text:
ET&C published highly cited, heavily downloaded Focus Articles and Critical Reviews in 2012. These articles highlight the latest research in the field, and have become a valuable asset to ET&C readers. The journal is now accepting proposals for Focus Articles for the second half of 2013 and would like to encourage SETAC members to submit their ideas and research. If you have an idea for a Focus Article, please contact ET&C's Editor-in-Chief Allen Burton at email@example.com. Critical Reviews can be submitted online throughout the year at mc.manuscriptcentral.com:80/etc.
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text:
Ohio State football talk.
That video has been posted a thousand times and I hope it gets posted a thousand more!
Troll hunter
Never saw the whole thing. Good stuff. They don't make em like that anymore...
"Do not pass me, just slow down - I can move right through you" Superchunk - Precision Auto.

subset: commoncrawl


text:
Bungie reveals Destiny's Achievements and Trophies
Posted on June 30, 2014 AT 01:03am
Bungie has revealed the full list of Achievements and Trophies that players will be striving for when Destiny launches this September.
A couple of notable Achievements/Trophies include "Suited for War," which requires players to kit out their character with Legendary or Exotic gear in every slot, and "Fully Weaponized," which asks players to fully upgrade a Legendary weapon. There are also rewards for fully upgrading each of the game's three classes.
During the weekly update, where this list was revealed, Bungie also commented on the rumors that the game cost $500 million to produce. "We're pouring everything it takes into Destiny to ensure it meets our fans' expectations, and our own," the developer said. "Activision is, too. But the budget for Destiny, including associated marketing costs and pizza Wednesdays, is nowhere near 500 million dollars."
Bungie also had something to say about the complaints regarding Peter Dinklage's voice acting in the recent alpha, stating that it has already been improved on for the upcoming beta. "Hey, we read the same forums, my fictional, question formulating friend! In fact, the Ghost dialog has already been updated for Beta. It will be updated again for launch on September 9th, as well.
"Funny thing about Alpha builds…they're not done. Please pardon our Moon dust!"
Destiny is set to launch September 9 for PlayStation 3, PlayStation 4, Xbox 360, and Xbox One.
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text:
Systems and methods for detecting false signals on a telephone line. The devices provide an additional verification for outbound calls which result in a disconnected or not in service signal that such signals are valid and not false tones being emitted by a device or service employed by the call recipient, The type of additional verification of the outbound call is dependent on the telephone network on which the outbound call is placed. For example, on an analog telephone network a drop in loop current may be detected indicting a connected call which is contrary to the disconnected or not in service signals being received. Such connected signals can be detected on digital networks by monitoring dedicated control data channels or through the use of data received through the use of in-band signaling. Once the additional verification had detected false tones the call can be process as if a connection occurred including routing such calls to an available agent or other handling process customized to handle such calls.
1. A method of detecting false signals on a telephone line, comprising:
placing an outbound call on a telephone line;
monitoring the telephone line for a detection of a service tone, wherein the service tone is a disconnected tone or a not-in-service tone;
detecting the service tone;
after detecting the service tone, determining, by a processor, that the outbound call is connected; and
upon the detection of a service tone and subsequent determination that the outbound call is connected, processing the outbound call as a false service tone call.
2. The method of
3. The method of
4. The method of
5. The method of
6. The method of
7. The method of
8. The method of
9. A call center for placing outbound calls, comprising:
a predictive dialer that places outbound calls on a telephone network;
at least one interface that connects the predictive dialer to a telephone network and routes connected outbound calls to a plurality of agents connected to the call center; and
at least one processor configured to:
detect a service tone on an outbound call, wherein the service tone is a disconnected tone or a not-in-service tone,
subsequent to detecting the service tone,
determine that the outbound call is connected, and
upon the detection of a service tone and subsequent determination that the outbound call is connected, process the outbound call as a false service tone call.
10. The system of
11. The system of
12. The system of
13. A system of detecting false signals on a telephone line, comprising:
at least one interface connecting to a telephone network;
a processor in communication with the at least one interface, wherein the processor is configured to execute computer executable instructions to:
place an outbound call on a telephone line;
monitor the telephone line for a detection of a service tone, wherein the service tone is a disconnected tone or a not-in-service tone;
detect the service tone;
after detecting the service tone, determine that the outbound call is connected; and
upon the detection of a service tone and subsequent determination that the outbound call is connected, process the outbound call as a false service tone call.
14. The system of
15. The system of
16. The system of
17. The system of
18. The system of
19. The system of
20. The system of
The present application claims priority to U.S. Provisional Patent Application Ser. No. 60/681,257, filed May 16, 2005, which is hereby incorporated by reference as if set forth fully herein.
The invention is related to the field of telephony and communications. More particularly, the invention relates to automated call distribution technology.
Existing technologies that automatically place outbound telephone calls, such as predictive dialing systems, are used by many companies to greatly improve the efficiency of their agents. Such systems are used by telemarketing companies, market research companies, and are heavily used by companies making collection calls. Such systems rely on hardware and/or software to determine the result of an outbound placed call. It is desirable that the system know the difference between a human answer, an answering machine, a busy signal, and a disconnected or not in service number recording in order to send only human answered calls to agents. The primary method for detecting that a call is being placed to a disconnected or not in service number is monitoring the line for the distinctive "tones" received or detected prior to the disconnected or not in service recordings. The frequency of these tones can be detected and the call can then be classified as disconnected or not in service.
Recently devices have been marketed to the general public, which are placed on their telephone line that falsely transmit the "tones" normally transmitted by the telephone network to signify a disconnected or not in service number. One such device is known as the Telezapper�, sold commercially by Privacy Technologies, Inc, a wholly owned subsidiary of Royal Appliance Manufacturing Co., a corporation located in the state of Ohio. While such devices have been marketed with the intent of stopping unwanted telemarketing calls, their use has caused problems for anyone placing calls to such telephone lines for legitimate business purposes such as to collect a debt owed by a person at the called number, or calls from companies with which the person has an existing business relationship, in which cases the called person may indeed wish to receive the call. Thus, there is an unsatisfied need for properly distinguishing between false tones transmitted by such devices and the valid tones sent by the telephone network.
The present invention is directed to systems and methods for detecting false signals on a telephone line. A method in accordance with an embodiment of the present invention includes the steps of placing an outbound call on a telephone line, monitoring the telephone line for a detection of a service tone and for a call connected signal. The monitoring can be performed in a variety of ways and may include monitoring for a drop in loop current on the telephone line, monitoring in-band signals for a signal indicative of a call connected signal, monitoring channel associated signals for a signal indicative of a call connected signal, or acquiring a signal from the control channel indicative of a call connected signal. When both a service tone and connected signal are detected in response to the outbound call, then the call is processed as a false service tone call. A false service tone call can be processed in a variety of ways including routing the call to an agent, playing a message on the telephone line or processing the call in substantially the same manner as a connected call. In accordance with an aspect of the present invention a service tone may be a disconnected service tone or a not in service tone.
The present invention is also directed to a call center for placing outbound calls which includes a predictive dialer, one or more processor that detect a service tone, detect a call connected signal, and determine when both detections have occurred on the same call, one or more interfaces that connect the call center to a telephone network and route calls to one or more agents that are connected to the call center at least in part on when both a service tone and a call connected signal are detected on the same call. Additionally, the call center also may include, or work in conjunction with, a router that routes a call from the call center to an agent based at least in part on when both a service tone and a call connected signal are detected on the same call. In an alternative embodiment of the present invention, the functions of the one or more processors can be performed by the single processor or two or more processors such as the call center's central processing unit, predictive dialer, or dedicated digital signal processors, ASICs, programmable ICs (PICs), etc.
Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein:
In accordance with an embodiment of the present invention, an exemplary system for implementing the present invention described below is illustrated in
These computer program instructions may also be stored in a computer-readable memory that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory produce an article of manufacture including instruction means that implement the function specified in the flowchart block or blocks. The computer program instructions may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions that execute on the computer or other programmable apparatus provide steps for implementing the functions specified in the flowchart block or blocks.
Accordingly, blocks of the flowchart illustrations support combinations of means for performing the specified functions, combinations of steps for performing the specified functions and program instruction means for performing the specified functions. It will also be understood that each block of the flowchart illustrations, and combinations of blocks in the flowchart illustrations, can be implemented by special purpose hardware-based computer systems that perform the specified functions or steps, or combinations of special purpose hardware and computer instructions. The inventions may be implemented through an application program running on an operating system of a computer. The inventions also may be practiced with other computer system configurations, including hand-held devices, multiprocessor systems, microprocessor based or programmable consumer electronics, mini-computers, mainframe computers, etc.
Application programs that are components of the invention may include routines, programs, components, data structures, etc. that implements certain abstract data types, perform certain tasks, actions, or tasks. In a distributed computing environment, the application program (in whole or in part) may be located in local memory, or in other storage. In addition, or in the alternative, the application program (in whole or in part) may be located in remote memory or in storage to allow for the practice of the inventions where tasks are performed by remote processing devices linked through a communications network.
The present invention will now be described more fully hereinafter with reference to the accompanying figures, in which like numerals indicate like elements throughout the several drawings. Some, but not all embodiments of the invention are described. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements, be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
With reference to
In the illustrative embodiment, the database 112 stores computer program code, and the program code can be designed to implement the process described below in connection with
The call center 106 uses the interfaces 110 and 114 to connect the call recipients 102 to the agents 118. The line interface 110 connects the predictive dialer 116 to a network 104, and the station interface 114 routes connected outbound calls to one or more agents 118 connected to the call center 106. In an alternative embodiment of the present invention, a router, located within or remote from the call center 106 may be used to route calls directly from the line interface 110 or predictive dialer 116 to the station interface 114 or directly to the agents 118 themselves. The call center 106 automatically places telephone calls over a network 104, such as the public switch telephone network (PSTN), cellular network, Internet, etc. through the use of one or more predictive dialers 116. The placed telephone calls can be transmitted over analog transmission lines, digital transmission lines, or both. It is also understood that such calls can be transmitted over a Voice over IP (VoIP) network. A predictive dialer 116 initiates a call and its associated hardware (e.g., a digital signal processor) monitors the progress of the call (e.g., connection to a person, connection to a machine, busy signal, no connection, or service tones such as disconnected tones, not in service tones or similar signals, etc.). The hardware used to monitor an initiated call may utilize the call center processor 108, the predictive dialer 116 itself, or hardware associated with the predictive dialer 116 such as dedicated ASIC chip(s), digital signal processor(s), PIC processors or an associated monitoring device controlled by the call center processor 108 as well as analog-to-digital and digital-to-analog converters depending on the transmission line being used. The network 104 routes the call to the appropriate call recipient 102. The call center 106 monitors the call over the line interface 110, and when the call center 106 determines that a call should be handled by an live agent it routes the call on the line to an agent 116 by way of the station interfaces 114.
In accordance with an embodiment of the present invention, when the call center 106 has detected that a dialed phone number results in service tones (e.g., disconnected tones, not in service tones, or similar signals), an additional verification is conducted to determine if the detected tones are authentic or false, that is, are they generated by the telephone network or by a device on the called party's line. This false signal detection is provided for by the processor 108, predictive dialer 116, or another processor such as a digital signal processor, dedicated ASIC, or PIC processor located within or remote from the call center 106 located in
In implementing the false signal detection process, which will be described below with reference to
If a call connected signal or indication is not observed, then the disconnect or not-in-service tones are treated as authentic and the call is processed accordingly. If a call connected signal or indication thereof is observed, then the service tones are treated as false service tones and the call is process accordingly, such as routing the call by the call center processor 108 to an available agent 118 over the station interface 114 for handling, playing a recorded message on the telephone line, or other call servicing that would be appreciated by one of ordinary skill in the art.
The detection of a call connected signal can occur in a variety of ways based in part on the type of network (analog or digital) the connection is operating. An example of how a "call connected" signal may be detected on an analog network can be a drop in loop current. In digital networks, a "call connected" signal may be acquired from the control channel in the group of dedicated digital lines being used. The control channel is a data channel that includes flags (or bits) that can be set or switched when a call has been connected. Another example of detecting when a call has been answered on digital networks involves the use of in-band signaling (e.g., allocating bits from the voice data transmission for signaling purposes and monitoring the allocated portion of the transmitted signal to determine if a call had been answered), and/or the use of channel associated signaling (CAS) protocol.
Next, if no signal or notification has been received or detected that would indicate that the call has been answered, then the tones may be presumed to be legitimate tones signifying the number is in fact either disconnected or not in service. In such a case, step 210 is invoked to classify and process the call appropriately (e.g., logging the detection of a disconnect or not in service signal for that particular phone number in a database, etc.).
However, if a call connected signal has been received or detected, then step 212 is invoked and the tones are classified as false tones transmitted by a device at the other end of the line and the call can then be handled as desired, such as based on a predetermined protocol. Examples of how a false signal call can be handled include (1) connecting the call to an available agent in the handling group for which the call was placed, just as one as if the call was answered by a person, (2) connect the call to an agent in a different agent specified group for special handling of these types of calls, (3) play a message on the line, (4) disconnect the call and mark the number and associated records as such for processing at a later time, (5) record the called number in a manner that signifies a false signal has been detected, and well as other suitable protocols for handling such calls. If the call is connected to an agent, then the agent may be notified by an audible whisper or screen interface message that the call resulted in false signals. These and other protocols can be used alone or in combination with one another.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
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Referenced by
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text:
Children whose parents separated or divorced when they were under age 5 had a fivefold increased risk of binge drinking at age 16 compared with children whose parents stayed together, a study including nearly 32,000 people showed. But the researchers also found that high levels of parental warmth before age 5 reduced the odds of excessive drinking by age 16, suggesting that instability and stress -- and not divorce itself -- affects children's drinking habits.
View Full Article in:Telegraph (London) (tiered subscription model), The
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text:
The grand Kushmas season is upon us! We're blazing the trees in celebration with the 1st Annual HMJ Kushmas Giveaway.
Everyday we announce a new awesome prize we're giving away on Instagram, Twitter, Tumblr, and Facebook. All you have to do is join us on your favorite social channels to win! Here are the rules:
It's Day 6 of the #HMJKushmas Giveaway and we're still going!!!
Today's prize is this awesome clear glass piece from Excellent Pipes.
Find the image below on Twitter, Facebook, Tu
Today we are giving away an awesome glass accessory prize package from our friends at Pipe Dream Pipe Clean, Pipe Wipes & Elevated Concepts' Lighter Buddy!
[420 Science] teamed up with visionary artist Stephan Peirce to create a line of tubes unlike anything you've seen before.The built-in jar provides a dual function. In addition to storage, the chamber creates a gentle swirling effect that aerates and cools the smoke. This results in a pleasing and refreshing hit. Kargo is made on 75x5mm heavy wall tubing and comes with a 4 ounce jar built into every pipe to store your favorite herbs
Made of thick and durable Pyrex glass. Equipped with a rubber grommet bowl and a carb hole. Plus, Free glass screens with every bong.
Featured Partner: Leafbuyer - The Best Marijuana Deals, Save At Your Local Dispensary
Leafbuyer is all about finding you the best ways to save money on your favorite cannabis products. Every day, hundreds of local Marijuana Dispensaries, Head Shops, and Grow Stores help us bring you deals and savings on anything you need. Whether you're looking to try the latest strain, find the perfect pipe, or start growing your own, Leafbuyer is your one stop shop to find the best marijuana deals in town. Businesses post new offers to Leafbuyer.com every day


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Cheap RBY to Modesto flights
Popular Flights from Ruby, Alaska
Top Ruby Flights to US Destinations
Popular Flights from Modesto, California
Top Modesto Flights to US Destinations
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RBY to MOD Route Map
Ruby to Modesto Flights, Stats and Airplanes
Route information for flights from Ruby AK to Modesto City County.
2,337.41 miles
MOD - Modesto City County Airlines
Browse the most popular airlines servicing the Modesto (MOD) airport. Visit their flights pages to learn more about the most popular routes these carriers fly.


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12:09 pm
Mon April 22, 2013
Tulsa Tower May Close Overnights
By KWGS News
Federal sequestration could have an impact soon on the Tulsa International Airport control tower. The Airport's Deputy Director Alexis Higgins says they have received notification that the tower may be closed from midnight to 5AM. Higgins stresses such a plan is only under consideration at this time.
Higgins says if that should come to pass it would have virtually no impact on commercial aviation. The last flights of the day arrive in Tulsa before midnight and there are no departures until after 6AM.
Federal sequestration has closed some control towers at regional airports because of the lack of funding to pay for air traffic controllers.


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News from WTOP
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Wed Apr 02nd, 2014
Roger Waldron, President, Coalition for Government Procurement.
The Senate's list of experts making recommendations for a 21st century government doesn't include any contractors or contracting groups. Agencies and think tanks will submit recommendations, but so far, no organizations that represent government's industry partners. Roger Waldron, president of the Coalition for Government Procurement, weighs in on In Depth with Francis Rose


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Fun for Ferrets
posted 7 years ago
We are using this in the house to get our ferret used to it untill spring when we can get outside. She has been great. Now lets get the snow melted!
Pros: This is just great
Cons: has to get used to it
9 of 9 people found this review helpful.
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Ferrets are officially adorable
posted 7 years ago
i saw a ferret walking around petsmart today in a harness similar to this and she was just the cutest thing i'd ever seen!! i really wish they would make harnesses smaller so that my little rat could have one-- the ferret seemed just fine with the idea of walking around the store on her leash. apparently, her brother didn't do so well! he had to stay in the car.
Pros: fit the ferret very well; didn't seem to mind walking in it
Cons: too big for my rat!!
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Guest Picks: Edwidge Danticat
Thursday, November 11, 2010
Edwidge Danticat stopped by The Leonard Lopate Show to share some of her favorite picks, and more.
What have you read or seen lately (book, play, film, etc.) that moved or surprised you?
Arcade Fire in concert in Quebec City
What are you listening to right now?
Arcade Fire "The Suburbs"
What's the last great book you read?
How To Read The Air by Dinaw Mengestu
What's one thing you're a fan of that people might not expect?
America's Top Model
What's your favorite comfort food?
Anything chocolate
Guests:Edwidge Danticat
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LAKELAND, Fla. (AP) — Justin Verlander hardly needed to win the American League Most Valuable Player and Cy Young Awards to boost his confidence.
"I am the same as I've always been," he said. "I have never been shy on confidence no matter what. I need my confidence and that's part of my ability to pitch."
Verlander allowed two runs and four hits in five innings Tuesday, making his fourth exhibition start as the Detroit Tigers beat the Atlanta Braves 7-2.
Verlander laughed when asked if expectations would be higher this year, coming off a season when he won the AL pitching triple crown by going 24-5 with a 2.40 ERA and 269 strikeouts. He said there's no difference between the current Verlander and the pitcher who went 11-17 in 2008.
Verlander struggled at times against the Braves, walking two and striking out four. Tigers manager Jim Leyland said he had no problem with Verlander pitching out of a jam for the first time this year.
"It gears up his intensity," Leyland said. "I don't like to see anyone put runners on base, but there's no problem."
After the Braves loaded the bases with no outs in the third, Verlander gave up a two-run single to Eric Hinske, then struck out Matt Diaz to end the inning.
"I was just trying to get my work in," Verlander said. "It was just time to take it to the next level and that's the first time this spring I have had to do that."
Ryan Raburn hit his sixth home run of spring training for Detroit.
Delmon Young hit his fourth homer and drove in three runs. Prince Fielder drove in a run and added two hits, boosting his batting average to .375.
Young had two hits and is hitting .485.
Braves starter Jair Jurrjens gave up seven runs — six earned — and eight hits in five innings. Bothered by the right knee injury that sidelined him last September, Jurrjens has a 10.13 ERA.
"I felt good," Jurrjens said. "They got some cheap hits in and there's nothing you can do about it. I felt good and I am happy with my control."
Braves manager Fredi Gonzalez said he wasn't jumping to conclusions about Jurrjens' struggles.
"We got five innings out of him," Gonzalez said. "He left a couple over the plate and they went out of the park. He's got two more starts."
Both starters struggled with the mound, and Jurrjens slipped in the fifth inning — causing Braves trainers to come out.
Braves closer Craig Kimbrel pitched one inning in his sixth straight scoreless appearance.
The Tigers reassigned C Bryan Holaday, IF/OF Eric Patterson, OF Jerad Head and OF Matt Young to their minor league camp.
Rick Porcello will start in a minor league game in Lakeland on Wednesday.
Pitcher Jacob Turner remains at least a week away from throwing due to tendinitis in his right shoulder.


text:
3.0 . . . Anyone?
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Join Date: Aug 2007
Posts: 113
Tom, hope you get back to the court soon.
I joined the same league as you and just had my first match, it was amazing. The only thing I am not sure is the players rating comparing to USTA rating, the league has players ranging from 3.0 to 4.5, don't know how they calculated that, and not sure if the ratings are accurate USTA ratings.
Originally Posted by
I'm a 3.0 to 3.25 level competitor also. However, my hitting partners are generally 3.5+ to 4.0+, as most sub 3.5 players don't hit solid shots consistently enough for good hitting sessions.
Why slow down on technique improvement? That doesn't make any sense to me. Practice with better players, grind on skills, join a league[*], and play matches at your competitive level. As you improve and win more matches, your rating will go up and you can compete in league play against ever higher rated players.
My excuse (
) for being a 3.0 competitor with 3.0 + strokes is that I'm typically twice as old as the people I play against, as well as being relatively inexperienced re total court time in my life. I've been sidelined for the past two weeks with a Crohn's disease flareup, but was improving before that, and I hope to continue to improve when I'm able to get back on the court again. I have no problem with bageling 3.0s and will continue to work toward being able to do that on a regular basis.
[*] I'm a member of Tennis Fort Lauderdale (tennisftlauderdale.com) which is affiliated with Tennis League Network (TLN). TLN began in Boston about 8 years ago and is now in several dozen metro areas. Check out Tennis Boston (tennisnortheast.com).
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text: Shlomi Steve Levy was recently interviewed by Bev Cline for an article which appeared in the Financial Post section of the National Post. In the article, Bev asks Shlomi about how Canadians should purchase US real estate and the special considerations that apply to such cross border purchases. Read the rest of this entry »


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Staff Reporter
Several IPS officers in the list
HYDERABAD: Twenty-five police personnel from the State have been selected for the prestigious President's Police medals, including several IPS officers, on the occasion of Independence Day for their distinguished services.
S. A. Huda, Additional Director General of Police, General Administration (Vigilance and Enforcement Wing), B. L. Meena, Deputy Inspector General of Police, Warangal Range, A. B. Venkateswara Rao, Joint Commissioner of Police, Central Armed Reserve, Headquarters, Ch. D. Tirumala Rao, Deputy Inspector General of Police, Special Intelligence Branch, Hyderabad, V. Naveen Chand, Superintendent of Police, East Godavari, G. Surya Prakash Rao, Joint Director, Anti Corruption Bureau, Hyderabad, P. Umapathi, Superintendent of Police, CID.
A.Sundar Kumar Das, Assistant Inspector General of Police, Administration, DGP Office, C. S. R. K. L. N. Raju, Superintendent of Police, CID, T. Saibaba, Deputy Superintendent of Police, ACB, Hyderabad, K. L. Devender Rao, Sub-Divisional Police Officer, Tenali, S. Natarajan, Deputy Superintendent of Police, CI Cell (Intelligence), V. S. R. Chowdhary, Deputy Superintendent of Police, ACB, Nellore.
K. Sudershan Reddy, Inspector, Andhra Pradesh Lokayukta and Upa Lokayukta, Hyderabad, V. Lingaiah, Sub-Inspector, CID, Mohammed Khaja Moinuddin, Sub-Inspector, SIB, B. Gopal, Sub-Inspector, Police Computer Services, Shaik Abdul Rasheed, Sub-Inspector, Gudur, Y. V. Ramana Rao, Sub-Inspector, First Battalion, APSP, Yosufguda, G. Lawrence Raju, Head Constable, Thotlavalluru police station in Vijayawada, T. Mohan Das, Head Constable, CSB, Vijayawada, K. Chandraiah, Head Constable, Karimnagar, P. Chenna Reddy, Head Constable, Nalgonda, K. Veeraiah, Head Constable, Warangal and K. Murali, Constable, Chittoor.


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Celtics on eBay: Red Auerbach's signed cigar
|08.19.11 at 4:55 pm ET
It's time to take you into the weekend with the latest edition of Celtics on eBay, which has now had four different names in four different weeks. Basically we ask, "Would you pay [a pretty penny] for [current or former member of the Celtics]' [eBay item]?" Confusing enough? Good. Let's get started.
This might be the first thing I would actually buy. It's a cigar. Signed by Red Auerbach. The greatest basketball coach in the history of basketball coaches (well, other than M.L. Carr). For 310 bucks. …
Or is it? I just spent an hour trying to figure out if the guy pictured signing the cigar is actually Red Auerbach or not. I still can't decide. Either way, I still might buy it.
Want to waste more time this weekend? Vote on these overpriced/awesome Celtics items from previous weeks:
- Ray Allen's Nike Air Jordan VIII shoes for $1,450 ("hell no": 162; "hell yes": 110)
- (Not) Tony Gaffney's 1-carat diamond earrings for $3,950 ("hell no": 86; "hell yes": 6)
- Arnie Risen's 1957 Celtics contract for $1,200 ("hell no": 35; "hell yes": 10)
Latest from Bleacher Report
- Why Celtics Will Be Better Than Expected in 2014-15
- Should Celtics Fans Buy or Sell Rondo's Hot Start?
- Lessons Learned from Celtics so Far
- Why Green Will Finally Succeed in Boston
- C's Big Offense Showing a Sign of Things to Come?
- Green's Transition to PF Could Reshape NBA Career Arc
- Could Rondo's Play Increase the Chances He's Dealt?


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Rajya Sabha MP and AICC Secretary Shantaram Naik on Thursday described the order banning Sri Ram Sene in Goa as 'toothless'.
"The ban order imposed by the Goa Government on Sri Ram Sene is without teeth, unless the order is followed by a regular order by the Central Government under the Unlawful Activities (Prevention) Act, 1967," Naik told reporters on Thursday.
Goa government has issued the order under section 144 of the Criminal Procedure Code but it will last only for two months and with great difficulty, for another six months. State Chief Minister Manohar Parrikar had announced on the floor of the House yesterday that Sene's entry in the state has been banned.
Naik said under Unlawful Activities (Prevention) Act, if the Central Government is of opinion that any association is unlawful association, it may, by notification in the Official Gazette, declare such association to be unlawful.
He said that Goa government has to prepare a case against Sene with proper documents including CDs with his speeches in Goa and outside, while demanding an immediate order declaring the outfit as unlawful.
The Congress leader said that if Goa CM is genuinely interested to prevent Muthalik from harming the social fabric of the state, then he should approach the Central Government under the Unlawful Activities legislation and prepare his dossier, which will not only convince the Home Ministry to pass necessary order but issue an order which will also be sustained before the Tribunal.


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Please use this identifier to cite or link to this item: http://hdl.handle.net/10355/953
|Resveratrol is a superinducer of the estrogen receptor [abstract]
|Beeman, Joseph; Myears, Hillary E.; Ghormley, Alison L.; Nagel, Susan C.
|University of Missouri-Columbia. Office of Undergraduate Research
|University of Missouri--Columbia. Office of Undergraduate Research
|Estrogens are steroid hormones that diffuse across the cell membrane and into the nucleus to bind with the estrogen receptor (ER). When bound, usually by the body's natural estrogen, estradiol, the ER binds with a specific DNA sequence found in the promoter region of estrogen target genes and is effectively able to regulate gene expression. Interestingly, many synthetic chemicals have been found to exhibit estrogen-like qualities because they are also able to interact with the estrogen receptor; these estrogen-mimicking compounds have been termed xenoestrogens. Billions of pounds of said chemicals are produced annually and can be found in many of today's widely used plastics, cosmetics and pesticides. Since xenoestrogens are capable of eliciting such significant biological alterations, like gene expression regulation, and are ubiquitous in nature, they pose an increasingly serious threat to public health. In this study, one known xenoestrogen, resveratrol, found in the skins of grapes and grape-related products, was tested for estrogenic activity. Immortalized MCF7 breast cells were transfected with an estrogen-sensitive reporter gene and then induced with a range of resveratrol concentration. After a twenty-five hour induction, the cells were screened for estrogenic response. It was found that resveratrol displays an atypically steep dose response and can be classified as a superinducer of the ER. Further investigation must still be done to determine what in vivo biological implications a xenoestrogen of this kind carries and will be performed using a transgenic estrogen-responsive mouse model.
Items in MOspace are protected by copyright, with all rights reserved, unless otherwise indicated.


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Francis Ford Coppola Encyclopedia
Inspiration + awesome web design + mind blowing packaging design + wine + customized moleskine + education + amusement? Does it really get any better? I don't even know where to start talking to you about how impressed and fascinated i am with the Francis Ford Coppola Encyclopedia site KnoWine.com ~ or the fact that they are making wine even more accessible by TEACHING people about it, with a playful site filled with videos, info, and a grade school feeling lined notebook paper on wooden desk feel! Super cool is that they sent people off to various regions for 30 days to learn about the wines and document their adventures in text, pics, and videos.
But seriously, lets talk packaging. Who knew making the "largest screwcap in all the land" could be so beautiful? As soon as i pulled the bottles out today i was already scheming ideas for what i could do with the gorgeous carafes... so, giant screwcap + awesome carafe bottle shape, it feels like an Erlenmeyer flask meets decanter... just enough glass details to keep it interesting (from the ridges to the text around the base... and that really sweet chemical structure of reservatrol, the antioxidant found in the skin of grapes)... not to mention the branding of using stamps with the countries the wines come from instead of labels! Oh, and then there's also the inspiring quotes on the lids of each bottle that can't not bring a smile to anyone's face when they are about to open it! And to top that, they sent over a few customized Francis Ford Coppola Encyclopedia customized moleskines! And they go beyond the usual cover art ~ they actually stickerize the inside cover with the stamp/labels from each region as well as stamp quotes and more randomly throughout!
I think they said it best: "Simply stated, wine is discovery. From how it's made and where it's made to why we drink it when we do, wine is not just wine." So take a look at MANY pictures on the next page, from close ups to the bottles and notebooks to screenshots and diagrams of their design choices.
TO PAGE 2 of "Francis Ford Coppola Encyclopedia"! ----->
Tags: gifts - packaging - review - science - shopping - wine


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Darcy Burner is the Executive Director of ProgressCongress.org and the Progressive Congress Action Fund, which work to connect the progressive movement, ideas, and Congress. She ran for the U.S. House of Representatives twice, and serves on the boards of NARAL Pro-Choice America, Campaign for a Livable World's PeacePAC, and the Congressional Progressive Caucus PAC.
Since 1945, health insurance companies have been allowed to collude to fix prices. Members of Congress will soon vote on whether to reinstate anti-trust provisions for the industry.


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MWC (Maybe We Can't) league fails again in SDSU controversy
Would J. Edgar Hoover invite Julius and Ethel Rosenberg to sit in on security meetings?
Would Gen. Washington text Benedict Arnold with his battle plans for Yorktown?
Would the Mountain West Conference allow a Brigham Young University athletic department employee to work in the replay booth during last Saturday's San Diego State-BYU football game?
The answer to the first two questions: Of course not. The answer to the third: Of course.
The MWC. The Maybe We Can't league. This is a conference that has blown its high-fallutin' expansion plans, blown its TV exposure by leaving ESPN (which kind of has national appeal) for The Mtn. (which at least entertained the Chilean miners), and now has failed to properly police its replay booth.
It's bad enough the Aztecs didn't play well at BYU. But to get screwed on an obvious replay call that eventually may have cost them the game due to the replay burlesque act that included one of the Cougars' own workers, is beyond unacceptable.
I'm not here to say the guy — in this case, Chad Bunn, BYU's video coordinator — did anything untoward. I don't know him. I wasn't in the booth, although being an SDSU alum, maybe I could have helped even things out.
But I do know they blew it when they failed to overturn an obvious missed fumble call that allowed the Cougars to go on and score what would be the deciding touchdown in their 24-21 win.
And I do know Bunn & Co. couldn't, declined to, or refused to look at TV replays that clearly showed BYU running back JJ Di Luigi fumbled the ball away.
Question: Why the hell was a guy on BYU's payroll allowed anywhere near that booth, let alone involved in a critical decision? (Unlike the NFL, where the on-field referee makes the final call on reviews, the college game relies on the people upstairs.)
After initially reviewing the situation and before the Bunn revelation, the MWC suspended the three replay officials for a week, a slap on the wrist for a kick to the groin. The Aztecs may not have gone on to win the game — they lost primarily because they allowed previously punchless BYU to hold onto the ball for 45 minutes — but these suspensions do nothing, really.
What good did Ed Hochuli's blown call do for the Chargers in Denver? Sure didn't give San Diego a win. And this sure doesn't give the Aztecs one. Hochuli forced the NFL to change a rule. This incident calls for the collegiately impossible — enforcing common sense and fairness.
I know it's only sports, but as in life, nothing gets fixed until something goes terribly wrong. In this case, were all the red flags in the laundry? Nobody saw a possible conflict of interest? Why didn't they make BYU coach Bronco Mendenhall referee?
Aztecs coach Brady Hoke recalls a similar incident in 2001 when he was an assistant to Lloyd Carr at Michigan.
"We got beat in a 1-second game at Michigan State and the timekeeper was a guy known as Spartan Bob," Hoke says of Bob Stehlin. The incident, known as "Clockgate," eventually led to the Big Ten adopting instant replay.


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Find Out How Women in Saudi Arabia Broke a New Guinness World Record Last Week on Susie's Big Adventure.
November 5, 2010
JEDDAH, Saudi Arabia, Nov. 5, 2010 /PRNewswire/ — Women in Saudi Arabia united for a common cause – Breast Cancer Awareness – this past week and in the process shattered an existing Guinness World Record. With the odds stacked against them, it seemed to be an impossible feat, but Susie was there and shares her experience on Susie's Big Adventure.
A yummy breakfast platter and interesting public art sculptures are featured this week on Susie's other blog, Jeddah Daily Photo Journal.
SOURCE Susie's Big Adventure
Source: newswire


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The transfer of R plasmids between bacteria has been well studied under laboratory conditions and the transfer frequency has been found to vary between plasmids and under various physical conditions. For the first time, we here study the expression of the selected plasmid mobility genes traD, virB11 and virD4 in the 45 kb IncU plasmid, pRAS1, conferring resistance to tetracycline, trimethoprim and sulphonamide, using an in vivo zebrafish infection- treatment model.
Three days after oral infection of adult zebrafish with Aeromonas hydrophila harboring pRAS1, elevated expression of pro-inflammatory cytokine (TNF α, IL-1β and IL-8) and complement C3 genes in the intestine coincided with disease symptoms. Tetracycline, trimethoprim and an ineffective concentration of flumequine given 48 h prior to sampling, strongly increased expression of plasmid mobility genes, whereas an effective dosage of flumequine resulted in lower levels of mRNA copies of these genes relative to placebo treatment. Following effective treatment with flumequine, and ineffective treatments with a low concentration of flumequine, with trimethoprim or with sulphonamide, the intestinal expression of immune genes was strongly induced compared to placebo treated control fish.
Treatment of zebrafish infected with an antibiotic resistant (TcR, TmR, SuR) A. hydrophila with ineffective concentrations of flumequine or the ineffective antimicrobials tetracycline and trimethoprim strongly induced expression of genes mediating conjugative transfer of the R-plasmid pRAS1. Simultaneously, there was a strong induction of selected inflammatory and immune response genes, which was again evident in fish subjected to ineffective treatment protocols. Our findings point to the essential role of therapeutic practices in escalation or control of antibiotic resistance transfer, and suggest that antibiotic substances, even in sub-inhibitory concentrations, may stimulate innate defenses against bacterial infections.
The zebrafish (Danio rerio) is a small tropical teleost that bridges the phylogenetic evolutionary gap between invertebrates and mammals in experimental biomedicine. It is evolutionarily closer to humans than fruit flies and nematodes, and is easier to work with and study than mice [1]. Recently, increased interest in using zebrafish for studies of human diseases as disparate as cancer, microbial infections and immune-pathological changes has evolved [2]. As an infection model, zebrafish have been employed for study of both human and fish pathogens [1,3-6].
Aeromonas hydrophila is a ubiquitous Gram-negative aquatic bacterium and opportunistic pathogen causing fatal hemorrhagic septicemia in several fish species including warm water and temperate aquaculture species [7-9]. In particular, A. hydrophila infections have been repeatedly reported from zebrafish facilities causing unusual [10] and sometimes high mortality rates [11]. Some strains of A. hydrophila have also been reported to be important human pathogens [12].
Conjugative R plasmids assigned to the IncU incompatibility group are widespread in environmental and fish pathogenic Aeromonas species worldwide [13]. An IncU representative, pRAS1, was detected in Aeromonas salmonicida from Norway [14]. This plasmid is very similar to an IncU plasmid derived from a human urinary tract pathogenic Escherichia coli in Eastern Germany as early as the 1970's [15]. The IncU pRAS1 has the potential to transfer between bacteria of diverse environmental compartments with high transfer frequency on solid surfaces [16].
The variable drug resistance region of IncU R-plasmids may contain a heterogenic collection of drug resistance genes and transfer systems that can mediate recombination and acquisition of additional resistance genes. In our study we used the 45 kb pRAS1 containing a class 1 integron, responsible for trimethoprim and sulfonamide resistance caused by dfr16 and sul1, respectively. In addition there is a Tn1721 transposon encoding tetracycline resistance by the Tet A determinant [14]. A highly conserved DNA backbone structure with a variable region encoding antibiotic resistance has been postulated for IncU group members [14]. The IncU plasmid pFBAOT6 (84.749 bp) was sequenced [17] and found to be almost identical with the IncU backbone of another plasmid RA3 (45.909 bp) [18]. Functional analysis of this broad-host-range IncU group of plasmids has demonstrated their self-transfer, replication and stable maintenance in alpha-, beta-, and gammaproteobacteria. The genetic functional transfer block of pRA3 consists of twenty-one different genes [18]. The mobility genes traD, virB11 and virD4 were selected from this functional block of the conjugative genetic system for analysis in this study.
The expression of a wide number of genes responsible for innate immune responses towards microbes in the intestine of adult zebrafish has been evaluated [19-23]. A recent study demonstrated the distribution of important innate antibacterial immunity mediators such as peptidoglycan recognition protein (pglyrp) and a factor that regulates neutrophilic cell densities and cytokines in the entire intestine of healthy zebrafish [24]. The bacterial pathogen recognition receptors (Toll-like receptors etc.) and signaling pathways activating the immune response (pro-inflammatory cytokines, hepicidin and heptoglobin etc.) are similar to those in mammals [25].
The aim of this study was, therefore, to assess the expression of transfer genes of pRAS1 caused by a pathogenic A. hydrophila in vivo in response to antibiotic treatments, while simultaneously monitoring selected inflammatory and innate immune system parameters.
Bacterial strains and growth conditions
Aeromonas salmonicida 718 (NVI 2402/89) originally isolated from the head kidney of diseased Atlantic salmon in 1989, harboring a 25-MDa conjugative IncU plasmid, pRAS1, mediating resistance to oxytetracycline, trimethoprim and sulfadiazine was used as the donor strain. A. hydrophila strain (F315/10), originally isolated from a skin ulcer of freshwater reared salmon was used as the recipient strain, prior to zebrafish challenge. Both strains were cultured at 22°C on 5% cattle blood agar [blood agar base no 2, Difco] for 48 h (A. salmonicida) or 24 h (A. hydrophila).
In vitro conjugation experiments
Conjugal transfer experiments were performed as described by Schmidt et al. [26]. In brief, donor A. salmonicida 718 (carrying plasmid pRAS1) and recipient A. hydrophila F315/10 strains were grown overnight in Luria Broth (LB) with shaking at room temperature. Overnight cultures were diluted in LB to approximately 108 CFU/ml. Volumes of 100 μl of donor and recipient culture, respectively, were mixed and placed on the surface of a sterile 0.45 μm filter [Millipore] placed on the surface of an LB agar plate and incubated for 24 h at 22°C. The resultant colonies were suspended by vortexing the filter in 1 mL LB, pelleted and re-suspended in 100 μl of the same medium. Serial dilutions were then spread onto selective Luria agar (LA) plates supplemented with tetracycline (10 μg/ml), trimethoprim (10 μg/ml) and sulphonamide (200 μg/ml) for selection of trans-conjugants after 24 h incubation at 28°C. In parallel, the total number of recipients was estimated on LA after 24 h incubation at 28°C, a temperature not permissible for the donor strain. Conjugal transfer frequencies were calculated by dividing the number of trans-conjugants by the number of A. hydrophila recipients. The frequency of pRAS1 transfer was 1.8 × 10-3. Transfer of the R plasmid pRAS1 was confirmed by plasmid profile analyses and determination of the resistance pattern of the trans-conjugants as described by Cantas et al. [27].
Plasmid isolation
The plasmids were isolated from trans-conjugants using a QIAprep Spin Miniprep kit [Qiagen, Hilden, Germany]. Plasmids were visualized under ultraviolet illumination following electrophoresis in 1% horizontal agarose gels and staining with ethidium bromide. Plasmid size was determined using BAC-Track supercoiled DNA markers [Epicentre].
Zebrafish, challenge procedure and treatment
The zebrafish experiment was carried out at the experimental animal unit of the Norwegian School of Veterinary Science (NSVS), a facility licensed by the National Animal Research Committee. The experiment was approved by the same committee in accordance with national Regulations on Animal Experimentation. Adult zebrafish (> 6 months, TAB line) were supplied by the Aleström Zebrafish Lab (AZL), Oslo, Norway. The fish were fed commercial dry feed (SDS400, Special Diet Services, Witham, Essex, UK), twice daily according to AZL standard operational procedures. Water temperature was maintained at 22 ± 1°C throughout the experiment. Forty-two adult zebrafish of mixed gender (22 male, mean weight 441 mg/20 female, mean weight 514 mg) were allocated into 21 experimental units (sterile one-liter lab bottles: 2 fish per unit × 3 replicates × 7 experimental groups). All fish were starved for two days prior to experimental infection. The fish were anesthetized by immersion in benzocaine (ethyl p-aminobenzoate, 0.34 mg/ml) [Sigma-Aldrich]. Each fish was laid on its side on a moisturized paper tissue and a 20 μl saline suspension of pRAS1 bearing A. hydrophila F315/10 (1.6 × 108 CFU/ml) was administered into the stomach, using a micropipette fitted with a sterile feline urinary tract catheter (n = 18 units). The same volume of sterile physiological saline solution (0.9% NaCI) was employed for intubation of the uninfected control group (n = 3 units). The fish were immediately returned to the respective experimental unit and feeding resumed (every 12 h) to evaluate the appetite during the post challenge period. For the remaining part of the experiment, the fish were kept under continuous visual monitoring, with absence periods of less than 1 h. After 24 h the infected zebrafish were bath-treated with the following antibiotics [Sigma-Aldrich] added to the water: tetracycline (20 μg/ml), trimethoprim (20 μg/ml), sulphonamide (20 μg/ml) and subtherapeutic (0.06 μg/ml) or therapeutic (2 μg/ml) concentrations of flumequine, respectively. Distilled sterile water (1 ml/L) was used as a placebo treatment while the infection control groups were untreated.
Sampling and culturing
To avoid mortality caused by the A. hydrophila infection prior to sampling, and to ensure maximum RNA preservation in bacteria sampled from the intestinal tract and in the intestinal tissue, fish from the challenged and control groups were observed every hour for three days following exposure. All fish were euthanized by decapitation at the end of the experiment. The abdominal cavity was opened by incision as described elsewhere Cantas et al. [28]. Entire intestinal samples were transversally sliced (< 0.5 cm) and immediately immersed in RNAlater [Invitrogen] for bacterial and tissue RNA stabilization. Kidney samples from each sacrificed fish were examined bacteriologically for the presence of systemic infection. Specimens were streaked on 5% cattle blood agar and Brocalin agar [Merck, Darmstadt, Germany] as described by Cantas et al. [28].
Gene expression
Total RNA from RNAlater-stored tissue samples was extracted using Trizol Reagent [Invitrogen, Carlsbad, CA, USA]. Sterile 5 mm steel beads [Qiagen, Valencia, CA] were added for complete bacterial lyses in a Qiagen TissueLyser [Qiagen, Valencia, CA], run at 30 Hz for 5 min. Further processing was performed with the RNeasy kit [Qiagen, Valencia, CA]. Complete removal of DNA was achieved by treating the supernatant from the RNeasy processed samples with RNase-Free DNase Set [Qiagen, Valencia, CA]. Gel electrophoresis was used to confirm that isolated RNA was intact while the concentration and purity of the RNA were quantified using NanoDrop® ND-1000 [NanoDrop Technologies, Delaware, USA].
Reverse transcription was performed with Superscript III Reverse Transcriptase [Invitrogen] following the manufacturer's instructions. cDNA amplifications were performed using previously published and novel designed specific primers [Table 1] by Primer 3 software [29]. Each primer (0.5 μl, 10 μM) was mixed with 18 μl of EXPRESS SYBR GreenER qPCR Supermix [Invitrogen]. Two μl template cDNA was used. Real-Time PCR tests were performed in duplicate with a Stratagene detection system [Stratagene, La Jolla, CA, USA] using optical grade 96-well plates, at the following conditions: 50°C for 2 min, 95°C for 2-10 min, 40 cycles of 95°C for 10-15 s and 55-60°C for 30-60 s. To determine the specificity of amplification, analysis of the product melting curve was performed after the last cycle of each amplification. Data was captured using Stratagene MxPro Mx3005P QPCR software.
Table 1. Primers employed for Real-Time PCR
Amplification efficiencies were measured with the formula of E = 10(-1/slope) by two-fold dilutions of cDNA as described by Bogerd et al. [31]. Expression of the plasmid target genes was normalized to dfr16, estimated to be the most stable endogenous reference gene on the plasmid for our in vivo experiment. The function describing the relationship between Ct (threshold cycle) and x (log copy number) for dfr16 was: Ct = -3.45x + 13.98; R2 = 0.99. The comparative CT method [2ΔCT method] was used to determine the expression level of analyzed genes [30]. The resultant fold units were calculated by dividing the normalized expression values with the placebo treated controls. Expression of the zebrafish inflammatory and immune response related target genes was normalized against expression of the housekeeping gene elongation factor 1 alpha (ef1α) [24] in challenged fish relative to sterile physiological saline solution intubated and placebo treated controls.
For absolute quantification of the total bacterial population of the gut, standard curves of 16S rDNA copy number were constructed using a PCR product of the 16S rRNA gene of Escherichia coli. The functions describing the relationship between Ct (threshold cycle) and x (log copy number) for total bacteria was: Ct = -3.19x + 53.66; R2 = 0.99, as used by Castillo et al. [32].
To better address the activity of the innate immune response in zebrafish during the A. hydrophila infection, the transcription levels of the immune mediators: TNF α, IL-1β and IL-8 (pro-inflammatory cytokines) and C3 (complement system, acute phase protein) were evaluated. Fold changes in mRNA levels post-challenge and treatment were calculated in relation to the average mRNA levels of placebo treated fish.
Statistical analysis
The effect of treatment on selected gene expression level was analyzed with Student's t-test as described by [33]. The results were expressed as mean ± SEM (standard error of the mean), based on variation between 6 adults per treatment group. Differences were considered significant at (*) p < 0.05, (**) p < 0.01 and (*** p < 0.001).
Clinical symptoms and re-isolation of A. hydrophila
No fish died within 3 days of the intubation challenge. All A. hydrophila inoculated zebrafish showed changes in external body color (pale, reddish coloration around gill covers), abnormal positioning in the aquarium (at the surface or near the bottom), increased gill ventilation frequency or lack of appetite within 24 h, while no such symptoms were seen in the uninfected control group. On termination of the experiment after 3 days, macroscopically visible ascites was observed in both the placebo treated fish and groups treated with ineffective antibiotics, whereas reduced clinical symptoms were noted in the group that had received effective treatment. Moderate to heavy growth of A. hydrophila in pure culture was detected from kidney samples of groups receiving placebo or ineffective treatments, whereas very low levels of A. hydrophila were isolated from groups of zebrafish exposed to effective antibiotic treatment [Figure 1].
Figure 1. Growth level median counts of A. hydrophila isolated from kidney samples of experimentally infected zebrafish, 48 h post antibiotic treatment (6 different treatment groups). Axis scale: absent = 0, very few = 1, few = 2, moderate = 3, rich = 4 and very rich = 5. Error bars represent ± SEM (6 adults per treatment group). Differences were considered significant at (**) p < 0.01 for total growth degree of placebo vs. other antibiotic treated zebrafish in each intestinal tissue analyzed.
Immune response of zebrafish to A. hydrophila
Compared to uninfected fish the transcription patterns of the innate immune response genes in placebo treated fish [Figure 2] were clearly raised and the transcription patterns of IL-1β (108 fold) and IL-8 (45 fold) genes were found to be substantially higher than TNF α (8 fold) and C3 (3 fold).
Figure 2. Relative pro-inflammatory cytokine and complement C3 genes expression levels across the entire intestine of A. hydrophila infected and placebo treated adult zebrafish after harvesting 3 days post-challenge. Expression levels are reported as fold change compared to average expression levels of uninfected (sterile physiological saline solution inoculated) control groups. Error bars represent ± SEM (based on variation between 6 adults per treatment group).
Comparing the gut microbiota related 16S rRNA gene copy number under different antibiotic treatments
The copy numbers of 16S rRNA genes in the digestive tract significantly decreased following treatment with inhibitory doses of flumequine. The copy numbers obtained from ineffective antibiotic treatment groups were similar to those observed in the placebo treated group [Figure 3].
Figure 3. Quantification of total gut microbiota in intestinal samples of experimentally infected and subsequently treated zebrafish based on 16S rDNA copy numbers. Error bars reflect ± SEM (based on variation between 6 adults per treatment group). Differences were considered significant at (***) p < 0.001 for total 16S rDNA copy numbers of placebo vs. other antibiotic treated zebrafish in each intestinal tissue analyzed.
Impact of antibiotic exposure on expression of the tra genes of pRAS1
The expression of traD, virB11 and virD4 was strongly induced by ineffective treatment (tetracycline, trimethoprim and sub-inhibitory levels of flumequine) and strongly reduced by treatment with effective concentrations of flumequine [Figure 4]. However, ineffective sulphonamide slightly reduced the expression of these genes.
Figure 4. Expression of three pRAS1 plasmid mobility genes in intestinal samples from adult zebrafish 48 h post treatment (72 h post experimental infection) relative to placebo treatment. Error bars represent ± SEM (based on variation between 6 adults per treatment group). Differences were considered significant at (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 for mobility gene expression levels of tetracycline vs. other antibiotic treated zebrafish in each intestinal tissue analyzed.
Immune responses following effective and ineffective treatments
Our results revealed a strong up-regulation of all four analyzed immune related genes after effective flumequine treatment. An induction of some of these genes was observed even after ineffective treatment with trimethoprim, sulphonamide and a sub-lethal level of flumequine, whereas ineffective tetracycline treatment apparently suppressed two of the innate immune response mediators [Figure 5].
Figure 5. Expression of selected inflammatory and immune response genes in the entire intestine of experimentally infected zebrafish 48 h post antibiotic treatment, relative to the expression in placebo treated fish (ref. Figure 2). Error bars represent ± SEM (based on variation between 6 adults per treatment group). Differences were considered significant at (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 for immune response levels of tetracycline vs. other antibiotic treated zebrafish in each intestinal tissue analyzed.
In this study, we have for the first time employed an experimental zebrafish infection- treatment model to mimic the conditions under which antibiotic resistance (mediated by a naturally occurring R-plasmid) transfer takes place in the intestinal microbiota during an infection caused by a resistant pathogen treated with effective or ineffective antibiotic treatments.
We were able to establish an infection with A. hydrophila resulting in disease symptoms similar to those previously described [10,11] but with no mortality 3 days post- infection, as intended in our study design. Rodriguez et al. [10] and Pullium et al. [11] observed per-acute cases of A. hydrophila infection with high mortality rates within a few hours possibly related to intraperitoneal injection of bacterial extracellular toxins and/or enzymes. In our study, re-isolation of the challenge organism from the kidneys of exposed fish supported the clinical findings and confirmed systemic infection. It has also been shown that A. hydrophila produces an array of virulence factors that induce strong inflammatory responses [34-36]. The induction kinetics of some of the zebrafish intestinal immune system genes revealed an Acute Phase Response (APR), that is the immediate host inflammatory reaction which counteract challenges such as tissue injury and infection [37]. In the current study A. hydrophila infection resulted in a clear increase in expression of the genes encoding the pro-inflammatory cytokines TNF α, IL-1β and IL-8. These cytokines are important inducers of APR resulting in increased production of Acute Phase Proteins (APPs) [38], such as C3. C3 is central in elimination of bacterial threats [39]. A systematic study of APR in zebrafish has shown striking similarities with mammals in function and induction of involved genes [25]. The fact that 1 IL-1β and IL-8 are highly induced while C3 remains moderately expressed is consistent with the expected expression profile at the early stages of infection (3 days in our case).
The composition of the zebrafish intestinal bacterial microbiota and its interaction with the host and the environment has previously been studied by cultivation and culture-independent methods [28,40]. In the present study this microflora and the experimentally introduced pRAS1 harboring A. hydrophila were impacted by various antibiotic treatments. Recent studies have shown that Real-Time PCR with species-specific or universal probes is an accurate and sensitive method for quantification of total bacterial populations as well as individual species from the intestinal contents [41-45].
In our study a broad spectrum of 16S rDNA primers were used since bacteria can have different genome sizes and different rrn operon copy numbers. There are different concepts for considering the rrn operon numbers in quantitative 16S rDNA-based experimental systems [43,44,46]. Ott et al. [47], have provided accurate and stable figures of similar bacterial concentrations in clinical samples with application of universal primers and specific probes. In the present study, 16S rDNA gene copy numbers were significantly decreased after effective flumequine treatment, whereas sub-lethal flumequine or the clinically relevant ineffective tetracycline, trimethoprim and sulphonamide treatments caused minimal change. The reduction in 16S rDNA gene copy number following treatment with flumequine might be the result of killing of pathogenic A. hydrophila and a disturbed and reduced commensal flora. In mammals and humans, it is well known that antibiotics can change the composition of the bacterial populations in the intestines [48-50]. Studies concerning the distribution of antibiotic resistant bacterial isolates in zebrafish facilities are, however, limited. Previous studies performed in our laboratory Cantas et al. [28], have shown a relatively low level of tetracycline (12-20%), trimethoprim (25-32%), sulphonamide (28-36%) and quinolone (0.5-4.8%) antibiotic resistant bacteria in the Gram negative cultivable gut flora in four different zebrafish facilities, one of which supplied the zebrafish for the present study. This would leave potential recipient flora for plasmid transfer in all treatment groups.
The minimal change in total 16S rDNA copy number following treatment with clinically relevant levels of tetracycline, trimethoprim and sulphonamide may be explained by multiplication of the resistant A. hydrophila pathogen due to the decreased competition following killing of the susceptible part of the normal intestinal microbiota.
The active involvement of the selected tra-genes in the DNA conjugation process is described [18]. The traD gene encodes an inner membrane protein with putative ATPase activity for DNA transport during bacterial conjugation. This protein forms a ring-shaped structure in the inner membrane through which DNA is passed to the transferosome [18,51]. However, it has been shown that the virB4 and virD11 genes may, in addition, mediate conjugative transfer via a C-terminal ATPase function during pili assembly which is more efficient on surfaces than in liquids [52,53]. pRAS1 is transferred approximately 1000× faster on solid surfaces compared to the frequency in liquid media [Kruse and Sørum 1994, unpublished data]
The genes of the conjugative transfer system studied i.e. traD, virB11 and virD4, were found to be differently expressed between the treatment groups. The expression of transfer genes was found to be low following sulphonamide and flumequine treatment, whereas treatment with a sub-inhibitory level of flumequine, clinical relevant levels of tetracycline and trimethoprim resulted in increased expression. Several factors have been proposed that could explain these differences; i) the susceptible gut microbiota was reduced in number leaving behind a variable number of potential conjugation recipients [54], ii) the donor potential and the genetic advantages/disadvantages of the specific plasmid in conjugating to the available recipient population [55], iii) the antibiotic itself might regulate the higher or lower expression levels of pRAS1 mobility genes resulting in possible different transfer frequencies. An increased transfer frequency induced by antibiotic exposures (tetracycline and trimethoprim) has been demonstrated for conjugal transfer of pRAS1 plasmid in sediment microcosm experiments [56].
A most remarkable result of the current study was the strongly increased expression levels of the selected plasmid transfer genes in the intestinal microbiota following treatment with tetracycline, trimethoprim (plasmid encoded resistance) and ineffective concentrations of flumequine. The low concentration of the quinolone flumequine was chosen to mimic the low concentration in the intestinal lumen when administering the drug intramuscularly or intravenously for treatment purposes, in in-appetent animals offered in-feed antibiotics, or by exposure to environmental residues from the water [55,57,58]. It has been shown that administration of sub-therapeutic levels can interfere with DNA replication (e.g. quinolones) [59,60], folic acid synthesis (e.g. trimethoprim) [61], protein synthesis (e.g. tetracycline) [62] as well as cell wall synthesis (e.g. β-lactams) [63] and may induce the so-called SOS response [64] which can promote acquisition and dissemination of antibiotic resistance genes [57,65]. Thus, our results reinforce the need for great caution in the use of SOS-inducing antibiotics to avoid induction of resistance transfer following antibiotic therapy. It is known that the LexA protein as part of the SOS response binds to the LexA box preceding the intI gene and thereby increasing the transcription rate of the intI gene resulting in an increased gene cassette exchange rate in the integron [66]. There is no recognized LexA box found close to the promoters of the traD, virB11 and virD4 genes of the pRAS1 plasmid sequence (data not shown). However, the occurrence of LexA targets in promoter sequence areas in vivo without the existence of a putative LexA box in the DNA sequence has been demonstrated. This indicates the assistance by an additional unknown factor in regulation of LexA gene expression in vivo [67].
An equally remarkable finding was the impact of antibiotic treatments on the expression of innate immunity genes. The decreased TNF α and C3 expression in the zebrafish's intestine after non-effective tetracycline treatment is in accordance with earlier reports [68,69] relating tetracyclines to posttranscriptional blockage of cytokine production [70]. Whereas, sulphonamide and trimethoprim treatments that have no impact on the growth of pathogenic A. hydrophila had little impact on IL-1β and IL-8, as expected. In contrast, the sub-inhibitory level of flumequine caused 40 and 20 fold increases in the expressions of IL-1β and IL-8, respectively. In addition effective flumequine treatment caused 200 and 100 times higher expressions of those genes, respectively. Hypothetically, this may be related to the immunomodulatory properties of those drugs [71,72] and in the diminished number (killed) of pathogenic A. hydrophila that can no longer depress the immune system by its virulence factors when the effective flumequine treatment was employed [73,74].
We have for the first time termed this clear, aggressive, immunological activity at the molecular level as 'Charged Immune Attack, (CIA)', which describes the inevitably strong revenge of the innate immune response against the weakened bacterial infection, as mediated by a short period with an effective antimicrobial treatment. The reason for this bias is not known, but both human and veterinary medical practitioners have observed that a single dose of antibiotics, sometimes surprisingly, may cure an infection. We think that the current results provide a glance into subtle and immediate effects of chemotherapy on the host's innate immune system that may be responsible for such outcomes. Further studies are needed to shed new light on the current findings and to clarify the underlying mechanisms.
For methodological reasons, most studies of in vivo conjugal plasmid transfer have been performed by adding donors and limited numbers of recipients in germ free animals [75,76] or by challenging conventional fish with genetically tagged bacteria [77]. To the best of our knowledge, this is the first report on the effect of antibiotic treatment of an infection on the expression of the tra genes of an R-plasmid harbored by the infecting pathogen and the early immune signals in a host model. Real-Time PCR technology offers a fast and reliable quantification of the mRNA production of any target sequence in a sample [78]. The results add information to our knowledge about development of antibiotic resistance in infected hosts including the clinical infection treatment and control scenario.
As expected the control of the A. hydrophila infection of zebrafish failed when tetracycline, trimethoprim and sulphonamide were used due to the R-plasmid (pRAS1) harbored by the pathogen. The same result was identified as expected when sub-inhibitory levels of flumequine were employed, whereas an effective dosage of flumequine reduced the clinical symptoms and controlled the pathogen and transfer of pRAS1. At the same time, the ineffective therapeutants tetracycline, trimethoprim and sub-inhibitory concentrations of flumequine increased the expression levels of plasmid mobility genes. The results should be taken into account by physicians and veterinarians when prescribing antibiotic drugs, underscoring the need to avoid risk for augmenting the transfer of genetic drug resistance elements to commensal microbiota.
This is the first combined in vivo study of antibiotic treatment on the innate immune system of the host and the conjugative activity of an R plasmid. A particularly valuable observation relates to the increased activity of the innate immune system caused by antibiotic exposure, even with ineffective drugs (R-plasmids) and at sub-therapeutic levels.
Authors' contributions
LC conceived the idea for the study, formulated the research hypothesis, designed the experiment, performed the fish infection studies, performed the sampling and data collection, carried out all bacteriological laboratory work including the quantitative Real-Time PCR tests, performed the statistical analysis and interpretation of the data, formulated the underlying causes and drafted the manuscript. PJM contributed to the study design and in vivo protocol, and supervised the zebrafish experimental infection trial. HS contributed to acquisition of funds, provided guidance to the formulation of the underlying hypothesis, supervision of the laboratory work and the interpretation of the data. All authors discussed the results, revised and adopted the manuscript.
This study was supported by Norwegian School of Veterinary Science. We thank Hanne Nilsen for donating Aeromonas hydrophila (F315/10) and the National Veterinary Institute, Norway for donating Aeromonas salmonicida 718 (NVI 2402/89). We also thank Samuel Duodu and Stine Braaen for technical support for quantitative Real-Time PCR assays. Finally we extend our thanks to Duncan Colquhoun and Arve Lund, for helpful support in reviewing the manuscript.
Disclosure statement
No competing financial interests exist.
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