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Author(s): Putnam, Michael C.J.
ISBN 10: 0691125376. See all ISBN's
Publisher: Princeton University Press
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The lives of Catullus and Horace overlap by a dozen years in the first century BC. Yet, though they are the undisputed masters of the lyric voice in Roman poetry, Horace directly mentions his great predecessor, Catullus, only once, and this reference has often been taken as mocking. In fact, Horace's allusion, far from disparaging Catullus, pays him a discreet compliment by suggesting the challenge that his accomplishment presented to his successors, including Horace himself. In Poetic Interplay, the first book-length study of Catullus's influence on Horace, Michael Putnam shows that the earlier poet was probably the single most important source of inspiration for Horace's Odes, the later author's magnum opus.
Except in some half-dozen poems, Catullus is not, technically, writing lyric because his favored meters do not fall into that category. Nonetheless, however disparate their preferred genres and their stylistic usage, Horace found in the poetry of Catullus, whatever its mode of presentation, a constant stimulus for his imagination. And, despite the differences between the two poets, Putnam's close readings reveal that many of Horace's poems echo Catullus verbally, thematically, or both. By illustrating how Horace often found his own voice even as he acknowledged Catullus's genius, Putnam guides us to a deeper appreciation of the earlier poet as well.
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ISBN 10: 0691125376, ISBN 13: 9780691125374, eISBN 10: 1400827426, eISBN 13: 9781400827428
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About Schools in Pymatuning North, PA
Contact a Coldwell Banker® real estate agent to learn more about the schools and school districts in Pymatuning North, PA.FIND AN AGENT
Pymatuning North, Pennsylvania is located in Crawford County. Nearby cities and towns include Linesville, Pymatuning Central, Andover, Harmonsburg, Conneautville, Conneaut Lakeshore, Hartstown, Conneaut Lake, Springboro, Pymatuning South, Adamsville, Jamestown, Atlantic, Geneva, Fredericksburg, Kerrtown, Kinsman Center, Albion, Jefferson, Meadville, Saegertown, Cranesville, Greenville, Roaming Shores, Conneaut, Venango, Rock Creek, Sheakleyville, North Kingsville, Woodcock, Edgewood, Platea, Austinburg, Blooming Valley, Orwell, Reynolds Heights, Orangeville, Ashtabula, Edinboro, Cambridge Springs, Cochranton, and Girard. Pymatuning North is a rural community with a population of 306. The median household income is $39,791. In Pymatuning North, 54% of residents are married, and families with children reside in 23% of the households. Half the population of Pymatuning North commutes 23 minutes or less to work, with 41% of residents holding white collar jobs and 59% residents holding blue collar jobs. The median age of homes in Pymatuning North, PA is 32 years, with 42% of those homes owned, 8% rented, and 50% not occupied.
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text:
- I Don't Like Kids. There. I Said It.
- Where Do Old Broads Hang Out? (An Oldie but Goodie)
- I'm Single and Happy...Why Does That Make Them So Mad?
- What's Wrong With Separate Bedrooms?
- Why I'm Done With Online Dating!
- How Can I Be Happily Single When I Hate Being Single?
- The Extraordinary Rise and Surprising Appeal of Living Alone
The Spinsterlicious Life
Below is a guest post from a very loyal reader of The Spinsterlicious Life. I think Dee is amazing: she's mart, funny, and adventurous. She's also tired of people who try to make her feel bad about being single. I'm happy to run her guest post:
Not long before the holidays last fall I went to get a manicure. Salon manicures are not something I usually do, but someone had given me a gift card for my birthday, so I made an appointment.
At first I sat in the chair across from my nail tech in awkward silence. Just when I thought she wasn't going to try and make conversation with me, she started asking me questions — first about my curly hair and then about my life.
Are you married?
(I knew where this was headed.)
How old are you?
That's when her eyebrows raised with concern. She didn't look up at me, but her expression said it all.
"Maybe you're too picky," she said shaking her head.
I just brushed it off and hoped for a subject change and that she'd get the second coat of my red polish on quickly. Some birthday present that turned out to be!
I read somewhere recently that people can't make you feel bad about something you don't already feel bad about. So if someone calls you crazy, it means nothing unless you buy into it yourself. Put another way, no one can make you feel bad without your permission. I was giving this stranger (and society at large) permission to make me feel bad. I felt bad because (and I hate to admit it) I have bought into the belief that it's shameful or embarrassing not to be partnered up. I don't so much care about the kid thing, but the relationship part can trigger single shame in me big time.
Single women are constantly bombarded with messages about finding/keeping a man. On TV sitcoms, romantic comedies, magazine and online articles, online dating commercials, family, friends, coworkers. It can be extremely hard to tune all of that chatter out and actually stop and listen to your own voice, your own inner wisdom that knows what's best for YOU. The radio static can get even fuzzier when close friends are fretting over their own single/childless state. They are fretting and anxious, so it can make you anxious and before you know it, you're stressed and life is passing you by.
I've decided I'm done. I'm done stressing. I'm done being anxious. I'm done waiting. I'm done fretting. I'm done analyzing and trying to figure it out. Most importantly, I'm done caring what society/family/friends/the media has to say about what I "should" do with my life. The more I thought about the woman in the nail salon, the angrier I became. Why was I letting complete strangers make me feel bad about my life?
I realized as I thought about it (and got angrier about it) that if I completely turn off the outside world, I really don't care if I get married and "settle down." It's just not a top priority in my life. When I stopped and asked myself if I actually want a relationship or if I just want to ease the social pressure, my truth is that more than anything I want to silence the social pressure, judgment and shame. I even reflected on whether I want kids (even though I've always been pretty sure that I don't) just to see how it feels in my body to sit with my decision – without the commentary playing in the background. And, nope, still don't want kids.
I've always known my own truth. I can just get sidetracked and thrown off of my Spinsterlicious game when the barrage of messages becomes too loud. When that happens I get in a panic. (Side note: I even briefly looked into adopting a child when a friend of mine was agonizing over her biological clock. That idea lasted five minutes.) I'm working on staying centered in my truth and not getting knocked down so easily. My aha moment came when I realized that I have the power to stop internalizing the messages.
So here I am ready to continue living my Spinsterlicious life. Only now I'm done with the anxiety. Because I know the anxiety is not about my own true desires, it's about peer pressure and a human need to fit in and be "normal."
Here's my plan: Go "deep single" (love that phrase) and live my life on my terms endeavoring to fully savor my freedom. Seek out new adventures. Continue to travel. Continue to decorate my apartment and make it my sanctuary. Continue to enjoy my work, hobbies and friends. If I meet a great guy along the way, great. But I'm no longer holding out hope that some guy needs to be "the one" so that I can quiet the anxiety. That's way too much pressure. I'll just nix the anxiety now, so that if he doesn't show up, I can live in peace.
I've been living a Spinsterlicious life all along. I just needed to get back in touch with me and remind myself that I got it going on!
How about you? Do you ever get sidetracked and question your own truth? What are your strategies for regaining your footing and getting centered?
"What made me think I wanted to climb a mountain?", I said to myself, out loud, in hour 3, Day 1 of the slow, extremely strenuous, even boring trudge toward the summit of Mount Toubkal in the High Atlas Mountain range of Morocco.
This was way more than I had signed up for. I was expecting a leisurely hike up a sloping incline with beautiful vistas every step of the way. What I got was the toughest physical challenge that was more than I could have imagined and certainly amazing views of a world I'd never seen but the scenery wasn't changing fast enough for me…every few hours, and I wanted more. It was mostly rocks.
And then it started to pour rain, a steady non-stop deluge for 6 hours. And no matter how much waterproofing you've done, in that kind of water, you're going to get wet. Very wet. Soaked through every layer of clothing, down to the skin. And even though you're drenched through and through, you have to keep going because there's nowhere else to go. You're on a mountain. So, all 31 of us kept trudging along step-by-step, though one woman had an even tougher time and had to finish this leg of the trek on a mule that was sent to "rescue" her. We were heading to the Neltner Refuge where we planned to spend the night. It was hours away.
Here's how I got myself into this situation. The TNS Global (where I work) parent company, Kantar, has a relationship with UNICEF. This trip was a fundraiser for UNICEF's Brighter Futures, with the money slated to help children in Malawi, Bangladesh, and Bolivia. It sounded amazing and I signed up immediately. I'm happy to say that our team raised about $110,000, exceeding our goal by approximately 30%.
So, back to the mountain climbing part. We finally made it to the refuge after about 8 hours. The place was full. 31 of us, plus probably another 30 climbers who were also crashing there. We slept 26 to a room. There were four showers. Two toilets that sometimes flushed and two of those hole-in-the-floor thingys they called toilets. Bring your own toilet paper.
We were greeted by a staff of several "locals" who came bearing gifts of mint tea and cookies, before they served us a hot dinner while we sat around the fire trying to kill the bone-deep chill we had from being soaked for so long. Our grumblings eased and we actually started to have fun, getting to know each other, playing games, and comparing our day's miseries.
Day two started at 6:30a. We were to complete the trek to Mount Toubkal's summit in 9 hours, 4-5 up and then the return. One guy in our group refused to go. He waited for us at the refuge. I didn't blame him. I (and a few others) didn't really want to go, either, but we
allowed ourselves to be cajoled into it. Plus, I didn't really come all that way to not try. I wasn't happy, though. The weather got worse, not better. After a few hours, we were turned back by the snow, ice, and wind. Many were disappointed. I wasn't. I was ready to turn around. In fact, a few of us turned back about an hour before the die-hards did. It was dangerous. My goals had been met: raised money, climbed a mountain, and had a new adventure. Reaching the top or not didn't matter to me.
So we spent another night in the refuge and then headed back down the mountain, which wasn't exactly a piece of cake either because it was slippery and muddy and as we got further down, we had to figure out how to cross the rivers that appeared out of nowhere from the melting ice and snow. But we stopped for a picnic lunch about 3/4 of the way down , which was pretty cool.
Our guides –Raheem, Omar, and Khalid– were beyond amazing. They safely –and with good humor– led 31 first-timers up and down a mountain in some of the harshest conditions the area has had in years. I love those guys.
Can something be awesome and horrible at the same time? Apparently… because that's what this was for me. Not 50:50,though. I really hated the climbing part; it was much, much tougher than many of us imagined it would be. But it was more awesome than it was horrible.
And the rest of the trip was a blast. Three days taking in the culture of Marrakesh, an overnight in Madrid, then back to home-sweet-home. What a magnificent experience! My Spinsterlicious Life.
A couple of years ago, right around the time the latest U.S. Census (and other studies) demonstrated the rise in single people, there began a similar growth in articles and blog posts arguing both sides of the issue: why being single is the new normal (and is ok) AND why being single is still a pathetic place to be.
If you Google 'why I'm not married' and/or 'why I'm still single', well over 1 million results will pop up. Clearly, this is a hot topic.
I started my blog –The Spinsterlicious Life— and wrote my book to address the former. I don't think that marriage is for everybody and I know that being single can be a darn good life. When I come across the many articles that take the opposite stance, I usually don't care. To each his (her) own, really.
But today, I came across a really stupid article called Girls, 5 Reasons Why You're Single and I thought "enough with this nonsense". The writer of this article does not know why I'm single. Part of me thinks this article might be joke because it is so asinine (and not particularly well-written).
According to this article, two of the reasons I and millions of other women are still single are:
"You don't take care of yourself.
Don't expect someone to take care of you when you don't even know how to look after your own self. There are certain physical standards that the (judgmental) society built. Like, you being sexy means you shouldn't be fat, or being pretty is having a fair complexion. You can be sweet, smart, cool and funny little cupcake but quality men will not give a second glance at you if you don't look alright. It's not saying that you have to live up to other people's expectations, but please love yourself first, before other people appreciate you.
You flirt too much
Whether you like it or not, the right kind of flirting is an essential skill but if you flirt like crazy, you may send the wrong signal and make you seem not-picky, which loses flirting's efficiency. It also projects that you do the same thing with other men, which is of course, an off for everyone."
I realize that by calling out this article, I'm giving the writer more attention. I'm just hoping that some single woman who isn't feeling particularly good about herself today doesn't stumble across this or similar articles and get her feelings hurt.
One of my favorite Maya Angelou quotes is "You alone are enough. You have nothing to prove to anybody." It's not particularly related to this topic…but then again, it is. Because it's always true, no matter your situation.
A few years ago, a nice young man offered me his seat on a crowded New York City subway. In his offer, however, he called me "ma'am." I was confused for a moment. I looked around to make sure he was in fact talking to me. I was surrounded by a bunch of school kids. He was talking to me.
Even though I was over 50, in my opinion I certainly wasn't a ma'am. I was youthful—even fly—in my skinny jeans, T-shirt, and strappy sandals. I thought that maybe he was a foreigner who doesn't quite have the American modes of greeting right. I asked him a question so I could hear him talk, and it was clear from his New York accent that he was right at home.
I was a bit annoyed. Not so annoyed that I refused the seat, but I was troubled that he thought I was of ma'am age. Maybe he was teasing me, so I looked back at him, waiting for a mischievous smile and wink. Nothing. Just a pleasant smile from a young man with good manners.
I didn't want to be addressed as ma'am. Miss would do just fine. Ma'am is my mother. Ma'am is a lady of 50 years or older, which technically I was, but I had a much fresher attitude . . . or so I thought.
When I think about that episode now, I laugh. Since then I have embraced my ma'amhood! Fast forward to a few months ago, when another nice young man called me ma'am and offered to get a flower pot off the shelf at Home Depot and even carry it to the checkout register for me. (And no, he didn't work there. ) I was happy to have him do it, even though the pot wasn't that big and I certainly could have carried it myself.
I have come to understand that there are some perks associated with being a ma'am. Young(er) guys offer to help me with all kinds of stuff that I don't want to do anyway (putting air in my car tires, carrying heavy stuff, hooking up my DVD to my new TV, even though that's not supposed to be part of the cable guy's offering). They're not just doing this because I'm a woman, they're doing it because I'm an older woman—a ma'am. I know because these kinds of offers have begun to increase.
One of my favorite parts of being ma'am, though, is that it allows me to share my opinion in ways that I couldn't before. Everybody's familiar with the stereotype of the older woman who is well-meaning but a bit of a busybody. I'm not a busybody, but I do usually have an opinion. Over the years, I've learned—much to my disappointment—that everyone isn't interested in my opinion. But I think I'm getting a little bit of a pass now, because people are more polite to ma'ams and less likely to take offense.
Giving unsolicited advice can be tricky because it's not always wanted. I was able to tell the guy who was doing some work on my house—whom I don't know that well—that he needed to change the profile picture on his business's website to one that was more professional. He was a little surprised at first, but I noticed later that he did follow my suggestion. There's a young woman I often see in the elevator in the building I work in. She seems bright and eager, but she dresses as if she's going to the club instead of work. I suspected this may be holding her back. So one morning I told her that. Let's just say she didn't seem exactly happy to hear this from me. I didn't care. I felt like the wise older woman helping guide a bright young thing. And I noticed a few weeks later that she had on a blazer over her short dress.
I sometimes use being called ma'am as a ticket to an easier life. Whenever someone calls me ma'am, I assume there's a shift in whatever paradigm ordinarily exists in the situation. The cop who pulled me over for speeding let me go with a warning and "be careful, ma'am" when I told him I was hurrying home because I didn't like being out so late.
In our culture, the young(ish) feel benevolent toward older people, so they often give us a pass in ways they may not for someone young. And I love it.
Except on a date. You can't call me ma'am if we're on a date. I was having dinner with a much too young man who called me ma'am, and it was clear to me at that point that this would be our last encounter.
A version of this post can also be found at Women's Voices for Change.
"You are so smart not to have gotten married." These are the words I heard from a dear long-time married-with-children friend. Her voice was strong and she sounded resolute; her husband had gotten on her very last nerve. She finished her comment with "and if you blog about it, you can use my real name. I don't give a *****!"
Well, I am blogging about it, but will refrain from using her name; no real reason to, and I wouldn't want it to embarrass either of us if in the future we regretted it. Plus, I'm not so sure she really means what she said, though I know she really meant it at that moment.
X and I have been friends for 25 years or so. We have so much in common, though we couldn't be more different on the subject of marriage. Marriage was always important to her, but not me. In fact, sort of like that Princeton Mom, she believes young women should lock down a life-mate sooner rather than later…like in college. I hate that idea.
Over the years, we've shared intimately the ups and downs of the life paths we've chosen: she mostly happily married and me mostly happily not married. Neither of us wants the other's life.
But she's always complained about her life way more than I do. I've never been quite sure how much she means it (or is she just easy to complain), but I do know that her life is waaayy more complicated than mine. She's gotta manage the household and the lives of her husband, two kids, and a dog…and there's a lot that goes with that. I manage me and the dog. Much simpler, and not that much goes wrong.
So, back to her "you're so smart not to have gotten married" comment. I felt for her at that moment but also kinda chuckled to myself. Over the 20+ years she's been married, she's been known to profess strong sentiments, sometimes about the kids but usually about the husband. And I chuckled because, perhaps not surprisingly, there's also the other side where she professes how she can't imagine her life any other way. She's never really used those words but I know it to be true.
Over the years, on a regular basis, I have heard from her:
– "I hate that MF."
– "He's so sweet, guess what he did."
– "I'm so glad he's out. It's so nice when it's just me and the kids."
– "He's one of the good ones."
– "Seriously. I'm leaving. I just can't take him anymore."
– "We had so much fun last night."
– "Seriously. I hate him".
And on and on. I guess she's probably not that different from most married women…although maybe just a little more honest. I think that's what marriage is like, isn't it? It's complicated. I don't like complicated stuff. And I do feel smart for knowing that marriage probably wasn't for me…because it's not for everybody.
Even though I know X is really pissed at him right now, I give her a month or two before she's all googly-eyed about him again. And that's how it should be, if she's going to stay.
(This post is sponsored by Oxytrol and BlogHer)
I love to travel. I want to go everywhere and I pretty much can because, as a single woman with no kids, my time and my money are my own and I get to spend both however I want. So traveling is what I do.
But I'm a low-key kind of traveler. I love to spend my vacations sleeping in until mid-morning, then walking the streets of whatever country I'm in, drinking in a café (or equivalent), enjoying whatever the locals drink, eating what they eat, and shopping for something representative of their culture.
So imagine my surprise when I signed up to join a bunch of coworkers on a trip to Morocco to hike the High Atlas mountains. I've never said to myself "Gee, someday I'd like to be on top of a mountain"…and, yet, in September that's exactly where I'll be!
Nobody who knows me is surprised that I'm going to Morocco. Everybody who knows me is surprised that I'll be trekking through mountains. That typically is not my kind of thing.
And I am super-excited. I don't consciously know what made me jump at this opportunity but I am a big believer that we…well, I…must continue to stretch, grow, try new things, do something that scares me a little bit. And this is it.
I'm in good enough shape to walk around anywhere, but being fit enough to trek for 6-8 hours a day every day for a week at a high altitude is a different kind of thing. So I've started training. I hate training. It's not interesting or fun, but it's what I need to do to make sure I can enjoy this trip in all its glory (and that the rest of the team doesn't vote to leave me on the side of a mountain because I can't keep up).
I never want to be in a rut and I'm always looking for a new way to celebrate my life and challenge myself. This time when I'm eating and drinking among the locals, it'll be in mountain village with the Berbers who live there. And I can't wait!
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One of the easy things about being fresh-and-young is that we don't even know how much we don't know. I –like so many young people—had lots of opinions on all kinds of things that had to be true simply because I thought them. Now that I'm young, primarily only in my head, I've managed to evolve my thinking on a few things. Like:
• Kids. I still don't want to raise one (or even be around one for too long), but I do often admire the mother-daughter dynamic I sometimes see between a grown woman and her grown daughter…assuming the daughter turned out to be my-definition-of-normal…which isn't always the case. But when she does, I think I might have liked that. I wish someone would come up with a way I could have a 24-year-old daughter without me having to do the previous 23 years.
• Husbands. Similar to the kid thing, above, I still haven't figured out a way to want to share my life with the same man in the same house every. single. day. but I often see 60+ year-old couples and like the way the way they take care of each other, communicate without talking, and just hang with each other. Of course, I'm excluding those couples who are miserable but just stayed together because they were too __________ (fill in the blank) to leave. And he would have to have his own place.
• Men Who Live With Their Mothers. That seems like the punchline to a much-cliched joke, doesn't it? It's not. For most of my dating life, I –like many others—made fun of grown men who lived with their mothers and assumed they were Losers, with a capital L. I'm talking really grown men, not guys in their 20s. But recently, I met two pretty great guys, both 60, who live with their mothers. They weren't losers, but a divorce, job loss, and heart attack in a short period of time could do in almost anybody. Life happens. I still don't want to go out with either of them, but I'm now smart enough to realize "there but for the grace of God…"
• Lying Can Be Good. I tend to believe that honesty really is the best policy most of
the time, but honesty can be tricky. Sometimes telling the truth can be hurtful…and it just may not be necessary. Today I ran into a famous actress who is on the other side of 50. The side that Hollywood doesn't really like. As far as I can tell, she hasn't worked in a long time. Partly, I'm sure, because Hollywood doesn't have much use for older actresses and partly (I imagine) because her face is so messed up. She's clearly had too many rounds of cosmetic surgery and her face looks crazy. I couldn't stop staring. In my head, I'm thinking "she looks ridiculous". What I said to her, though, is "You look great." And she beamed as she said "thank you." So part of me is thinking "why did you tell that lie?" But the smile on her face made me know I had done the right thing. I can't begin to imagine what it must be like to wake up every morning and look at that face. Maybe my little white lie made it feel just a bit better for a minute or two.
My book, The Spinsterlicious Life, has 20 Life Lessons I've learned in all my years as a single woman. Maybe it's time to update it with a few more. And what about you? What long-held beliefs have you modulated with the wisdom of time?
One of the things I love about being single is that I have the freedom to take off on an adventure whenever it suits me (after checking in with my bank account, of course). Traveling is one of my passions; I've been many places –22 countries—and there are many more I need to get to.
I'm excited about my next adventure, even though it's a few months away. I'm going with some of my colleagues to trek through the High Atlas Mountains in Morocco, finishing up in the fabulous historic city of Marrakech.
I'm a little surprised at myself because I don't believe I've ever said or even thought "Gee, someday I'd like to be on top of a mountain." But when the opportunity presented itself, I jumped at the offer right away. A few years ago, I took a day trip to Morocco (Tangiers) from Spain and wished I had spent more time there, and now is my chance. I was enticed by the culture, the food, the bright colors everywhere and I knew I wanted to come back for a real visit.
Marrakech sounds amazing, and this trekking thing is an opportunity for me to challenge myself in a way I've never done. It's not exactly mountain climbing, I think it's more like mountain hiking…but I have to train for 3 months, so it ain't no joke. We'll be doing things like hiking through the mountainside villages of Morocco for 6-8 hours/day hauling "stuff" in our backpacks…sometimes sleeping in hotel-like places, and sometimes outside. (Note to self: ask Kim if you can borrow her sleeping bag). I've never done anything like this, but I'm game for a new experience.
We are partnering with UNICEF and raising money for kids in Malawi, Bangladesh, and Bolivia as part of their Brighter Futures venture. Each of us is raising $2500. This is an opportunity for me to do something for others in a pretty big way. This fundraising is also as an opportunity to even the score with all the parents I know from whom I've bought whatever their kids were selling that I didn't want. Girl Scout cookies, bake sale goodies, wrapping paper, chocolate turtles, raffle tickets…the list is sizable.
If you want to contribute, you can do so here–> http://crowdrise.com/EleanoreWells. No amount is too small. If you have any advice for me on hiking, high altitudes, sleeping outdoors, blisters on feet, getting along with people I don't know well when we're tired and cranky, and/or how not to succumb to the native charm and buy too much stuff I don't really need, well, please leave your thoughts in the Comments section.


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I couldn't find it while searching through various threads , so sorry if this has alreayd been posted but I couldn't find it.
I have a few questions about them.
How do I get them ?
What are they ?
What classes can have them ?
In what campaign can I get them ?
well I can't think of any more , I'm sure I had more questions about them , ooh well.
Thanks in advance.


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1223 Mangrove Ave 899-7273 Fax: 899-0863 1141 Forest Ave 345-7155 Fax: 345-7141 2760 Esplanade 566-9605 Fax: 566-9607
Monday-Friday 6:00-5:30 Saturday 6:30-5:00Closed Sunday for loafin'
Great Harvest Bread Co. is a crew of fun-loving bakers, sandwich makers and more importantly "people-people". We love what we do and we enjoy our community. We are your neighborhood bakery where friends and family meet to shop, eat lunch or enjoy a cup of delicious Peet's coffee. We take pride in serving the freshest most flavorful baked goods, bread, sandwiches and coffee you've ever tasted. We're family owned and community focused. Stop by for a free slice today!
Because freshly milled, whole wheat tastes phenomenal, and fresh whole grain bread is life-enhancing. We use premium wheat bought from family owned farms located in the finest wheat growing region in the world -- Montana. And we only use pure and simple ingredients in our products. You won't find additives, preservatives or any ingredients you can't pronounce. Learn more about whole grains!


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Paramus, NJ 07652
Paramus, NJ 07652
March 23, 20124.0
This is such a trendy place in you want to impress a date. The Asian fusion cuisine is innovative and creative. Take some time and enjoy your drinks in the bar/lounge area before getting seated. Just be prepared to wait a little bit if you can during the busy hours. Also the prices are a bit more expensive but you pay for service, food and ambiance. It's a great change of pace if you are looking for something different.
December 26, 20104.0
This place was needed in Bergen County. It is an interesting lounge-type setting but with all seating rather than a dance floor, and an older crowd that you wouldn't generally see in the other club-type places in the area.
Overall, the service was excellent and the food and drinks were quite good indeed. The servers were well trained and could intelligently discuss all the dishes and specialty beverages, and make honest recommendations. The atmosphere was romantic and fun, although the noise level was high.
We were there primarily for drinks (fantastic selection), but we ordered some appetizers to munch on. We split a pumpkin and frisse salad to start, which was quite unique and yummy. We also split two huge sushi rolls: the rainbow roll and the dragon roll. Both were excellent.
One warning: all the ingredients are not listed on the menu. People with allergies should specifically inquire when ordering.
March 1, 20093.0
November 20, 2012
My wife and I love Chakra and the food is always great. Last Saturday, however, we had service problems. We waited so long after our meal that we had to skip desert and head home to relieve the babysitter. Not what we expected after a…
September 9, 2011
went early on Friday night to dinner with my husband. We are normally disappointed with many of the restaurants in Jersey. We started with the grilled octopus on a bed of white bean puree - it was amazing. If you have ever eaten octopus…
These dishes from the Chakra menu are contributed by Menuism users directly, as part of a restaurant review, or as part of an image upload.


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Improving the signal-to-noise ratio
I have you all "beat." Got mine yesterday = $.07!!!
Mr. Money Mustache - Bing Rewards
2.0 TDI "BHW" engine partout - @tdiclub @vwvortex
BMW is getting some sweet upgrades now
Texts Between Men: I make a concerted effort to always taste awful / borderline poisonous
Lunch is on me!!!!
OT - did anyone get an email yesterday on a proposed settlement of a class action suit against Ticketmaster on order processing fee?
Hell yeah. I am going to get mucho cash back from those bastardsDon't expect your credits quite yet, though. Credits won't be issued prior to April 15, 2012, and should come within 30 days after Final Approval of the settlement (currently scheduled for May 29, 2012).
If you can't measure it, you can't understand it; if you can't understand it, you can't control it.
If you can't measure it, you can't understand it; if you can't understand it, you can't control it.
Everyone pool their money into a paypal account. Use money to buy something awful off eBay motors. Make viral meme "I got ripped off by eBay and all I got was 1/1000th of this crappy car.
Shutup and drive yo cah'!
Mine was for $2.16, and my husband's was about $38
$600 and change
Not me personally, work.
1.50 here
i got around $2.60
"you will automatically receive, discount codes (Codes) which can be used for future purchases for U.S. events from Ticketmasters Website. For each transaction that you made during the Class Period, you will receive one code via email for a $1.50 discount, up to a maximum of 17 codes.
@SSLByron, @leftlanenews
Domestic Muscle Owners Not-So-Anonymous
31.49 Hell Yeah!!!
I didn't but my in-laws got $189.XX check. Saw the check with my own eyes past Sunday.
2007 Mazda 3 s Grand Touring
$493 and change, myself, what do I win? Those scammin' bastards sure get a lot of money from me.
I think ours ended up being like $500 or so, at work. Actually had to look into it and see if it was legit.
My presence is a present.
2.77, i feel like a douche even cashing it
Proud owner of a $1.71 check here! Still sitting on my nightstand.. One of these days, I'll make my way to the credit union and deposit it.
$16.47 here. Similar threads on several other boards I go to. Must have cost them millions & millions.
Buddy of mine got a little over $750.
$ 0.98
I can add that to the pennies I've been faithfully putting in a jar for eight years and finally afford that large baja chicken Quiznos sub w/ bacon.
If you have a 5 cylinder, you don't need a stereo.
i got mine on Thursday for a whopping .09
i also got the stink eye from the bank teller when i asked for it back in newly minted copper
my garage: www.defyperception.net


text:
LIMA — For many area veterans, talking about their experiences in combat is often difficult because civilians are unable to relate personally to their experiences.
"There was a time when I came back that I didn't want to talk about it either," said Russell Reynolds, a Vietnam War veteran living southwest of Lima. "When we came back, it was unpopular, and nobody wanted to hear about it."
For Reynolds, who attributes his ability to talk freely about his Vietnam experience to the spiritual healing he has experienced as a Christian, his experience in the military began in much the same way as many others who fought in that conflict: through the draft.
"I can still remember coming home and my mom and dad had a somber look on their faces," he said. "They said, 'You've got some mail on the TV.' I went in and saw the letter."
Reynold's uncle, who had served in the Navy, encouraged him to gain a skill through his military training that could be used in civilian life, so Reynolds took welding training in Maryland after joining the Army.
"The only thing was I had to volunteer to go to Vietnam," he said.
Serving with the 704th Maintenance Battalion, 4th Infantry Division, Reynolds was sent to LZ Oasis, located 23 miles southwest of Pleiku, near the Cambodian border. While Reynolds ended up serving in Vietnam for 18 months, one event early in his deployment is permanently seared in his memory.
"Mother's Day will never be the same for me," he said. "On Mother's Day 1969, I had been in the country a little over 30 days. At about 1 a.m. we started hearing the sounds of rockets and mortars. Those of us who were sleeping had to go out to a bunker. People came around asking if people would take ammo to the bunker line. So my best buddy and I got out, crawled all over people and took ammo to the bunkers all night."
That night saw a battalion of enemy soldiers attack Reynolds' base. By the time the smoke cleared in the morning, the base held, but 11 soldiers were killed, with many others wounded.
"I can still remember lining up at the mess hall and looking over at the helipad where 11 filled body bags were laid out waiting to be picked up by helicopter," Reynolds said. "There were 11 mothers who lost their sons that day."
After serving in Vietnam for 18 months, Reynolds returned to the States, faced with the difficult challenge of readjusting to life outside the military.
"I was very angry," he said. "I used to be more easy and quiet, but after I got back, everything was a fight."
That anger led to alcohol abuse and multiple failed marriages, finally leading to a turning point.
"In 1982, I hit rock bottom," Reynolds said. "That's when I surrendered my life to the Lord, and God started healing a lot of things. Right now at this present time, he's working on purging that stuff out."
Instead of welding chain link fence posts or tank parts, Reynolds used his skills to weld together the cross that stands in a pond at New Hope Christian Church on Baty Road.
"I always hope that when I give my testimony, it can help someone who went through the same things," he said.
In talking about his wartime experiences, Reynolds is not looking to draw attention to himself, but rather to honor those who never made it home.
"They talk about the Greatest Generation, but I believe that in every generation, those who answer the call to service are the greatest of their generation," he said.


text:
--(0.03)-Transaction-related costs (3)
-0.02-0.03Benefit related to insurance recovery (4)
---(0.02)Termination of bridge financing (5)
---0.07Legal settlement (6)
-0.04-0.04Amortization of: Legacy intangible assets (7)
0.010.010.030.03NextRx-related intangible assets (8)
0.04-0.10-EPS from continuing operations, adjusted$ 0.65$ 0.42$ 1.80$ 1.30EPS from discontinued operations, as reported$ (0.01)$
-$ (0.04)$
-Non-recurring items: Impairment and other charges (9)
0.01-0.04-EPS from discontinued operations, adjusted$
-Total EPS, adjusted$ 0.65$ -$ 0.42$ 1.79$ 1.30 The Company is providing diluted earnings per share excluding the impact of non-recurring charges and intangibles amortization in order to compare the underlying financial performance to prior periods. (1)Integration-related costs include those costs directly related to the acquisition of NextRx, primarily comprised of transition services, integration, site closures and severance costs of $15.1 million ($9.5 million net of tax) and $62.7 million ($39.5 million net of tax) included in cost of revenues for the three and nine months ended September 30, 2010, respectively.
Additionally, the Company incurred integration-related costs of $12.3 million ($7.7 million net of tax) and $22.7 million ($14.3 million net of tax) included in selling, general and administrative expense in the three and nine months ended September 30, 2010, respectively. (2) Non-recurring benefit relating to an amendment of a client contract of $30.0 million ($18.9 million net of tax) is included as an increase to revenue for the nine months ended September 30, 2010. This amount was originally accrued in the NextRx opening balance sheet and, in accordance with business combination accounting guidance, the reversal of the accrual was recorded in revenue, since it
|SOURCE Express Scripts, Inc.
Copyright©2010 PR Newswire.
All rights reserved


text:
Latest News
EXCLUSIVE DETAILS: NYC Housewives Host Premiere Parties For New Season...
Reality's leading ladies of New York City are putting on their best party dresses for the season 4 premiere this week!
Ramona Singer and Alex McCord are host… READ MORE
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Fans love Real Housewives of New York for it's non-stop drama and endless catfights, so when news spread Jill Zarin would be a no-show at Ramona Singe… READ MORE
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It looks like Real Housewives of New York will finally stop those nasty rumors that the show's season premiere was pushed back because it was "… READ MORE
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One of New York fashion week's hottest tickets was Real Housewives Of New York star Jill Zarin's launch for her sexy new shapewear line –… READ MORE
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The house husband everyone loves to hate — Simon Van Kempen — will soon have his own reality show to showcase his quirky clothes and eccentric p… READ MORE
PHOTOS: OMG Gaga And Sizzling Sofia In The Best And Worst
Stars bundled up braved the rain and the frigid temperature on Tuesday, scattered around the world dressed in the best and worst duds. RadarOnline.com has … READ MORE
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First it was Carrie Bradshaw, now the Real Housewives of New York are swapping Manhattan for Morocco.
Leaving New York City behind, Kelly Bensimon, Jill Za… READ MORE
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Jennifer Connelly may have A Beautiful Mind, but the brunette bombshell does not have a flawless sense of fashion! The leading lady hit the red carpet of the … READ MORE
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Real Housewife of NYC Alex McCord is revealing the secret to married bliss in a new book she's writing about relationships, she tells RadarOnline.c… READ MORE
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BOISE -- If you are a Jeep owner, listen up.
After some argument, Chrysler has decided to act on the government's request to recall nearly three million SUVs.
On Tuesday, the company announced it would be recalling Jeep Grand Cherokees with model years between 1993 and 2004, as well as Libertys from 2002 to 2007.
This comes after concern that if one of these Jeep models was rear-ended in a crash, a fire could ignite at the fuel tank.
KTVB spoke with Peterson Stampede in Nampa about this, and they say wait for a Chrysler card in the mail to see if you're impacted.
If you've got a vehicle that falls into the criteria, then call the dealer. Call us and find out what we do or don't know. But right now, just today this happened, so we don't have all the details. The biggest thing is to make sure we have the correct information, said Service Director Mert Logue.
So if you've moved since you've registered your car, call a nearby Jeep dealership to update your address.
Join the Conversation
To find out more about Facebook commenting please read the Conversation Guidelines and FAQs
Jeep issues recalls on certain models after fire concern
Jeep issues recalls on certain models after fire concern
BOISE -- If you are a Jeep owner, listen up.


text:
WD WD3000BKFG 300 GB 2.5" Internal Hard Drive - SAS - 10000 rpm - 16 MB Buffer - 40 Pack - WD3000BKFG-40PK
Learn more about WD WD3000BKFG 300 GB 2.5" Internal Hard Drive - SAS - 10000 rpm - 16 MB Buffer - 40 Pack:
Manufacturer: Western Digital
Mfg Part#: WD3000BKFG-40PK
Sku: 222260414
See more in Internal
WD S25 SAS hard drives provide up to 600 GB of high performance storage suitable for both enterprise server and enterprise storage applications. The ultra-fast 6 Gb/s SAS interface enables optimum data reliability, availability, and scalability. In an environment where space and power are limited, the WD S25 drives are an effective solution for high performance computing solutions such as power-constrained data centers, database transaction processing, and rack, tower, and blade servers.


text:
DOBSON — Cooperative Extension Agent Whitney Sprinkle's Summer Explosion program Crafting for a Cause set its sights on living up to its creed by encouraging 4-H members to pledge their "hands to larger service."
The program, held Tuesday at the Cooperative Extension Center, was divided into a morning class for children from age 5 to 8 years and an afternoon session for 4-H members ages 9 to 14 years. Each session lasts two hours. Sprinkle said the morning group would get to make rainbows out of cotton balls and colored paper to take home to give to someone who means a lot to them.
Other items the group made included clothespin and regular wreaths, picture frames which will be given to local nursing homes and aprons tentatively earmarked for The Children's Center of Surry and Yadkin.
"The goal we have in mind is to allow them to express their creativity and get them used to helping others," said Sprinkle. "The rainbows are made using the same rules but they all turn out looking a bit different." She said the concept was greeted with enthusiasm, even by members of the program who are boys.
Sprinkle said the group is also looking forward to The Blind Side slated for August 6 at the Davie County Cooperative Extension Service Center. Activities will include participants learning about braille and some of the technology used by the blind as well as learning etiquette to those who are blind or use a service dog.
"It's important for them to learn about disabilities. Most may have never seen a person who is disabled," Sprinkle said. "We are going to have several trainers of service dogs present to talk about what they do and the children will get to work with some of the Exceptional Program kids to create sunshine smiles for them to take to their first day of school."
Summer Explosion is a youth program with classes designed to be fun and educational with hands-on activities. Classes are typically set up by age range. It is offered through the North Caroline Cooperative Extension Service. Interested persons may get information on joining a 4-H club or the Summer Explosion program by calling 336-401-8025.
David Broyles may be reached at email@example.com or on Twitter@MtAiryNewsDave.


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The completion of the Plasmodium falciparum genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions.
We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST), including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons versus 5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6%) with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions.
Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively spliced transcripts, and that more genes than currently predicted have one or more additional introns. It is therefore necessary to annotate the parasite genome with experimental data, although obtaining complete cDNA sequences from this parasite will be a formidable task due to the high AT nature of the genome. This study provides valuable information for genome annotation that will be critical for functional analyses.
Malaria parasites infect and kill millions of people in the tropics each year [1,2]. Efforts to develop vaccines have so far failed to produce any effective vaccine. Additionally, drug-resistant parasites are spreading quickly, particularly parasites resistant to chloroquine, leading to a recent resurgence of malaria in many developing countries [3,4].
To facilitate our understanding of parasite molecular biology and development of drugs and vaccines, the genome of the malignant human malaria parasite Plasmodium falciparum was sequenced and published in 2002 [5]. The genome sequence provides a basis for various genome-wide approaches such as microarray and proteomic analyses [6-9]. Unfortunately, the majority of the genes in the P. falciparum genome were predicted using computer software, with ~60% of the predicted genes encoding hypothetical proteins [5]. Although software 'trained' with well characterized genes and improved strategies have provided relatively accurate gene prediction [10,11], the accuracy of gene prediction of this organism is unknown. It is therefore necessary to verify the predictions with complementary DNA (cDNA) sequences, particularly for eukaryotic organisms that have introns in their genes. Indeed, full-length cDNA clones from many species from Drosophila to human have been collected and characterized [12-16], providing important information for verification of genes in a genome and for studying gene functions. Recently, when a high-density array was used to survey transcribed exons, up to 30% of the detected transcripts were found to be unannotatd even in the well characterized Drosophila genome [17].
P. falciparum has a unique genome with a very high AT content (~82% of AT) [5] that presents various difficulties for studying gene structure and gene function. The extremely high AT content in non-coding regions (up to 99%) is often an obstacle to obtaining sequences from introns, 5' and 3' untranscribed regions (UTR), and intergene sequences. P. falciparum DNA is often unstable in bacteria, making it almost impossible to obtain full cDNA clones from genes larger than 5 kb for expression or other analyses. Approximately 50% of the genes in the P. falciparum genome were predicted to have introns flanked by the conserved eukaryotic GT-AG intron-exon splice sites [18,19]. The parasite genome also has many large open reading frames (ORF) that likely encode large transcripts; however, introns imbedded in the ORF cannot be ruled out [20]. The elements regulating gene expression such as promoters and polyA recognition sites seen in other eukaryotic cells may not function properly in this parasite due to the high AT content in noncoding regions [21].
Expressed sequence tags (EST) from malaria parasites, particularly P. falciparum, have been obtained previously [19,22-27]. The first survey of P. falciparum EST produced 389 tags from 550 random cDNA clones [22]; and the number of EST was later increased to ~2,500 [23]. More recently, 2490 single random sequences were obtained from a library enriched for full-length cDNA [19], which were updated to 11424 sequences covering 1357 predicted genes [27]. cDNA sequences from the full-length cDNA clones (mostly sequences from 5' UTR) identified new genes and multiple transcript initiation sites in some genes, but it appeared that no efforts were made to obtain complete cDNA sequences from full-length cDNA clones. In this report, we constructed various cDNA libraries from mixed blood stages, including three cDNA libraries with different sized inserts enriched for full-length transcripts and sublibraries that contain smaller clones after digestion of the initial inserts with restriction enzymes. We also used synthetic oligonucleotides to extend sequences deep into coding regions. We obtained a total of 17332 clean EST. Comparison of our EST, the EST in public databases, the predicted coding sequences (CDS), and genomic DNA sequences identified 393 genes that may be incorrectly predicted.
Results and Discussion
cDNA libraries and DNA sequencing
Collection of EST from P. falciparum has been reported previously, and searches of public databases found 21305 P. falciparum EST in PlasmoDB [28,29] and GenBank, contributed by various research groups [19,23,27] (Washington University, unpublished). The majority of EST collected previously were short sequences from single sequencing reads. To obtain longer cDNA sequences, we used two different approaches–primer-walking and construction of sublibraries of restriction enzyme-digested DNA clones–to extend sequence reads into the cloned DNA. Three different libraries, each with three sublibraries of different insert sizes, were constructed using polymerase chain reaction (PCR) products after 11 cycles of amplification (Additional file 1 and 1). The first library contained cDNA clones directly from 5'-enriched cDNA inserts, which were divided into groups of large (> 3 kb), medium (1–3 kb), and small (< 1 kb) insert sizes (Additional file 1). Unfortunately, we were not able to obtain sequences from either 5' or 3' ends of many clones from this library, probably due to polyA or polyT sequences in non-coding regions, suggesting that these clones may contain full coding sequences. We then constructed sublibraries with DNA inserts digested with restriction enzymes BamHI or Sau3A before cloning into the vector (Additional file 1).
Additional file 1. Construction of cDNA libraries. The procedures for cDNA librabry construction and sequencing are summarized in diagram (A). PCR products were separated on 1% agarose gel (B) and DNA fragments >3 kb, 1.5–3 kb, and <1.5 kb were eluted from gel blocks. M, molecular weight marker; lane number 8–18 on the gel were products from PCR amplification from 8 to 18 cycles. Eluted DNA fragments were cloned into trip-1EX2 vector that were transfected into bacteria. For construction of sub-libraries, the DNA were first digested with BamH1 or SAU3A and cloned into the same vector. DNA amplified from the vector was sequenced directly.
Format: PDF Size: 81KB Download file
This file can be viewed with: Adobe Acrobat Reader
Sequence trimming and contig assembly
A total of 28416 sequence runs–including 10656 from 'full-length' libraries, 10368 from BamHI-restricted libraries, 7392 from Sau3A-digested libraries, and 4,800 runs from primer walking–were performed. From the sequence runs, we obtained 17332 EST 100 base pairs (bp) or longer [GenBank EL492722-EL510074] after trimming and vector sequence cleaning (see Methods). Because of difficulty in obtaining sequences from AT-rich sequences in non-coding regions and sequences with polyA tails, most of the sequences were from digested libraries or from the 5' ends of the undigested libraries. The trimmed EST from our libraries were assembled into 2548 contigs and 2671 singletons with an average size of 473.4 bp and an average qual value of 64.7. When our EST were assembled with EST in public databases, we obtained 5220 contigs and 5910 singletons with an average size of 520 bp.
Genome-wide cDNA coverage
To determine patterns of genome-wide gene expression and locations of EST on chromosomes, we assembled our EST and the public EST with 5485 predicted CDS in PlasmoDB (version 5.2) and displayed them on the physical chromosomes (Figure 1). When assembled using CAP3 [30] (21 bp overlap and 85% identity), 3857 CDS were assembled with EST contigs. When the sequences were aligned using Blast and methods described previously [31], 3792 CDS were identified by the same EST with cutoff values of at least 100-bp long and 95% identity. The two methods produced almost identical numbers of hits on predicted CDS. This percentage of genes (~70% of total predicted genes) with EST coverage is a little higher than those detected using a 70mer oligonucleotide array (~60%) [6]. Among those EST matching CDS, approximately 42% (or ~1700 genes) were matched by EST from both our collection and those in public databases.
Figure 1. Diagram of the 14 P. falciparum chromosomes showing positions of potentially expressed genes. Expressed sequence tags (EST) from our libraries or from public databases were assembled against predicted coding sequences in PlasmoDB; genes that matched our EST only (green), EST already in public databases (red), or both (yellow) are displayed according to gene order on the chromosomes. Those in white are CDS that were not covered by any EST. Approximately 70% of the 5485 predicted CDS were matched with one or more EST.
Alignment of cDNA to predicted genes on physical chromosomes allowed us to identify chromosomal regions that are transcriptionally active or silent. Our results show that genes located at telomere or subtelomere regions of many chromosomes (for example, genes at ends of chromosomes 7 and 10) do not have matching cDNA or are largely silent (Figure 1). The chromosome ends of P. falciparum are highly variable, consisting of many multigene families such as rifin, stevor, and var [5]. Although the functions of the proteins encoded by rifin and stevor are still uncertain, the var gene family has been shown to encode variant proteins (PfEMP1) that can mediate parasite adhesion to receptors on host endothelial cells [32-34]. Different observations on the expression of the genes at chromosomal ends have been reported using microarray hybridization, with one reporting silent chromosome ends [6] and another suggesting expression of genes from chromosome ends [7]. Because microarray is based on probe-target hybridization, cross hybridization among probes from members of gene families could produce false-positive signals under some hybridization conditions. Our data are consistent with results showing that RNA transcripts from only a small subset of these genes could be detected in intraerythrocytic stages [6]. Additionally, there are regions in the middle of the chromosomes with genes that do not have cDNA coverage (Figure 1).
Full-length cDNA sequences and discovery of new introns
One of our goals was to collect complete cDNA clones and sequences from the P. falciparum genome. Unfortunately, we encountered difficulties in sequencing highly AT-rich regions, mostly 5' and 3' UTR, and obtained only 199 contigs that cover the entire ORF of 87 predicted genes, with predicted ORF sizes ranging from 126 to 2709 bp (Additional file 2). Among the 87 genes, 21 (~24%) were predicted incorrectly (or mismatched), with 18 genes having 23 additional introns and 3 genes with cDNA sequences running into predicted introns. Of the 23 new introns, 21 were found 5' of the predicted ATG, suggesting either additional exons or introns in the predicted non-coding regions. Assembly of our EST and those in public databases increased the number of genes 'fully' covered by EST to 356, with 85 (~24%) genes having mismatched introns (Table 1; Additional file 2). If we assume an error rate of gene prediction for the whole genome similar to that seen in the 356 fully covered genes, we would expect 1316 genes (24% of 5485 genes) being predicted erroneously. This is quite a large number of predicted genes that may have to be re-annotated, which argues for efforts to experimentally annotate the genome using full-length cDNA sequences.
Additional file 2. Genes with predicted coding regions fully covered by EST and confirmation of predicted introns. Aligned cDNA and predicted CDS sequences can be viewed by double clicking the hyper-linked gene names in the Excel file.
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Table 1. Predicted coding regions that were covered fully by cDNA and their mismatched introns
Approximately half of the P. falciparum genes (53.9%) were predicted to contain introns [5]. Our data suggest that the percentage of genes with introns will be higher than the predicted 54%. Among the 21 genes found to have new introns in cDNA, 10 were predicted to have no introns, and one gene predicted to have only one intron actually had none. This represents a net gain of 9 genes with introns among the 87 genes (or ~10%). Among the 85 genes with mismatched introns from the 356 genes with full coverage of predicted coding sequences, 21 genes gained introns (~5.9%), Based on these data, we can predict that about 60% to 65% of the genes in the P. falciparum will have one or more introns. Of interest, the majority (> 90%) of the new introns were found at 5' and 3' UTR or within 100 bp from a predicted ATG or stop codon, suggesting additional exons or changes of start or stop codons. It is also possible that the proposed genome sequence contains insertion/deletion errors causing apparent frameshift. Automatic prediction algorithms would then have to find an intron/exon border adding one spurious intron.
Alignment of our cDNA contigs with predicted CDS also identified 78 genes, although not fully covered by our cDNA sequences, with 88 introns either missed by computer prediction or predicted incorrectly (Additional file 3). Among them, 26 genes have 38 introns missed by computer prediction; 25 genes have falsely predicted introns (i.e., they do not exist); 22 genes have 25 introns larger than predicted; and 11 genes have 13 introns smaller than predicted. There are also three predicted genes (PFA0175w, PFB0610c, and PFL2160c) that have cDNA sequences extending into their neighboring genes (PFA0180w, PFB0605w, and PFL2155w, respectively). These predicted gene pairs are 200 bp or less apart on the chromosomes. It is likely that the 3' UTR of the genes will be longer than 200 bp, particularly for gene pairs PFB0610c/PFB0605w and PFL2160c/PFL2155w with ORF in opposite orientations. Similarly, assembly of our and public EST with predicted CDS and genomic DNA increased the number of genes having incorrectly predicted introns to 305, with 152 new introns found and 182 introns having sizes different from those predicted (Table 2; Additional file 3). These genes will require further experimental verification with complete cDNA sequences.
Additional file 3. Genes with cDNA sequences not matching predicted CDS perfectly. Aligned cDNA and predicted CDS sequences can be viewed by double clicking the hyper-linked gene names in the Excel file.
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Table 2. Genes having introns that do not match those predicted in public databases
Confirmation of conserved GT-AG intron splicing sites and alternatively spliced introns
All the introns confirmed by our cDNA sequences have typical eukaryotic GT-AG splicing sites except a few genes that have potential 'introns' lacking GT-AG. These atypical 'introns' could be due to deletion during cloning in bacteria. For example, a 497 bp gap was found at 32 bp 5' of the ATG in gene MAL13P1.130, but no GT-AG sites were found in the gap. Gaps without GT-AG sites can be due to either deletion during cloning in bacteria or sequencing errors, although it cannot be ruled out that some introns may not have the conserved GT-AG sites. To investigate this possibility, we designed PCR primers flanking the 497-bp gap in MAL13P1.130 and confirmed the absence of the 497 bp gap (Table 3 and data not shown). Similarly, gene PFL0290w has a gap of 287 bp without GT-AG sites within the predicted ORF; we could not confirm the gap, either. It is clear that gaps without GT-AG sites are unlikely to be true introns. This observation also shows that sequences, including coding regions with relatively high GC content, can be deleted during cloning in bacteria.
Table 3. PCR verification of selected introns that were alternatively spliced
Alternative splicing has been well documented in many organisms [35,36] including malaria parasites [37-39]. We noticed that many predicted introns were covered with EST contigs that may or may not have the predicted introns, suggesting potential alternatively spliced introns (Table 3; Additional files 2 and 3), in addition to some cDNA that showed introns of different sizes; however, we could not rule out that those cDNA contigs without introns were from contaminated genomic DNA sequences. To verify these introns, we synthesized primers to amplify some alternatively spliced introns suggested by the cDNA sequences (Table 3). The majority of these introns (except four that have different intron sizes) were either present or absent in sequence alignments, e.g., contigs with some sequences running into the predicted introns. Results from PCR confirmed 29 alternatively spliced introns out of 42 genes tested, including genes with more than two forms of transcripts (Figure 2; Table 3).
Figure 2. PCR products confirming alternatively spliced introns. Oligonucleotide primers flanking selected predicted introns that might be alternatively spliced were amplified from genomic DNA (G lanes), reverse-transcribed mRNA of mixed asexual stages (C lanes), and mRNA controls of mixed asexual stages (without reverse transcriptase, R lanes). Genes with alternatively spliced introns are as marked; M, 100 bp DNA ladder. Note that more than two bands were amplified from PFE1540w, PF13_0220, and PF13_0224.
Antisense transcripts
Antisense transcripts are present in the cDNA collections. Because of our cDNA cloning strategies (digestion with restriction enzymes), the orientation of our cDNA clones was not preserved; however, there were transcripts with introns that had conserved GT-AG intron splice sites in the orientation opposite to the predicted genes (Table 2; Additional file 3). These transcripts matched the genomic DNA sequences but with introns having the conserved GT-AG sites in the opposite direction, suggesting antisense transcripts. Of interest, DNA sequence encoding gene PFL1420w (predicted as human macrophage migration inhibitory factor homolog) was matched by two cDNA contigs, one in sense and the other in antisense orientation. The sense sequence had an intron that matches the predicted intron with conserved GT-AG splicing sites. The antisense contig also had an intron with conserved GT-AG sites, but was 121 bp smaller than the predicted sense intron (Figure 3). Translation of the antisense sequence produced a polypeptide with 84 amino acids that had good homology with N-terminal sequence of myosin IXA protein, which could represent a new gene. The presence of these antisense cDNA is consistent with previous reports of antisense transcripts in the parasite [40,41], but the functions of the these transcripts are largely unknown.
Figure 3. Diagram of exon/intron structures of predicted gene PFL1420w and cDNA contigs covering the gene. FC (forward contig) is a sense transcript with an intron matching the predicted intron. RC (reverse contig) is an antisense transcript having a smaller intron with GT-AG sites in the opposite direction. The line on top represents plus strand genomic DNA. Dashed lines are introns; heavy lines are predicted exons or ORF.
Functional classification
The EST contigs matching CDS predictions were grouped as functional categories according to GO molecular functions. As expected, the majority of the genes with functional assignments were housekeeping genes (Figure 4; Additional file 4). Almost all genes with functional assignment among the 356 genes fully covered with EST (likely representing genes relatively small and highly transcribed) were housekeeping genes encoding proteins related to transcription, translation, and other basic cell functions such as ribosomal proteins (41), histone proteins (7), or proteasome proteins (7) (Additional file 2). Based on this observation, we can predict that the majority of the 171 hypothetical genes in Additional file 2 are likely housekeeping genes.
Figure 4. Functional categories of expressed genes covered by all EST. A total of 3862 genes matched by EST were sorted according to GO molecular functions with P values < 0.0001 on sequence matches. The majority of the genes encode housekeeping proteins involved in DNA/RNA and protein binding, enzyme catalytic activities, transcription, translation, signal transduction, and transport activities.
Additional file 4. EST contigs that do not match P. falciparum CDS and genomic DNA. For all EST contigs and linked files in additional file 4, please go to [42].
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Potential new genes
There were also contigs and EST sequences that match neither the nuclear genome nor the mitochondrial and plastid genomes (Additional file 4). Some of these sequences might be parasite DNA sequences that were not represented in the finished P. falciparum genome. Similarly, there were sequences that match genomic DNA but not the predicted CDS. These sequences could represent new genes or non-coding sequences of intergenic/intron/UTR that require further investigation. For sequence information, linked files and detailed annotation for all the EST contigs, please visit [42].
There are also many predicted ORF larger than 5 kb in the P. falciparum genome. The sizes of these large ORF/genes are probably off the limit of cloning stability in bacteria and in vitro extension capability of reverse transcriptase. In addition, high AT content in the DNA is an obstacle for obtaining good-quality DNA sequences from PCR products. More efforts with new strategies will be required for obtaining full cDNA sequences for the large genes.
Although our EST data are still limited, this work obtained 17332 high-quality cDNA sequences that almost double the current EST collection in public databases. Our effort to extend sequences into cDNA clones allows us to assemble some relatively long cDNA sequences and to correct some erroneously predicted introns. Our data suggest that considerably large numbers of genes in this parasite genome may have incorrect intron/exon predictions, arguing for more efforts to collect complete cDNA sequences and reannotate the genome with cDNA sequences. This study also confirms the conserved eukaryotic intron splice site (GT-AG) at the parasite introns, shows the presence of relatively large numbers of alternatively spliced and antisense transcripts, and reveals silence loci at subtelomeric regions of many chromosomes. The cDNA sequences presented here will provide useful resources for genome annotation and analyses of gene expression.
Parasite culture and RNA extraction
P. falciparum isolate 3D7 was cultured as described [43,44]. Parasite mRNA was extracted from mixed asexual stages using the Micro-Fast Track mRNA isolation kit (Invitrogen).
Construction of cDNA libraries
PCR-based cDNA libraries were constructed using a SMART cDNA library kit (BD-Clontech) as previously described [45]. After reversed transcription using polyT primer, the cDNA were amplified for 11 cycles with primers attached to the 5' capping sequences (5'-GCAGTTGTA TCAACGCAGAGTGGCCATTACGGCCGGG-3') and 3' polyT tail. After separation of the PCR products on 1% agarose gel, DNA inserts of large (> 3 kb), medium (1–3 kb), and small (< 1 kb) sizes were eluted from the gel and cloned into Trip-lEX2 vector for trasnfection of XL1blue cells (BD-Clontech). Additional libraries with inserts digested with BamHI and Sau3A were constructed similarly (Additional file 1).
Sequencing cDNA clones
Plaques were randomly picked and transferred to a 96-well PCR plate (PGC Scientifics) containing 43 μl of SM buffer per well. Each phage sample (5 μl) was used as a template in PCR amplification of the insert using 5' primer PT2F1 (5'-AAGTACTCTAGCATTGTGAGC-3') and 3' primer PT2R1 (5'-CTCTTCGCTATTACGCCAGCTG-3') flanking the cloning sites. For libraries restricted with BamHI or Sau3A, PBKF (5'-ACGGCCAGTGAATTGTAATAC GAC-3') and PBKR (5'-ACAGGAAACAGCTATGACCTTGAT-3') were used in PCR amplification. PCR setups included 30 μl H2O, 4.0 μl of 10× buffer, 0.4 μl dNTP (10 mM), 0.15 μl (5 U/μl) Tag polymerase, 0.25 μl of each primer (50 μM), and 5 μl phage solution. The amplification conditions were: 94°C for 5 min; 35 cycles of 94°C for 1 min, 56°C for 10 s, 52°C for 10 s, 60°C for 2 min; and a final extension at 60°C for 5 min. PCR products were treated with 1 μl of ExoSAPIT (United States Biochemical) at 37°C for 15 min and 80°C for another 15 min. Treated PCR products (5 μ) were used in cyclesequencing reaction using BigDye terminator chemistry. The primers for sequencing were PT2F3 (5'-TCTCGGGAAGCGCGCCATTGT-3'), T719 (5'-TAATACGACTCACTATAGGG-3'), or T320 (5'-GAAATTAACCCTCACTAA AG-3'). Sequencing cycles were as follows: denaturing at 94°C for 2 min; 25 cycles at 94°C for 20 s, 52°C for 5 s, 50°C for 5 s, and 60°C for 3 min; and a final extension at 60°C for 5 min. After cleaning with Sephadex 50 beads packed in a multiscreen 96-well cleaning plate (Millipore), the products were analyzed on an ABI3730×l automatic DNA sequencer. To extend the cDNA sequences, 4800 oligonucleotide primers were synthesized based on sequences obtained and used to extend sequences that could not be reached using primers from the vector.
DNA sequence trimming and assembly
Sequence runs were first base called and assigned quality scores using Phred [46,47] and then trimmed using Lucy [48] to remove sequences shorter than 100 bp or with Phred quality scores lower than 20. Vector sequences and polyA/T were also removed. The trimmed sequences were assembled using CAP3 [30] with 21-bp overlap and 85% identity; the quality of the assembled sequences was inspected visually using Sequencher 4.5 (Gene Codes) and Blast [49]. For sequences having mismatches with predicted CDS (indicating potential incorrect intron/exon predictions), genomic sequences covering the whole predicted coding region plus 1 kb from 5' of start codon and 1 kb from 3' stop codon were downloaded and assembled with EST and CDS. After assembly, the intron/exon junctions were visually inspected and adjusted to ensure proper alignments, particularly for intron splice sites, as software frequently fails to align the A-Trich sequences properly. For BamHI- and Sau3A-digested libraries, some artificial clones from ligation of unrelated DNA fragments were identified and trimmed accordingly after Blast search of the mismatched sequences against the parasite genome sequence.
Locations of each cluster on the assembled chromosomes and the relationships of clusters with each computer-predicted CDS were displayed with Artemis [50]. Sequence annotation, comparison, classification, and functional annotations were performed as described [31] using various software and databases.
bp, base pair(s); cDNA, complementary DNA(s); CDS, coding sequence(s); EST, expressed sequence tag(s); ORF, open reading frame(s); PCR, polymerase chain reaction; UTR, untranslated region(s).
Authors' contributions
FL participated in library construction and in sequencing; HJ and JD participated in sequencing trimming and analysis; JM participated in sequence alignment and PCR; JGV participated in library construction; JMCR participated in sequence clustering, database search, analysis, and manuscript writing; X-z S conceived the project design and participated in analysis and manuscript writing.
This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank Dr. Tetsuya Furuya for help in PCR and sequencing and NIAID intramural editor Brenda Rae Marshall for assistance.
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text:
Robstown softball tops Sinton 14-13 in eight innings
Robstown advances to region quarterfinals to face Columbia
SINTON - In a crazy game that took nearly 3 ½ hours, Robstown outlasted Sinton 14-13 in eight innings to sweep the Pirates in the Class 3A area playoffs Saturday night.
Aside for counting for 27 runs on 30 hits, the sides combined for 12 errors, 20 walks and five lead changes in the back-and-forth contest before the Cotton Pickers advanced to the region quarterfinals where it will face Columbia.
"Whenever we play Sinton, it is always a tough game," Robstown coach Sart Perez said. "We knew they were a team that will hit the ball and they never give up. Our girls never gave up either. I kept telling them anything can happen in softball and tonight that was the case."
Tied at 8 heading into extra frames, Robstown seemingly took control with six runs in the top of the eighth.
Vicki Gutierrez, who had three RBI, and Tina Gonzalez reached on a pair of errors to start the inning. Gutierrez scored on a single to left-center field by Mia Tamez, who had a game-high four hits, while Gonzalez came home on a wild pitch by Megan Castillo.
After Tamez was safe at home on a wild pitch, the big blow in the inning was a two-run single to center by Sabrina Salazar off Mary Christene Chapa. Salazar, who had left the game earlier with a facial cut, crossed the plate for Robstown's final run as Lexi Reyna reached on an infield error.
Sinton rallied in the bottom of the eighth with doubles by Traci Cantu, who had three RBI, and Alex Morin leading to the first run. After Angelina Villarreal walked, Morin scored on a single by Katrina Ortiz. Villarreal later scored on a passed ball, while Ortiz and Shelby McCabe, who walked, tallied on wild pitches.
After Castillo was caught in a run down between third and home, Salazar ended the game with a stellar defensive play. The junior, who had a handful of errors after returning following her injury, knocked down a hard-hit ball and beat Lexi Martinez, who was forced from the rubber after being hit in her pitching hand, at first with a strong throw.
"I had made some errors when I first came back in the game," the shortstop said. "But after picking up my offense, it helped me get back into the game defensively.
"A win is a win at this point no matter how it looks. We knew they would play hard."
Sinton coach Leslie Balerio was pleased with her team's effort in battling back despite losing an 8-6 lead in the seventh when Robstown scored on a wild pitch and an error.
"The girls showed a lot of character tonight," said Balerio, whose team had just three seniors. "They fought until the end tonight.
"I told them before coming to bat in the eighth to do it for the seniors and yourself. Don't come out with any regrets."
ROBSTOWN 14, SINTON 13 (8 inn.)
Robstown 003 201 26- 14 12 3
Sinton 040 013 05- 13 8 9
WP – Lauren Sepulveda. LP – Megan Castillo. 2B – Robstown: Vicki Gutierrez. Sinton: Angelia Villareal, Teresa Cantu, Alex Morin. Highlights – Robstown: Cassi Sanchez 2-5, 3 runs, Sabrina Salazar 2-5, 2 RBI, 2 runs, Vickie Guiterrez 1-3, 3 RBI, 2 runs, Mia Tamez 4-5, 2 RBI, 2 runs . Sinton: Teresa Cantu 2-4, 3 RBI, 3 runs, Stefani Garcia 2 runs, 3 BB; Angelina Villareal 1-3, 2 BB, 2 runs, RBI.
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