested in. Meanwhile, enjoy browsing and please let us know if you think anything is missing or if there are any ways we can improve our website or our service to you.


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Herbal Digestive Care Capsules
Product Description
In order to meet the specific preferences of clients, we have introduced highly effectively Herbal Digestive Care Capsules. These capsules are widely used for improving the digestion system of the body to avail relief from the digestion problems. The provided capsules are processed by our ingenious professionals using quality-assured natural herbs and contemporary methodology. Furthermore, we offer these Herbal Digestive Care Capsules to the clients in different packaging options at industry leading prices.
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Office Number 302, 3rd Floor, Saki Vihar Road, Near Chandi Wali Petrol Pump, Chandi Wali, Powai, Mumbai, Maharashtra, 400072, India
Phone :91-22-28576262


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It was a hot (90 degrees), sunny day in the mountains for our score meet. The area is undergoing extensive thinning (logging) activity and our course design had to be adjusted accordingly. Should be better for us in the long run after the logging debris is cleaned up and the dust settles. Also new single track mtn. bike trails seem to appear daily and old roads either get gated or "decommissioned". Huckleberries were sparse due to unusual weather conditions, and the bears hungry!
Runners were allowed 90 mins. to get as many controls as they could, starting individually. The six easy controls in the meadow near the start were worth one point each (and also made up the beginner course), the rest were worth 2 points, with a one point per minute overtime penalty. The score format allowed participants to select which controls to try for and their route to them, it also would allow one to give up on a control if they couldn't find it and move on to other controls. Some of the controls gave people some trouble with ZZ on the west side of the map being a real mystery with Andy and Elijah never finding it and Sergey making a rare 7 min. error there. Most other participants found it easily. Sergey also had a problem with his next control XF because he hit the trail west of where he thought he was and went west by mistake instead of east. He then tried to climb straight up to the upper trail which was probably a bad choice because that steep hillside is very dense with logging debris and thick bushes. On the right is Andy's route as recorded by his GPS (firmly placed in his back pocket during the course), they went in a clockwise direction.
We can see by Andy's GPS that we need to adjust some of the trail locations a bit, but the control locations are pretty good. Bill and his two dogs decided to get a good workout and go for all the controls and came in just a bit late. David brought his friend Steve to try orienteering for the first time and he went for the gusto. His inexperience caused him to be unable to find several controls, but his athleticism got him to all corners of the map, still a very impressive showing for a first timer. Bob helped me out by checking four of the farthest out controls on bike to make sure they hadn't disappeared since being placed several days earlier.
It was good to hear that several of our CTOC members are planning on going to the Sierra 3-day A meet coming up soon, it will be a great orienteering experience! Thanks to Andy, Ole, Jeff D., Bill, and Jeff B. for control pick up help.
- Karin Didisse
Score Course - 32 pts. possible 7.3km 210m. climb (all)
Name - Time (rel. to limit) - Points - Final Points
Sergey Velichko, -19, 32, 32
Ole Bergset, 0, 29, 29
David Murray, -2, 28, 28
Jeff Black, +2, 29, 27
Andy & Elijah Hill, 0, 17, 17
Jeff Decker, +2, 18, 16
Steve Raeder, -1, 15, 15
John Murray, +3, 15, 12
Bob Didisse (bike), 0, 8, 8
Bill Pilcher, +38, 32, -6
Beginner Course 1.8km
Dondi Black & Toby dog 26:43
August 23, 2011
Bear Basin Results 2011
Posted by Karin at 11:18 AM
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Declaration Of Independence Quotes: Here Is The Huge Variety Of Quotes, Images, Messages, Wishes And Many More For Independence Day For Your Dear And Near One's Scroll Down To See
1. I wish that Independence Day will make our leaders work instead of fight.
2. Even as individuals become families and families become communities, and communities become nations, so eventually must the nations draw together in peace.
3. Nigeria is the most populous country in Africa. If Nigeria succeeds at democratic governance it will be an anchor for all of West Africa. Africa needs a strong Nigeria — Ed Royce
4. Be an example of a Filipino who loves our nation so much.
5. Nations and governments come and go, but people remain, and therefore people are the ultimate foundation of what is real and worthy.
Declaration Of Independence Quotes
Table of Contents
6. Well, Nigeria has played a constructive role in peacekeeping in various parts of West Africa. But unless and until Nigeria itself is democratic and respects human rights, it too may well be a source of much greater instability as political repression limits the ability of the people of Nigeria to achieve their full potential — Susan Rice
7. Celebrate this day with a silent prayer of thanks to our heroes who made it possible for us to live in a nation free from the regime.
8. President Thomas J. Whitmore: Nuke them. Let's nuke the bastards!
9. The life of nations no less than that of men is lived largely in the imagination.
10. Each one of us, and, indeed, all those who aspire to national leadership must bring their own visions, views, and styles to the business of reforming Nigeria, and the search for solutions — Ibrahim Babangida
Quotes From The Declaration Of Independence
11. We are free because we can move and decide on our own.
12. We can live a peaceful life without any intrusion. Just always remember that this freedom entails responsibility that we will pass on to our children.
13. Russell Casse: I picked a hell of a day to quit drinkin'.
14. Patriotism is, fundamentally, a conviction that a particular country is the best in the world because you were born in it…
15. There is no country in the world with the diversity, confidence and talent and black pride like Nigeria — Binyavanga Wainaina
Thomas Jefferson Declaration Of Independence Quotes
16. Our government cannot resolve issues because our leaders have their differences.
17. President Thomas J. Whitmore: Nobody with any sense can believe me as the president.
18. If this country is ever demoralized, it will come from trying to live without work.
19. Youth is the hope of the future. Make this true by being a good example for the next generation.
20. David Levinson: What I'm trying to say is–if you stay I'll hurt you.
Quotes From Declaration Of Independence
21. Do you know why our heroes sacrificed their lives for us?
22. The propitious smiles of heaven can never be expected on a nation that disregards the eternal rules of order and right which Heaven itself has ordained.
23. You can be one by doing the simple deed out of kindness without asking for anything in return or doing it merely for no reason at all.
24. One person is nothing. Two people are a nation.
25. Russell Casse: Alright, you alien assholes. In the words of my generation…UP YOURS!
26. Take time to look back and examine if you have lived a life like a hero to your fellow
27. It is because they value independence so much and has loved our country more than their lives.
28. Do your part. Have an Independence Day with meaning.
Famous Quotes From The Declaration Of Independence
Related Tags: Famous Quotes From The Declaration Of Independence, Quotes From Declaration Of Independence, Thomas Jefferson Declaration Of Independence Quotes, Quotes From The Declaration Of Independence, Declaration Of Independence Quotes


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Did you know that a rested body equals beautiful skin?
If you sleep less than 6 hours a night, you are likely to look dull and tired. In general, it is advised that you sleep at least 8 hours per night. This is especially important now than ever because we are constantly dealing with a lot of stress and the body needs its rest. Read more on how the lack of sleep can affect your skin here.


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Capacity and Know-how for Big and Small Commercial Concrete Jobs
As a contractor or developer, you know construction delays cost money. You need a professional, reliable concrete supplier who can get you what you need, when you need it. We have the capacity and experience to meet the needs of even the biggest projects.
We work with our commercial clients to meet all of their concrete needs, from concrete blocks and slabs to stamped concrete to concrete coatings and finishes. Have a last-minute order you need filled? Give us a call. Did the other ready mix concrete supplier fail to show? We can help.
Because we have little staff turnover, you won't hear a new voice on the phone every time you call. You won't have to continually "retrain" us on your ready mix concrete needs. We'll learn your preferences and work hard to build a long-term relationship.
Learn more about how Graestone Ready Mix can help keep your projects on time and on budget. Give us a call at 604-888-1415 or email firstname.lastname@example.com for more information.


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Yet less attention is paid to the parents who enable the behavior. How?
They help rent the house, pay for it, draw up legal contracts dealing with sharing costs and behavior codes, and sometimes serve as chaperones (which doesn't always go well). When kids break those codes and get arrested — arrest rates soar in beach towns during this time — their parents pay for lawyers to get them out of trouble.
During a hearing before a Senate committee last week, Kavanaugh presented calendars he had kept from his high school years, one of which shows days marked off for Beach Week. A senator asked him about a comment in his school yearbook in which Kavanaugh called himself the "biggest contributor" to the "Beach Week Ralph Club," and the judge responded that "Ralph" was code for vomiting. "I'm known to have a weak stomach," he added.
But it's not just Beach Week when parental involvement — or lack of it — has been an issue. Kavanaugh has been accused by a professor of sexually assaulting her at a party when they were both in high school, he at the elite private all-boys Georgetown Preparatory School and she at the elite private all-girls Holton-Arms School.
The accusation by Christine Blasey Ford involves a party where parents were nowhere to be found — a common factor in various incidents we have heard of over the years about high school students getting into trouble at alcohol- and drug-fueled parties.
It goes back a long way, as evidenced by a 1990 letter about that very subject from the heads of some prestigious schools, including the one Kavanaugh attended. According to this Washington Post article:
The headmasters at seven of the Washington area's most prestigious private schools have written a letter to the parents of all students warning them that students are regularly throwing large, unsupervised parties where "excessive drinking and sexual license are common."In what the headmasters called a rare joint effort, the letters, which were mailed Thursday, asked parents to step up supervision of their children to prevent them from attending or throwing weekend parties that are open to almost anyone and where alcohol is easily available.The letter was written jointly "to give it more impact," said Malcolm Coates, headmaster at Landon School in Bethesda. "The fact that seven schools decided it was enough of a problem to address it is significant."Individual schools have confronted the issue before. At the beginning of the school year, for example, Georgetown Preparatory School in Rockville held a conference with parents to discuss the problem of unsupervised parties and similar activities.
On July 4, 2007, there was this Washington Post article:
When police showed up recently at a Walt Whitman High School graduation party, three young people were drinking in a vehicle parked outside the Bethesda home. Then three more teenagers walked up with a six-pack in a bag. While the police were dealing with them, the mother came outside, saw the officers and ran back in.Montgomery County police wrote dozens of citations against the minors who were found to have been drinking at the party. The party-hosting parents were given two civil citations each, carrying fines of up to $1,500 per infraction.The outcome for the Bethesda parents was considerably less severe than for a Charlottesville area mother and stepfather who recently began serving 27-month jail sentences for hosting an underage drinking party. In Virginia and the District, parents who host such parties can be charged with contributing to the delinquency of a minor, a misdemeanor that can carry jail time. In Maryland, hosting an underage drinking party is punished with a civil penalty, payable with a fine, even for multiple offenses.The stark contrast in punishments is just one inconsistency in a patchwork of conflicting legal practices and public attitudes about underage drinking parties. Even at a time of strong concern about youth drinking and drunken driving, police and prosecutors say parents in the Washington region are rarely held responsible — criminally or civilly — for allowing teenagers to gather at their homes and consume alcohol. That's in large part because it's difficult to prove that the adults provided alcohol or condoned its use.
In 2016, a legislator from Montgomery County, Md., proposed a bill that would have raised the penalties for parents who host underage drinking parties, including jail time. What happened to the bill? It got watered down, as this Post article explains:
Never before had Sen. Robert A. Zirkin, chairman of the Maryland Senate's Judicial Proceedings Committee, stopped in the middle of a hearing to take a vote on a bill.But after listening to emotional testimony from two fathers whose teenage sons were killed by an underage drunk driver, Zirkin (D-Baltimore County) accepted a motion to approve a bill that would stiffen the penalties for adults who provide alcohol to people younger than 21 — including a provision that would send such adults to jail.The vote, taken in February, was unanimous. Some onlookers in the hearing room cried. Others applauded.The bill named for slain 18-year-olds Alex Murk and Calvin Li seemed destined that evening to sail through the legislature. But it was dramatically weakened just days before the General Assembly adjourned this month....[O]thers say the amended legislation, which awaits approval by Gov. Larry Hogan (R), no longer does what it was originally intended to do: discourage parents from hosting parties for teenagers where alcohol is provided.
And then there is Beach Week.
As Susan Coll, author of the acerbic, hysterically funny 2010 novel "Beach Week," said in an email:
Make the mistake of visiting just about any beach on the Eastern Shore in early June, for example, and you might see, as I did a couple of years ago, kids falling off bicycles, or stumbling around drunk. A friend not familiar with this particular tradition looked at me, puzzled, when I explained casually that no, there was not some sort of contagious virus in the air felling teenagers, it was just Beach Week — business as usual around these parts. Kavanaugh even boasts about his Beach Week drinking in his high school yearbook, outing himself as the biggest contributor to the Beach Week Ralph Club, meaning, presumably, that he did a lot of throwing up.
Apart from the Kavanaugh saga and what he did or didn't do in high school, Coll's novel starkly shows just how much parents feed into the Beach Week zeitgeist by enabling their children to attend — and sometimes escape consequences — for their behavior.
In 2013, Doug Gansler, then the attorney general of Maryland and a candidate for governor, was caught in something of a Beach Week scandal. He was photographed in what looked like a drunken teenage party at a Delaware beach house. He was chaperoning, he said — one of the ways that parents try to keep their kids from getting arrested at Beach Week. Parents whose kids stayed in the house with Gansler's son had agreed — as is common — on a set of rules for the week, including:
- "NO ONE goes out alone; boys should always stay in pairs."
- "NO DRIVING! All car keys will be held by chaperones. Chaperones will drive when necessary."
- "No climbing, hanging or jumping on or from balconies."
- "No hard liquor or controlled substances may be consumed."
Coll was inspired to write the book — which tells a tale of parents who come together to make it possible for their daughters to attend Beach Week, how the girls get arrested, what the chaperone parents do — by events observed over many years of living in Montgomery County.
I'm publishing a little bit of the novel so you can understand the extent of parental participation:
Excerpted from BEACH WEEK by Susan Coll, published by Sarah Crichton Books/Farrar, Straus and Giroux. Copyright © 2010 by Susan Coll. All rights reserved.
They'd been gone all day — graduation was held in an auditorium downtown, and the expedition had required major logistical planning in order to park and get the grandparents situated, and then they'd fought rush-hour traffic to get back to the restaurant where they'd reserved a table.Leah had just corralled everyone into the backyard for cake when Charles appeared at the patio door, his face pale. In his hand were several envelopes that he'd evidently just collected from the box on the front porch."What is it?" Leah asked, waving the match in the air to put out the flame."Nothing. It can wait.""You look pretty upset, Dad. What is it? We can handle it.""Probably best not to do that right now."Jordan went over and grabbed one of the envelopes from his hand. It was thin, like a college rejection letter, except that the return address said "Department of Justice, Chelsea Beach, Delaware." The other envelopes all had the same return address."Charles?" Leah asked tentatively.Jordan ripped open the letter. "It's a summons. They want me to appear as a witness in the case of State v. Cherie Long and"—she opened the other envelopes—"as a witness in the case of State v. Dorrie Glover and in the case of State v. Courtney Greene . . . and in the—""We get the idea," said Leah."When is all this?"Charles grabbed the papers from Jordan. "One on August eighth, another on August ninth . . . August tenth, August eleventh . . .""All week?" Leah asked. "Are they insane? There must be some way to get out of this. It's an unreasonable burden. How can Jordan be expected to be there for an entire week?""I've heard it's easy to wriggle out of, Mom. And probably all the charges will be dropped by then anyway, what with everyone having their lawyers and stuff.""I think we should make the best of this," said Charles."Let's embrace this thing and stop fighting it.""What do you mean?""I mean let's go to the beach. Let's have our own Beach Week.""I don't know, Dad. Your behavior wasn't exactly exemplary.""Hey, I just got caught up in the moment. I mean, have you ever played beer pong? I got a little carried away, but—""You played beer pong, Dad?" Jordan asked, incredulous."What's beer pong?" asked Leah."We can play at the beach. I'll teach you.""Dad, I don't see that happening, somehow."The phone rang from inside."Let it go," said Leah. "We're just about to have cake." But her mother had already answered it and was calling to her."Take a message, Mom," Leah said."I did," her mother said, returning to the patio. "It was some woman named Janet Glover. She said there's a meeting tomorrow night at the home of Alice Long . . . something to do with Beach Week? Collecting checks to pay a lawyer or something?I said you'd call her back.""Speaking of checks," said Charles. "Did you ever get—""Please let's not go there right now," said Leah. "Let's just enjoy this moment."
And then there is this:


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Yate & District Athletics
Club Kit and Clothing
This page contains details of club kit and clothing. You should allow a minimum of 10 working days for your order to be printed/embroidered and collated. You can collect your order from any of our three branches or have it despatched to your home address. Any of the items on this page can be viewed in more detail in our main catalogue by using the code number given in the garment description. The main catalogue can be accessed from the navigation bar in the left hand column.


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1848 BBQ is a Brownsville, Texas-based barbecue joint that focuses on bringing you the best low and slow cooked food with local and regional flare.
Located in the downtown district of Brownsville, Texas, on historic Palm Boulevard, 1848 BBQ celebrates great food, great drinks, and our great local culture.
A project by La Leña LLC, 1848 BBQ aims to pay homage to Texas BBQ while staying true to our outstanding local culture. In fact, our name 1848, is a nod towards the year Brownsville was founded, and we live by this name. Everything we cook and do is based on our city, our region and our state. We know Brownsville deserves the best, so we brought you the best BBQ using the highest quality ingredients on the market and the cooking methods we know and love.
You've seen us grow from a small storefront and tent to our new dine-in area and carry out section, and we continue growing daily because of your loyal patronage. Thank you!
If you haven't had a chance to experience 1848, drop by and give our food a taste. I'm sure you'll agree; this is the best BBQ in the state of Texas.
Discover Dining in Brownsville - November 2018
Come and see what you have been missing!#DiscoverBrownsville and visit all its exciting locations, even those featured in national publications such as Texas Monthly. From retail to dining, and all the quality of life in between. You cannot miss it! Follow Discover Brownsville to learn more!www.discoverbrownsville.org#DiscoverDining #RioGrandeValley #RGV #Texas #TourismPosted by Discover Brownsville on Monday, November 26, 2018


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Previously, we described a gene cluster of Enterococcus faecalis OG1RF that produced an antigenic polysaccharide when cloned in Escherichia coli. The polysaccharide antigen was not detectable in E. faecalis strains, however. Here, we show by reverse transcriptase-PCR that the 16 genes in this region are transcribed in OG1RF. Gene disruption of orfde4, encoding a putative glycosyl transferase, and orfde6, a putative dTDP-rhamnose biosynthesis gene, generated two OG1RF mutants. The mutants showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. In addition, two mucoid E. faecalisisolates from patients with chronic urinary tract infections were found to produce the polysaccharide antigen.
Polysaccharides (PS) of bacterial pathogens play important roles during infection. The capsule ofStreptococcus pneumoniae is considered its most important virulence factor, since it enables the organism to persist in the host by conferring resistance to phagocytosis (6, 8). Capsular PS from Staphylococcus aureus types 5 and 8 were shown to induce cytokine release from human epithelial and endothelial cell lines (22). The serotype f PS (a rhamnose-glucose polymer) of Streptococcus mutans was found to stimulate the release of tumor necrosis factor α (23). Furthermore, being the dominant immunogens on the bacterial surface, some PS (e.g., the O antigens of gram-negative bacteria and capsules of streptococci and staphylococci) have been used as the basis for both vaccine development and serological typing of clinical isolates.
Enterococci are a leading cause of nosocomial infections and account for 5 to 15% of infective endocarditis in the United States (14), with most clinical isolates being Enterococcus faecalis. The development of multiple antibiotic resistance in enterococci in recent decades has posed a serious threat to effective therapy and raises awareness of the need for a better understanding of the pathogenicity of enterococci. Members of our group previously reported that a cloned gene cluster (epa, encoding proteins involved in the biosynthesis of an enterococcal PS antigen) of E. faecalis OG1RF produced a PS in Escherichia coli(27). The PS reacted with sera from four patients with enterococcal endocarditis. Sequence analysis showed that epagenes were similar to those involved in the biosynthesis and export of PS, including genes for rhamnose biosynthesis, glycosyl transferases, and ATP-binding cassette transporters. Insertion mutations in three of the genes, orfde4, orfde5, and orfde8, abolished immunoreactivity of the E. coli clone (27). However, we were not able to detect the PS antigen in several E. faecalis strains. Early studies of E. faecalis showed that some cell wall PS were antigenic and might be used for serological typing (1, 9, 19, 21). Neither the chemical compositions nor the genetic basis of these PS have been elucidated. In addition, Bottone et al. (2) recently described the first isolation of mucoid E. faecalis strains from patients with chronic urinary tract infections.
The biosynthesis of PS in many bacteria is regulated. The production of colanic acid in E. coli is elevated under certain growth conditions, such as in chemically defined media with high concentrations of phosphate and with high carbon-to-nitrogen ratios and temperatures below 25°C (24, 25). Alginate production inPseudomonas aeruginosa is turned on in the lungs of cystic fibrosis patients (18). Since the synthesis of PS is a costly process, using ATP and sugars that would otherwise provide energy for cell activities, regulation is not surprising. Furthermore, since PS play specific roles in infection, it may also be advantageous to regulate their production during the stages of infection (25). Possibly the production of PS in E. faecalis is regulated.
In this study, we demonstrate that even though no antigen is detected,epa genes are transcribed in OG1RF with at least three transcriptional start sites. Furthermore, OG1RF mutants with disruptions in two of the genes showed a slightly higher 50% lethal dose (LD50) and a statistically significant delay in killing in a mouse peritonitis model. In addition, two recently reported mucoid E. faecalis strains showed positive reactions with specific antibodies against the PS produced by theE. coli clone.
Bacterial strains, plasmids, and culture conditions.Strains and plasmids used in this study are listed in Table1. E. coli cells were grown in Luria-Bertani broth or on Luria-Bertani agar with appropriate antibiotics overnight at 37°C. Enterococci were grown in brain heart infusion (BHI) broth or on BHI agar (Difco) overnight at 37°C for routine purposes unless otherwise stated. The following antibiotics for selection of E. coli recombinants were used: chloramphenicol (CM) (25 μg/ml) and kanamycin (KM) (50 μg/ml). A concentration of 2,000 μg of KM per ml was used for selection of E. faecalis mutants. Serum samples were collected from patients withE. faecalis endocarditis infections and had high titers against E. faecalis strains (26). E1 and E2 are two mucoid E. faecalis isolates kindly provided by Edward J. Bottone at the Division of Infectious Diseases, the Mount Sinai Hospital, New York, N.Y. E1 is constitutively mucoid, while E2 is mucoid after incubation at room temperature but not at 37°C (E. J. Bottone, personal communication).
DNA manipulations and transformation of E. coli.DNA preparation, purification, restriction digestion, agarose gel electrophoresis, and ligation were performed using standard methods (17) or following the manufacturer's instructions, unless otherwise stated. Routine preparation of competent E. colicells and transformation of DNA into E. coli were performed by a one-step procedure (5).
Preparation of total RNA.The RNeasy Mini Kit (Qiagen, Santa Clarita, Calif.) was used to extract total RNA from E. faecalis, with slight modifications. All the solutions and utensils were treated with diethylpyrocarbonate (DEPC) to eliminate RNase. A single colony was inoculated into 5 ml of BHI broth and incubated at 37°C overnight with shaking. A fresh 5-ml culture was then inoculated with 50 μl of the overnight culture and incubated with shaking for 4 h. The cells were harvested and resuspended in 100 μl of TE (10 mM Tris, 1 mM EDTA [pH 8.0]) containing 3 mg of lysozyme per ml. After incubation at room temperature for 20 min, 10 μl of 10% sodium dodecyl sulfate (SDS) and 350 μl of buffer RLT (Qiagen) were added and the mixture was vortexed vigorously. Subsequent steps were performed according to the instructions from the kit. After elution with DEPC-treated H2O, the sample was treated with 5 μl of RNase-free DNase (Promega) at 37°C for 30 min and then boiled for 10 min to inactivate the DNase. After phenol-chloroform extraction and ethanol precipitation, the sample was again resuspended in DEPC-treated H2O. The RNA concentration was measured in a spectrometer at 260 nm.
RNA RT-PCR.Murine leukemia virus (MuLV) reverse transcriptase (RT) (Perkin-Elmer) was used to synthesize cDNA for PCR. The Perkin-Elmer protocol for reverse transcription, with modifications, was used. A 20-μl reaction mixture contained 4 μl of MgCl2 (25 mM), 2 μl of 10× PCR buffer II (Perkin-Elmer), 2 μl of each dNTP (10 mM), 0.5 μl of RNase inhibitor (20 U/μl), 1 μl of reverse primer (20 μM), 0.5 μl of MuLV RT (50 U/μl), 25 ng of total RNA, and DEPC-treated H2O. For the DNA control, 5 ng of OG1RF genomic DNA was used instead of total RNA. When MuLV RT was not used, the volume of DEPC-treated H2O was increased correspondingly. The reaction was carried out at 42°C for 30 to 60 min, and then the mixture was heated at 95°C for 5 min in a Perkin-Elmer 9600 thermal cycler. The subsequent PCR was performed according to the Perkin-Elmer protocol. To each reverse transcription reaction mixture, 4 μl of 25 mM MgCl2, 8 μl of 10× PCR buffer II, 65.5 μl of H2O, 1 μl of each primer (20 μM), and 0.5 μl of AmpliTaq DNA polymerase (5 U/μl) were added. PCR cycling was done with the following conditions: 24 to 30 cycles of 96°C for 1 min; 94°C for 30 s, 50 to 55°C (based on the melting temperatures of the primers) for 30 s, and 72°C for 30 s, followed by 10 min at 72°C and holding at 4°C. The primers used in RT-PCR are listed in Table2.
Sequencing of RT-PCR products and sequence comparison.The RT-PCR products were gel purified by using the Qiaquick Gel Extraction kit (Qiagen). After elution with 30 μl of H2O, 10 μl of each DNA solution was subjected to electrophoresis through an 0.8% agarose gel to check the bands. For sequencing, 3.5 or 7 μl of each DNA solution was used as the template and the upper primers were used as sequencing primers. The sequences were compared to each corresponding open reading frame (ORF) by using the Bestfit program of Genetics Computer Group (Madison, Wis.).
Cloning intragenic DNA fragments of orfde4 andorfde6 into plasmid pTEX4577.Plasmid vector pTEX4577 was derived from pBluescript SK(−) containing a KM resistance marker selectable in both E. coli and E. faecalis(15). Intragenic DNA fragments of orfde4 andorfde6 were PCR amplified from chromosomal DNA of OG1RF with primers containing the recognition sequence for BamHI, as follows: GW345, GW346, GW347, and GW348 (Table3). Each PCR mixture contained 2 μl of OG1RF chromosome DNA (5.6 ng/μl), 10 μl of 10× PCR buffer I (containing 15 mM MgCl2; Perkin-Elmer), 2 μl of each dNTP (100 mM; Perkin-Elmer), 1 μl of each primer (20 μM), 77 μl of H2O, and 1 μl of AmpliTaq DNA polymerase (5 U/μl; Perkin-Elmer). PCR cycling was performed in a Perkin-Elmer 9600 thermal cycler, as follows: 96°C for 1 min; 94°C for 20 s, 55°C for 20 s, and 72°C for 20 s (30 cycles); 72°C for 10 min; and holding at 4°C.
After amplification, a small aliquot of each reaction mixture was analyzed by agarose gel electrophoresis to determine the size of each product. The remaining reaction mixtures were purified using the Qiaquick PCR purification kit (Qiagen). After BamHI digestion of the purified PCR products and subsequent purification by the Qiaquick PCR purification kit to remove the BamHI restriction enzyme, each fragment was ligated toBamHI-digested pTEX4577 with T4 ligase (New England Biolabs). The ligation mixtures were transformed into DH5α and plated on LB agar-KM (50 μg/ml) with isopropyl-β-d-galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). Four KM-resistant white colonies were picked after overnight incubation at 37°C and analyzed by restriction enzyme digestions. A plasmid containing the intragenic fragment of orfde4 was designated pTEX5177, and one containing the orfde6 intragenic fragment was designated pTEX5178. Plasmids pTEX5177 and pTEX5178 were further subjected to DNA sequencing to verify the inserts.
Generation of OG1RF mutants with disruptions inorfde4 and orfde6.DNA from plasmids pTEX5177 and pTEX5178 was prepared from 250 ml of overnight cultures using the standard alkaline-SDS method and purified in a CsCl-ethidium bromide gradient, followed by dialysis against 10 mM Tris, pH 8.0, and precipitation with ethanol (17). The final DNA concentration was adjusted to 2.5 μg/μl with sterile H2O. Preparation of OG1RF competent cells and electroporation of DNA into OG1RF were carried out as described previously (15), with slight modifications. For each electroporation, 5 μg of DNA and 50 μl of OG1RF competent cells were mixed and added to an ice-cold 0.1-cm gap electrode Gene Pulser/E. coli Pulser Cuvette (Bio-Rad). One pulse was given at 1.25 V with 400-Ω resistance and 25-μF capacity. Immediately after the pulse, 1 ml of Todd-Hewitt broth (THB)–0.25 M sucrose was added to the cuvette. After incubation at 37°C for 2 h, the cell mixture was centrifuged and the cell pellet was resuspended in 0.2 ml of THB–0.25 M sucrose and plated onto THB–S0.25 M sucrose agar containing 2,000 μg of KM per ml. Colonies appearing within 36 h of incubation were streaked onto BHI agar with 2,000 μg of KM per ml. Clones that grew up after overnight incubation at 37°C were further analyzed by colony PCR and Southern blot analysis.
Colony PCR of OG1RF mutants.A single colony was picked with a 200-μl sterile pipette tip and resuspended in 100 μl of sterile H2O. GW378 and GW379 are oligonucleotides directing outward DNA synthesis from the ends of the KM resistance gene of pTEX4577. Their sequences are as follows: GW378, 5′ GTGATATTCTCATTTTAGCC; and GW379, 5′ GACTTACTGGGGATCAAGCC. GW378 and GW379, and primers to orfde4 and orfde6, GW301 and GW303 (Table 2), and GW345, GW346, GW347, and GW348 (Table 3) were used in different combinations for PCR. PCR mixtures contained 1 μl of the cell suspension, 4 μl of PCR buffer I (Perkin-Elmer), 1 μl of a mixture of the four dNTPs (2.5 mM [each]), 4 μl of each primer (1 μM), 25.5 μl of sterile H2O, and 0.5 μl of AmpliTaq DNA polymerase (5 U/μl; Perkin-Elmer). PCR cycling was performed in a Perkin-Elmer 9600 thermal cycler with the following conditions: 96°C for 1 min; 94°C for 20 s, 55°C for 20 s, and 72°C for 1 min (30 cycles); 72°C for 10 min; and holding at 4°C. One of each of the colonies showing the expected PCR products was designated TX5179 or TX5180 for interruptions in orfde4 and orfde6, respectively, and was subjected to Southern blot analysis for verification.
Southern blot analysis of OG1RF mutants.Genomic DNA from OG1RF, TX5179, and TX5180 was prepared based on a procedure described for E. coli (7). Briefly, enterococcal strains were grown in 5 ml of BHI broth with appropriate antibiotics at 37°C overnight. The cells were harvested, washed once with 5 ml of J buffer (0.1 M Tris-Cl, 0.1 M EDTA, and 0.15 M NaCl [pH 8.0]) and resuspended in 0.16 ml of J buffer. The suspension was then treated with 20 μl of freshly made lysozyme solution (10 mg/ml in 0.25 M Tris-Cl, pH 8.0) at 37°C for 20 min, followed by 1 μl of RNase A (34 mg/ml; Sigma) at 37°C for 10 min and then 70°C for 3 min. After this, 16 μl of 30% Sarkosyl was added and the mixture was incubated at 70°C for 20 min and then at 37°C for 1 h. Then 0.4 mg of proteinase K was added, and the solution was incubated at 37°C for 2 to 4 h. Another 0.4 mg of proteinase K was added at the end of the incubation, and the solution was dialyzed against 0.01 M Tris (pH 8.0)–0.01 M EDTA (pH 8.0)–0.15 M NaCl at 37°C overnight. The DNA solution was extracted once with phenol-chloroform and once with chloroform, and dialyzed against TE for several hours at room temperature. The concentration of DNA was then determined in a fluorometer.
For Southern blot analysis, 3 μg of genomic DNA was digested withEcoRI and XbaI and subjected to agarose gel electrophoresis. Blotting was performed using Hybond-N+nylon membrane (Amersham) and 0.4 N NaOH solution according to the manufacturer's instructions. DNA of plasmids pTEX5177 and pTEX5178 was digested with BamHI, and the insert fragments were purified using the Qiaquick Gel Extraction kit (Qiagen). The Random Primers DNA Labeling System (GIBCO BRL) was used to label the insert fragments with [α-32P]dCTP (Amersham). Hybridization was carried out at 65°C according to manufacturer's instructions.
Determination of growth curves and stability of single crossover disruptions in TX5179 and TX5180.Overnight cultures of TX5179, TX5180, and OG1RF were diluted 100-fold with fresh BHI broth and incubated at 37°C with shaking. Klett units were measured every hour until the stationary phase was reached. Aliquots (100 μl) of each culture were taken every hour, and 100 μl of 10−4 to 10−8 dilutions of each sample were plated onto BHI agar plates. Colonies were counted after incubation at 37°C for 24 h.
TX5179 and TX5180 were grown in BHI broth without antibiotics at 37°C with shaking. Aliquots (100 μl) of each culture were taken after 24 h of growth, and 10-fold serial dilutions were made. Dilutions (10−5-, 10−6, and 10−7-fold; of each sample in 100-μl volumes) were plated on BHI and BHI-KM (2,000 μg/ml) agar plates and incubated at 37°C overnight. Colonies were counted, and the numbers from BHI agar plates and BHI-KM agar plates were compared.
Examination of OG1RF mutants in a mouse peritonitis model.Testing of the mutants was performed as described previously (20). OG1RF and the mutants were incubated in BHI broth overnight at 37°C with shaking. The cells were harvested by centrifugation, washed with chilled 0.85% saline solution, and resuspended in 20 ml of ice-cold 0.85% saline to reach a density of ∼5 × 1010 CFU/ml. A series of twofold dilutions were then made with 50% sterile rat fecal extract to obtain four to five inocula for injection into mice. Meanwhile, 10-fold serial dilutions were made and aliquots of 10−6, 10−7, and 10−8 dilutions were plated onto BHI agar plates with proper antibiotics to obtain the actual titer. Outbred ICR female mice, four to six weeks old (Harlan Dawley, Houston, Tex.) were used. Each was injected intraperitoneally with 1 ml of cell suspension with a syringe with a 25-gauge needle. Observations were made every 3 h, and the number of surviving mice was recorded.
The LD50 was determined as described by Reed and Muench (16). Survival statistics were computed using the Kaplan-Meier method and compared by log rank using StatView (Abacus Concepts, Inc., Berkeley, Calif.). Spleens were recovered from dead mice injected with the highest inocula, homogenized, and suspended in 3 ml of chilled 0.85% saline. Serial dilutions of each spleen suspension were plated onto BHI agar plates and, for TX5179 and TX5180, also onto BHI agar plates containing 2 mg of KM per ml. Colonies were counted after incubation for 24 h at 37°C.
Elution of antibodies specific to the PS.Antibodies were eluted as described previously (27). Briefly, cell lysates of TX5159 were treated with proteinase K, applied to SDS-polyacrylamide gel electrophoresis gels, transferred to nitrocellulose membranes, and incubated with one of the patient serum samples. Elution was carried out using 100 mM glycine (pH 2.5); the solution was neutralized with a 1/10 volume of 1 M Tris (pH 8.0) and stored at −20°C until used.
Immunoblot analysis of bacterial colonies.Colonies were inoculated onto LB or BHI agar plates containing antibiotics and incubated at 37°C or room temperature for 24 h. NitroBind nitrocellulose transfer membranes (Micron Separations, Inc., Westborough, Mass.) were used to lift the colonies. Subsequent steps were performed as described previously (27).
Detection of mRNA transcripts from epa genes in OG1RF.To determine if the ORFs in the epa region were transcribed, RT-PCR was performed. In the reactions using RNA as the template, only those reaction mixtures containing both RT andTaq polymerase yielded PCR products. Reaction mixtures containing only Taq polymerase and no RT (negative controls) did not yield PCR products (data not shown). This confirmed that the PCR products were derived from mRNA templates and not from contaminating DNA. In many cases a single RT-PCR band of the expected size was visualized after agarose gel electrophoresis (orfde3, orfde4, orfde5_6,orfde8, orfde9, orfde10,orfde12, orfde15, and orfde16). However, in some cases a band of the expected size as well as bands of other sizes were observed (orfde2, orfde5,orfde6, orfde7, orfde11,orfde13, and orfde14). Varying the RT-PCR conditions and designing new primers to different regions of the ORFs in the latter case improved the results with orfde5,orfde6, orfde11, and orfde13 but not with orfde2, orfde7, and orfde14. PCR controls using DNA instead of RNA (without RT) yielded only bands of the expected sizes.
To verify that the RT-PCR products observed on agarose gels were the expected products, each band of the correct size from orfde2to orfde16 was gel purified and subjected to DNA sequencing. The DNA sequences were compared with the sequences of each corresponding ORF. The results showed that each product had the correct sequence (data not shown). This indicated that all 16 genes in theepa region are transcribed. To examine the nature of the bands of incorrect sizes, some of them were also gel purified and subjected to DNA sequencing. None of the sequences examined showed any homology to the corresponding ORFs, although sequences resembling those of the primers could be found at the ends. Thus, these bands do not represent RNAs derived from an epa gene.
Generation of OG1RF mutants.Although PS could not be detected in E. faecalis, sera from patients with enterococcal infections contained anti-PS antibodies (27). If the PS was produced only during infection, it may be a virulence factor. To test this, mutations were made in orfde4 and orfde6, both necessary for PS production in E. coli. Intragenic fragments of orfde4 and orfde6 were cloned in pTEX4577, resulting in plasmids pTEX5177 and pTEX5178, respectively. In pTEX5177, the orfde4 intragenic fragment was in the same orientation as that of the T7 promoter, while in pTEX5178, theorfde6 fragment was in the orientation of the T3 promoter.
DNA of pTEX5177 and pTEX5178 was electroporated into OG1RF to generate single crossover mutants. Four resistant colonies for orfde4and one for orfde6 were obtained. Colony PCR of these five clones produced bands of the expected sizes (data not shown). To verify the disruptions, one clone for orfde4 and one fororfde6, designated TX5179 and TX5180, respectively, were further analyzed by Southern blot hybridization (Fig.1A). The results showed that the 5.7-kbEcoRI band in OG1RF was split into two bands of about 6.5 and 4.2 kb in TX5179 and into two bands of 7.2 and 3.9 kb in TX5180, while the 9.4-kb XbaI band was split into two bands of about 12.5 and 2.3 kb in TX5179 and into two bands of 9.6 and 5.2 kb in TX5180. These sizes are as expected from a single crossover atorfde4 or orfde6 (Fig. 1B).
Growth curves of TX5179 and TX5180 and stability of single crossover disruptions.To determine if the growth of mutants TX5179 and TX5180 differed from that of wild-type OG1RF, the optical turbidity (in Klett units) and bacterial titers were measured (Fig.2). TX5179 and TX5180 grew slightly slower than OG1RF and had slightly lower cell densities at stationary phase. The bacterial titer for OG1RF after growth for 24 h was (2.20 ± 0.28) × 109 CFU/ml, while the titers for TX5179 and TX5180 were (1.65 ± 0.13) × 109CFU/ml and (1.80 ± 0.27) × 109 CFU/ml, respectively, similar to the turbidity of each culture (Fig. 2). Thus, the insertion mutations did not affect growth.
The numbers of colonies of TX5179 and TX5180 on BHI agar plates were comparable to those on BHI-KM agar plates (1.24 × 109versus 1.4 × 109 for TX5179 and 1.52 × 109versus 1.45 × 109 for TX5180) after growth for 24 h in BHI medium, indicating that the insertion mutations in the chromosomes of TX5179 and TX5180 were stable.
Comparison of mRNA transcript levels in OG1RF and TX5180.To determine the effect of gene disruption on the transcription of downstream genes, total RNA was extracted from OG1RF, TX5179, and TX5180 after 4 h of growth in BHI broth. Extraction from OG1RF and TX5180 yielded 300 to 400 ng of RNA per μl, whereas for unknown reasons, no RNA was obtained from TX5179 in two attempts. RT-PCR was performed on total RNA from OG1RF and TX5180, and transcription of a number of ORFs (orfde5 to orfde11,orfde13, and orfde15) was measured. After RT-PCR, the intensity of each band from TX5180 was compared to that of its corresponding band from OG1RF. The results indicated that the amount of mRNA transcripts of orfde7 to orfde10 in TX5180 was greatly reduced, while that of transcripts of orfde5,orfde6, orfde11, orfde13, andorfde15 was not (Fig. 3A). A band was observed for orfde6 because the region between the primers (GW303 and GW304) used in RT-PCR was not affected by the insertion. This suggests that there is a polar effect of the insertion on transcription of some epa genes.
To examine the level of discrimination of RNA concentration in the RT-PCR system, total RNA (25 ng/μl) of OG1RF was diluted in a series of 10-fold dilutions and 1 μl of each dilution was subjected to RT-PCR with reaction and cycling conditions the same as those used for comparing RNAs of TX5180 and OG1RF. Primers GW301 and GW302 fororfde4 were used (Fig. 3B). The results showed that bands were observed with 25, 2.5, 0.25, and 0.025 ng of total RNA, but not with 2.5 pg of total RNA. The intensity of the bands decreased with the amount of RNA. Thus, under the conditions used, the system was able to discriminate a 10-fold difference in the amount of total RNA. Furthermore, the difference in the levels of intensity of bands in lanes 1 and 10 of Fig. 3B, indicative of a 1,000-fold difference in the RNA level, appeared to be of the same order of magnitude as the reduction in transcription of orfde7 to orfde9 in the mutant TX5180.
Examination of OG1RF mutants in a mouse peritonitis model.To test if the disruptions in orfde4 and orfde6 had any effect on the virulence of OG1RF, TX5179, and TX5180 were examined in a mouse peritonitis model. In an initial experiment, a group of six mice was used for each of the four inocula made by twofold serial dilutions of overnight cultures with sterile rat fecal extract. Bacteria were recovered from the spleens of dead mice injected with the highest inocula of OG1RF, TX5179, or TX5180, and their titers on BHI agar plates and BHI-KM agar plates were determined. The titers for OG1RF, TX5179, and TX5180 on BHI plates were 3.6 × 108, 1.2 × 108, and 1.6 × 108 CFU/ml, respectively. The numbers of colonies from BHI plates were comparable to those from BHI-KM agar plates for both TX5179 and TX5180, indicating that the disruptions were stable in vivo.
The range of inocula used in this experiment did not allow the calculation of LD50, since the bacterial titers for OG1RF and the two mutants were not closely matched. However, when the data (not shown) were analyzed for the time to killing, TX5179 and TX5180 showed statistically significant delays in killing compared to that of OG1RF (Table 4, rows 1 and 2).
To further examine the effect of the mutations on the survival of mice, sixteen mice were used for each of the two mutants, with inoculum sizes of 4 × 108 CFU for TX5179 and 5 × 108 CFU for TX5180 (Table 4, row 3). Six mice were used for OG1RF at 3.5 × 108 CFU. The survival of the 16 mice injected with TX5179 and TX5180 was compared with that of the six mice injected with OG1RF. The percentage of surviving mice was plotted against time (Fig. 4). The two mutants resulted in a higher percentage of surviving mice and delayed killing compared with that of OG1RF. The P values were <0.0001 for TX5179 versus OG1RF and 0.0002 for TX5180 versus OG1RF, consistent with the results obtained in the first experiments.
To compare the LD50 values of OG1RF and the mutants, the experiment was repeated using a slightly different range of inocula and six mice for each inoculum. The two mutants showed higher LD50 than OG1RF (Table 4). These results demonstrate that genes in the epa locus contribute to virulence in the mouse peritonitis model.
Reaction of two mucoid E. faecalis isolates with anti-PS antibody.Two mucoid E. faecalis isolates from patients with chronic urinary tract infections (2), E1 and E2, were tested for reaction with anti-PS antibody. E1, E2, and OG1RF were streaked on BHI agar plates and incubated at room temperature or 37°C for 24 h. The colonies were lifted onto nitrocellulose membranes for immunoblotting with the antibody eluted from the E. coli clone expressing the PS antigen (Fig.5). The results showed that E1 grown at both room temperature and 37°C reacted, E2 reacted only after incubation at room temperature, and, as found previously, OG1RF did not react at either temperature. The temperature dependence of the immunoreactivity paralleled that of mucoidy, suggesting that the PS being overproduced in the mucoid strains is in fact the antigen.
In a previous study, members of our group described a PS antigen biosynthesis gene cluster (epa) of E. faecalis(27). In this report we demonstrate that although theepa antigen is not detectable in laboratory-grown E. faecalis cells, the genes within epa are transcribed inE. faecalis and disruptions in two of the genes (orfde4 and orfde6) caused a statistically significant delay in killing in a mouse peritonitis model.
The E. coli clone containing the epa gene cluster, TX5159, was previously found to react with sera collected from four endocarditis patients infected with E. faecalis in different regions in the United States, suggesting that the PS was produced in all of these patients (27). However, we were able to detect the antigen neither in E. faecalis strain OG1RF under different growth conditions (including incubation in the peritonea of mice) nor in several other E. faecalis clinical isolates or laboratory strains. To determine if the genes withinepa were transcribed, we performed RT-PCR analysis of the total RNA of OG1RF grown in BHI medium for 4 h. Each band of the correct size was confirmed by sequencing the purified PCR products. The results indicated that the orfde2 to orfde16genes were transcribed. However, it is not clear whether the amount of mRNA represents a low basal level that must be induced for the production of the PS or whether this is sufficient mRNA for PS production.
To study the effects of mutations in this region, two mutants, TX5179 (in orfde4) and TX5180 (in orfde6), were generated. Members of our group previously showed that insertions inorfde4 abolished antigen production in the E. coli clone TX5159 (27). Transposon mutagenesis also indicated that one of the putative rhamnose biosynthesis genes,orfde8, was required for the immunoreactivity of TX5159, andorfde6 is predicted to encode the first enzyme in this rhamnose biosynthesis pathway.
Since the mutants were generated via single crossover events, resulting in two partial copies of orfde4 or orfde6, the stability of the crossovers was determined. The disruptions appeared to be stable, since very few cells became KM sensitive after growth in plain BHI broth for 24 h or incubation in the peritonea of mice for at least 12 h. The mutants grew slightly less well than OG1RF; however, the difference appeared to be minor.
To examine if the disruptions had polar effects on transcription, RT-PCR was performed to measure the mRNA levels from several ORFs. The transcription of orfde7 to orfde10 in TX5180 was drastically reduced, while that of orfde11,orfde13, and orfde15 was not (Fig. 3A). This suggested that the orfde6 to orfde10 genes were in one transcriptional unit and that their transcription was independent of that of orfde11 and other downstream genes (Fig. 6). This is consistent with the results of DNA sequence analysis, which suggested that a promoter-like sequence was found upstream of orfde11 (27). In the study previously conducted by members of our group, analysis of Tn7 transposon mutants indicated that insertions inorfde3 or between orfde3 and orfde4did not affect the immunoreactivity of TX5159, and an insertion inorfde4 was complemented by a plasmid, pTEX5175, that contains only orfde4 and orfde5. These results suggested that there should be transcriptional start sites upstream oforfde4 and orfde6 (27). Thus,epa should contain at least three transcriptional start sites (Fig. 6).
To test if the disruptions had any effect on the virulence of OG1RF, the two mutants were examined in a mouse peritonitis model. TX5179 and TX5180 appeared to be less virulent, as indicated by a high LD50 as well as to have delayed killing compared to that of OG1RF. The attenuation in the virulence of TX5179 and TX5180 in the mouse peritonitis model could reflect an involvement of this region inE. faecalis virulence. Considering the slightly slower growth of the mutants, the P values were calculated using higher initial inocula of the mutants versus OG1RF. In the case of TX5179, for example, a statistically significant P value (0.0034) was obtained when results from TX5179 with an inoculum of 6.6 × 108 CFU were compared with those from OG1RF with an inoculum of only 2.3 × 108 CFU. This suggested that the delayed killing was not due to the differences in growth rates.
The recent report of four mucoid encapsulated E. faecalisisolates from patients with chronic urinary tract infections by Bottone et al. (2) shed new light on this project. Two of the isolates, E1 and E2, were examined and showed positive reactions with the anti-PS antibody. E1 reacted after incubation at both room temperature and 37°C, while E2 reacted only after incubation at room temperature. These results suggested that the antigenic PS was overproduced in the mucoid strains.
The reasons that no antigenic PS was detected in wild-type E. faecalis remain to be determined. One possibility is that it is expressed only under certain conditions. In P. aeruginosa, the production of alginate is suppressed by genes in the muclocus until the organism invades the lungs of cystic fibrosis patients. The muc region was mapped to a separate locus from that of the biosynthesis genes (3, 4, 11, 12, 18). Therefore, it is possible that a negative regulatory region is also present in E. faecalis at a different location from epa and suppresses the PS production in E. faecalis. Under certain conditions, such as chronic infections or attachment to heart valves, the suppression could be turned off. The PS could then be produced, generating antibodies in the patients. By this model, in E. coli TX5159 carrying the cloned epa locus, the negative control region is absent or not functional and PS production is permitted. Since the orfde2 to orfde16 genes were transcribed in OG1RF grown in BHI medium, it is not clear if the level of transcription of these genes reflects a basal level produced under the negative control. Alternatively, the epa locus could be posttranscriptionally regulated or the target(s) of regulation could be outside of epa.
A second possibility is that some function(s) required for the synthesis or export of the PS is not present in the strains examined. For example, the capsulation gene cluster (cap) ofHaemophilus influenzae type b is on a compound transposon flanked by two insertion element-like (IS1016) elements. The type b cap locus consists of a duplication of capwith an unduplicated small bridge region containing bexAbetween the two large repeats. Recombination between the large repeats can result in the deletion of bexA, leading to a capsule-deficient phenotype (10). If this were the case inE. faecalis, the lost function(s) must have been complemented in TX5159 by E. coli genes outside therfb region (E. coli rfb was previously shown not to affect antigen production). A variation of this possibility is that there is a hot spot in this region for transposon or conjugative plasmid integration and/or excision. In support of this hypothesis, comparison of the OG1RF gene cluster with preliminary genome sequences of E. faecalis strain V583 recently released by the Institute of Genome Research showed that all epa genes are present in V583 but that the V583 orfde10 is interrupted by conjugative transposon-like sequences (unpublished data). We think this explanation is less likely, however, since the insertion mutations in the epa locus of OG1RF led to reduced virulence, suggesting that the PS functions during infections.
The possibility that the antigenic PS is produced in OG1RF but is different or modified compared to the antigen made in E. coli also cannot be excluded. The antibodies generated during infection may be directed against a degradation product of the native molecule. The partial PS may be the antigen made in E. coli. In the mucoid strains, the overproduction of the native PS may also provide enough of the partial molecule to be detected, whereas in the other E. faecalis strains tested, there would not be enough to detect.
In conclusion, we report here that genes orfde2 toorfde16 in the epa region were transcribed in OG1RF and that disruptions in orfde4 and orfde6caused statistically significant delayed killing and a higher LD50 in a mouse peritonitis model. There are at least three transcriptional start sites hypothesized in epa and genesorfde6 to orfde10 are in one transcriptional unit. In view of the regulation of the production of many bacterial PS and the fact that E2 reacted only with the eluted antibody after incubation at room temperature, it is possible that E. faecalis also has adopted ways of manipulating the production or display of the antigenic PS. Comparisons of the genetic organizations of the epa region in OG1RF and in the mucoid E. faecalis strains and of levels of gene expression and isolation of the PS material and analysis of its chemical composition will yield information invaluable for addressing many of the issues discussed above. Further study on the involvement of epa in virulence should have important implications for prevention and treatment of enterococcal infections.
We are grateful to Edward J. Bottone at the Division of Infectious Diseases, the Mount Sinai Hospital, for providing us with the mucoid strains, and to Robert Rakita and Bei Wang of the Division of Infectious Diseases, Department of Medicine, and Monjula Chidambaram of the Department of Microbiology and Molecular Genetics at the University of Texas Medical School at Houston for helpful advice and technical support.
This work was supported by grant AI 33516 from the NIH Division of Microbiology and Infectious Diseases to B.E.M.
Editor: E. I. Tuomanen
- Received 22 July 1999.
- Returned for modification 8 September 1999.
- Accepted 9 November 1999.
- Copyright © 2000 American Society for Microbiology


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Mitt Romney has been appealing to four blue-leaning states much more aggressively this week, launching ads totaling $1.8 million in Minnesota and New Mexico this past Wednesday. In an attempt to "buy" the traditionally Democratic states, the pro-Romney Super PAC Restore Our Future has been dumping money into advertising in these crucial states. PAC and Super PAC money has been flowing freely in a last minute attempt to gain with undecided voters.
In a tit-for-tat of funding, every state in which the Romney campaign infuses cash, Obama support follows suit, and vice-versa.
According to opensecrets.org, the super PAC Restore Our Future has raised a total of $131,692,123 in support of Romney. In comparison, the highest fundraising PAC for Obama, Priorities USA, has raised $63,897,639, less than half of Restore Our Future.
Both the Romney and Obama campaigns are trying to garner last minute support throughout the country. The Romney campaign held two separate fundraising events in Michigan in October. Romney was not in attendance, but Ann Romney and VP candidate Paul Ryan stood in. The Obama campaign held no events in Michigan, but the first fady was campaigning for the president in the state, as he was working on Sandy relief efforts on the East Coast.
This last push for support might be too late in the campaign cycle; as of November 2, one third of voters in swing states will have already cast their ballots. The states most in question now are: Iowa, New Hampshire, Nevada, Florida, Virginia, Wisconsin, and of course, Ohio. In these states, nearly 32 percent voters say they have already cast their vote.
The Rasmussen Reports Daily Presidential Tracking Poll for November 2 shows the race tied, with President Obama and Governor Romney attracting support from 48 percent of voters nationwide. One percent prefers some other candidate, and three percent remain undecided.November 2 Ramussen Daily Presidential Tracking Poll credit:ramussenreports.com
The Washington Post released a preliminary electoral vote count on November 2 as well. This poll shows President Obama leading with 186 "solid" electoral votes, compared to Romney's 170 "solid" electoral votes. The poll further shows that 51 electorates are leaning towards Obama, while 36 are swayed in Romney's direction. Ninety-five are still considered a "toss up."
It would seem that Governor Romney's elevated outside spending, as compared to President Obama, would reflect a significant gap between the two candidates. However, as it stands now, this is an exceptionally close race, so it will not be surprising to see PACs and super PACs supporting either kicking into higher gear, even in the race's last two days.


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People get frustrated trying to figure out when to update which software. As a rule of thumb, we don't buy into brand new issues of software until they've been out for at least 6 months. So, for example, right now we're warning our clients to stay away from Internet Explorer 9. Microsoft is notorious for throwing their software out there half-baked. BUT there are lots of cases where you SHOULD update, and that includes normal windows updates, driver updates, other standard security updates from vendors like Adobe, Java (Sun), etc. WATCH OUT because a lot of times they bundle some crappy package with it, like McAfee Security Scan. To avoid it, look for a checkbox along the way and UNCHECK IT. Another good reason to buy new releases of software is for your database(s). If you have critical business or personal info on a database (like Quickbooks, for example), you should get the new version every 2-3 years to insure that your data gets converted to the latest, greatest. Otherwise, if you wait too long, you might not be able to upgrade.
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We live in a fast-paced world. We always have something to do, someone to see, trips to take and Facebook or Instagram to fill up the few moments in between (or the bathroom – c'mon, you know I'm right). The problem with this lifestyle is it gives us little – if any – time to add something new. Even if that something new is incredibly important.
So, when you get pregnant, what new activities do you have to squeeze into your over-packed life? Well, the baby needs stuff, right? So you have to go shopping, at least a "few" times (diapers and such). And you're probably interested in how a whole person will exit your body so you get the basics with an inexpensive, couple-hour hospital class (because hospitals are experts, at birth, right). And then you get back to running to and fro and living your normal life until your little person decides to appear.
What's wrong with this picture, you ask? Isn't this what everyone else does?
If you're a typical American, then yes. But what is wrong with this? Oh so much.
Birth is one of THE pivotal moments in your story. This isn't just another appointment on your calendar. This is the moment you become a parent. (Of one, two, or more!) And when that happens, everything changes. You have to make decisions that affect another person – possibly for their whole life. You have to trust yourself and your instincts because everyone has a different opinion on every subject. And you have dig down deep, finding strength and courage you never realized you had. That's because this will be hard; the hardest beginning to any relationship you've ever had. And it requires preparation. So here's what you'll need:
You need knowledge.
Do you know what NORMAL birth looks like? It's nothing like the movies. You need to know what activities can prepare you for labor and birth. You need to understand how your body works and what helps it work best! And, you need to fill up your brain with accurate information to force out all the myths and what-ifs! (The best way to do this with an out-of-hospital class!)
You need support.
Your and your partner both need to know how to work together during labor. How to relax, how to cope with the intensity (no one realizes how intense labor actually is), and how to work with your body and not against it. Get a doula! Find a provider who actually supports (not tolerates) normal birth and have lots of conversations about your birth and your situation.
You need centering.
This is not a new-age, hippy-dippy type of centering. Your mind needs to be free of fear. Your emotions must be stable (or as stable as pregnancy hormones can make them). And your spiritual compass must be aligned. I'm a follower of Jesus and I believe God created women to birth babies – in most cases – beautifully and without complication. But, our world and our culture can get in the way of that process. So, don't allow your frantic pace of living to distract you. Make time to sit calmly and quietly. Think of your baby's birth as more than a speed bump on the road of life.
And to prepare for your birth, your baby and parenthood:
1. Set aside time every day to prepare mentally, physically and emotionally for your birth.
2. Know what procedures, policies and people can negatively and positively affect your birth and take steps to avoid or implement those TODAY!
3. Hone your instincts for birth – which will help hone your instincts for parenting.
4. Don't waste time. Every moment is precious as you prepare for your baby's arrival.
Looking for a doula in the Omaha area? Kate will provide individualized support and guidance to help you achieve your most amazing birth!


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Already have been 1 month since I started to send my files to webimmune in order to stop the annoyings system menssages about being infected by a virus. The result? Nothing. So I have to disable the security software I paid for in order to run simple software that I KNOW for a fact is fine and has been on my computer without incident for years. Please let me know when the trusted program list is available because then I may renew my subscription.
Did you get a response from them? Usually they respond immediately and often clear the detection within hours assuming that it isn't an obvious risk.
What method were you using to report the incorrect findings?
There wont be any major announcement regarding such a feature change but it most likely will be mentioned somewhere in these forums.Message was edited by: Ex_Brit on 02/06/10 2:44:39 EDT PM
I used webimmune, emailing system and 'send to mcafee'. Yes I got a response and it said they couldnt conclude anything.
You should have replied to that email disputing the finding.
Did that and didnt got an answer
Send a new email in that case with the header "False" minus the "" followed by their reference number.
Nah thank you, already did that and aint going to that all over again, I bought this program to have fewer concerns, but so far it just gave me work. Im just going to wait for the trusted list realease, hope it wont take long.
We Moderators having been begging them to reintroduce it for at least 7 years now. They may be listening at last as I heard some favourable rumours regarding next year's version, although I can't promise it of course.
It should be in beta in a couple of month's time hopefully.
Corporate Headquarters
2821 Mission College Blvd.
Santa Clara, CA 95054 USA
Consumer Support | Enterprise Support | McAfee.com


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|Gold member
Does it matter what version of iWay is installed on remote servers? We will be upgrading our environment to version 7.6.2. Our person responsible for upgrading the DB2 remote server was told that 7.6.2 is not supported on our DB2 server, but 7.6.0 is listed as supported on the matrix. Will it cause any problems to have the main WF server on iWay 7.6.2 and the DB2 server on iWay 7.6.0?This message has been edited. Last edited by: Suzanne,
|Platinum Member
What do you mean by DB2 remote server, do you mean the DB2 adapter?.
WF7.1.4 Prod/Test, MRE, self serve, DM
|Gold member
Our environment is configured so that iWay runs on the main Web Focus server and the DB2 server, we create a remote server on our WF server that points to the DB2 adapter. (We create the DB2 MFDs on the DB2 instance and then create the MFD on the WF server by pointing to the MFDs on the DB2 server.)
|Platinum Member
So you are using a hub sub architecture. My experience with these is that they work most of the time even though Information Builders does not recommend it. We have a 7.1.4 WebFOCUS server talking to 7.1.1 server with out any issues.
WF7.1.4 Prod/Test, MRE, self serve, DM
I know I am chiming in a bit late on this but my experience has been that as long as the major release (ie. 7.6) is the same, you should be ok. I know IBI does always recommend that they be the same however.
WF Reporting Server 71x, 76x
We're in the process of upgrading from 7.6.1 to 7.6.2 and have experienced issues when running a flow developed on 762 on a 761 server. 762 adds alot more to synonyms and dataflows. 761 developed process also gave us some issue because they did not have the require setting in the fex. not an easy one but we've paid the price for assuming the change will be seemless.
|Platinum Member
I think you should double check what is supported (I can not member when we dropped a dbms version in an SP). If you start at the URL http://techsupport.informationbuilders.com/tech/iway/iway_cri_cfg_toc.html and navigate down to 760 and 762 you will see nothing dropped on the windows platform.
In general, when it comes to upgrading, upgrade the most back-end component first. Some times the front end tools require new facilities in the back-end (expansions to the SYSCOLUMN table for example). Most of our testing is done on level versions. But we do test new major versions (76x) with the latest prior client (71x) a few times a year to make sure nothing snuck in that would disable a tool.
Brian Suter
VP WebFOCUS Product Development
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California is rich in its diversity with bustling streets and an abundance of people. The state of California is known for its restaurants, city life, and beaches. Oceanside is a popular town in Southern California. It is well-known for having exquisite beaches and a wide array of restaurants. People are itching to get a taste of city life in Oceanside. Residential properties in Oceanside feature a full view of the beach and its busy streets, making them ideal for families and beach-goers. However, the price of housing in the town is quite expensive. This is why mortgage companies in the area are numerous. Residents tend to rent their houses., but if you are dreaming of owning residential property in Oceanside, consult with a professional mortgage broker near Oceanside.
Now, there are a lot of ways that a professional mortgage broker can help. They can give professional advice on which options to consider. However, some mortgage companies may give you bad advice and lead you to unsuitable options. This can cause you to lose money. Here are some tips on which you may consider before partnering with a mortgage broker.
The mortgage broker must know what he/she is talking about. This will ensure the integrity of the process. Misleading information may cause you to lose money.
They must have options and consider the best solutions for you from adjustable- to fixed-rate home loans to refinancing. Considering all options may benefit you in the long run.
You must see to it that the mortgage broker you partner with has connections. Connecting yourself with borrowers and lenders may give you a better loan option.
The mortgage broker must have knowledge and expertise in the business. The mortgage broker must see to it that their reputation as a broker lives up to its title.
As a future homeowner in Oceanside, there are a lot of factors that you must consider when you are going to partner up with a mortgage broker. Integrity, reliability, options, and interest rates are just some factors that may lead you to a better investment. If you are one of those people who is dreaming of residential property in Oceanside, Michael "Mike" Severino can help you.
Mike Severino is the founder and president of Severino Financial. Severino Financial is a mortgage company that is located in Vista, California, that offers a host of options for loans. We also offer competitive rates, making it affordable for borrowers. Here are some reasons you might consider us.
1. Reliable Assistance
Assisting you is our top priority. We consider all the options available to you. This will ensure the best and most suitable option for your situation. We also establish connections with different borrowers and lenders making the process fast.
2. Quality Assurance
Our office adheres to high standards. Our clients are assured that our process is fast and efficient. Applying for a home loan takes a lot of time and funds. We assure you that your investments are in order.
3. Guaranteed Assessment
We assess your situation and provide you options that are reliable and affordable. This guarantees you results that are both satisfactory and hassle-free. We help you by analyzing your situation and connecting you with reliable and trusted lenders for solutions.
4. Experienced Professional
With 20 years of accumulated knowledge as a mortgage broker, Michael Severino guarantees that you will receive quality service. Michael Severino will always lend a hand assisting you, providing information, and assuring you that you get the best deal.
We understand that you are looking for the most valuable, affordable, and appropriate mortgage for your situation. The experts at Severino Financial are more than willing to help you get the best mortgage deal. If you're looking for a reliable mortgage broker, you may call us at (760) 598-9366 or visit our office located at 2605 Campanero Ave., Vista, California.


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Because "I Am" as "I 'm Not " , also entered in our expressions fashion to express what we feel when we feel , what concerns us but what displeases us. "I am" and "I 'm Not " have become part of our language to freely express our opinions, our favorites , our solidarity and our beliefs for some. In summary this App is the new symbol of freedom of expression
Download fast application " I Am - I 'm Not " and instantly share your mood, feeling, opinion etc of the moment.
Long live freedom of expression


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I spent the weekend making some terrariums for christmas gifts. Terrariums are great for offices or any small space that needs a little green beauty added to it and they are very very easy to maintain. I made a few extra in case any of you are interested in having one of your own. Email me and I'll send better pictures and pricing. Local purchases only.
Tags: fancy seeing you here, fancyseeingyouhere, sarah hanks, Sarah Hanks Ltd., terrariums, terrariums for sale


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Overview: Financial Transaction Systems @ Stanford
On this page:
- Key Functions of the Oracle Financials System
- Modules Used Campus-wide
- Modules Used Only by Central Office Administration
- Staying Informed
Key Functions of the Oracle Financials System
Oracle Financials is the core of Stanford's Accounting System, allowing the University to conduct and manage its financial affairs. Key functions of the Oracle Financials system include:
- Processing transactions (reimbursements / expense reports, purchases, payments, billings, receipts and journals);
- Receiving activity from the PeopleSoft system related to student accounts and payroll, recording it in the proper accounts, and sending accounting information back to the PeopleSoft and Time and Leave systems;
- Receiving accounting entries from other "feeder" systems (Development, Inter-department billings, etc.);
- Applying "burdens" (indirect costs, fringe benefits);
- Creating and posting allocations such as endowment payout;
- Tracking activity in funds, allowing for reporting to sponsors and donors, and departmental management of funds and expenditures.
Modules Used Campus-wide
Several hundred to several thousand school and department users across campus use one or more modules of the Oracle Financials System. Included in the table below are links to:
- Module overviews outlining roles and responsibilities, how to get access, types of transactions, available training, and more.
- Index pages where you will find How To instructions, job aids, etc., pertaining to the use of each module.
|Module Name
|Module Purpose & Description
|Index Pages with Related Quick Steps & Job Aids
|Commitment Management System (CMS)
A custom designed module used by school and department staff to forecast and manage available funds by tracking actual expenditures, accounting for anticipated expenditures and running "what if" scenarios.
See Commitment Management System (CMS) web site.
|Funds Management
|Expense Requests
Allows processing and approval of expense reports / reimbursements, advances, Travel Card charges, non-PO payments and petty cash replenishments
• Buying and Paying
|Gift Transmittals
A custom designed module used by school / department staff and the Office of Development to enter, approve and track gifts made to the University
A custom designed module that allows authorized users to initiate modifications to Expense or Revenue Control information documented in the 9/1 University Consolidated Budget.
See Overview: About the iBudgets Module of Oracle Financials.
A custom designed module that allows users to initiate transfers among PTAs (Project, Task, Awards) or across Expenditure Types or Object Codes.
|Internet Procurement (including SmartMart)
Allows campus users to initiate purchases of goods and services on behalf of their departments and business units.
See About the Internet Procurement Module of Oracle Financials.
|Buying and Paying
|Labor Distribution
Allows authorized users to distribute and adjust payroll earning allocations, which are paid by the PeopleSoft Payroll system, to the appropriate accounts (PTAs).
See Overview: About the Labor Distribution Module of Oracle Financials.
A custom designed module used to verify, edit, search, approve, or withdraw charges made against Stanford Purchasing Cards (PCards).
|PTA Maintenance
A custom designed module used to update PTA attributes for existing non-sponsored and miscellaneous accounts receivable accounts (PTAs)
|PTA Manager
A custom designed module used to request, approve and track non-sponsored and miscellaneous accounts receivable accounts (PTAs)
Provides authorized users a means of tracking specific moveable assets and the ability to report on the status of these assets.
For more information, see Property Training, Property Management Office.
|Tidemark Budgeting System
|Used to plan, prepare, review, and submit business unit annual budgets
|About Tidemark Budgeting
Modules Used Only by Central Office Administration
Certain modules of Oracle Financials are used only by Central Office Administration staff within Financial Management Services (i.e., Controller's Office, Procurement, Treasurer's Office, Global Business Services, and Financial Management Consulting & Support), Research Administration (i.e., Office of Sponsored Research and Research Financial Compliance & Services), the Property Management Office, and Land Buildings & Real Estate. These core modules include:
- Accounts Payable
- Accounts Receivable
- Capital Accounting and Debt Management System (CADMS)
- Cash Management
- Enterprise Asset Management
- eBusiness Tax
- eSourcing
- Fixed Assets
- General Ledger
- Grants Accounting
- Purchasing
Staying Informed
To receive email communications regarding changes and updates to Oracle Financials modules and Oracle Financials Reporting, subscribe to any of the email lists below.
|Module Name
|Click a link below to subscribe
|Commitment Management System (CMS)
|Note: The CMS user mailing list includes all staff with access to CMS.
|Internet Procurement
|Expense Requests
• Subscribe to reimburse-users@lists
|Labor Distribution
• Subscribe to LD-adjusters@lists
|Oracle Business Intelligence (OBI)
|Note: The OBI user mailing list includes all staff with access to OBI Financial Reporting tools
For the latest information about Sunflower, visit the Property Management Office web site.
Check Archived Bulletins to view communications organized by financial activity and date.


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Our Impact is Widening Overseas
We started HOBKNOBS with a vision to serve maximum possible people. Through our journey, we excelled in our products, relished appreciation and enjoyed passion. Our customers have always given us a lot of love and admiration. With our passion, vision, knowledge and skills, we have taken our products to the global market of hardware fittings. We have capitalised our business in exports to fulfil the requirements of clients around the globe. We have done business with the reputed brands and well-known stores in Australia, Canada, Middle East, the U. S. A. and many other Western European countries. They have become our consistent retailers, and regular investors in our products. We export not just in bulk packaging but in ready-to-retail packaging as well. We have tie-ups with various dealers and wholesalers around the globe.


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FORT ST. JOHN, B.C. – The fight against illicit drugs in B.C. is getting harder by the day.
Illicit drug deaths in B.C. have hit a staggering high most notably in the month of November according to new statistics released today by the B.C. Coroners Service.
128 people died from use of illicit drugs during November, an average of more than 4 a day. The previous high for a single month was 82 in January, 2016.
Community Interviews with Moose FM
In northern B.C., numbers continue to rise. There has been 38 deaths so far in 2016 compared to 25 last year.
Also alarming is that when it comes to Illicit Drug Overdose Death Rates by Health Services Delivery Area per 100,000 from 2007 to 2016, northeast B.C. has the 3rd highest rate per capita. The northeast currently sits at 23.3 while the Thompson Cariboo sits at 23.7 and Vancouver has the highest with 26.5.
From January to the end of November 2016, there has been 755 apparent illicit drug overdose deaths. This is an increase of 70.4% over the number of deaths occurring the same time period last year.
Fentanyl continues to be present in a high percentage of deaths. From January 1 through October 31, 2016, fentanyl was detected in 374 cases, about 60% of all illicit drug deaths. That is almost triple the number of fentanyl-detected deaths for the same period last year.
The B.C. Coroners Service during a conference today stressed the importance of those who are using drugs to be somewhere where medical attention can be administered immediately.
"With the number of deaths remaining so high, the BC Coroners Service continues to stress the importance of harm-reduction measures that need to be followed by anyone using any illicit drugs or accompanying anyone who is using. These include never using alone, having naloxone and medical help readily available when using, using an overdose-prevention site or supervised-consumption site wherever possible, and knowing the signs of an overdose and calling 911 immediately."


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There's only one way to escape the city.
Life in New Liberty is tough, but it's the only life Marjoram has ever known. At fourteen, she lives with her parents and wears a breathing mask to survive the polluted city streets on the way to school. Crucible Station offers a better life to any citizen smart enough to receive an invitation, but Marj won't leave her family. When she is thrown into a detestable government home because her family can't feed her, Crucible Station is the only way out–if she is clever enough to pass The Trials.
From the Author:
Whether Crucible Station is selected or not, all who nominate will get a free copy of the eBook. Instructions will be in the author's thank you letter.
About the Author:
Julianne Q Johnson writes in a little Indiana house when her two cats, two ferrets, and one fiancé will let her. Her favorite stories to write are modern-day, realistic worlds with a supernatural or paranormal element. She enjoys taking otherwise ordinary characters and giving them a paranormal gift or putting them in fantastical situations to see how they will handle it. Julianne loves cheap coffee and expensive chocolate. Find out more at JulianneQJohnson.com
Categories: Science Fiction / Fantasy
Tags: author julianne q. johnson, crucible station, crucible station book, crucible station kindle scout, crucible station scout, julianne johnson, julianne johnson scout, julianne q johnson crucible station, julianne q. johnson, julianne q. johnson scout


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Fail-Safe System
Signaling System
Reliable fail-safe is pursued from both the hardware and software side.
When developing railway signaling systems that ensure safe traffic, Toshiba enhanced fail-safe design reliability, and at the same time used general purpose information technology with network technology. We offer an electronic interlocking unit and an electronic railway crossing controller that have achieved high performance, high reliability, high functionality and a compactness and reduced weight, as well as the advanced WireLess Train Control system WELTRAC™.
Electronic Interlocking Unit
General purpose processors are used in the logic unit, and through task synchronizing triple system parallel processing and majority logic control, safety and reliability are improved. Also, when altering or updating interlocking logics, both the new and old logics can be installed in the system, enabling them to be switched over quickly and testing is also easy.
Electronic Railway Crossing Controller
Light weight and compact design can be done with Toshiba's proprietary circuit technology. Dual system comparison verification method is used, so safety is improved as well as preventing malfunctions due to wind or unstable relays.
This is a CBTC (Communication Based Train Control system) which complies with IEEE1474.1, and communication between ground and a train is carried out through a high-speed and large-capacity general purpose wireless system.
Benefits & Value
Electronic Interlocking Unit
- Many maintenance support functions such as live program switching, CPU switching without stopping control and in-operation module exchange.
- PU and various modules can be replaced without powering-off the unit.
Electronic Railway Crossing Controller
- Compact and light weight design. External size is 280(W)x180(H)x220(D)mm, and mass is approximately 7 kg.
- Standardization that supports single track and double track typical combinations.
- The user can adjust an appropriate alarm time according to the site conditions.
- The system controls intervals between trains without relying on fixed-blocks.


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2019 Piedmont Regional Beef Conference
Find this article at:
The date for the 7th Annual Piedmont Regional Beef Conference has been announced. The conference will be held on Thursday, March 7, 2019, at the North Carolina Cooperative Extension, Guilford County Center in Greensboro, NC.
This year's conference will feature cattle handling expert, Dr. Ron Gill from Texas A&M AgriLife Extension and Dr. Deidre Harmon from NC State Extension. Participants will have the opportunity to learn cattle handling and stockmanship skills as well as fescue pasture establishment and renovation techniques through classroom and demonstration sessions. There will also be a trade show featuring 20+ industry partners that offer feed, equipment, seed, fertilizer, cattle services, animal health, and more.
You will not want to miss this event!!
Early registration is $15 or registration at the door is $20.
Register online or through the mail by February 25.
For More information, contact Lauren Langley (firstname.lastname@example.com), 336-570-6740.


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MATRIX VISION becomes part of the Balluff Group
MATRIX VISION GmbH is now part of the sensor and automation specialist Balluff (head office in Neuhausen auf den Fildern, Germany.
Neuhausen a.d.F. – 14 September 2017 – After several years of development partnership, MATRIX VISION GmbH of Oppenweiler near Backnang is becoming part of the sensor and automation specialist Balluff. The shareholders of MATRIX VISION and the Balluff management signed the relevant agreements in August. MATRIX VISION, with its 100 employees, will still operate independently and with an unchanged brand in the field of image processing. The managing directors Uwe Furtner and Erhard Meier will continue to manage the company at its site in Oppenweiler. Balluff now holds 75 percent of the shares in the company, with the remaining 25 percent being retained by the previous shareholders. They all agree: "Machine Vision will grow in importance in the future against the backdrop of Industry 4.0. As part of the Balluff Group, we will be able to service this growing market better, both nationally and internationally. The resulting long-term opportunities for our company are enormous, giving our workforce a clear perspective for the future."
Extending the portfolio in the machine vision area
"In the future, our product portfolio will complement the range of the Balluff Group in a field in which Balluff has played a relatively small role till now: Machine Vision," explains Uwe Furtner, Technical Director of MATRIX VISION. The company, which was founded in 1986, specializes in development and sales of frame grabbers, standard industrial cameras, intelligent cameras, video sensors, embedded systems and software in the field of industrial image processing. "For four years, we have been closely cooperating with MATRIX VISION in a development partnership," adds Balluff Managing Director Florian Hermle. "One of our two camera-based product lines is from MATRIX VISION. So we already know that we are well-suited to one another and our product lines are optimally matched." Furthermore, MATRIX VISION also attaches great importance to the subject of software. As Hermle explains: "MATRIX VISION lets us extend both our product portfolio in the area of camera-based sensor systems and our software development capacities.
Furtner agrees: "Balluff and we are an excellent match. We already know each other very well from several years of collaboration in the development partnership. Our product ranges do not overlap, but rather complement each other perfectly." Furtner also identifies great potential for MATRIX VISION within Balluff Group in terms of sales and services for customers. "Many of our customers have production sites worldwide and expect local support from their suppliers. At some point MATRIX VISION would have run into difficulties on that score, simply due to our size, but with the Balluff sales and service network, we now have access to a whole new range of possibilities."
Link: http://www.balluff.com/en/de/news/press-news/balluff-family-is-growing/
About Balluff
Founded in 1921 in Neuhausen auf den Fildern, Balluff and its 3,550 employees enjoy an international reputation for innovation, quality and cross-sector experience in industrial automation. As a leading sensor and automation specialist, the family-owned company in its 4th generation offers a comprehensive portfolio of innovative sensor, identification and network technologies and software for integrated system solutions. In 2016 Balluff Group reported revenues of around EUR 378 million. In addition to its headquarters in Neuhausen, Balluff has sales, production and development sites around the globe, as well as 37 wholly owned subsidiaries worldwide. This guarantees customers swift worldwide product availability and a high standard of local support and service.
About MATRIX VISION
Founded in 1986 in Oppenweiler near Backnang, MATRIX VISION is one of the major providers of image processing components in the German-speaking market. As a pioneer in the field of vision, the company offers a wide range of frame grabbers, industrial cameras, intelligent cameras, video sensors, embedded systems and software in the industrial image processing sector. MATRIX VISION also develops customized solutions for special requirements ranging from individual components to fully rounded functional units. MATRIX VISION employs 100 people and generated revenues of EUR 15 million in 2016.


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By Woodrow Bellamy III | March 9, 2017 Send Feedback | @WBellamyIIIAC
Gogo test aircraft. Photo: Gogo.
Gogo's latest in-flight connectivity (IFC) technology is ready to enter service within the business aviation market, as the FAA has issued Supplemental Type Certification (STC) for its Gogo Biz 4G system. The 4G technology was designed as an upgrade for Gogo's existing IFC technology featured on in-service business jets, leveraging the U.S.-based Gogo network of 250 air to ground (ATG) towers and fiber backhaul.
According to Gogo, the 4G technology is capable of enabling streaming video and audio in a business jet cabin, with multiple users connected to the router. Inside one line replaceable unit (LRU), Gogo packed a dual-band 802.11ac Wi-Fi service, advanced router, cabin management system (SMS) integration capability and its Gogo Vision in-flight entertainment (IFE) server.
The 4G box is also compatible with the Inmarsat Classic and SwiftBroadband, Iridium, and Jet ConneX networks.
Additionally, Gogo says operators that equip their aircraft with its 4G Biz system will be eligible for an upgrade path to the next generation ATG network when it reaches completion in 2018.