text:
Country-Fried Steak With Mushroom Gravy
Total Time
Prep 15 mins
Cook 10 mins
Adapted this recipe from Cooking Light Annual Recipes.
Ingredients Nutrition
- 3 tablespoons nonfat milk
- 2 large egg whites
- 1⁄3 cup all-purpose flour
- 1⁄2 teaspoon onion powder
- 1⁄2 teaspoon salt
- 1⁄4 teaspoon garlic powder
- 1⁄4 teaspoon black pepper
- 4 (4 ounce) sirloin cube steaks
- 2 teaspoons vegetable oil
- 2 cups mushrooms, quartered
- 2 1⁄2 tablespoons all-purpose flour
- 1⁄4 teaspoon salt
- 1 (14 ounce) can reduced-sodium fat-free beef broth
- Combine 3 tablespoons milk and egg whites in a shallow dish, stirring with a whisk. Combine 1/3 cup flour and next 4 ingredients (1/3 cup flour through pepper) in a shallow dish. Working with 1 steak at a time, dip in egg mixture then dredge in flour mixture. Repeat procedure with remaining steaks.
- Heat oil in a large nonstick skillet over medium-high heat. Add steaks and cook 3 minutes on each side or until browned. Remove steaks from pan and keep warm.
- Add mushrooms to pan and sauté 3 minutes. Combine 2 1/2 tablespoons flour, 1/4 teaspoon salt, and broth, stirring with a whisk. Add broth mixture to pan. Bring to a boil and cook 1 minute, stirring constantly. Spoon over steaks.
- Serve with mashed potatoes.
Most Helpful
Very tasty, thank you. Meduim-high was too hot for my stove. I served atop mashed potatoes.
This is so easy and good. I made it exactly as stated and it turned out perfect. Very tasty.


text:
Brad Pitt's publicity team is furiously
denying UK tabloid reports the movie hunk has wed
Angelina Jolie in a secret ceremony.
Britain's Daily
Star claims the couple exchanged vows in a recent Buddhist
ceremony, attended by a few family and friends, but Pitt's publicist
Cindy Guagenti insists the story is "completely not true."
She states, "They've used a wedding picture from (movie)
Mr. and Mrs. Smith and run with this story on the
front page. There's simply no truth to this whatsoever."
Simpsons, Hilton and Richie
Up for Razzies?
HOLLYWOOD - Next year's Golden Raspberry Awards is set to be a
catfight between Hollywood's top party girls, with Jessica and
Ashlee Simpson, Paris Hilton and Nicole Richie the frontrunners
to land Worst Actress dishonors.
Insiders claim the foursome is the favorites to fight it out to
be named the Worst Actress of the Year.
Full story
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Unauthorized duplication in part or whole strictly prohibited by
international copyright law.
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subset: commoncrawl


text:
Free Books for Windows
Free Books for Windows (TM) provides you curated lists of the best free books daily. 1) Daily Lists of the best new free books. 2) Curated so you get the best offers and deals. V2.0.4: 1) Changed App Name to 'Free Books for Windows'. 2) Improved performance of Books View page. V2 - 1) Added Back Button when in Blog View. 2) Added Genre filtering so you can see only books in your Chosen Genres. Please Note: Genre filtering only works in 'Books View'. It does not work in 'Blog View'. ******* V1.1: Added Refresh button so you can refresh manually and ensure you get the latest lists. ******* Important: Please check the prices at Amazon (TM) before buying. Book prices can change any time of the day so PLEASE check the price at Amazon (TM) BEFORE buying. Important: Please make sure the book is free for ALL users, and not just for Amazon PRIME (TM) Subscribers. The App comes with several strong features - 1) Browse and buy within the App. By using WebView we let you buy the free books and book deals you want within the app itself. 2) Easy Navigation. 3) Lists of Books you can quickly browse through. 4) Information about Genre, Price, Ratings. We open the book page right in the app so you can read reviews and make a decision in the app itself. 5) Blog View to view as a blog or Books List view to navigate book by book. The simple, elegant design and easy usability makes it fun to use. You don't have to be a rocket scientist (or even a catapult scientist) to use it. Tested and works fine on - 1) PCs (tested on a 23.6" PC). 2) Laptops. 3) Surface RT (TM). The App Supports Filled and Snapped Views. For the best user experience please use it full-screen or in Filled View. You need enough screen space to be able to browse book details. Do give our app a try - You'll love it! Please Note: 1) Surface RT (TM) and Windows (TM) are trademarks of Microsoft Corporation. We only use the name for identification purposes i.e. to let you know that we tested this app on it and it's compatible. All rights in and to the Microsoft Marks are Microsoft's exclusive property, and any goodwill generated by our use of any Microsoft Mark will inure to Microsoft's exclusive benefit. We will not take any action that is in conflict with Microsoft's rights in or ownership of any Microsoft Mark. 2) Amazon (TM), Kindle (TM), Amazon PRIME (TM) are trademarks of Amazon.com, Inc. or its affiliates. We only use them for identification purposes i.e. so users know what our App is about. All rights in and to the Amazon Marks are Amazon's exclusive property, and any goodwill generated by our use of any Amazon Mark will inure to Amazon's exclusive benefit. We will not take any action that is in conflict with Amazon's rights in or ownership of any Amazon Mark.More
- Daily Free Book Lists
- Best Free Kindle Books Daily
- Public Domain Books
- Supports Surface (TM)
- Supports ARM
- Supports Desktops and Laptops
- Simple, beautiful design
- Optimized for Simple and Easy Use
- Browse and Buy from Inside the App
- Supports Filled and Snapped Views (Full Screen is recommended)
- Fast and Easy to Use
App details
- Publisher
7 DragonsMore
- Category
Books & referenceMore
- Approximate size
2.71 MBMore
- Age rating
For ages 12 and upMore
- Supported operating systems
Windows 10Windows 8.1
- Supported processors
x86, x64, ARMMore
- Supported languages
English (United States)
- Learn more
- Additional terms
- More
- Capabilities
Access your Internet connection
- Report this app
Thanks for reporting your concern. Our team will review it and, if necessary, take action.Sign in to report this app to Microsoft
Version notes
In this update (V2.0.4) we added: 1) Name Change. 2) Performance Improvement for Books View page. In the previous update (V2) we added: 1) A Back Button in 'Blog View' for going back when in Browser. 2) A Genre Filtering option. Just press the Genres button when in Books View. Please Note: Genre Filtering only works in Books View. ******* In the previous V1.1 update we added the following: 1) A Refresh button so users can refresh any time. 2) Added fallback servers in case main server fails.
Free Books for Windows
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1-10 of 89 reviews
A bookworm Finding a New Menu
I like being able to browse and load my pc so I can read what ever when ever.More
- Was this helpful?
- Yes (2) Yes (2)
- No (0) No (0)
I LIKE THAT IT GIVES ME THE CHACE TO READ BOOKS WITHOUT HAVING TO PAY AND ITS REALLY FAST.More
- Was this helpful?
- Yes (10) Yes (10)
- No (2) No (2)
Getting Started....
The jury is still out, but it seems like a great app. I am going to try my skills on writing and publishing a book that I have been thinking about doing for years. It's about time I put my thoughts on paper. Angela aka SpiritWolfMore
- Was this helpful?
- Yes (1) Yes (1)
- No (5) No (5)
Refreshingly easy to use
This app provides a daily list of recommended free Kindle books on Amazon.com. It also provides a click-thru to the book on Amazon's site. The first time I used it, I found 12 books of interest, all free. The only problem I found was that the window into Amazon was too narrow, forcing me to scroll back and forth to read book descriptions and reviews. I'm sure that this is a problem that the developer can remedy, maybe by making the window easily resizable.More
- Was this helpful?
- Yes (3) Yes (3)
- No (1) No (1)
Free Books for Windows
A fun and easy way to add to my Kindle collection.More
- Was this helpful?
- Yes (2) Yes (2)
- No (2) No (2)
I got into this space with NOOK in mind and, BOOM, I get switched to Kindle by Microsoft Magic. What? No password for this switch-a-roo?More
- Was this helpful?
- Yes (2) Yes (2)
- No (2) No (2)
Free Kindle Books-Website/Windows Program Review.
I like the program, due to the fact that you are able to long into Amazon/Kindle website within the program. After a person sign's in Amazon/Kindle website, a person would look for certain type of books on a regular basis. However, you would think that the program would show recommendations on the left side, based on the last several searches that the account holder used.More
- Was this helpful?
- Yes (2) Yes (2)
- No (2) No (2)
Very Useful and Convenient App
Not only is this a convenient app but it is used everyday. Daily updates are very effective. This app provides much needed information in one convenient place. I would recommend this app.More
- Was this helpful?
- Yes (1) Yes (1)
- No (0) No (0)
Useful and Practical for Kindle Users
I really enjoy the manner in which this app helps in browsing the Kindle Markets free book selection. It does what it says it will do and runs pretty smoothly. I think it could benefit from a bit of color and jazz in the UI, but it gets the job done. Thanks to the developer, it's helped me find a lot of interesting books.More
- Was this helpful?
- Yes (0) Yes (0)
- No (1) No (1)
Free Kindle Books
Really enjoy owning this app. It's easy to use and a great way to find interesting, free kindle booksMore
- Was this helpful?
- Yes (1) Yes (1)
- No (0) No (0)
- Was this helpful?
- Yes () Yes ()
- No () No ()


text:
Cartoon Passover Seder Plate - Porcelain W/dish Liners
This item has been shown times.
Cartoon Passover Seder Plate - Porcelain W/dish Liners:
Add some fun to your Seder with our CARTOON SEDER PLATE. Border shows march from slavery to freedom, while cartoon vignettes illustrate the imponderables of the Seder (breathing fire from the horseradish, chicken and egg question, sour punim from the bitter herbs and more!). The seder plate is made of porcelain, measures 10.25" in diameter and INCLUDES 6 plastic bowls (3" diameter) for the traditional foods. Gold rim, center matzoh colored circle for your kiddush cup placement. Comes boxed.
Please read terms and policies below before making your purchase.Buyer to pay 3lb. priority shipping plus $1 handling per plate. Flat rate shipping is available if that is less expensive. Please scroll down to 's shipping calculator and enter your zip code - you will see the total amount required for shipping. Payment will be made via PayPal only. Payment is due within 7 days of sale end.
We look forward to a wonderful trading experience.
Please be sure to read the information below before making your offer/purchase. The key to a successful transaction is communication. Please ask questions before placing a offer or buying an item.
SALES TAX: 8.625% iscollected onanysale shipped to a New York address.SHIPPING:United States Postal Serviceonly.Delivery Confirmation tracking on all parcels. Email for combined shipping quotes. Orders returned unclaimed, wrong or incomplete address will be reshipped to the customer only at the customer's expense.
- International Shipping: No international sales at this time.
- Shipping Damage: Notify us immediately. Keep all packagingmaterials to present toUSPS with the claim form we will send you.
REFUNDS and RETURNS:Returns are not accepted for holiday items, jewelry and clothing/accessories unless defective. THIS IS A HOLIDAY ITEM.
- Defective merchandise:
- Defect does not apply to tarnish on silver, slight differences between the color you see on the computer screen and the item you receive, etc.
- New item with a manufacturer's defect: we will refund shipping both ways.
- Used items arenot perfect.Defect will mean "significantly not as advertised".
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Cartoon Passover Seder Plate - Porcelain W/dish Liners:
Judaica Groom Green Glass Wedding Cup "if I Forget Thee, O Jerusalem..."
Wine Goblet Sterling Silver Kiddush Cup Artisan Filigree Handmade Judaica -id743
New Pretty Horseradish And Saltwater Serving Set For Passover With
Vintage Israel Naaman Tiberias & The Lake Of Galilee 1970 Porcelain Plate
H.quality Wash Cup Natla Netilat Yadayim Hand Washing Judaica .steinls Steele
Judaica - Vintage, Kiddush Goblet And Saucer, Collectible, Silver


text:
- Swimmer Ryan Lochte reveals one of his worst dates - US Weekly
- Get the latest on Dr. Dre's arrest - TMZ
- Despite rumors, Eddie Murphy's daughter is not pregnant - Gossip Cop
- The Vampire Diaries star Michael Trevino steps out for a good cause - Just Jared Jr
- Are Blac Chyna and Kylie Jenner on good terms? - Radar
- Blue Ivy is just as stylish as her mom Beyonce! - Lainey Gossip
- This Narcos star confirms he's not coming back for another season of the show - The Hollywood Reporter
25 August 2014 at 4:51 PM
Gerard Butler Can't Keep His Hands Off His Mystery Girl!
About this photo set: Gerard Butler got hands on with a beautiful mystery girl while meeting up in a parking lot by the beach on Wednesday (August 20) in Malibu, Calif. The 44-year-old… Read More Here
Photo #: 3183145
Posted to Bikini, Gerard Butler
powered by ZergNet


text:
May I know which Javascript function detects when user press the X (close) button? I tried using the onbeforeunload event but seems like this works when user clicks the Back, Forward, Refresh, Close Window or Home button.
I want it to be only for the X (Close Window) button. How can I do so?


text: Danny Smith wrote: > --- Danny Smith <danny_r_smith_2001@...> wrote: > From: Danny > > I would like to submit patch, adding this file to win32api (after > testing the prototypes). Should these declarations be removed from > winsock.h or left there and guarded with something like #ifndef > _MSWSOCK_EXT? I would prefer that they match the documentation. It's gives a means by which we can answer definitively why a prototype or macro is defined in a particular header. However, if our users (this includes the Cygwin users) have grown accustomed to finding these in winsock.h then we'll have to guard them in both files. Earnie. _________________________________________________________ Do You Yahoo!? Get your free @yahoo.com address at http://mail.yahoo.com


text:
Members of British Columbia's notorious Red Scorpions gang have been operating a drug-trafficking network in central and southern Alberta for two years after escaping violence and threats in their home province, police say. Cory James Lesperance, a 29-year-old B.C. man who police say is a Red Scorpion member, is alleged to be at the centre of this drug distribution ring in Calgary, Airdrie and Red Deer.
Accused Surrey Six killer Matt Johnston held up six fingers as he told his gangmate about the murders right after they happened, B.C. Supreme Court heard Tuesday.
After Jonathan Barber was shot to death in May 2008, alleged killer Troy Tran admitted to an associate that he mistakenly believed he hit Jamie Bacon because all "white guys" look the same. Tran, a Calgary gangster linked to the Fresh Off the Boat Killers (FK), was regularly in B.C. in the company of United Nations leader Clay Roueche and others in the gang behind a lengthy conspiracy to murder the Bacon brothers and their Red Scorpion associates.


text:
Technical issue
Members may be unable to log into the site. We're working to resolve the issue asap, thanks for your patience.
1000s OF RATINGS
A reliable baby monitor gives you peace of mind if your living areas are out of earshot of the baby's bedroom or you're a heavy sleeper. Our baby monitor buying guide will help you work out which kind you need.
We test many different types including:
Our report will tell you which models have:
Our interactive comparison tool also helps you filter results based on sound range score and features (such as whether they play music). And our Recommended list will help you see quickly which models come out on top.
List of brands we tested in this review.
Recommended retail as of January 2015.
enter value/s in increments of 1 between 5 and 399
A baby monitor needs to score well for both ease of use and sound performance to be a Recommended product. We don't factor picture performance into our overall score because some monitors are audio-only and aren't directly comparable, but we do take it into account when making our recommendations. You can view the picture performance scores separately.
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Whether the baby monitor is audio-only, combines video and audio functions, or is app-based.
Made up of sound performance (70%) and ease of use (30%). Picture performance results are listed separately.
Note: Some Wi-Fi enabled baby monitors use a smartphone or tablet as the carer device. Where applicable, our scores are based on the app that is associated with the product.
enter value/s in increments of 1 between 0 and 0
We look at
ease of using the controls on both baby and carer unit
ease of setup including handling, positioning and adjusting settings
ease of accessing the batteries and connecting the adaptors.
(A) Uses tablet/phone as carer unit.
How well the baby unit and carer unit can maintain acceptable sound monitoring at various distances with obstacles (walls) separating them.
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text: Hi and contrats to Jonathan, > > KDE 3.4 also includes DNS-SD using mDNSResponder-87 from apple. Is the > > Apple Public Source Licence 2 likly to cause any problems? It seems to > > have the approval of the FSF. > > Boh, why is KDE using Apple's stuff instead of howl? howl uses _old_ zeroconf implementation forked from Apple which also breaks licensing ( http://bugs.debian.org/cgi-bin/bugreport.cgi?bug=289856 ). Regards, ismail


text:
|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|"sam rash (JIRA)" <j...@apache.org>
|[jira] Commented: (HADOOP-6762) exception while doing RPC I/O closes channel
|Fri, 19 Nov 2010 18:00:15 GMT
[ https://issues.apache.org/jira/browse/HADOOP-6762?page=com.atlassian.jira.plugin.system.issuetabpanels:comment-tabpanel&focusedCommentId=12933890#action_12933890 ] sam rash commented on HADOOP-6762: ---------------------------------- todd: where are we on this? you said some gridmx test in the mapred project needs updated based on this? shall we commit this and file a mapreduce jira? > exception while doing RPC I/O closes channel > -------------------------------------------- > > Key: HADOOP-6762 > URL: https://issues.apache.org/jira/browse/HADOOP-6762 > Project: Hadoop Common > Issue Type: Bug > Affects Versions: 0.20.2 > Reporter: sam rash > Assignee: sam rash > Attachments: hadoop-6762-1.txt, hadoop-6762-10.txt, hadoop-6762-2.txt, hadoop-6762-3.txt, hadoop-6762-4.txt, hadoop-6762-6.txt, hadoop-6762-7.txt, hadoop-6762-8.txt, hadoop-6762-9.txt > > > If a single process creates two unique fileSystems to the same NN using FileSystem.newInstance(), and one of them issues a close(), the leasechecker thread is interrupted. This interrupt races with the rpc namenode.renew() and can cause a ClosedByInterruptException. This closes the underlying channel and the other filesystem, sharing the connection will get errors. -- This message is automatically generated by JIRA. - You can reply to this email to add a comment to the issue online.
|View raw message


text:
Group Booking Terms and Conditions
Because you are making a Group Booking, there are additional Terms and Conditions that apply. Please read them and click 'I Accept' below to continue with your booking
Hostel Specific Terms & Conditions
The remainder of the amount due will be charged to your credit card 1 week prior to arrival. Should numbers or room selection change, the per person deposit times the number of people who actually travelled will be deducted from the total accommodation charge levvied by the hostel.
Cancellation / No Show & Reduction in Numbers Policy
The following group types are not accepted by this hostel: Junior / Primary School, Business Trip, Stag/Hen/Bachelor Party If you have misrepresented the type of group you are booking for, your booking may be cancelled by the hostel.
Any cancellations resulting from agreement to these terms and conditions under false pretences will result in loss of deposit.
General Group Booking Terms
By making a group reservation through Hostelworld.com you agree to the following terms and conditions as set by Hostelworld.com and as set by the hostel in question.
- In order to make a group reservation on Hostelworld.com it is necessary to pay a deposit. The balance is due directly to the hostel according to their payment conditions.
- The booking deposit is non-refundable
- Hostelworld.com are not responsible for changes or amendments to your reservation once it has been processed. All correspondence with regard to changes to your booking need to be dealt with directly with the hostel.
- Cancellations for reservations must be made directly with the hostel and must conform with the individual hostels cancellation policy (see below)
- It is your responsibility to pay the balance of your accommodation cost to the hostel in accordance with their payment conditions (see below). Failure to do so may result in your reservation being cancelled by the hostel.
- Individual hostels may have special booking conditions relating to group stays, please read the hostel booking conditions carefully before making your reservation.
É necessario accettare i termini e condizioni per confermare la prenotazione.
Vanno a ruba
Descrizione della struttura
Ultima recensione
" Belissimo hostello, personale eccellente! arrivati sul isola "Ilha Grande" con il traietto che colleca Mangaratiba con l' isola, subito dopo il nostro arrivo siamo andai a cercare l'hostello per il vilaggio di Abraao, fate atenzione perche si cono 2 Biergarten Hostel. cmq ci siamo orientati facilmente e abbiamo trovato quello che avevamo prenotato. colazione normale ma personale moltro ospitale che parla in inglese. bel posto molto calmo! "- Anonimo
Applicazioni di Hostelworld per cellulare
Prenota dove vuoi e quando vuoi con le nuove app per dispositivi mobili di Hostelworld.


text:
Beyonce, Jessica Simpson and American Idol: Album Grades Are In
Kids around the country are heading back to school. But they aren't the only ones being graded as summer turns to fall.
Multiple artists have released new albums over the last couple weeks. The critical folks at Entertainment Weekly have taken the time to grade each CD - and those from The Gossip figured it was our duty to present such estimations to readers everywhere:


text:
We're tempted to offer three cheers for the League of Women Voters and faculty members at three Kansas public universities for developing a course to instruct college students on how to register to vote. After all, college students who are American citizens — and other
Downzoning an almost 11-block area east of City Park isn't just a good idea, it's one that's years overdue. What's more, the failure to take advantage of the present opportunity could doom the area to further degradation. The plan that gained —
Though it matters immensely to those most affected, the death of a U.S. soldier in combat isn't always noticed in America. After all, more than 4,000 U.S. military personnel were killed in the last Iraq war. But a single death stood out Thursday
It was news earlier this month when the state of Kansas hired an efficiency expert to scrutinize the state budget. Unfortunately, it was news for the wrong reason. That's because the $2.6 million being spent on a firm that has a spotty record in other
Phill Kline is at it again, trying to blame others for his legal troubles. Not content to have abused his authority as Kansas attorney general, he is now suing the entire Kansas Supreme Court and just about anyone else who had anything to do with
Kansas Secretary of State Kris Kobach has gained the power to prosecute cases of alleged voter fraud, and now he's using it. Earlier this month, he filed three election fraud cases in two counties, accusing the defendants of illegally voting in Kansas while casting
A tip of the cap to Riley County Attorney Barry Wilkerson, who was named the prosecutor of the year by his peers this past week. Mr. Wilkerson, who is the top law-enforcement official in Riley County, was lauded by the Kansas County and District Attorney'
It's tough to know whether Manhattan will end up with federal money to cut flood risk along Wildcat Creek. What we do know is that the community has tried pretty hard. There's a $1 billion chunk of change left over from the federal disaster
It was striking to read the news account earlier this week of U.S. Rep. Tim Huelskamp's appearance in Leonardville in the same edition that The Mercury published an opinion column by David Brooks. Huelskamp, in his appearance here, rattled on about how awful
The candidates for president on the Democrat side all took the stage earlier this week in their first debate. "Candidates" is really not the right word, because, frankly, this whole thing comes down to Hillary Clinton. Yes, Bernie Sanders has surged in the polls and
You can download and print:
Terms of Service | Privacy Policy | The Manhattan Mercury, 318 North 5th Street, Manhattan, Kansas, 66502 | Copyright 2012
Reproduction of any kind is prohibited without written consent.


text:
ScriptSpot is a diverse online community of artists and developers who come together to find and share scripts that empower their creativity with 3ds Max. Our users come from all parts of the world and work in everything from visual effects to gaming, architecture, students or hobbyists.
Register for free and be part of the community!
Simple script to batch clean material slots... by slot name or by Class used in slot (eg NormalBump etc)
It shows a list of material classes + used slots, (only used slots will be listed). Then you can select what you need to clean and press Clean
Must-have plugin for fast modeling! Now with ProBoolean.
Fast 1-click boolean operations with multiple objects:
-Union selected objects.
-Subtract selected objects from the first selected object.
-Subtract the last selected object from the other selected objects.
-[ProBoolean] Union selected objects.
-[ProBoolean] Subtract selected objects from the first selected object.
Textures2materials is a very handy plugin which can turn multiple textures into materials at once (into multi-material or into multiple material slots). It also checks if your textures have a bump versions and load them as bump. "Must have" plugin for any 3D designer.
Materializer (materials equalizer) is the fastest way to create materials mixing different templates. It works like equalizer but for materials!
Example: To make glossy wood press the 'Wood' button and then add a little bit of plastic.
Every time you hit 'Set' selected material will be updated without removing of maps.
Currently only V-RAY supported.
PLACE PEOPLE (Commercial script)
PLACE PEOPLE is a 3ds Max plugin written in MaxScript that allows the user to place characters created with the 3ds Max 'Populate Toolset' anywhere, following a 3D spline path, and be glued to any surface, in really few mouse clicks.
V-Ray Material Presets Pro is a powerful material collection for 3ds Max and V-Ray. It will includes shaders like: Metal, Glass, Leather, Liquid, Ceramics, etc.SIGERSHADERS V-Ray Material Presets Pro version 4.0.0
Problem: The filesize of scenes suddenly increases dramatically. This slows down the workflow since every manual-save and autobackup-save takes long and it fills your fileserver with maxfiles that are very big.
Reason: 3ds max adds notetracks and reactor-collisions to the scene. A common workaround for this problem is ctrl+N (New Scene: Keep Objects and Hierarchy) or to merge your scene into an empty new scene.
I am wrinting for those who work with MAX+V-RAY combo and have several render nodes.
I'm sure you have encountered many DR and Backburner problems, that you could only solve by restarting the V-ray spawner or BB server.
I used to do this with remote desktop access, or in worse case, manually back then, but now I have started to develop a system that makes this a lot easier.
Please watch the video introduction.
If you are interested you can register at: https://rendernodemonitor.com/register
This script aims to replace standard 3dsmax's Batch Render and imitates Houdini Output Node workflow. Using it you can specify rendering parameters - frame size, frame number, output path - directly to camera object, so every camera in scene 'knows' what it intended to render. Additional functionality is Lighting Assignment - you can specify which light will be turn on or off for selected camera during the rendering.
New features:
This is major update of the script, so it may not be as stable as before. Backup scenes or cameras at least before running this version. More information can be found here - 1.10b Multiple presets for each camera
Previous updates:
Here are some details: http://usakhelo.github.io/batchcam/batchcam-1.9.20.html
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Updated Brown fly-fishing book published
June 17, 2014 Southwest Minnesota State University Chemistry Professor Dr. Jay Brown has updated his Camden State Park fly-fishing book. more »»
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Karin Elton
July 22, 2016 After our one-day (and two nights) stay in San Francisco, my sister Dorene and I started the drive back to Eugene across the Bay Bridge onto Interstate 5 once again. more »
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Suddenly feeling alone, suddenly feeling brushed aside.
Never did ask, but I wouldn't mind going along for this ride.
They're all lies about me. I'm not the bad guy. I'm the right guy.
Your high eye ain't helping your eye sight. Yeah this hoody's dark as night, but I'm still Mr. Right.
Alone again, time to find out who truly is your friend.


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Vineyard artist Abe Pieciak is opening a pop-up gallery for his art business, Lure Fish, at Nevin Square in downtown Edgartown.
By all accounts, the 69th Martha's Vineyard Striped Bass and Bluefish Derby was a major success with records all around. But the number of striped bass was dramatically lower, with only 288 weighed in.
Monday, October 20, 2014
The 70th annual Martha's Vineyard Striped Bass and Bluefish Derby begins Sunday, Sept. 13 and continues through Saturday, Oct. 17, 2015. For more information, visit the derby's website.
Bluefish carried the day this year at the Martha's Vineyard Striped Bass and Bluefish Derby, with two men taking home the grand prizes for bluefish caught from the shore and from a boat. The derby saw a record 3,282 registrants.
The last weigh-in Saturday night saw Helena Kirschenbaum win the grand slam with her striped bass while Seb Keefe brought in a shore bonito to take over top spot on the leader board. And then it was over. The derby awards ceremony is today at Farm Neck.
Thursday, October 16, 2014
Tuesday, October 14, 2014
A heroes' welcome greeted wounded soldiers arriving Sunday for the sixth annual American Heroes Saltwater Challenge. Each year, wounded veterans visit the Island to fish the Martha's Vineyard Striped Bass and Bluefish Derby and to enjoy fellowship and Vineyard-style relaxation.
The veterans are hosted by Bob and Sarah Nixon, owners of the Menemsha Inn, Beach Plum Inn and the Home Port Restaurant.
© 2016 Vineyard Gazette


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Announcement Original oil paintings, cards and prints of cats and other creatures.
Original oil paintings, cards and prints of cats and other creatures.
Julie O'Connell on May 27, 2016
I LOVE my "Naughty Charlie" print, couldn't be more happy!!!??. My collection of Charlie (and Willie) prints keeps growing and has turned my wall into a beautiful collage of Heidi's magnificent works!
Laurel Glasco on May 24, 2016
This cat looks a lot like my Linnet. It is a lovely piece of art and I will enjoy it every day. Thank you, Heidi!
Laurel Glasco on May 20, 2016
This is the sweetest picture. It makes me feel happy every time I see it. Thank yoi for the card. I will frame it also! Your art is charming, Heidi! I love all your cats.
Cats and other animals are what I love most, and come to me naturally as my subject matter. My whole life, I've enjoyed animal art; I was captivated by Louis Wain's anthropomorphic cat art and the Mainzer postcards as a child, and as I matured, Franz Marc became my favorite. Today, I'm proud to be carrying on the centuries-old tradition of cat and animal art in my own style.
A self-taught artist, I painted for many years while pursuing my archaeology career. Once I had small children at home, I had the crazy thought that perhaps I could sell my artwork on the internet; in 1999 that was a pretty far-fetched idea. I went ahead and built a website and amazingly, my paintings sold! Never went back... Over the years, I've added reproductions of my paintings in the form of small prints and notecards.
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Heidi Shaulis
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The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase and has multiple functions residing in its different domains. In the present study, we examined the role of the hypervariable hinge region of P protein in viral RNA synthesis and recovery of infectious VSV by using transposon-mediated insertion mutagenesis and deletion mutagenesis. We observed that insertions of 19-amino-acid linker sequences at various positions within this region affected replication and transcription functions of the P protein to various degrees. Interestingly, one insertion mutant was completely defective in both transcription and replication. Using a series of deletion mutants spanning the hinge region of the protein, we observed that amino acid residues 201 through 220 are required for the activity of P protein in both replication and transcription. Neither insertion nor deletion had any effect on the interaction of P protein with N or L proteins. Infectious VSVs with a deletion in the hinge region possessed retarded growth characteristics and exhibited small-plaque morphology. Interestingly, VSV containing one P protein deletion mutant (PΔ7, with amino acids 141 through 200 deleted), which possessed significant levels of replication and transcription activity, could be amplified only by passage in cells expressing the wild-type P protein. We conclude that the hypervariable hinge region of the P protein plays an important role in viral RNA synthesis. Furthermore, our results provide a previously unidentified function for the P protein: it plays a critical role in the assembly of infectious VSV.
Vesicular stomatitis virus (VSV) is the prototypic member of the family Rhabdoviridae in the order Mononegavirales. VSV is an enveloped virus with a negative-stranded RNA genome of 11,161 nucleotides. The viral envelope consists of a lipid bilayer with the spike glycoprotein protruding out and the matrix (M) protein lying underneath the lipid bilayer. The viral genome which is present in the virion core is tightly encapsidated by the nucleocapsid protein (N protein) to form the ribonucleoprotein complex (6). The viral ribonucleoprotein serves as the template for transcription and replication by the viral RNA-dependent RNA polymerase, which is a complex of the large protein (L protein) and phosphoprotein (P protein). The L protein is the catalytic subunit of the polymerase complex responsible for polymerization of nucleotides, capping, methylation of capped mRNAs, and polyadenylation of mRNAs (reviewed in references 1 and 45). P protein, on the other hand, acts as an accessory subunit of the viral polymerase (1, 45). In addition to having a role in polymerase functions, it binds to the L protein and stabilizes it from proteolytic degradation (7, 17), it complexes with the newly synthesized N protein for the efficient encapsidation of nascent RNA (16, 36, 42), and it interacts with terminal sequences of viral genome for viral RNA synthesis (29, 30).
The P proteins of the two serotypes of VSV, Indiana (VSVI) and New Jersey (VSVNJ), are small (265 and 274 amino acids, respectively) and acidic in nature. Although these two proteins share only 30% sequence homology (19, 21, 26, 41), mutational and functional studies have identified four distinct structural regions (see Fig. Fig.1A).1A). Three of these structurally homologous domains (22, 41, 44) have been the targets of recent mutational studies to more precisely define their role in viral genome transcription and replication. Domain I, which encompasses the amino-terminal half of the protein (amino acid residues 1 through 150 for VSVI or 1 through 137 for VSVNJ) is a highly acidic region that contains residues for the constitutive phosphorylation of P protein. Phosphorylation of specific amino acid residues (serine-60, threonine-62, and serine-64) in domain I by cellular casein kinase II is important for transcriptional activity of P protein (2, 3, 12, 14, 22, 39, 48, 49). However, replication functions of the protein are unaffected by the phosphorylation status of this domain (39). Domain II, spanning approximately residues 210 through 244 in VSVI and 231 through 247 in VSVNJ, is phosphorylated (10, 12), although the identity of the kinase responsible for this modification remains elusive. This domain interacts with L protein to form an active transcription complex with N-RNA template (10). Unlike the domain II of VSVNJ, which regulates transcription in vitro (10), the phosphorylation of residues in domain II of VSVI does not appear to have a role in transcription in vitro or in vivo (20, 27, 35). However, the phosphorylation of residues in this domain has been shown to influence the function of the P protein in replication (27). Using an infectious clone of VSV (31), we recently demonstrated that the phosphorylation of residues in both of these domains is indispensable for VSV growth (13). Although individual mutations at various phosphorylation sites in either domain of P protein did not adversely affect virus recovery or growth, multiple mutations that resulted in the complete loss of phosphorylation in either domain I or II were detrimental to virus recovery (13).
The extreme carboxy-terminal region, containing 21 to 25 residues, is known as domain III. The last 11 amino acids of this domain are highly conserved and contain a number of basic residues that are important for P protein function in mediating the binding of P protein to N-RNA template (15).
The fourth region of P protein, which links domain I and domain II, is a stretch of approximately 50 to 60 amino acids, spanning residues 150 through 210 (11, 22). A comparison of the amino acid sequences of the P protein of several isolates of VSVNJ representative of the natural genetic diversity suggested the highest degree of variability within this region (4). The function(s) of this hypervariable region, often referred to as the hinge region, is largely unknown. It has been shown that the hinge region is not required for the association of P protein with N protein, by using a yeast two-hybrid system (50), or with L protein, by using in vitro interaction studies (17). The exact role of this hinge region of P protein, if any, in viral RNA synthesis and/or the life cycle of VSV has not been addressed.
To obtain a better understanding of the function of the hinge region of the P protein, we employed two different but complementary approaches in this study: transposon-mediated insertion mutagenesis and deletion mutagenesis. The abilities of the mutant P proteins to support viral RNA synthesis and recovery of infectious VSV were examined. Our results indicate that the majority of the sequences within the hinge region are dispensable for viral RNA synthesis and generation of infectious VSV. However, amino acids spanning residues 201 through 220 are required for viral RNA synthesis. Furthermore, our data reveal a new function for the P protein: the hinge region of the protein is required for assembly of infectious VSV particles.
Baby hamster kidney (BHK-21) cells used in this study were maintained in Eagle's minimal essential medium containing 5% fetal bovine serum (FBS) and 100 units of penicillin, 20 units of streptomycin, and 20 units of kanamycin per ml of growth medium. VSV (Indiana serotype, San Juan strain) was propagated in BHK-21 cells. Recombinant vaccinia virus (vTF7-3) expressing bacteriophage T7 RNA polymerase (18) was grown and titrated by plaque assay in BHK-21 cells. Production and purification of defective interfering T (DI-T) particles (32), which are of the copy-back type, have been described previously (40).
The P coding region from pET-P (39) was amplified by using Pfu polymerase (Stratagene) and P(+) and P(−) primers (13) digested with KpnI and PstI and was cloned into pGEM-3 vector (Promega) under the control of T7 RNA polymerase promoter. The resulting plasmid encoding the wild-type (wt) P protein was used as the template for Tn5-based in vitro mutagenesis using an EZ::TN in-frame linker insertion kit as per the instructions of the manufacturer (Epicentre). This Tn5-based insertion mutagenesis system allows random and single-hit in-frame insertion of a 48-nucleotide linker in the target DNA (23). Plasmids carrying insertions can be selected on the basis of kanamycin resistance encoded within the Tn. Following an in vitro transposase reaction, transformants were selected on kanamycin- and ampicillin-containing LB agar plates. Miniprep DNAs of individual clones were digested with NotI to release the kanamycin cassette, religated, and used for another round of transformation and selection on ampicillin-containing plates. The P coding region containing Tn-mediated insertions were identified by restriction mapping and sequence analysis. From an initial panel of insertion mutants that were identified, eight mutants spanning the hypervariable region and its flanking areas with insertions in approximately every 7 to 15 amino acid intervals were selected (Fig. (Fig.1B).1B). The mutants were designated PTn136, PTn148, PTn159, PTn167, PTn180, PTn189, PTn196, and PTn220, where the number refers to the amino acid position at the site of insertion in the P protein. From sequence analysis, it was observed that the Tn-induced insertions occurred in all three open reading frames (ORFs): PTn136, PTn148, PTn159, PTn180, and PTn220 contained insertions in ORF1; PTn167 and PTn189 contained insertions in ORF2; and PTn196 contained an insertion in ORF3. It must be noted that the insertion resulted in duplication of three amino acids of the target sequence at the site of insertion. Therefore, the Tn-induced in-frame insertions led to insertion of a total of 19 amino acids into the P protein.
The deletions in the hinge region were generated by overlapping PCR using DNA encoding the wild type P protein (Pwt) as the template. For each deletion, two fragments were amplified from either side of the desired region to be deleted, using primers designed to include overlapping sequences flanking the region to be deleted. The deletion mutants were then generated in a second round of PCR by amplification with P(+) and P(−) primers (13) using both the PCR-amplified fragments as templates. The final PCR product was cloned into pGEM-3 vector under T7 RNA polymerase promoter and sequenced to make sure that desired deletions were introduced into the clones and no other errors were present. The five small deletion mutants were designated PΔ1, PΔ2, PΔ3, PΔ4, and PΔ5, with amino acids 131 through 150, 151 through 170, 171 through 190, 191 through 210, and 211 through 220 deleted, respectively. In a similar manner, we also generated two other larger deletion mutants, PΔ6 and PΔ7, with amino acids 151 through 190 and 141 through 200 of the P protein deleted, respectively.
The plasmids pN, pP, and pL, carrying the coding sequences of N, P, and L proteins of VSV under the control of T7 RNA polymerase promoter, as well as the plasmid pVSVFL(+) containing an antigenomic-sense full-length VSV sequence, have been described previously (31, 38, 40). Construction of the plasmid p10BN, encoding an antigenomic-sense VSV minigenome, has also been described earlier (28, 33). The mutant P coding regions were replaced with wt sequence in the full-length VSV genome plasmid pVSVFL(+) using the EcoRV sites that flank the P gene as described earlier (13).
BHK-21 cells grown in 12-well plates to 90 to 95% confluence were infected with vTF7-3 at a multiplicity of infection (MOI) of 10 PFU/cell. Following adsorption for 45 min at 37°C, cells were washed with phosphate-buffered saline (PBS) and transfected with 1 μg of either wt or mutant P protein plasmids by using Lipofectamine2000 (Invitrogen) and Dulbecco's modified Eagle's medium (DMEM) without serum. At 4 to 6 h posttransfection, cells were washed twice with DMEM containing 5% FBS and incubated with 1 ml of the same medium for 16 to 18 h. The cells were then starved for 45 min at 37°C with 1 ml DMEM without methionine and cysteine and then labeled with 60 μCi/ml of Expre35S35S protein-labeling mix (NEN Life Sciences, MA) in 0.4 ml of the medium described above for a period of 2 h at 37°C. Following radiolabeling, the cells were washed twice with cold PBS and lysed with 200 μl of radioimmunoprecipitation assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (100 μg/ml) and leupeptin (1 μg/ml) as described previously (40). After clarification at 16,000 × g for 5 min, the lysate was incubated with rabbit anti-P antibody (1:500) overnight at 4°C with constant rotation. A 100-microliter slurry of protein A-Sepharose (Amersham) (4 mg protein A-Sepharose in 100 μl RIPA buffer) was added to each sample and incubated for 2 h at 4°C with constant rotation. The immunoprecipitates were washed three times with RIPA buffer and finally resuspended in 20 μl RIPA buffer. The samples were then mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled for 4 min, and analyzed by SDS-PAGE. The proteins were detected by fluorography as described previously (13).
For coimmunoprecipitation (co-IP) studies, BHK-21 cells infected with vTF7-3 as described above were cotransfected with 1 μg each of wt or mutant P protein plasmids along with pN or pL as described above. Following radiolabeling as described above, cells were washed twice with cold PBS and scraped into 200 μl co-IP buffer (0.1 M HEPES, pH 8.5, 0.05 M NH4Cl, 7 mM KCl, 4.5 mM manganese acetate, 0.25% NP-40) (8) containing phenylmethylsulfonyl fluoride (100 μg/ml) and leupeptin (1 μg/ml). The cell lysates were incubated on ice for 15 min and then clarified by centrifugation at 16,000 × g for 5 min at 4°C. Clarified lysate was used for immunoprecipitation with either rabbit anti-P polyclonal antibody, anti-N monoclonal antibody 10G4 (5), or polyclonal antibody to the NH2-terminal of L protein (47) for 18 h at 4°C with constant rotation. Subsequent analysis of proteins was performed as described above except that for all steps, the co-IP buffer was used in place of RIPA buffer.
To analyze the viral proteins in infected cells, BHK-21 cells were grown in six-well tissue culture plates to 90 to 95% confluence. Cells were infected with either wt or mutant virus at an MOI of 10 PFU/cell and incubated in 2.5 ml fresh medium containing 2% FBS for 4 h at 37°C. The cells were starved for 1 h with 0.6 ml of DMEM without methionine and cysteine and radiolabeled for 2 h as described above. The cells were washed and lysed in 300 μl RIPA buffer. One-tenth of the lysate was analyzed by SDS-PAGE, and the proteins were detected as described above. Western blot analysis of unlabeled proteins was performed as described previously (13).
BHK-21 cells were grown in six-well tissue culture plates to 90% confluence. To examine transcription activities of mutant P proteins, cells were infected with vTF7-3 at an MOI of 10 PFU/cell and subsequently transfected with 2.5 μg of p10BN, 1.5 μg of pN, 0.5 μg of pL, and 1 μg of wt or mutant pP plasmid by using Lipofectamine2000 as described above. At 4 h posttransfection, cells were washed twice and incubated with 2 ml of fresh medium containing 5% FBS for 16 h. RNAs were radiolabeled in the presence of actinomycin D and analyzed by electrophoresis in agarose-urea gel as described previously (13). To examine the replication activities of mutant P proteins, cells infected with vTF7-3 and transfected with pN, pL, and wt or mutant P plasmid were superinfected with DI particles and RNAs were radiolabeled and analyzed as described previously (13). Quantitation of the transcription and replication products was carried out by densitometric scanning of the fluorograms with a VersaDoc system using ImageQuant software (Bio-Rad). Normalized levels of transcription were determined as described before (39) by using the following formula: (levels of transcription/levels of replication) × 100. Viral RNAs synthesized in infected cells were also analyzed as described previously (13).
BHK-21 cells were grown in 60-mm tissue culture dishes to 80 to 90% confluence. Cells were infected with vTF7-3 at an MOI of 10 PFU/cell. Subsequently, cells were transfected with 4 μg of pVSVFL(+) encoding full-length viral genome with wt P protein or mutant P proteins, 6 μg of pN plasmid, 2 μg of pL plasmid, and 4 μg of pP plasmid by using Lipofectamine2000. At 4 h posttransfection, cells were washed twice and incubated with 2 ml of fresh medium containing 5% FBS for 48 h. Culture supernatant was harvested, clarified at 16,000 × g for 20 min, passed through a 0.45-μm syringe filter to remove vaccinia virus, and used to infect fresh BHK-21 cells. If virus was recovered from transfected cells as evidenced by development of cytopathic effects (CPE) during subsequent passage and also by indirect immunofluorescence of infected cells, the culture supernatants from transfected cells and amplification passage (P1) were titrated by plaque assay. Stocks of mutant viruses were prepared by infecting BHK-21 cells with a plaque-purified stock virus at an MOI of 0.1 PFU/cell. To confirm the identity of mutant viruses, BHK-21 cells were infected with an MOI of 10 of each virus. At 6 h postinfection, total RNA from cells was isolated by using Trizol (Invitrogen). Reverse transcriptase PCR was performed using Moloney murine leukemia virus reverse transcriptase and Taq DNA polymerase (Invitrogen). PCR products were analyzed on agarose gels and directly sequenced to determine the presence of the desired mutation.
Single-step growth kinetics of mutant and wt viruses in BHK-21 cells were determined essentially as described previously (13).
BHK-21 cells infected with wt virus or mutant viruses from transfected-cell supernatants were fixed with acetone-methanol (1:1) at −20°C and incubated with a monoclonal anti-N antibody 10G4. The cells were then incubated with the secondary antibody, anti-mouse Alexa-488 (Molecular Probes) and examined by a Nikon fluorescence microscope attached with a camera (Optronics). The images were processed by using Photoshop software.
BHK-21 cells grown to 80% confluence were cotransfected with two plasmids: (i) pCI-T7, a derivative of the plasmid pCI-Neo (Promega), in which the gene encoding the neomycin phosphotransferase was replaced with the gene encoding the T7 RNA polymerase under the control of cytomegalovirus promoter in the plasmid, and (ii) the plasmid pP. At 24 h posttransfection, approximately 75% of cells expressed the P protein, as judged by the immunofluorescent staining of the cells. The cells were then superinfected with culture supernatant containing the VSV PΔ7 mutant (VSV-PΔ7). After 48 h postinfection, culture supernatants were collected and VSV-PΔ7 particles were titrated by plaque assay in cells expressing the P protein by using the two-plasmid system as described above.
The role of the hypervariable hinge region (Fig. (Fig.1A)1A) of the VSV P protein in viral genome transcription and replication is not known. To examine its role, we generated a series of Tn-induced insertion mutants in this region of the P protein and determined the activities of the mutant proteins in viral RNA synthesis. From an initial panel of mutants, we selected eight mutants with insertions within the hinge region and the flanking sequences. Each mutant contained an insertion of 19 amino acids, and the insertions were approximately 7 to 15 amino acids apart (Fig. (Fig.1B).1B). Each insertion maintained the ORF of the P protein. In addition, each mutant contained only one copy of the insert. To determine if the insertion resulted in viable and stable proteins, we examined the expression of these proteins in transfected cells. Accordingly, BHK-21 cells were transiently transfected with plasmids encoding either wt or mutant P proteins following vTF7-3 infection and subsequently analyzed the radiolabeled proteins by immunoprecipitation with anti-P protein antibody and SDS-PAGE. As can be seen from Fig. Fig.1C,1C, each of the mutant P proteins was easily detected. The mutant P proteins possessed slightly reduced electrophoretic mobility compared to the wt P protein. In addition, the mutant proteins exhibited minor differences in electrophoretic mobility, a result which is most likely due to differences in the amino acid compositions of the insertion sequences, since the mutants contained insertions in each of the three reading frames. It should be noted that the majority of the mutant proteins were detected at levels greater than those of the wt P proteins. The reason(s) for this is unclear but could be due to changes in antibody reactivity. Pulse-chase analysis showed that the mutant proteins were as stable as the wt P protein (data not shown).
In order to examine the effect of insertion mutation on P protein function in viral RNA synthesis, we used DI particles to determine the replication activities of various P mutants. The DI particle genomes are only replication competent and therefore are used to assess the replication activities of the mutant P proteins in the absence of transcription. BHK-21 cells infected with vTF7-3 were transfected with the plasmids encoding N, L, and wt or mutant P proteins. Cells were subsequently superinfected with DI particles, and replication of DI RNA was examined by radiolabeling in the presence of actinomycin D and analyzing the total RNA by electrophoresis. Results from such an experiment (Fig. (Fig.2A)2A) show that the mutant proteins supported replication of DI RNA to different extents. A quantitative analysis (Fig. (Fig.2B)2B) of data from three independent experiments revealed that the level of replication supported by PTn136 and PTn180 was significantly reduced with less than 20% activity of the wt protein. PTn148, PTn159, and PTn189 supported replication at levels 20 to 50% of the wt protein. PTn167 and PTn196 proteins were 60 to 85% as active as the wt protein. Interestingly, one insertion mutant, PTn220 was completely defective in supporting replication.
To examine if the mutant proteins possessed altered transcription activities, we used a transcription- and replication-competent minigenome template encoded in the plasmid p10BN (28). RNA generated from this plasmid by T7 RNA polymerase is a positive-sense antigenomic RNA which must undergo replication to generate genome-sense RNA that serves as the template for transcription to generate NΔL mRNA. To perform the experiment, BHK-21 cells infected with vTF7-3 were transfected with the minigenome plasmid p10BN and the plasmids encoding N, L, and wt or mutant P proteins. At 16 to 18 h posttransfection, the cells were radiolabeled in the presence of actinomycin D. Total radiolabeled RNA was isolated and analyzed by electrophoresis. The synthesis of NΔL mRNA was used as a measure of transcription. Results presented in Fig. Fig.2C2C show that the mutant P proteins supported different levels of transcription relative to that of the wt P protein. Since transcription of NΔL mRNA was dependent on replication of the template, we normalized the levels of transcription relative to the levels of replication as described previously (39) and in Materials and Methods. Normalized levels of transcription would therefore measure the transcription activities of the mutant proteins relative to that of the wt P protein. A quantitative analysis of normalized levels of transcription from three independent experiments (Fig. (Fig.2C)2C) revealed that the majority of the insertions had no significant adverse effects on the transcription of the minigenome. Only two mutants, PTn136 and PTn180, possessed transcription activities that were 40 to 60% of that of the wt protein. Thus, the results from the studies described above suggest that insertions within the hypervariable hinge region have negative effects mostly on viral RNA replication. It should be noted that the transcription activity of PTn220 could not be directly evaluated due to the defect of PTn220 in supporting replication.
Since Tn-induced insertions within the hinge region or sequences flanking the hinge region affected the P protein function in viral RNA synthesis to various extents, we were interested to examine further the role of this region in P protein function by generating deletion mutants spanning this region. To this end, we generated a series of deletion mutants of P protein: PΔ1, PΔ2, PΔ3, PΔ4, and PΔ5, with amino acids 131 to 150, 151 to 170, 171 to 190, 191 to 210, and 211 to 220 deleted, respectively (Fig. (Fig.3A).3A). All the deletion mutants expressed P proteins with sizes consistent with the extents of deletion (Fig. (Fig.3B).3B). To analyze the effect of these deletions on replication activity of the proteins, DI RNA replication in cells expressing the mutant proteins was examined. Results (Fig. (Fig.3C)3C) show that PΔ1, PΔ2, and PΔ3 were active in supporting replication of DI RNA, whereas PΔ4 and PΔ5 were unable to support detectable levels of replication. A quantitative determination of the levels of replication supported by these mutants from three independent experiments (Fig. (Fig.3D)3D) revealed that PΔ1 is about 42% as active as the wt protein. PΔ2 and PΔ3 proteins possessed 77% and 67% activity of the wt protein. PΔ4 and PΔ5 proteins were completely inactive. These results suggest that the hypervariable region of the P protein plays a critical role in the function of the P protein in replication.
We also examined the transcription activities of the deletion mutant P proteins to determine if these proteins have defects in viral RNA transcription. The experiment was performed as described in the legend to Fig. Fig.2C,2C, and the result from one of the experiments is shown in Fig. Fig.3E.3E. When the normalized level of transcription from three independent studies was calculated, it was observed that PΔ2 and PΔ3 were nearly as active as the wt protein, whereas PΔ1 possessed only 28% activity of the P protein. As expected, PΔ4 and PΔ5 proteins were completely inactive in transcription, since in this assay, transcription was dependent on replication of the antigenomic-sense viral RNA template.
Since some of the mutants with 20 amino acids deleted from the hinge region (PΔ2 and PΔ3) resulted in almost fully functional proteins, we were interested to determine if larger deletions within this region would perturb the overall structure of the protein, rendering the mutant proteins less active or inactive in function. We generated two larger deletion mutants in which 40 (PΔ6) or 60 (PΔ7) amino acids from the P protein were deleted (Fig. (Fig.3A).3A). These proteins were easily detected in transfected cells (Fig. (Fig.3B).3B). Interestingly, such large deletions in the P protein did not have major adverse effects on replication, as the levels of replication supported by these proteins were 60 to 70% of that of the wt protein (Fig. 3C and D). Normalized levels of transcription also remained nearly unaffected by the deletion of these regions from the P protein (Fig. (Fig.3E3E).
Since certain insertion (PTn220) and deletion (PΔ4 and PΔ5) mutants of P protein were nonfunctional in viral RNA synthesis, we wanted to examine whether the interactions of these proteins with N and L proteins were affected. To perform these studies, plasmids encoding wt or mutant P proteins were cotransfected along with either the plasmid encoding the N or L protein into cells infected with vTF7-3. Following radiolabeling of the proteins, cell extracts were prepared and interactions of N or L proteins with the wt or mutant P proteins were examined by co-IP of P protein with antibody to either the N or L protein. Wt P protein could not be immunoprecipitated with anti-N (Fig. (Fig.4A,4A, top panel, lane 1, and C, lane 1) or anti-L (Fig. 4B and D, lanes 1) antibodies, demonstrating that these antibodies were specific only to the N or L protein, respectively. However, anti-N antibody could immunoprecipitate both N and P proteins from cell lysates of cells coexpressing both proteins (Fig. 4A and C, lanes 4), suggesting that wt P protein interacts with the N protein in cells expressing both proteins. PTn220, which is nonfunctional in viral RNA synthesis, and PTn196, which is functional, were found to interact with N protein, as these proteins could be immunoprecipitated with the N antibody (lanes 5 and 6). It should be noted that PTn220 comigrates with N protein, and therefore, in the gel shown in Fig. Fig.4A4A (top panel), it could not be resolved from N protein. However, Western blot analysis of unlabeled and similarly immunoprecipitated proteins demonstrated the presence of PTn220 (Fig. (Fig.4A,4A, bottom panel, lane 5). In a similar study, we showed that PTn220 also interacts with the L protein as PTn220 could be coimmunoprecipitated with anti-L protein antibody (Fig. (Fig.4B,4B, lane 5). Thus, these results show that the interactions of PTn220 with the N and L proteins remained unaffected by the insertion, even though the protein was nonfunctional in viral RNA synthesis.
In similar co-IP studies, we observed that PΔ4 and PΔ5, which are nonfunctional in viral RNA synthesis, and PΔ2, which is functional in viral RNA synthesis, interacted with the N protein (Fig. (Fig.4C)4C) and the L protein (Fig. (Fig.4D).4D). Taken together, these studies suggest that insertions within or deletions from the hypervariable hinge region of the P protein have no effect on interactions with N or L protein.
From the insertion and deletion mutagenesis studies described above, we had identified mutants in the hypervariable region that were defective in viral RNA synthesis. In addition, several mutants with insertions in other regions of the P protein were also found to be nonfunctional (S. C. Das and A. K. Pattnaik, unpublished data). Since the P protein is modular with distinct domains, we were interested to determine if two inactive mutant proteins, when coexpressed, could reconstitute the activity of the P protein. Using pairwise combinations of various inactive mutant P proteins, we were unsuccessful in reconstituting the activity of P protein in either transcription or replication (data not shown), indicating that trans complementation using pairs of inactive P mutants does not occur in this system. However, during our studies, we observed that PTn220, which is completely defective in supporting transcription and replication, when coexpressed with the wt P protein, inhibited the activity of the wt P protein, suggesting that PTn220 may be a dominant-negative mutant. To examine this possibility, we analyzed transcription in cells coexpressing PTn220 and Pwt proteins. The data presented in Fig. Fig.5A5A show that coexpression of a fixed amount of Pwt with various amounts of PTn220 resulted in significant inhibition of transcription (lanes 6 to 8). This inhibitory activity was more evident when levels of transcription supported by a given amount of Pwt were compared with levels of transcription supported by the same amount of both proteins together. The level of transcription with 4 μg of total plasmids (2 μg Pwt plus 2 μg PTn220) was about fivefold less than that with 4 μg of Pwt plasmid (Fig. (Fig.5A,5A, compare lanes 3 and 7). Similarly, the amount of transcription with 6 μg of total plasmids (2 μg Pwt plus 4 μg PTn220) was approximately 12-fold less than that obtained with 6 μg of Pwt (Fig. (Fig.5A,5A, compare lanes 4 and 8). Furthermore, when 2 μg of Pwt was used with increasing amounts of PTn220, increased inhibition in a dose-dependent manner was observed (Fig. (Fig.5A,5A, compare lane 2 with lanes 6 to 8 and the corresponding histograms in Fig. Fig.5B).5B). These results demonstrate that PTn220 inhibits the RNA synthesis activity of Pwt protein in vivo and therefore acts as a dominant-negative mutant of P protein function.
It should be noted that similar studies with PΔ4 and PΔ5 mutants indicated that these proteins do not function as dominant-negative mutants (data not shown).
In order to examine whether recombinant VSVs encoding deletion mutants of P protein can be recovered, we subcloned the mutant P protein coding regions in place of the wt P coding sequences in the plasmid encoding the full-length VSV genome (31). We chose to use the functional mutant P proteins, PΔ2, PΔ6, and PΔ7, in which 20, 40, and 60 amino acids were deleted, respectively, spanning the hypervariable hinge region of the P protein. Plasmids encoding full-length VSV genome containing the deletion mutant P proteins were used to recover recombinant VSVs from transfected cells as described previously (13). Recombinant VSVs with deletion mutants of P protein (VSV-PΔ2, VSV-PΔ6, and VSV-PΔ7) could be recovered from transfected cells, as evidenced by examining the cells infected with the transfected-cell supernatants for CPE and performing indirect immunofluorescence using anti-VSV antibody. Although VSV-PΔ2 and VSV-PΔ6 viruses could be amplified to high titers by passaging of the transfected-cell supernatants, VSV-PΔ7 virus could not be amplified by passaging of the supernatants similarly. Further studies on VSV-PΔ7 virus, which appeared to have a defect in growth, are reported in the next section.
Since the overall titers of these mutant viruses (VSV-PΔ2 and VSV-PΔ6) were low compared to that of wt VSV, we wanted to examine the growth kinetics of these viruses. Determination of single-cycle growth curves by using plaque-purified virus stocks showed that both VSV-PΔ2 and VSV-PΔ6 exhibited remarkably slower growth kinetics than VSV-Pwt virus (Fig. (Fig.6A).6A). The final highest titers of these viruses reached approximately 3 × 107 and 3 × 106 PFU/ml, respectively, which were nearly 10- and 100-fold less than that obtained for VSV-Pwt. Interestingly, both mutant viruses possessed small-plaque phenotype. On average, the size of the plaques generated by plaque-purified stocks of VSV-PΔ2 and VSV-PΔ6 were approximately 1.26 mm and 0.64 mm in diameter, respectively, compared to the size of 1.99 mm for VSV-Pwt virus (Fig. (Fig.6B6B).
To determine if the plaque size and viral growth kinetics correlated with the extent of viral macromolecular synthesis in infected cells, we examined viral protein and RNA synthesis in cells infected with these viruses. Our results show that the overall synthesis of viral proteins (Fig. (Fig.6C)6C) or viral RNA (Fig. (Fig.6D)6D) was not significantly different from that obtained with VSV-Pwt virus-infected cells. These results indicate that in these mutant viruses, the extent of viral protein and RNA synthesis did not correlate with the viral growth kinetics or plaque size.
During recovery of viruses encoding deletion mutants of the P protein, we consistently observed that VSV-PΔ7 virus could be detected in the supernatants of initially transfected cells as evidenced by the observation of CPE and immunofluorescent detection of viral proteins in cells subsequently infected with the transfected-cell supernatants (Fig. (Fig.7A).7A). However, further passage of the culture supernatants to amplify the virus resulted in complete loss of PΔ7 by passage 3, and the cells did not exhibit cytopathic effects nor could the viral proteins be detected by immunofluorescent staining. In addition, attempts to detect VSV-PΔ7 in transfected-cell supernatant by plaque assay were unsuccessful. It is possible that PΔ7 protein, which is competent to support high levels of transcription and replication of the viral genome (Fig. (Fig.3),3), may have some inherent defect in packaging of the viral genome into particles. The wt P protein synthesized from the support plasmid used in the initial transfection may have been responsible for generating VSV-PΔ7. As VSV-PΔ7 was passaged into naïve cells, the wt P protein may have been diluted with each passage, resulting in the complete loss of VSV-PΔ7 by passage 3. We reasoned that if this was true, it may be possible to amplify VSV-PΔ7 to high titers by passaging the virus in cells expressing the wt P protein. Furthermore, it may also be possible to detect infectious units by plaque assay in cells expressing the wt P protein.
To examine these possibilities, BHK-21 cells were transfected with two plasmids: one encoding the T7 RNA polymerase under cytomegalovirus promoter and the other encoding the wt P protein under T7 RNA polymerase promoter. P protein expression was detected in nearly 70 to 75% of the cells at 24 h posttransfection (data not shown). When VSV-PΔ7 virus from transfected-cell supernatant was used to infect the cells expressing wt P protein, distinct plaques on cell monolayers could be seen by 20 to 24 h postinfection (data not shown). Generation of these plaques indicated that the wt P protein facilitated formation of VSV-PΔ7 particles, which could undergo multiple rounds of infection of surrounding cells to generate the plaque.
To determine if VSV-PΔ7 generated from transfected cells could be amplified in cells expressing wt P protein, the transfected-cell supernatant was used as inoculum to infect BHK-21 cells expressing the wt P protein as described above. For a control, the same inoculum was also used to infect BHK-21 cells not expressing the wt P protein. At 48 h postinfection, culture supernatants from the infected cells were collected and the titers of VSV-PΔ7 in each were determined by using the plaque assay as described above. Results obtained from three independent experiments (Fig. (Fig.7B)7B) indicate that VSV-PΔ7 in cells expressing wt P protein was considerably amplified and the titers on average reached 7 × 106 PFU/ml, which is 10,000- to 20,000-fold higher than the titers obtained from cells not expressing the wt P protein.
In the experiments described above, it is possible that VSV-PΔ7 could have potentially recombined with sequences containing the wt P gene and generated a wt VSV that could grow to high titers as observed. However, by plaque assay in cells not expressing wt P protein, we were unable to obtain plaques of these viruses (data not shown), indicating that recombination to generate wt VSV did not occur. Furthermore, we also analyzed viral mRNAs synthesized in cells infected with these viruses and found that VSV-PΔ7 synthesized PΔ7 mRNA with electrophoretic mobility consistent with the deletion (Fig. (Fig.7C).7C). With the same multiplicity of infection, the levels of mRNAs synthesized in VSV-PΔ7 virus-infected cells expressing the wt P protein were greater than those synthesized in wt VSV-infected cells not expressing the plasmid-driven wt P protein (Fig. (Fig.7C,7C, compare lanes 2 and 3). This is likely due to the expression of two functional P proteins in the system that could drive higher levels of synthesis of mRNAs, since similar levels of mRNAs were synthesized in cells not expressing the plasmid-derived wt P protein (Fig. (Fig.7C,7C, compare lanes 2 and 4).
In this study, by using a series of insertion and deletion mutants, we examined the role of the hypervariable hinge region and the flanking sequences of VSV P protein in viral RNA synthesis and recovery of infectious particles. Our results suggest that some of the sequences in this central region are required for the activity of the protein in viral RNA synthesis. Furthermore, our studies demonstrate that sequences present in this region play a critical role in the assembly and generation of infectious VSV, thus delineating a previously unidentified role for the P protein in the life cycle of this virus.
The hinge region of the P protein links two functionally well-characterized domains of the protein, namely, domains I and II, which have been shown to play pivotal roles in viral RNA transcription and replication, respectively (20, 27, 35, 39, 49). It is maintained throughout evolution in all vesiculoviruses, though with much flexibility and considerable variability. In addition, compared to the hinge region in VSVNJ, the region in Chandipura virus is longer and that in VSVI is shorter (37). One postulated function of this hypervariable region is that it acts as a spacer to maintain the correct spatial arrangement of the two active domains of the protein (4). Experimental demonstration using in vitro transcription studies in which the function of the P protein was shown to be affected by alteration of the spatial organization of the two functional domains (I and II) through disruption of the hypervariable region (9, 11) is consistent with this interpretation. Analogous to the VSV P hinge region, putative spacer-like regions within other transcription-associated proteins with modular structures, e.g., yeast transcriptional activators, have been described (25, 34, 43).
Although this hinge region of the P protein shows considerable sequence plasticity, it is not necessarily without any role in the biology of the virus. Our mutational studies provide for the first time a definitive role for this domain in the function of the protein in viral RNA synthesis. Tn-mediated insertions showed that positioning of the insertions at different regions affected the transcription and replication activity of P protein to various extents. Insertions at some positions (amino acids 136 and 180) reduced the replication activities to <20%. The lower activities might be due to the perturbation of the functional domains which might affect their contacts with various components of the replication machinery. It is unlikely that the sequence of the insertion per se is responsible for the observed reduction in transcription and replication, as some of the insertion mutants (PTn196) retained nearly full activities in viral RNA synthesis. Although the activity of the P protein was affected by the insertions, there was no apparent correlation between the level of activity and the position of the insertion in the hinge region relative to either of the functional domains. The reason(s) for this is not clear at this time. It should be noted that although the insertion of 19 amino acids at various positions within the hinge region affected P protein function to various extents, none of the insertion mutants was fully defective. On the other hand, the insertion of larger sequences (for example, enhanced green fluorescent protein) at some of these positions led to completely inactive P protein, whereas functional P proteins could be obtained by the insertion of enhanced green fluorescent protein at other positions (Das and Pattnaik, unpublished data).
We and others have previously shown that phosphorylation in domain I residues is the major determinant of transcription activity of P protein with no significant adverse effects on replication (39, 49), whereas phosphorylation of domain II residues is required for replication but not for transcription (20, 27, 35, 39). In the case of the insertion mutants within the hinge region, both replication and transcription functions of the P protein were affected (Fig. (Fig.2).2). These results suggest that although domain I and II are involved in discrete functions of the protein, the spatial arrangement of the functional domains relative to each other is important for the overall function of the P protein.
Our results from studies with the deletion mutants not only further strengthened the notion that proper spacing of the domains is important for optimal activity of the P protein but also revealed that sequences within this region are required for its function. Mutants of P protein (PΔ1, PΔ2, and PΔ3) from which 20 amino acids, spanning residues 131 to 150, 151 to 170, or 171 to 190, from the amino-terminal part of the hinge were deleted possessed significant levels of activity, whereas mutants from which 20 or 10 amino acids, spanning residues 191 to 210 or 211 to 220 (as in PΔ4 and PΔ5), from the carboxy-terminal part of the hinge region were deleted resulted in fully defective P protein (Fig. (Fig.3).3). From further deletion mapping studies, it became clear that the region between amino acids 140 and 201 is able to tolerate deletions without significant adverse effects on viral RNA synthesis. It is possible that the deletion of residues in the hinge region could bring the functional domains I and II closer to each other and potentially influence the activity of the protein by steric hindrance of the functional domains as has been seen for PΔ1, PΔ2, PΔ3, PΔ6, and PΔ7 mutants. Alternatively, since the activity of a polypeptide is determined by its properly folded structure (46), the removal of the hinge region may have an adverse effect on the three-dimensional structure of the P protein, resulting in reduced activity. We conclude that the length of the hinge region is critical for optimal functioning of P protein. This observation is consistent with previous results obtained from various in vitro transcription studies in which disruption of the linkage of the P protein functional domains negatively affected the viral polymerase activity (9, 11).
The observations that the removal of 20 (residues 191 through 210) or 10 (residues 211 through 220) amino acids from the hinge region led to completely nonfunctional P proteins (PΔ4 and PΔ5), whereas the removal of 60 (residues 141 through 200) amino acids resulted in a functional protein (PΔ7), suggesting that residues 201 through 220 play an important role in the function of the protein. The exact role that this region plays in the function of the P protein is currently a matter of speculation.
The insertion and deletion mutants that are nonfunctional did not show any obvious loss of interactions of P protein with the other known components of the viral replication machinery, namely, the N and the L proteins. This observation is in agreement with previous data, obtained with a yeast two-hybrid system, in which it was shown that the acidic N-terminal domain I and the basic C-terminal domain III of P protein are involved in the interaction with the N protein, whereas the hinge region is not required for the interaction (50). The L protein binding region on P protein has also been located at the N-terminal acidic domain (17). It must be emphasized here that the insertion and deletion mutants that maintained their interactions with N and L proteins still lost their transcription activities. It is possible that this region of P protein spanning residues 201 through 220 may be involved in interactions with other unknown components of the viral replication machinery or with other cellular components required for viral RNA synthesis.
The observation that PTn220 acts as a dominant-negative mutant and inhibits the function of the wt P protein is quite interesting. Since PTn220 is not defective in its interaction with the N or L protein, it could therefore compete with Pwt for binding to the replication machinery resulting in the inhibition of viral RNA synthesis. Alternatively, since P protein exists in oligomeric form in infected cells (20), PTn220 may interact with the wt P protein, forming P oligomers that are inactive in polymerase functions. On the other hand, it is intriguing that PΔ4 and PΔ5 do not function as dominant-negative mutants. Both of these proteins interacted with the N and L proteins and were somewhat expected to function as dominant-negative inhibitors of P protein function. It is possible that these deletion mutants had lost their interactions with the cellular proteins involved in viral RNA synthesis, thereby not affecting the formation of functional viral polymerase complexes. Further studies using these mutants may lead to an understanding of this phenotype.
The adverse effect of the deletion in the hinge region on virus growth is remarkable. The viruses with 20 and 40 amino acids removed from the hinge region (VSV-PΔ2 and VSV-PΔ6) grew to titers that were approximately 10- and 100-fold less, respectively, than that of the wild-type VSV (Fig. (Fig.6),6), even though the RNA synthetic functions of the proteins were not adversely affected (Fig. (Fig.3).3). What is more interesting is that both mutant viruses exhibited small-plaque morphology. The sizes of the plaques generated with the mutant viruses were significantly smaller than those obtained with wt VSV. The small-plaque phenotype does not appear to depend on the extent on viral macromolecular synthesis, since approximately similar levels of viral proteins and mRNAs were synthesized in infected cells. These results suggest that the extents of viral protein and RNA synthesis are not the sole determinants of viral growth kinetics and plaque size. Other factors, such as localization of viral nucleocapsids at appropriate sites for assembly and interactions of nucleocapsids with other viral proteins involved in viral assembly, in particular, with the M protein may have been affected in VSV-PΔ2 and VSV-PΔ6 viruses, resulting in retarded growth and small-plaque phenotype.
Perhaps one of the most interesting aspects of the present study is the observation that VSV-PΔ7, which has 60 amino acids (residues 141 through 200) deleted from the P protein that was still functional in viral RNA synthesis, was highly debilitated in growth and could not be amplified with multiple passages. Passaging of the virus in cell culture resulted in loss of the virus, even though the virus was initially recovered from the transfected-cell supernatants. We suspected that the mutant P protein may have lost the ability to generate infectious VSV that is competent for multiple rounds of passaging. Initial recovery of VSV-PΔ7 in transfected-cell supernatant may have been due to the presence of wt P protein synthesized from the transfected P plasmid provided in the support plasmid mix. Since the P protein possesses redistributive properties (24), passaging of the virus may have resulted in dilution of the P protein, eventually leading to the complete loss of wt P protein and therefore, loss of VSV-PΔ7. This contention is supported by data from two experiments. First, VSV-PΔ7 could be amplified to high titers by passaging the virus in cells expressing the wt P protein (Fig. (Fig.7B).7B). Second, when the plasmid encoding PΔ7 was used in the support plasmid mix for recovery of virus, VSV-PΔ7 virus could not be detected in supernatants of transfected cells as examined by immunofluorescent staining of cells infected with the supernatant (data not shown). We conclude from these studies that, in addition to having a role in viral RNA transcription and replication, the P protein also plays a role in viral assembly. These two functions are separable, since the deletion of certain regions led to a protein that is defective in viral assembly while maintaining significantly high levels of RNA synthetic activity. From examination of the deletion mutants used in this study, it appears that the region of P protein spanning amino acids 191 through 200 may be responsible for this new function. The role of P protein in VSV assembly is not known at this time. It may mediate the interaction of the viral nucleocapsid with the M protein either directly or indirectly for the assembly of particles. Alternatively, it may be involved in the transport and proper localization of the viral nucleocapsids to the sites of viral assembly. Further detailed studies will be necessary to provide an understanding of the mechanism of action of the P protein in viral assembly.
In summary, using Tn-mediated insertion mutagenesis and deletion mutagenesis, we have shown that the hinge region of the P protein plays a role in viral RNA synthesis. Furthermore, our studies demonstrate a new function for the viral P protein: it is involved in assembly of infectious VSV particles.
We thank Debasis Nayak, Angela Martinsen, and Laura Pickering for assistance in the construction of some of the mutants. We also thank You Zhou, Center for Biotechnology, University of Nebraska-Lincoln, for help in fluorescence microscopic studies.
This paper is publication no. 14918 of the Agricultural Research Division of the Institute of Agriculture and Natural Resources of the University of Nebraska-Lincoln.
This investigation was supported by Public Health Service grant AI-34956 from the National Institutes of Health and also in part by P20RR15635 from the Centers of Biomedical Research Excellence program of the National Center for Research Resources, National Institutes of Health.
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SYLVANIA, Ohio — Laura Diaz almost chose to walk away from golf to spend more time at home with her family.Now she's making the decision to juggle both jobs seem like a shrewd move."I love being a mom; it's the greatest thing in the world," she said after following a 9-under 62 with a solid 69 on Friday to maintain a three-shot lead through 36 holes at the Marathon Classic."It's hard for me when I have to choose. So I made a list. My family is first, and then golf comes second and we work everything out in between."Chasing her first win since 2002, Diaz is at 11-under 131, three shots ahead of Lee-Anne Pace and Lydia Ko.Diaz led by four shots over Pace and Austin Ernst after the first round.She was pleased her game was sturdy enough to hang on to the lead despite being in the heat of contention for the first time in several years."It was just a challenge because I haven't been in this position in a very long time," Diaz said. "For sure, I haven't had cameras on me in a long time."So she took the opportunity to say hello to her kids — 8-year-old son Cooper and 4-year-old daughter Lilly."(The cameras) get in my face and I don't really know what to do," she said. "I said, 'Hi, kiddos. I love you.' That kind of stuff."She made sure the cameras stuck around with her strong play down the stretch.After a nine-birdie, no-bogey effort in the first round, she showed signs of faltering early on Friday.She had her first bogey of the tournament on the sixth hole to fall back into a tie with Pace and Ko, but then regained her touch after getting par on the next six holes.Starting at the 13th, she birdied four of the next six, including the par-5 closing hole.Diaz was a rising star early in her career, winning twice in 2002 and playing on four Solheim Cup teams. After she married Kevin Diaz, she found that she liked being a wife and mother more than traveling the world playing golf. She hasn't been in the top 20 of a tournament since 2010.Before the season she was torn between coming out on tour and staying home so as not to miss any of the daily routine. In the end, she decided to keep playing."(It was) knowing that I could manage being a mom and play. I think that's something I've struggled with, for, well eight years," she said. "When I only had one (child) I think I got to a point where I was OK. But with two you've got twice as much to get done.Page 2 of 2 - Pace, a South African who is an alum of the University of Tulsa, is looking for a breakthrough win on American soil. After a 68, she couldn't contain herself."I'm very happy about (my) position," said Pace, who has won eight times on the Ladies European Tour. "I would like to be in contention on the weekend. That's when the fun starts, right?"The 17-year-old Ko is bidding for a second LPGA Tour title as a pro to go with the two she won as an amateur. She shot a second consecutive 67, turning in 1 under and then posting three birdies on the inward nine.The key, she said, was to keep her emotions in check."I try and be calm. You know, that's what you have to do," she said, sounding far older than a rookie. "Being overly happy or overly angry, that's not going to help me."Lurking another shot back at 135 are 2012 Marathon winner So Yeon Ryu and Rebecca Lee-Bentham, both of whom shot 67s.Ryu was grouped with U.S. Open winner Michelle Wie and last week's Women's British Open champion Mo Martin. A large gallery followed the threesome."It was really great to be playing with the two major champions," Ryu said. "I felt like I was one of the other major champions."Brittany Lang had the day's low round (66) and was at 136 along with Kayla Mortellaro. Cristie Kerr (67) led the pack of six golfers at 137.Defending champ Beatriz Recari of Spain followed a 70 with a 68 and was at 138 along with Martin, who shot a 71. Wie missed the cut by seven strokes.
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