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The Supreme Court of Kentucky will hear civil cases that originated in Magoffin and Pike counties when it convenes Wednesday, April 27, in Frankfort. Proceedings are open to the public and will take place in the Supreme Court Courtroom on the second floor of the state Capitol at 700 Capitol Ave.The public may also observe oral arguments via the Supreme Court live stream on the Kentucky Court of Justice website. Oral arguments are available online as they occur in real time and are not available as archives. The Supreme Court is the state court of last resort and the final interpreter of Kentucky law. Seven justices (bios) sit on the Supreme Court and all seven justices rule on appeals that come before the court. The justices are elected from seven appellate districts and serve eight-year terms. A chief justice, chosen for a four-year term by fellow justices, is the administrative head of the state's court system and is responsible for its operation. The Supreme Court may order a ruling or opinion to be published, which means that the ruling becomes the case law governing all similar cases in the future in Kentucky.WEDNESDAY, APRIL 27, 201610 AM2015-SC-000408-MRJOHN DOE, NO. 1, ET AL V. HON. EDDY COLEMAN, JUDGE, PIKE CIRCUIT COURT, ET AL. View case briefsSummary: "Writ of Prohibition. Disclosure of the identity of an anonymous internet speaker."Pike Circuit Court, Judge Eddy ColemanAttorney for Appellants: Lawrence R. WebsterAttorneys for Appellee: Richard A. Getty and Danielle Brown11 AM2015-SC-000572-DGE CHARLES HARDIN, M.D. V. JOHN MONTGOMERY, ET AL.2015-SC-000575-DGEMAGOFFIN COUNTY BOARD OF ELECTIONS, ET AL. V. JOHN MONTGOMERY, ET AL. View case briefsSummary: "Elections. Issues involve deviations from the election statutes, especially with respect to applications for and counting of absentee ballots, and the impact upon the validity of the 2014 election for Magoffin County Judge Executive."Magoffin Circuit Court, John David PrestonAttorneys for Charles Hardin, M.D.: Eldred E. Adams, Jr. and James Lee DeckardAttorney for John Montgomery: Gordon B. LongAttorneys for Magoffin County Board of Elections, Renee Arnett-Shepherd, Magoffin County Clerk, Carson Montgomery, in his official capacity as a member of the Magoffin County Board of Elections, Susie Salyer, in her official capacity as a member of the Magoffin County Board of Elections, and Justin Williams, in his official capacity as a member of the Magoffin County Board of Elections: Jason Michael Nemes and Matthew Cory Williams
1001 Vandalay Drive, Frankfort, KY 40601


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Shuttle Services
Shuttle Services
Attention riders: Now you can track the Shuttle!
This spring, Rowan University introduces Shuttle Tracker, an app for iPhone and Android devices that enables users to see, in real time, where the campus shuttle is so they can make the most of the shuttle service.
Live Shuttle Tracker Access
iOS App Store OR Android Marketplace
Shuttle Tracking on a Desktop
The FREE app, collects data from shuttle-mounted GPS units and displays it on a map so riders can track the vehicles' locations. The app also features SMS text messaging of location information.
- Download Shuttle Tracker for iOS OR Download Shuttle Tracker for Android
- Search for "Rowan University" in Transit Systems
- Select the "Routes" icon and choose the shuttles you want to follow
Maps and Shuttle Information
Effective Jan. 19, 2016, Rowan University will run three shuttle routes: Internal, Campus-to-Campus (Glassboro-Camden) and South Jersey Technology Park (SJTP). Shuttle schedules are available and services are free to Rowan students, faculty and staff with valid Rowan ID.
Internal Shuttle Times & Stops
The Internal Shuttle serving the main campus will run from 7:00 a.m. to 1:00 a.m., Monday through Friday, during fall and spring semesters. A second shuttle will run from 7:00 a.m. to 4:15 p.m to reduce wait times to no more than 20 minutes. The Internal Shuttle route was revised for 2016 and includes additional stops and times.
Campus-to-Campus Times & Stops
The Campus-to-Campus (Glassboro-Camden) locations for spring semester are Robinson/James Halls and the Camden Academic Building. This shuttle runs Monday through Thursday from 7:15 a.m. to 11:00 p.m. and on Friday from 7:15 a.m. to 5:00 p.m. (except for Glassboro outbound, which runs until 6:00 p.m.).
South Jersey Technology Park Shuttle Times & Stops
For service to the SJTP, please catch the Campus-to Campus shuttle at stop #9-Robinson/James Halls. Not all Campus-to-Campus shuttles stop at SJTP. Please review the SJTP shuttle schedule for times and details.
South Jersey Public Transportation
For service away from campus we have partnered with the South Jersey Transportation Authority (SJTA). For $1, riders may take SJTA's Pureland East-West Community shuttle to/from:
- Avandale Park and Ride in Sicklerville
- Glassboro
- Mullica Hill
- Swedesboro
- Pureland Industrial Complex and points between stops
The SJTA shuttle drops off riders on Main Street near Rowan Boulevard Apartments (Pureland stop #5), one block from Rowan's Internal Shuttle routes 1 and 19. Visit the Pureland East-West shuttle site for schedules and more information.
The Pureland East-West shuttle also connects with NJTransit routes 313, 316, 400, 401, 402, 408, 410, 412, 459, 463, and 551. Transfers are available between the NJTransit routes and the Pureland East-West shuttle, providing a free or discounted ride.
For More Information
Please contact John Aderinto in the Division of Student Life at email@example.com.
Additional Contacts
South Jersey Transportation Authority
856-614-1072 After 5:00 p.m. call 856-993-8048
Rowan University Shuttle Coordinator, John Aderinto
Savitz Hall, Room 206
Glassboro, NJ 08028
856-256-4671 (Office) 856-562-5189 (Cell)
Camden Campus Office of the Public Safety
Glassboro Campus Office of the Public Safety
856-256-4992 Emergency contact 856-256-4992
- Camden Campus
- Glassboro
- Regional
- Northeast
- Glassboro Campus
- RowanSOM Stratford
- Commencement

subset: commoncrawl


text:
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Can you find your way out of the asteroid maze in Captain Cowboy?
Don't let greed get the best of you as you try and find your way out of a labyrinth in Captain Cowboy.
Christine Chan
| January 15
You have to be fast to find the right path in Hyper Maze Arcade
Don't get lost in this endless maze.
Christine Chan
| February 4
Guide Ive through the twisted green mazes of puzzle game Poison Ive
Who knew poison ivy could be so useful?
Daniel Celeste
| August 7
Help An Innocent Millipede Pursue A Career In Aviation In Millie
It's always been Millie's dream to attend the local Aviation School. Can you help her get there?
Daniel Celeste
| April 3
Dotard's Escape Is A Prison Break Unlike Any Other
With randomized, trap filled mazes, this game will challenge even the best of gamers.
Daniel Douvris
| June 9
Maze+ Twists And Winds Its Way Into The App Store
Get online and competitive with Maze+'s puzzling action, and earn yourself some clothes, cash, and pride.
Luke Patrick
| September 4
Race Others From Around The World To The Finish Line With Maze+
Just released by Secret Headquarters, Inc is Maze+, one of the first 3D multiplayer maze games for the iPhone and iPad. Race through several different mazes and earn gems so you can move up and receive special items in the game.
Susan Megrund
| August 31
Navigate Your Way Through Mazes And Beat The Clock With Revolve Ball
Revolve Ball brings mazes on the iPad and iPhone to a whole new level. For $.99 you're sure to get a kick out of all 40 levels. Whether you're racing to get your best time or battling gravity be sure to read ahead and find out more.
Greg Young
| May 17
Gears Is A Graphic Adventure That Is Worth Checking Out
This game creates an amazing challenge as you navigate a maze through different platforms and gears. This unique app is enjoyable and brings a refreshing option to the iPhone and iPad.
Susan Megrund
| May 8
1001 Crystal Mazes To Challenge Your Brain
Your brain will not lack for challenges with this great puzzle game.
Hannah Camacho
| April 29
Brutal Labyrinth: Where Fire Is Your Friend
Brutal Labyrinth is similar to other accelerometer controlled maze games, and adds a spark or two along the way. But is there enough to differentiate it from other labyrinth games?
Samuel L. Lytle
| March 8
Getting Through A Maze With Your iPhone
Getting through a maze isn't what it used to be. Thanks to the iPhone, some have found an easier way to the maze's exit. Read more...
Bryan M. Wolfe
| October 7
Your Kids Can Find Bigfoot And Play With Him Too
If you have kids, you're familiar with Fisher-Price. Now, one of the largest makers of toys is bringing children's entertainment to the iPhone with Bigfoot the Monster for iPhone.
Scott Grizzle
| July 31


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The City/Suburban Wine Divide
Although it sits just north of the Bronx, Westchester County is a world apart from New York, especially when it comes to wine and wine drinkers. Or so it would seem.
William Rattner has a particularly good vantage point. Billy (no one calls him William) has been the wine director of chef Peter Kelly's Westchester- and Rockland-based restaurant group for nearly two decades and is widely considered one of Westchester's top "wine gurus." (His good friend Glenn Vogt of Crabtree's Kittle House in Chappaqua is the other "guru" most commonly cited.)
Mr. Rattner presides over four restaurants: X2O-Xaviar's on the Hudson in Westchester and Xaviar's at Piermont, Restaurant X and the Freelance Café and Wine Bar in Rockland County. When I caught up with him at X20 in Yonkers last week, Mr. Rattner had just returned from a trade-sponsored wine trip to Italy. Mr. Rattner takes such trips on occasion and often finds himself in the company of large teams of sommeliers. That's one of the differences between New York and the suburbs, said Mr. Rattner: There just isn't the budget for wine teams in the suburbs. He described his wine team as "myself."
The suburbs do, however, have a few advantages. One is proximity to local wineries. Mr. Rattner makes his own wine with grapes he sources from the Hudson Valley. The 2008 Greendale White ($40) on the wine list at the Freelance Café is a wine Mr. Rattner crafted from Chardonnay grown in Columbia County and a little bit of Riesling from the Finger Lakes. Mr. Rattner is currently working on a Cabernet Franc—possibly a blend of Hudson Valley and Long Island grapes.
Another asset is access to local private cellars. There are wines on the X20 list that Mr. Rattner has purchased from a local college professor, whose cellar Mr. Rattner said he has visited "every three months since I was a child." (I assume this to be an exaggeration; Mr. Rattner, a former actor, favors dramatic pronouncements.)
Mr. Rattner bought several wines from the professor's cellar, including a 1969 DRC Richebourg that he's priced at a reasonable $2,100. In fact, Mr. Rattner has fielded quite a few calls about this particular bottle, including a recent one from San Francisco. "They wanted me ship the bottle but I told them that we want them to enjoy it in our dining room," he said.
I asked about suburban versus city palates and their respective spending habits. Do city diners drink more imported wines than suburbanites, and do they spend more? (X2O draws a large number of New Yorkers—for Mr. Kelly's food as well as for the view: The restaurant is rather dazzlingly set right over the Hudson, with water views. It's also right across from the train station, a 25-minute ride from New York.)
"I see more of a difference in how they eat," replied Mr. Rattner. "A New Yorker might order three courses—that's not a suburban tradition." Otherwise, he said he hasn't noticed much of a difference between the two. "There's a tremendous wine sense in Westchester," he added, as if to countermand any clichés about suburban ignorance. Or cheapness. In fact, he said he'd seen an increase in wine spending. The average sale at X20 is about $70 a bottle, though it's a bit lower in Rockland.
Suburbanites do tend to ask for guidance a bit more, Mr. Rattner added, though there are both city and suburban diners who drink only "life raft" wines. Life raft wines? "I can't disclose the names or I won't be able to visit those winemakers in California," he said with a laugh.
There are a fair number of such wines—well-known California Chardonnays and Cabernets—on the various Xaviar restaurant lists, though there are a good many more obscure, imported wines, too. The more interesting, and reasonably priced, wines tend to be imported. (Life rafts don't come cheaply, it seems.) In fact, when my dining companion and I were trying to decide between whites, they were all from the "old world" side of the list.
We selected three possibilities and asked Mr. Rattner to choose: the 2007 Raats Chenin Blanc from South Africa ($45); the 2007 Inama Du Lot Soave ($58) and the 2009 Ferret Pouilly Fuisse ($68). The Soave, Mr. Rattner answered definitively. He had just visited the region and he'd loved it. Plus, he doesn't get to share much Soave—it's often a tough sell for city and suburban diners alike.
Mr. Rattner thought of one more thing that city dwellers and suburbanites share: They all prefer the south side of the restaurant. "Everyone likes to sit facing New York," he said.


text: Joseph Marc 4122 Matte Blue eyeglasses are refreshingly fascinating. This cool blue frame features a striking matte acetate finish, paired with rounded lenses and a classic keyhole cutout at the bridge. The temples have a subtle transparent orange base with dark blue stripes lining the surface, completed with silver Joseph Marc branding.


text:
, Volume 19, Issue 3, pp 339-349
First online:
Are youth BMI and physical activity associated with better or worse than expected health-related quality of life in adulthood? The Physical Activity Longitudinal Study
- Katya M. HermanAffiliated withSchool of Kinesiology & Health Studies, Queen's University Email author
- , Wilma M. HopmanAffiliated withClinical Research Centre, Kingston General Hospital, and Department of Community Health and Epidemiology, Queen's University
- , Cora L. CraigAffiliated withCanadian Fitness & Lifestyle Research InstituteSchool of Public Health, University of Sydney
Rent the article at a discountRent now
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Body mass index (BMI) and physical activity (PA) affect health-related quality of life (HRQL); however, the long-term impact of youth BMI and PA on adult HRQL is unknown. We investigated the relationship of youth BMI and PA to adult HRQL 22 years later.
Subjects included 310 participants aged 7 to 18 in the 1981 Canada Fitness Survey, followed up in 2002–2004. The associations of youth BMI and leisure time PA to adult HRQL were examined, comparing to age- and sex-adjusted Canadian SF-36 norms.
Bivariate analyses revealed positive associations between youth overweight and mental aspects of adult HRQL, but little association with physical aspects. In logistic regression adjusting for adult BMI and other covariates, overweight youth were 7 times more likely than healthy weight youth to score at/above the norm on both mental health (MH) and bodily pain, and almost 18 times more likely on the mental component score (MCS). Youth BMI was also positively associated with general health (GH), social functioning, and role emotional. Removing adult BMI from the models led to attenuated associations with mental HRQL and no association with GH. Longitudinal BMI status change was explored, and findings supported the main regression results. Youth PA was not associated with adult HRQL.
Youth overweight conveyed a long-term positive impact on several aspects of adult HRQL, and this impact may be both direct and indirect through BMI change and the effect on adult BMI. Youth PA had no long-term impact on adult HRQL.
KeywordsBody mass index Body weight Exercise Adolescent obesity Longitudinal studies Mental health Quality of life SF-36 BMI change Reversal paradox
Reference tools
- Title
- Are youth BMI and physical activity associated with better or worse than expected health-related quality of life in adulthood? The Physical Activity Longitudinal Study
- Journal
Quality of Life Research
Volume 19, Issue 3 , pp 339-349
- Cover Date
- 2010-04
- 10.1007/s11136-010-9586-8
- Print ISSN
- 0962-9343
- Online ISSN
- 1573-2649
- Publisher
- Springer Netherlands
- Additional Links
- Topics
- Keywords
- Body mass index
- Body weight
- Exercise
- Adolescent obesity
- Longitudinal studies
- Mental health
- Quality of life
- BMI change
- Reversal paradox
- Industry Sectors
- Authors
- Katya M. Herman (1)
- Wilma M. Hopman (2)
- Cora L. Craig (3) (4)
- Author Affiliations
- 1. School of Kinesiology & Health Studies, Queen's University, Kingston, ON, K7L 3N6, Canada
- 2. Clinical Research Centre, Kingston General Hospital, and Department of Community Health and Epidemiology, Queen's University, Kingston, ON, Canada
- 3. Canadian Fitness & Lifestyle Research Institute, Ottawa, ON, Canada
- 4. School of Public Health, University of Sydney, Sydney, NSW, Australia


text:
California Pacific Medical Center
Doctors 1 - 3 of 3
Michael Katz, M.D.Sutter Pacific Medical Foundation
1580 Valencia Street
Suite 601
San Francisco, CA 94110-4422
(415) 600-6400
Elliott K. Main, M.D.3700 California Street
Suite G330
San Francisco, CA 94118
(415) 600-6388
Specializing inMaternal & Fetal Medicine
Please contact physician's office to see if they are accepting new patients.
Obstetrics and Gynecology
Please contact physician's office to see if they are accepting new patients.
Carl E. Otto, M.D.Sutter Pacific Medical Foundation
3700 California Street
Suite 4360
San Francisco, CA 94118
(415) 600-6400
Doctors 1 - 3 of 3
Want to See More Options?Need to see more options? You can remove your filters to show all Maternal & Fetal Medicine Doctors at California Pacific Medical Center.
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text:
We prospectively studied the safety of replacing intravenous delivery systems, including those used in total parenteral nutrition, at 72- compared with 48-hour intervals in 487 patients. Although the prevalence of contamination of intravenous fluid was higher in administration sets replaced at 72-hour intervals (10/664,1.5%) than in sets replaced every 48 hours (6/710,0.8%), the difference is not statistically significant. Contamination in both groups was almost exclusively with small numbers of coagulase-negative staphylococci (range, 1 to 27 colony-forming units/mL); no contaminated infusion was associated with clinical signs of sepsis or concordant bacteremia. Contaminants were recovered less frequently from peripheral venous infusions (0.6%) than from infusions used for central venous access or hemodynamic monitoring (1.5%) or total parenteral nutrition (3.6%); infusions in an intensive care unit were more frequently contaminated (2.5%) than infusions on medical and surgical wards (0.9%). These data indicate that extrinsic contamination of intravenous fluid is a rare cause of endemic nosocomial septicemia, and for most infusions it is unnecessary to routinely replace delivery systems more frequently than every 72 hours.
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text:
our mailing list. It's so wizard.
DJ Clark Kent
12 April, 2013 2:37 am
New freestyle from Maffew Ragazino produced by DJ Clark Kent.
17 January, 2013 6:01 am
On the first day his debut album LONG.LIVE.A$AP hit retailers, A$AP Rocky went up to The Late Show with David Letterman for a special performance. With DJ Clark Kent, A-Trak, and araabMUZIK on the boards, Rocky performed the title track and 'Wild For The Night'.
11 December, 2010 2:23 pm
Nice interview here with legendary producer DJ Clark Kent who speaks on what materials he likes on his sneakers, Camp Lo passing on the beats Ski had made for Reasonable Doubt, and the first time he met Jay-Z and Puffy (before he was Puff Daddy).
Search HipHopSite.com
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text:
My main research interests are in memory and memory disorders, particularly in those kinds of memory problems that occur as a result of normal or pathological aging or as a consequence of brain damage or injury. My students and I are particularly interested in understanding the relation between memory and executive functions and how they interact during different cognitive tasks. We are also developing rehabilitation techniques to help people with memory impairments improve their memory performance by taking advantage of preserved processes to compensate for those that have been damaged or lost. Current projects include studies of source memory, prospective memory, and self-referential processing. We are also studying the effects of social interaction and mindfulness on memory in older adults, and the effects of age-related hearing loss and hearing aids on cognitive function. We have a long-standing and continuing longitudinal study of changes in memory and executive function as people age and of the individual difference variables that might affect these changes. I teach courses in memory and memory disorders and in cognitive aging.
Elizabeth Glisky


text:
I really love this system!
But as the progress of our surveys go on, I realise a
"missing feature" on Invitation-Only controlled surveys.
Some of my users didn't bother to answer the survey on
the first email notification. (Was sent during the
christmas holidays... might be the reason, hehe).
Nevertheless; it would be great to have a feature to
RESEND the invitation email WITHOUT regenerating the
invitation code.
If I now use the action "Send Invitation Code to
Selected", UCCASS will generate a new invitation code.
The old code (that might still be in the users email
inbox) will not work any longer, and we generate a lot
of "disturbance"...
Best regards,


text:
Scammers posing as debt collectors are sparking a reminder from the Kansas Attorney General about a person's rights when it comes to owing money.
The Kansas Insurance Department initially alerted people about the scam. A caller demands your bank account information and cell phone number, saying you owe on a loan plus interest. When you ask for the caller's contact information, the call suddenly ends.
Attorney General Steve Six reminds people that under the Fair Debt Collection Practices Act, a debt collector can't call you before 8 am or after 9 pm.
They also can't be harrassing or abusive; use deceptive or misleading methods; or refuse to disclose their identity. They also must contact you in writing within five days of the initial contact.
Six reminds people to not give out personal information over the phone. If in doubt about the legitimacy of a call, contact the Consumer Protection Division at 800-432-2310 or file a report online at www.ksag.org.


text:
During a class field trip, Kim learns about an aunt she never knew she had (even morseo, looks exactly like her) who allegedly pulled 'the crime of the century'. Determined to save the family name, Kim goes on an incredibly weird adventure filled with amazing coincidences.
!!This episode features examples of:
* AllJustADream: The end of the episode reveals that the whole thing was just a dream that Dr. Drakken was having, so now it's unknown if any of the characters ancestors were actually real.
* ContrivedCoincidence
* GenerationXerox
* LetUsNeverSpeakOfThisAgain: This is what Drakken declares after Kim saves him and Shego from the exploding Electro Static Illuminator.


text:
Hello I been thinking about how to do a regular check on a system to assess the state of health of the systems. I am really turning into a system admin gradually and I already manage about 10
ubuntu servers. I have set up zabbix already but I believe either myself or a script should be run every month to just check on the servers.
Is it how it's done in real life
if yes what are the ways to achieve that?
is there any suggestion anyone feels sharing with a young system admin?
THanks for your advices


text:
Top Definition
videos or photos or views of up a woman's skirt.
you can get a good upskirt view if you have a mirror attatched to your shoe.
by laura December 27, 2003
Trending Now
what kids with camera phones do in school
Jarad Morgon took a crazy upskirt shot of Jen Hunsinger
by Trey December 14, 2004
A video or photograph of up a woman's skirt. Some have underwear, others don't, they reveal the vagina.
The scandalous recent upskirt of Paris Hilton has been hitting the news lately
by kilometeros July 10, 2008


text:
Rebecca Mead wrote an upbeat profile of Benjamin Millepied in the Aug. 5, 2013 issue of The New Yorker.
Only the opening is available online now for non-subscribers, but the issue is also available on newsstands.
This site uses cookies. By using this site, you agree to accept cookies, unless you've opted out. (US government web page with instructions to opt out: http://www.usa.gov/optout-instructions.shtml)
Bejamin Millepied Profile in The New YorkerAug 5, 2013
Started by Helene , Aug 01 2013 08:24 AM
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text:
Email addressVerified
Phone number(▒▒▒) ▒▒▒- ▒▒ 16
658 Friends
Reviewed4 Reviews
About Me
- School
- Portland State University
- Work
- The Rialto
- Terry's Airbnb Symbol
Hey, I'm Terry!
Portland, Oregon, United States · Member since March 2011
Todd and I are both in our mid-40s, have been married for 6 months, have two dogs and three cats and live in Portland, Oregon where he is a tile-setter and I am the general manager of a restaurant/bar in a historical building downtown. We're not loud, are pretty easy going, and clean up after ourselves. Todd is a baseball fan, I am training for my first 5K, we love paintings, sculpture, history, good coffee, and the New York Times.


text:
Find a copy online
Links to this item
purl.access.gpo.gov Adobe Acrobat Reader required
usda.gov link no longer works.
Find a copy in the library
Finding libraries that hold this item...
|Additional Physical Format:
|Online version:
Nutrition and your health.
[Washington, DC] : U.S. Dept. of Agriculture : U.S. Dept. of Health and Human Services : [For sale by the U.S.G.P.O., Supt. of Docs.], 2000
|Material Type:
|Government publication, National government publication, Internet resource
|Document Type:
|Book, Internet Resource
|All Authors / Contributors:
|United States. Department of Agriculture.; United States. Department of Health and Human Services.
|0160503760 9780160503764
|OCLC Number:
|Cover title.
"Aim for fitness, build a health base, choose sensibly ... for good health."
Shipping list no.: 2000-0371-P.
|39 pages : color illustrations ; 28 cm.
|Series Title:
|Home and garden bulletin, no. 232.
|Other Titles:
|Dietary guidelines for Americans
Dietary guidelines for Americans, 2000
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Related Subjects:(7)
- Diet -- United States.
- Nutrition -- United States.
- Nutrition Policy.
- Diet.
- Nutritional Physiological Phenomena.
- United States.
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by ifprilibrary updated 2011-11-03


text:
'The Twilight Saga: Breaking Dawn - Part 2' Los Angeles Premiere
Kristen Stewart, Robert Pattinson, Taylor Lautner, and the rest of the cast of the final installment turn out for the world premiere.
Kristen Stewart, Robert Pattinson, Taylor Lautner, and the rest of the cast of the final installment turn out for the world premiere.
r2 d2: There are many things I have in-question after watching the DNC speakers. On one hand, Hillary states all the wonderful things she will change, then touts how many years she has served. Why weren't all of these wonderful changes already made, then (is she saying she needs more time)?. Then Bill Clinton spoke about a beautiful ove story with Hillary, in what sounded like a "love at first sight", and how he has never admired or respected any other person more than he does her. But, I long associate Bill with having one affair after another-- and throughout his speech I could not help but to keep thinking about Monica Lewinsky, as opposed to the "love of his life" he was speaking about. Michelle Obama spoke about her "black daughters" on the White House lawn...and, then how a "black boy" commented on Barack hair. I found it very strange her repeated references that kept putting "black" before them. And, I am still recalling how Michelle said she was not proud to be an American in the past-- leaving me still curious, as to what made her now suddenly proud?


text:
We are excited to announce that over the next few months Favored Nations will be overhauling it's website while preparing an amazing slate of new releases for 2016!
To insure you don't miss a beat we would like to invite you to sign up for an exclusive All Access VIP Pass (Just click on the pass). This will entitle you entry to the Backstage section of the new site where you will be able access excusive content, receive special offers only for VIP members, free downloads, special on demand podcasts, special discounts from our new store and much more.


text:
I have used GNU ddrescue to copy a crashed WD external disk over to an identical one. ddrescue told me that 9 MB out of 2TB could not be rescued, so I think that's pretty good.
Anyway, being a Mac disk, I connected the new disk to a Mac and ran Disk tools, which told me that the filesystem needed repairs. When clicking Repair, it first successfully (or so it says) repairs the filesystem and then gets stuck on something like
Updating Boot Support Partitions for the volume as required
I have no idea what to make of this. How long should I wait? Can I cancel it safely?


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According to the Dalmatian Club of America (DCA), the beginnings of the breed go so far back that researchers cannot agree to their origin. The one thing that is agreed upon is that its distinctive looks have not changed over the centuries. The spotted dogs frequently accompanied gypsies, which is probably why its history takes it to so many different regions.
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From memory you had to do something in WP to enable a new printer. Let's see if google can help?
THIS is from Corel about this version.
From there we go to http://apps.corel.com/support/downloads/printersearch.asp
I have a LexMark X1270 that is less than a year old. I usually use MS Word but I have several legal documents that are in WordPefect 11 and it won't print anything. It prints Word, wordpad, notepad, excell, images etc. Just not WordPerfect. I emailed LexMark support about this but have not as yet received a reply. I was able to print WordPerfect from this computer when I had an HP printer, which I no longer have. Any ideas.


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Tuberous sclerosis
Other Names for this Disease
- Tuberous sclerosis complex
- Tuberous sclerosis 1
See Disclaimer regarding information on this site. Some links on this page may take you to organizations outside of the National Institutes of Health.
autosomal dominant pattern of inheritance and can be caused by mutations in the TSC1 or TSC2 gene.[1] Treatment depends on the symptoms in each individual case and may include medications or surgery.[2]Tuberous sclerosis is a genetic disorder characterized by the growth of numerous noncancerous (benign) tumors in many parts of the body. Common signs and symptoms include patches of unusually light-colored skin, areas of raised and thickened skin, and growths under the nails. Tumors on the face called facial angiofibromas are also common beginning in childhood. Tumors may cause developmental problems (e.g., seizures, hyperactivity, aggression, learning problems, autistic-like behaviors). Some tumors can cause serious complications (e.g., those affecting the brain, heart, or kidney). Tuberous sclerosis complex has an
Last updated: 5/19/2016
- Tuberous Sclerosis Complex. Genetics Home Reference. December 2013; http://www.ghr.nlm.nih.gov/condition/tuberous-sclerosis-complex.
- Northrup H, Koenig MK, Pearson DA, Au K-S. Tuberous Sclerosis Complex. GeneReviews. September 3, 2015; https://www.ncbi.nlm.nih.gov/books/NBK1220/.
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Basic Information
- Genetics Home Reference (GHR) contains information on Tuberous sclerosis. This website is maintained by the National Library of Medicine.
- MedlinePlus was designed by the National Library of Medicine to help you research your health questions, and it provides more information about this topic.
- The National Institute of Neurological Disorders and Stroke (NINDS) collects and disseminates research information related to neurological disorders. Click on the link to view information on this topic.
- The National Organization for Rare Disorders (NORD) has a report for patients and families about this condition. NORD is a patient advocacy organization for individuals with rare diseases and the organizations that serve them.
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- Medscape Reference provides information on this topic. You may need to register to view the medical textbook, but registration is free.
- The Monarch Initiative brings together data about this condition from humans and other species to help physicians and biomedical researchers. Monarch's tools are designed to make it easier to compare the signs and symptoms (phenotypes) of different diseases and discover common features. This initiative is a collaboration between several academic institutions across the world and is funded by the National Institutes of Health. Visit the website to explore the biology of this condition.
- Online Mendelian Inheritance in Man (OMIM) is a catalog of human genes and genetic disorders. Each entry has a summary of related medical articles. It is meant for health care professionals and researchers. OMIM is maintained by Johns Hopkins University School of Medicine.
- Orphanet is a European reference portal for information on rare diseases and orphan drugs. Access to this database is free of charge.
- PubMed is a searchable database of medical literature and lists journal articles that discuss Tuberous sclerosis. Click on the link to view a sample search on this topic.
Other Names for this Disease
- Tuberous sclerosis complex
- Tuberous sclerosis 1
See Disclaimer regarding information on this site. Some links on this page may take you to organizations outside of the National Institutes of Health.


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4800 Oxford Street
A location in Bethesda
Commute to Downtown North Bethesda
Most errands require a car.
About your score
4800 Oxford Street has a Walk Score of 36 out of 100. This location is a Car-Dependent neighborhood so most errands require a car.
4800 Oxford Street is a nine minute walk from the MARC BRUNSWICK - WASHINGTON at the GARRETT PARK MARC eb stop.
Nearby parks include Waverly Ave, Rock Creek Park and Porcupine Woods.
Explore how far you can travel by car, bus, bike and foot from 4800 Oxford Street.
Rail lines:
0.4 mi
Red Metrorail Red Line
0.5 mi
Bus lines:
5 Silver Spring-Twinbrook Station
0.1 mi
37 Potomac-Wheaton Metro Station
0.1 mi
6 Parkside/Grosvenor-Montgomery Mall
0.2 mi
46 Mont.College-Rockv-Medical Center
0.4 mi
96 Grosvenor-Rock Spring-Montg Mall
0.4 mi
Car shares:


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The endosymbiotic bacterium Wolbachia is being investigated as a potential control agent in several important vector insect species. Recent studies have shown that Wolbachia can protect the insect host against a wide variety of pathogens, resulting in reduced transmission of parasites and viruses. It has been proposed that compromised vector competence of Wolbachia-infected insects is due to up-regulation of the host innate immune system or metabolic competition. Anopheles mosquitoes, which transmit human malaria parasites, have never been found to harbor Wolbachia in nature. While transient somatic infections can be established in Anopheles, no stable artificially-transinfected Anopheles line has been developed despite numerous attempts. However, cultured Anopheles cells can be stably infected with multiple Wolbachia strains such as wAlbB from Aedes albopictus, wRi from Drosophila simulans and wMelPop from Drosophila melanogaster. Infected cell lines provide an amenable system to investigate Wolbachia-Anopheles interactions in the absence of an infected mosquito strain. We used Affymetrix GeneChip microarrays to investigate the effect of wAlbB and wRi infection on the transcriptome of cultured Anopheles Sua5B cells, and for a subset of genes used quantitative PCR to validate results in somatically-infected Anopheles mosquitoes. Wolbachia infection had a dramatic strain-specific effect on gene expression in this cell line, with almost 700 genes in total regulated representing a diverse array of functional classes. Very strikingly, infection resulted in a significant down-regulation of many immune, stress and detoxification-related transcripts. This is in stark contrast to the induction of immune genes observed in other insect hosts. We also identified genes that may be potentially involved in Wolbachia-induced reproductive and pathogenic phenotypes. Somatically-infected mosquitoes had similar responses to cultured cells. The data show that Wolbachia has a profound and unique effect on Anopheles gene expression in cultured cells, and has important implications for mechanistic understanding of Wolbachia-induced phenotypes and potential novel strategies to control malaria.
Author Summary
Wolbachia are bacteria that infect many insect species, but do not infect Anopheles mosquitoes. These mosquitoes transmit Plasmodium parasites, which cause malaria in humans. Wolbachia infection in Aedes aegypti mosquitoes reduces their ability to transmit diverse pathogens including viruses, nematodes and bird malaria parasites. Wolbachia-induced stimulation of the mosquito's innate immune system has been suggested as a mechanism conferring this pathogen interference. Since no Wolbachia-infected Anopheles mosquito strain exists, we used infected cultured Anopheles cells to examine the effect of infection on Anopheles gene expression. Wolbachia had a profound influence on Anopheles gene expression. Many of the genes regulated by Wolbachia have been seen in other studies to influence Plasmodium levels in mosquitoes, but interestingly and in contrast to other mosquitoes, many of the host genes were suppressed rather than induced.
Citation: Hughes GL, Ren X, Ramirez JL, Sakamoto JM, Bailey JA, Jedlicka AE, et al. (2011) Wolbachia Infections in Anopheles gambiae Cells: Transcriptomic Characterization of a Novel Host-Symbiont Interaction. PLoS Pathog 7(2): e1001296. doi:10.1371/journal.ppat.1001296
Editor: Kostas Bourtzis, University of Ioannina, Greece
Received: August 13, 2010; Accepted: January 18, 2011; Published: February 17, 2011
Copyright: © 2011 Hughes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by NIH/NIAD grant R21AI070178 and a Johns Hopkins Malaria Research Institute (JHMRI) pilot grant to JLR. GLH was partially supported by the JHSPH Jane Welsh Russell Scholarship and a JHMRI post-doctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Wolbachia are alpha-proteobacteria that infect a range of arthropods and nematodes, and are possibly the most common endosymbiotic bacteria on the planet. In their arthropod hosts, Wolbachia induce a variety of reproductive manipulations that enhance the fitness of infected females compared to their uninfected counterparts [1]. Wolbachia have recently been shown to interfere with pathogen infection and transmission in both naturally-infected and artificially-transinfected insects [2], [3], [4], [5], [6], [7]. These phenotypes make Wolbachia-based control strategies an attractive option to minimize the impact of arthropod-borne diseases and insect pests [8], [9].
Anopheles mosquitoes transmit human malaria, a devastating disease that kills approximately 2 million people per year, and are naturally uninfected with Wolbachia [10], [11], [12]. Transfer of Wolbachia into cultured Anopheles gambiae cells and transient somatic infection of adult female mosquitoes demonstrates that the bacteria can survive in this species, suggesting that the Anopheles genus may be amenable to stable infection [13], [14]. Although several novel Wolbachia-mosquito associations have been created using a variety of transinfection techniques, no stable Wolbachia-infected Anopheles line has been developed [15], [16], [17], [18], [19], [20]. The development of such a strain may open the possibility for Wolbachia-based control strategies for malaria. Indeed somatic infections of the wMelPop strain reduce oocyst levels in the murine malaria model, Plasmodium berghei [7]. However the global effects of Wolbachia on Anopheles and the interplay within the tripartite association of the human malaria Plasmodium parasites and the mosquito host are currently unknown.
Novel phenotypes are sometimes observed upon transinfection of Wolbachia into novel insect hosts [15], [21], [22]. In the artificially infected wMelPop-Aedes aegypti strain (wMelPop CLA), Wolbachia limits infection by a broad range of pathogens including dengue virus, filarial nematodes and Plasmodium [2], [3]. The mode of action for pathogen resistance is uncertain, however two mechanisms have been postulated; immune activation of the host by Wolbachia and/or metabolic competition between the bacteria and the pathogen. Evidence for both hypotheses was observed with a range of immune genes up-regulated in wMelPop-infected Ae. aegypti [2], [3] and the finding that dengue virus only persisted in Wolbachia-uninfected cells of the insect [3]. A similar phenotype was observed in some infected Drosophila strains where Wolbachia infection induced refractoriness to multiple RNA viruses [4], [5]. Interestingly, a previous study using naturally infected hosts found that Wolbachia seems to be able to evade the host immune response in Drosophila and Aedes albopictus [23], suggesting Wolbachia-induced immune activation may be more likely in novel rather than co-evolved Wolbachia-host associations.
Within Anopheles mosquitoes, there is a conserved immune response towards foreign bacteria and Plasmodium [24]. By using multiple methods such as co-feeding, injection or removal of microflora, bacteria have been seen to mediate Plasmodium infection levels in the Anopheles host [25], [26], [27], [28], which is thought to be due to the bacteria priming the host immune response. Interestingly, Gram-negative bacteria elicit a greater response compared to Gram positive, although there are species-specific differences [25], [27]. If Wolbachia (a Gram-negative bacterium) evokes a similar response and up-regulates the basal immunity in infected Anopheles, infection may confer an anti-Plasmodium phenotype. Some evidence for this has been shown in somatically-infected mosquitoes infected with rodent malaria [7].
The generation of Wolbachia-infected Anopheles cell lines allows the investigation of Wolbachia-Anopheles interactions in the absence of a stably-infected mosquito strain [14]. Cell lines provide a platform whereby Wolbachia host lineages can be generated with relative ease, and allow the exploration of both natural and artificial Wolbachia host interactions [29], [30], [31]. To investigate the effect of Wolbachia infection on global patterns of Anopheles gene expression we performed microarray analysis on both wAlbB (from Ae. albopictus) and wRi (from Drosophila simulans) infected Anopheles gambiae Sua5B cells compared to uninfected cells. We validated microarray results in vitro, and in vivo for a subset of differentially expressed genes in somatically-infected adult female mosquitoes.
Wolbachia infection of Anopheles cells resulted in the regulation of 690 genes relative to uninfected Sua5B cells (False discovery rate (FDR) P<0.05, ≥ 2.0 fold-change (FC)) (Table S1). When comparing Wolbachia strains, 255 genes were uniquely regulated by wAlbB infection, while 331 were regulated specifically by wRi infection (Figure 1A). Of the 104 genes regulated by both strains, the majority (74 genes) were down-regulated, 11 were similarly up-regulated and the remainder had alternating regulation patterns between the two Wolbachia strains (Figure 1B). Interestingly, we observed a greater number of genes regulated by wRi compared to wAlbB even though the cell infection density of wRi was much less than wAlbB (wRi∼10% cells infected, wAlbB >90% of cells infected) [14]. It is possible that since wRi was purified from live flies, it has a greater impact that wAlbB which was purified from another cell line [14]. Of the regulated genes, a diverse range of functional classes was represented with a large proportion being genes of unknown or diverse function, which was consistent for both Wolbachia strains. Among the genes assigned to specific known functional classes, immune-, transport- and metabolism-related transcripts were the most abundant categories regulated by Wolbachia (Figure 1C). Strikingly, over 75% of the immune related transcripts were down-regulated, which was consistent between both strains. Overall, down-regulation was a common theme, with only redox/stess/mitochondrial (RSM) and replication/transcription/translation (RTT) classes not down-regulated in wRi infected cells and RTT in wAlbB. Microarray data is available at gene expression omnibus (accession number GSE23215) [32].
A. Venn diagram of 690 Anopheles transcripts which display differential expression due to wAlbB or wRi infection. 104 transcripts were common to both strains, while 389 were down regulated and 320 up regulated due to Wolbachia infection. B. Scatter plot of regulated significant genes (>2 fold regulation; False discovery rate P value <0.05). Blue dots represent significant genes regulated by wRi only, red regulated by wAlbB only and purple, genes commonly regulated. C. Number of genes in each functional classes class up or down regulated in response to either wAlbB or wRi infection. Genes were classified into groups; transport (TRP), replication, transcription and translation (RTT), redox, stress and mitochondrial (RSM) proteolysis and digestion (PROT), metabolism (M) cytoskeletal and structural (CS) and immune (I) depicted in the first column, and diverse (D) and unknown (U), in the second column.
qPCR validation of microarray genes and comparison to whole mosquitoes
To gauge the accuracy of the microarray data, we selected a subset of genes to validate by quantitative real-time PCR from cell culture. Eight genes, (HSP20, HSP90, HSPDnaJ, cold-shock protein, cecropin, Serpin11, Filamin, TEP3) with varying expression profiles, regulated by both Wolbachia strains were evaluated. These genes spanned a variety of functional classes including defensive and immune genes that may be relevant to Plasmodium infection and potential Wolbachia-mediated reproductive phenotypes. qPCR results corroborated the array data and had a positive linear correlation (R2 = 0.9595) when comparing the log2 values using both gene expression techniques (Figure 2A).
A. Log2 fold change for selected An. gambiae genes (HSP20, HSP90, HSPDnaJ, cold-shock protein, cecropin, Serpin6, Filamin, TEP3) comparing microarray and QPCR methods. B. Comparison of Anopheles gene expression in response to Wolbachia in cell culture and whole mosquitoes. Expression of 6 genes from wAlbB in Sua5B cells analyzed using microarrays (MA) compared to wAlbB somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). C. Microarray data from wRi infected Sua5B cells compared to wMelpop somatically-infected whole mosquitoes 15 days post injection (N = 5 mosquitoes/treatment). qPCR gene expression is a ratio of Wolbachia infected (wAlbB or wRi) to Schneider's injected control. Error bars represent maximum and minimum range of expression.
Wolbachia has been shown to persist, disseminate, and replicate in injected adult Anopheles mosquitoes [13]. We injected live female mosquitoes with Wolbachia to determine if the effect of infection on gene regulation in vivo was consistent with results observed from infected cell cultures. Several immune related transcripts and other genes, which potentially convey interesting phenotypes and had varying expression profiles identified in cell culture, were assessed. When comparing wAlbB regulation in cells and mosquitoes, the direction of regulated expression was similar (Figure 2B), although, not surprisingly, the intensity of expression varied leading to a lack of significant correlation (data not shown). The loss of the wRi-infected cell line prevented a direct comparison to somatically infected mosquitoes, however, this array data was compared to wMelPop-infected mosquitoes. wMelpop and wRi both infect Drosophila and are classed in supergroup A. When making this comparison, again we observed that the direction of gene regulation was similar (Figure 2C), but the intensity of expression varied. Notably, the intensity of two genes, the LRR-like transcript and FBN9 is greater in the cells compared to the whole mosquito. This may be explained by the hemocyte-like character of the cell line or Wolbachia strain-specific variation. Nevertheless, the similarity in the direction of gene regulation in vivo and in vitro suggests that the effect of Wolbachia in the cell line may be applicable to whole mosquitoes.
Comparison to other systems
We compared Wolbachia-regulated Anopheles transcripts identified in this study to genes regulated by wMelPop in Aedes aegypti [2] and by bacterial infection in A. gambiae [26]. Fourteen A. aegypti homologues were identified from differentially expressed Anopheles transcripts in response to Wolbachia infection, with five having an immune related function (Table S2). When comparing these results, 75% of both wRi and wAlbB regulated homologs displayed a similar direction of expression. Similarly, when comparing Wolbachia-regulated transcripts to those of regulated by bacterial infection in A. gambiae, 15 homologs were regulated by Wolbachia. (Table S3). Most of these homologs were of unknown function.
In comparison to other studies using Drosophila cell culture systems to examine the influence of Wolbachia on host gene expression, we find a dramatically elevated number of identified regulated Anopheles genes compared to Drosophila. In Drosophila S2 cells, 263 genes had a 1.2 fold change due to Wolbachia infection, however when the more common ≥2 fold criteria was used, very few regulated Drosophila genes were identified [33]. At the proteomic level, only four proteins, all host antioxidant proteins, were elevated in Wolbachia infected Ae. albopictus Aa23 cells [34]. A lower Wolbachia titer may account for the subtle gene regulation in wRi infected Drosophila cells [33], although the infection density of wRi in infected Anopheles was similarly sparse [14]. Alternatively, the mild effect of Wolbachia on gene regulation in Drosophila and protein expression in Ae. albopictus could be due to previous co-evolution between the Wolbachia strains and their naturally infected hosts.
Effect of Wolbachia on transcription of Anopheles genes potentially affecting pathogen transmission
The most striking effect observed for both wRi and wAlbB infections was the general suppression of heat shock protein transcripts (HSP20, HSP70, HSP90, HSP-DNAJ). Cells infected with wAlbB had a dramatic suppression of these genes with 5 out of the top 6 most down-regulated genes (FC −31 to −16). Similarly, these genes were down-regulated by wRi, albeit to a lesser extent (to −5.3). Presenting a similar pattern of regulation, multiple HSPs were down-regulated by wRi infected Drosophila S2 cells [33]. In vivo, it has been shown that Wolbachia-infected flies have altered expression of HSP, which in turn affects Wolbachia-induced reproductive phenotypes [35]. HSPs have also been implicated in Anopheles-pathogen interactions. Elevated levels of HSP20 were identified in An. gambiae heads after infection with P. berghei [36]. If this protein assists transmission, either directly or indirectly, the antagonistic actions of may potentially reduce P. berghei sporozoite infection. Additionally, knockdown of a heat shock proteins (HSC70B) via injection of dsRNAi in conjunction with O'nyong nyong virus (ONNV) significantly reduced the lifespan of adult mosquitoes as compared with the control [37]. We speculate that if this expression pattern translates to in vivo Anopheles infections, Wolbachia-induced down-regulation of HSPs may modulate vector competence of ONNV or shorten mosquito lifespan.
Metabolic and other genes.
Wolbachia regulates a suite of genes involved in Anopheles metabolism, with most of these transcripts being down-regulated by infection. Although the heterotrophic needs of Plasmodium and mosquito growth factors required for parasite development are not well understood in the insect, changes in transcription of metabolism genes which alter the mosquito environment may affect Plasmodium growth. Infection of Sua5B cells with wAlbB drastically reduces phosphoenolpyruvate carboxykinase (PEPCK) transcripts 26 fold. In response to P. falciparum, PEPCK is up-regulated in the mosquito [38], [39]. Carbonic anhydrase, which catalyses the reversible hydration of carbon dioxide to bicarbonate, is down-regulated in wAlbB-infected cells by 2.6 fold. In many mosquitoes, inhibition of this enzyme results in a reduction in pH of the mosquito midgut [40]. Moreover, carbonic anhydrase inhibitors in P. falciparum reduced parasite survival in the human blood stages and have been suggested as targets of anti-malarial drug design [41], [42]. The effect of these host derived enzymes on parasite development is unknown, however changes in regulation between mosquito and Plasmodium suggest that further examination of these genes is warranted to determine their affects on parasite development. Although not strictly metabolism related, laminin and collagen are components of the basal lamina, which are interrelated with parasite invasion [43], [44], [45]. Both laminin (FC −2.1, −3.8) and collagen (FC −4.4) are down-regulated by wRI infection. RNAi knock down of laminin lead to a substantial reduction of oocysts in mosquito midguts [44] possibly due to laminin inhibiting the melanotic encapsulation of oocysts [46].
Immunity-related transcripts.
Many Anopheles genes associated with arthropod immunity were regulated by Wolbachia infection. Genes within all the broad categories of immunity (pathogen recognition receptors, signaling amplification cascades, immune signaling pathways, and effector molecules) were regulated. Immune genes up-regulated by both infections included CLIPs and antimicrobial peptides (AMP), while serpins (SRPN), and a leucine rich repeat (LRR) were induced by wRi and fibrinogens (FBN) and thioester-containing protein (TEP) were induced by wAlbB (Figure 3). More striking were those immune genes down-regulated by infection. wRi significantly suppressed expression of class C scavenger receptors, Gram-negative binding proteins (GNBP), FBN, CLIP, SRPN, LRR-containing genes, a TEP, effector proteins involved in phagocytosis and a lysozyme (Figure 3). The wAlbB strain down-regulated genes of similar functions, however in the class of effector molecules, this strain had more of an influence on peroxidases rather than AMPs (Figure 3).
Anopheles immune networks regulated by wRi (A) and wAlbB (B). Pathways are models of the IMD and Toll pathways [81] and components of the melanization regulatory module [51] divided into the 4 broad categories of immune molecules. Blue color represents induction, while yellow color represents suppression. The intensity of coloring is proportional to the intensity of expression. Regulation is depicted to a maximum fold change of ±4. Some transcripts were greater than ±4 regulated. Abbreviations: LLR leucine rich repeats; FBNs fibrinogens; TEPs thioester containing proteins; GNBPs Gram-negative binding proteins; CTLs C type lectins; CLIPs clip-domain serine protease; PGRPs peptidoglycan recognition proteins; SRPNs serpins; CEC cecropins; Def defensins; PPO Prophenoloxidase; PO phenoloxidase; LYS lysozmyes.
In addition, other immune-associated apoptosis and detoxification transcripts were regulated by infection. Brennan et al. [34] identified Wolbachia-induced host antioxidant proteins in cell culture. In contrast to the enrichment of these genes at the protein level, a peroxiredoxin transcript was down-regulated 2.1 times by wAlbB and 11 times by wRi. Likewise, superoxide dismutase was down-regulated in wRi-infected cells (FC −2). Additionally, eight glutathione S transferases were regulated. Two of these were co-regulated by both strains, while 3 were induced and 3 suppressed in wRi. The level of regulation for these genes was approximately 2–3 fold, however one transcript was suppressed 19-fold by wRi compared to uninfected cells. Taken in total, these data suggest that Wolbachia can significantly affect cellular defense, detoxification and immunity in An. gambiae cells, and that expression of many of these defensive genes is suppressed rather than induced. These results contrast with observations of up-regulation of the majority of immune-related transcripts in stably-infected Ae. aegypti mosquitoes, which have reduced capacity to transmit pathogens [2], [3]. Gene expression of a small subset of immune genes were characterized in response to wMelPop infection of a different An. gambiae cell line (Mos55), where they were up-regulated, suggesting a potential difference between Anopheles cell lines or Wolbachia strain-specific variation [7].
Although pathogen interference occurs in naturally infected hosts, there is evidence that the transfer of Wolbachia to a new host is a catalyst for pathogen interference, illustrated by wAlbB inducing dengue resistance in a novel host, Ae. aegypti, yet not conferring interference in it's native host, Ae. albopictus [6]. The effects of tripartite relationship of Wolbachia-Anopheles-Plasmodium are relatively unknown, however, recently wMelPop somatically infected into Anopheles was seen to decrease P. berghei oocyst levels, with evidence that TEP1 may involved in the process [7]. Many of the regulated defensive genes we identified have been shown to directly or indirectly affect Plasmodium infection in Anopheles, either positively or negatively. TEP3 was dramatically up-regulated (FC 7.6) in response to wAlbB. Similar up-regulation is observed when mosquitoes are fed a blood meal, either uninfected or infected (P. berghei), or challenged with bacteria [47], [48]. TEP1, a protein similar to TEP3, has been shown to be an important molecule involved in the melanization and anti-Plasmodium response across the Anopheles genus [49], [50]. Looking at genes involved in the immune signaling cascade, CLIP7A, a suppressor of melanization, was suppressed by both wAlbB (FC −5.2) and wRi (FC −2.6), which may confer an anti-Plasmodium phenotype as seen in knock-down experiments of this gene [51]. In contrast, the gene galectin, which is up-regulated in response to P. berghei infection and immune challenge by Micrococcus luteus, had conflicting strain-specific responses: up-regulated by wAlbB (FC 9.1) but down-regulated by wRi (FC −3.5) [52].
In contrast to genes that may abate Plasmodium infection, a suite of genes were also regulated in ways that may elevate parasite levels in infected mosquitoes. For example we observed down-regulation of many CLIPs. Reverse genetic techniques have shown that both CLIPB4 and CLIPB8 are involved in the melanization process, where knock-down of these genes ablates melanization [53]. In double knock-down (KD) experiments, reducing transcripts of both CLIPB4 and CLIPB8 in tandem with CTL4 partially interferes with P. berghei ookinete melanization [51]. Using over-expression, up-regulation of cecropin was shown to decrease Plasmodium levels in Anopheles [46]. Expression of both SRPN18 (FC wAlbB −3.2, wRi −3.6) and TEP15 (FC wAlbB −3.5, wRi −2.1) is suppressed by both Wolbachia strains and although the specific function of these molecules has not been identified, these classes of molecules are associated with immunity [48], [54]. In Ae. aegypti, TEP15 is one of the most strongly induced genes in response to KD of Cactus, the negative regulator of the Toll pathway [55]. In addition, GNBPB1, which was also down-regulated by both strains (FC wAlbB −5.2, wRi −6.0), is strongly induced by parasite invasion of the midgut and bacterial challenge [52], [56], [57]. In contrast to our study, GNBP was induced in Aedes mosquitoes infected with wAlbB and wMelpop [2], [6].
In terms of a general response to bacterial infection, we see the regulatory transcriptional factor for the Toll pathway (Rel1) down-regulated 2.3 times by wRi infection. We observed an up-regulation of caspar (FC 2.2), the negative regulator of the IMD pathway in response to wRi. PGRP-LA expression was suppressed 3.2 times by wRi. In Drosophila, PGRP-LA is likely to be a hemocyte transmembrane protein [58], while other PGRPs activate negative feedback loops in the IMD pathway [59], [60]. A similar long transcript PGRP (PGRP-LC) in An. gambiae controlled proliferation of gut microbiota, which subsequently influenced Plasmodium infection [61]. When all three PGRP-LC isoforms were silenced simultaneously, mosquitoes challenged with Staphylococcus aureus had induced expression of cecropin and defensin. In Drosophila, silencing of PGRP-LC by RNAi induced expression of diptericin, cecropin A1, and attacin A, but these effector molecules were not regulated due to depletion of PGRP-LA [62]. Here we see similar independent regulation of attacin which was up-regulated 3.3 times in wRi infected cells, while defensin is also up-regulated by wAlbB (FC 2.3). Interestingly, attacin was found to inhibit the outer membrane synthesis of Escherichia coli in the giant silk moth, Hyalophora cecropia [63]. Thus, we may be observing an active defensive response from Anopheles to prevent Wolbachia infection.
The general pattern of immune gene down-regulation appears to be a Wolbachia-specific phenomenon in this cell line. In addition to Wolbachia, Sua5B cells can support infection of additional intracellular bacteria such as Rickettsia [64]. We used qPCR to test selected immune-related genes (cecropin1, defensin1, gambicin and immune-responsive serpin-related protein [IserpF1]) in Sua5B cells that had been infected with a taxonomically and phenotypically diverse array of Rickettsia species: R. typhi (typhus group), R. felis (transitional group), R. montenensis and R. peakockii (both in the spotted fever group). R. typhi and R. felis are human pathogens, while R. montenensis is non-pathogenic. R. peakockii is a non-pathogenic vertically-transmitted tick endosymbiont. While there was variation between bacterial species and the gene tested, all four Rickettsia induced expression of most tested immune genes (up to 12-fold induction), including the endosymbiont R. peakockii (Figure S1). These results suggest that the natural response of Sua5B cells to intracellular bacterial infection is immune up-regulation, and that Wolbachia is suppressing this response. It should be noted however that Wolbachia exist in a potentially protective host vacuole, while Rickettsia are free in the cytoplasm.
Wolbachia influence on reproduction-related genes
Wolbachia-induced CI expression is associated with abnormal decondensation of the paternal pronucleus during fertilization, epigenetic factors, and/or problems during embryogenesis. Xi et al. [33] observed that in wRi-infected Drosophila cells, the gene angiotensin converting enzyme (Ance), which is involved in spermatogenesis, was up-regulated by Wolbachia infection in cells and flies, and was potentially involved in the CI phenotype. In our study, the six Anopheles homologues of Ance on the microarray were not affected by Wolbachia infection. We screened our data for other significantly regulated genes associated with cytoskeleton formation/function, epigenetic modification, gametogenesis or embryonic development. Multiple cytoskeleton-associated genes, genes associated with chromatin formation and remodeling and genes associated with embryogenesis and cell division were regulated by both infections.
We identified multiple genes that may be linked to the CI phenotype. Transcription of a Kazal-like serpin was enhanced dramatically due to Wolbachia infection (FC wAlbB 13.1, wRi 5.3). Kazal domain-containing proteins identified in animals have a diverse array of functions. A Kazal-like serpin was found to inhibit both gelatinolytic activity of sperm and the proteolytic activity of sperm extracts to vitelline coat in prawns [65], while in mice, a serine protease inhibitor Kazal-type-like protein bound to sperm, enhancing motility and suppressing sperm capacitation [66]. Although in these two species the function of the Kazel-like serpin is varied, it has the commonality that it interferes with sperm–oocyte interactions. Up-regulated (FC 2.3) in wRi-infected cells, crooked neck (crn) transcripts are involved in embryogenesis. In it's recessive form, crn is lethal to embryos, while heterozygotes display a crooked phenotype [67]. In both Drosophila and humans, crn has been implicated in the mRNA splicing process and is thought to be a pre-mRNA splicing factor [68], [69]. Another gene induced by wRi (FC 3.1), otefin, codes for a nuclear laminin which is essential for germ cell maintenance in Drosophila [70]. A further candidate protein, Dumpy-30 (Dpy-30) is expressed in spermatids in Drosophila, and mutations or knockout of the male-specific dpy-30L2 gene results in male sterility as mutant sperm have impaired motility and fail to accumulate in sperm storage organs of females [71]. In Anopheles cells, wAlbB up-regulates (FC 2.0; significant at unadjusted P<0.05) Dpy-30, and although the effect of over-expression is unknown, this could potentially have a role in the CI phenotype. Serine active site containing (Serac1) mediates sterility in mice [69] and is up regulated (FC 2.4) by wRi infection. TEP15, suppressed by both strains (FC wAlbB −3.5, wRi −2.2), may influence reproduction. TEP15 is a male accessory glands protein and is transferred to female in the mating plug [72]. It would be interesting to determine if Wolbachia-induced regulation of these transcripts is Anopheles specific or common to other insect species infected with Wolbachia.
In addition to these genes, heat shock proteins were dramatically down regulated by both bacterial strains, but the effect was most dramatic by wAlbB. HSPs have been associated with sperm production and are inferred to be involved in CI [35], [73]. A range of chaperone proteins were also up-regulated by wRi, including a cold shock protein (FC 4.8) multiple DNAJ heat shock proteins (FC 3.3, 2.1), GrpE protein (FC 2.7), and a ubiquilin-1 gene (FC 2.3).
Pathogen related phenotypes
Other identified regulated genes may have behavioral implications for infected Anopheles. It has been reported that some older wMelPop-infected Ae. aegypti mosquitoes have "bendy" and "shaky" phenotypes [74], [75]. The proboscis of "bendy" individuals is flexible and unable to penetrate the skin [74]. Mosquitoes with the "shaky" phenotype have a jittering action of the insect body [75]. Here, we have identified genes that may elucidate these phenotypes at the molecular level. Both Wolbachia strains suppress the defective proboscis extension response (dpr) gene (wAlbB −3.3, wRi −2.6). Moreover, this gene is also down regulated in Wolbachia-injected mosquitoes (wAlbB −3.0, wMelPop −2.4; Figure 2B & 2C). In Drosophila, dpr is part of a gene family encoding predicted cell adhesion molecules that contain two Ig domains [76]. It is possible that a reduction in cell adhesion causes plasticity in the proboscis leading to the "bendy" phenotype. In addition to reduced dpr transcripts, Wolbachia down-regulated numerous other cell adhesion genes. Interestingly, dpr also has been shown to be required for the proper timing of male courtship [76], and given that Anopheles have elaborate swarming courtship behaviors in the wild, Wolbachia infection may have the potential to alter reproductive success.
Sestrins (Sesn), a family of conserved proteins, accumulate in cells in response to stress and are inhibitors of target of rampamycin (TOR) that prevent age-related pathologies [77], [78]. In wAlbB-infected cells, we see a down regulation of Sesn (FC −3.5). In Drosophila dSesn-null mutants, age related degeneration of muscle was observed in the form of cardiac malfunction and abnormal skeletal muscle [78]. Possibly, suppression of Sesn in wMelpop infected Ae. aegypti is related to the "shaky" phenotype [75]. Moreover, it would be interesting to correlate Sesn levels in both Drosophila and Ae. aegypti infected with wMelpop, which display life shortening and age related pathologies [17], [79], to determine if Sesn plays a role in life shortening from this strain of Wolbachia. The "shaky" and "bendy" phenotypes are more prevalent in older Wolbachia infected Aedes mosquitoes [75]. If the genes identified here confer the "bendy" and "shaky phenotypes in a Wolbachia-infected Anopheles mosquito, these effects could be more influential on malaria transmission compared to direct pathogen interference.
Wolbachia-infected mosquito cells provide a tractable platform to characterize Wolbachia-Anopheles transcriptomic interactions in the absence of a stably-infected mosquito strain. Using this system, we identified a suite of Anopheles genes regulated by two divergent Wolbachia strains. As a general theme, Wolbachia have a profound effect on transcription of many host defensive genes, possibly to facilitate and maintain intracellular infection. These data may give insights into the transfer of Wolbachia into novel hosts, Anopheles-Wolbachia interplay, interaction with pathogens transmitted by Anopheles and other Wolbachia-induced phenotypes such as reproductive manipulations.
Materials and Methods
Cell culture
Wolbachia-infected (wRi and wAlbB) and uninfected Sua5B cells were generated and cultured as previously described [14]. Both cell lines were >30 passages post-infection at the time of experiments. Cell line transcriptome expression was assessed using the Affymetrix Anopheles/Plasmodium GeneChip. Processing of samples for microarray analysis was performed by the Johns Hopkins Malaria Research Institute Gene Array Core Facility (JHMRI-GACF), using standard Core protocols as described below.
RNA extraction
Cells were harvested, washed, resuspended in PBS, flash frozen in liquid nitrogen, and stored at −80°C. Homogenization and lysis of cells was performed with Lysing Matrix D (Qbiogene) in Trizol LS reagent (Invitrogen) by rapid agitation in a FastPrep 120 Instrument (Qbiogene) for 15 seconds at speed setting 5. Homogenates were subsequently processed according to the manufacturer's (Invitrogen) protocol with the following minor modifications. Two microliters of 5 mg/ml glycogen was used as a carrier for overnight isopropanol precipitation, and all centrifugation times were increased to 15 minutes. RNA pellets were resuspended in Nuclease-free water. Further purification was performed using the Qiagen RNeasy Mini kit, according to manufacturer's recommended protocol. Quantitation of RNA was performed using a NanoDrop spectrophotometer, and quality assessment determined by RNA Nano LabChip analysis on an Agilent BioAnalyzer 2100.
Affymetrix GeneChip protocols
Processing of templates for GeneChip Analysis was in accordance with methods described in the Affymetrix GeneChip Expression Analysis Technical Manual, Revision 5. Double stranded cDNA was synthesized from 5 micrograms of total RNA using the GeneChip Expression 3′ amplification reagents one-cycle cDNA synthesis kit (Affymetrix), and subsequently column-purified using the GeneChip Sample Cleanup Module. Biotinylated cRNA was synthesized from the double-stranded cDNA by in vitro transcription (IVT) using the GeneChip Expression 3′ amplification reagents for IVT labeling (Affymetrix), according to the manufacturer's recommended protocol. Resultant cRNAs were purified by column purification with the GeneChip Sample Cleanup Module (Affymetrix), and quantified. 15 micrograms of cRNA were fragmented by metal-induced hydrolysis in fragmentation buffer (250 mM Tris acetate pH 8.1, 150 mM MgOAc, 500 mM KOAc) at 94°C for 35 minutes. Quality of pre- and post-fragmentation cRNAs was assessed by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100. Hybridization cocktails were prepared as recommended for arrays of "Standard" format including incubation at 94°C for 5 minutes and 45°C for 5 minutes, and centrifugation at maximum speed for 5 minutes prior to pipetting into the GeneChips (Affymetrix Plasmodium/Anopheles). Hybridization was performed at 45°C for 16 hours at 60 rpm in the Affymetrix rotisserie hybridization oven. The signal amplification protocol for washing and staining of eukaryotic targets was performed in an automated fluidics station (Affymetrix FS450). Arrays were scanned in a GeneChip 3000 7G laser scanner with autoloader (Affymetrix) at an emission wavelength of 570 nm and 2.5 µm resolution. Intensity of hybridization for each probe pair was computed by GCOS software.
Data analysis
Detailed analysis was performed with Genomics Suite Software, version 6.4 (Partek). GC-RMA algorithm defaults were used for background correction (GC-RMA), normalization (Quantile), and summarization (median polish) of probesets. Analysis of variance (ANOVA) was performed with linear contrasts for each Wolbachia treatment (strain) vs. control. Gene lists were developed based on 2 fold or greater gene expression and a False Discovery rate P<0.05 criteria. Lists were annotated manually. Immune gene networks were developed using Pathvisio2 [69].
qPCR verification of expression analysis
Using qPCR, microarray data were validated using infected cell cultures and also somatically-infected mosquitoes. Live female mosquitoes (2 days post emergence) were immobilized on ice and transferred to an electronic cold plate. Mosquitoes were injected with Wolbachia (wMelpop or wAlbB) or Schneider's medium as described previously [13]. Although a standard protocol was followed for Wolbachia preparations, titers were not explicitly standardized. Injected mosquitoes were incubated at 19°C for 2 days before transfer to 28°C (80% humidity) insectary and were provided with access to a 10% sucrose solution through a cotton wick. After 15 days, mosquitoes were collected and RNA was extracted using TriReagent (Ambion) following manufactures guidelines. For verification of microarray data, total RNA was extracted from Sua5B cell lines (uninfected, wAlbB-infected, or wRi-infected) using the RiboPureTM kit (Ambion) following the manufacturer's instructions. RNA from cells or mosquitoes was DNase treated (Ambion) and cDNA synthesized using superscript III (Invitrogen) following manufactures guidelines. qPCR was performed in triplicate on an AB 7300 Sequence Detection System using the QuantiTect SYBR Green PCR Kit (Qiagen). Analysis was performed using Sequence Detection Software v.1.3 (ABI). Relative quantitation was completed by normalizing gene of interest to the ribosomal protein S7 gene (primers listed in Table 1) and data analyzed using the comparative Ct method (ΔΔCt method) [80].
Accesion numbers
The following is a list of genes and their ENSEMBL or affymetrix accession numbers which are listed in the text: HSP20 AGAP005547, HSP90 Ag.2R.417.0_CDS_a_at, HSPDnaJ AGAP007565 AGAP001810, Cold-shock protein AGAP005641, Cecropin3 AGAP000694, SRPN11 AGAP001377, Filamin, AGAP004335, TEP3 AGAP010816, LRR-like AGAP004017, FBN9 AGAP011197, HSP70 AY137766.1_s_at, PEPCK AGAP003350, Carbonic anhydrase AGAP010052, Laminin AGAP001381 AGAP004993, Collagen AGAP009201, Peroxiredoxin AGAP011824, Superoxide dismutase AGAP010517, glutathione S transferases AGAP004164 AGAP004163 AGAP000761 AGAP009194 AGAP009193 AGAP004173 AGAP000165, CLIP7A AGAP011792, Galectin AGAP012529, CLIPB4 AGAP003250, CLIPB8 AGAP003057, Cecropin1 AGAP000693, TEP15 AGAP008364, GNBPB1 AGAP004455, Caspar AGAP006473, PGRP-LA AGAP005205, Attacin AGAP005620 ANCE AGAP009751 AGAP009756 AGAP009757 AGAP004563 AGAP007622 AGAP004563 AGAP007982, Kazal-like serpin AGAP011482, Crooked neck AGAP001879, Otefin AGAP007603, Dpy-30 AGAP007884, Serac1 AGAP011044, GrpE AGAP011150, ubiquilin AGAP004294, Defective proboscis extension response AGAP001242, Sestrin AGAP007169.
Supporting Information
Figure S1.
Supplementary Figure S1 and associated methods.
Table S1.
List of Anopheles genes significantly regulated by Wolbachia infection.
Table S2.
Common Wolbachia-regulated genes between Anopheles and Aedes.
Table S3.
Common genes regulated by Wolbachia and bacterial challenge in Anopheles.
We would like to thank the Johns Hopkins Malaria Research Institute Gene Array Core Facility (JHMRI-GACF) for assistance in with microarrays. The expertise, facilities, instrumentation, and software for Affymetrix GeneChip experimentation and analyses are provided and supported by the Johns Hopkins University Malaria Research Institute. We thank Scott O'Neill for his gift of the wMelPop Wolbachia strain.
Author Contributions
Conceived and designed the experiments: G. Hughes, X. Ren, J. Rasgon. Performed the experiments: G. Hughes, X. Ren, J. Ramirez, J. Sakamoto. Analyzed the data: G. Hughes, X. Ren, J. Ramirez, J. Bailey, A. Jedlicka, J. Rasgon. Contributed reagents/materials/analysis tools: J. Sakamoto, J. Rasgon. Wrote the paper: G. Hughes, J. Rasgon.
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text:
The tiller version of the FX 1750 SC, this model is loaded with smart features including convenient variable seating options, durable VEC construction, and excellent power and performance with a 17–foot hull.
HULL & DECK: Integrated Fiberglass Floor; Fixed Cleats (4); Grab Handles; Built in Fuel Tank; Deck Reinforcement for Rod Holder; Color Band w/Sport Graphic.
COCKPIT & BOW AREA: Bow Storage Compartment (4) w/Latches; Drink Cup Holder (4); Aerated Stern Livewell (29 gal); Bow Casting Platform; Stern Casting Platform; *Lockable Command Center for Fish Finder/Graph (Fits 2-5" or 1-10" Graph); Center Storage Under Forward Casting Platform; Trolling Motor Battery Storage Compartment (12/24 Volt) (Located Under Mid Rod Storage Locker).
SEATING: Fishing Seats (2) w/5 Bases.
ELECTRICAL: Auto Bilge Pump; Horn, Navagation/Anchor lights; Bow Panel (12/24 Volt); Lowrance Fish Finder Locator; Aerator Pump; Gauge Package Includes: Fuel, Tachometer, Rocker Switches & Courtesy Lights; Trolling Motor (Bow) MinnKota 55lb (12V) w/Slide Plate.
TECHNICAL: Five to Life Warranty, VEC Construction, NMMA Certified, Meets or Exceeds USCG Regulations.
TRAILER: Custom Color Matched FX 2300 lbs w/Swing Tongue & Diamond Plated Fenders. Trailers Standard for US and Canada Only.


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