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Well done! Only it's made by Squadafroin, not Firemage
I'd say Watery Grave by Firemage
Well done! Only it's made by Squadafroin, not Firemage
My bad, I mixed them up.
All for a Night's Sleep by Morrgan
All For A Night's Sleep, a good short but sweet one.
Someone else post a pic, OK? Thanks.
Dock Land
Me: 'Hmm, would that be Dock Land Purgator?'
Other Me: 'Indeed it would Purgator, well played Sir.'
Me: 'May I say, what a decidedly attractive hairdo you are sporting today, P.'
Other Me: 'Why thank you P, so kind of you to notice. I've been going to that new place on the High Street. Prices are a little steep, but the results are defintely worth it. Heads are turning, as they say.'
Me: ' I wish I could afford those prices. A pudding bowl and some safety scissors is about all my wallet will stretch to these days, I'm afraid.'
Other Me: ' Quite. Now, about posting that screenie, dear boy...'
Me: 'Oh, I think I'll let someone else do the posting if that's ok ? My leg is giving me a bit of gyp.'
Last edited by Purgator; 13th Jun 2018 at 10:09.
Ok, here we go:
Answer: Between These Dark Walls
Shadow Politics by Skacky?
Edit: Nope!
Oh, that's definitely Between These Dark Walls.
Edit: Yes!
I'll have a screenshot in a bit...
Curse of the Ancients
Last edited by Grandmauden; 13th Jun 2018 at 12:09.
Coterie of Smokers by Christine?


text:
* Last night things looked bad. VERY bad. Hotter was satting in the low nineties on six liters of humidified oxygen, CT scans were showing possible fluid build-up around the donor kidney, and he sounded...well, I'll spare him a public sharing of the details, but he sounded awful, y'all.
* I made myself a stiff drink and went to bed, where for the first time in almost a week I slept eight hours straight.
* While I was passed out, Hotter was getting his act together in a big way over at Local Teaching Hospital; last night's MRI showed NO fluid around the kidney, decreased fluid around the liver (to the point where they're no longer worried about that), and he is now OFF oxygen support entirely (with a pulse ox of 95, which isn't great but is better than he was managing on an arse-load of oxygen yesterday).
* At this point he is still there because a) they want to see how it goes when he eats something, which aside from some ramen on Sunday he hasn't really done since Thursday and b) before they have him eat something, they want to do a few more tests including an ultrasound of his abdomen, because when one of their peeps falls apart in such spectacular fashion the transplant team likes to leave no stone unturned (for which I applaud them--I want ANSWERS and when I do bring Hotter back home I want to feel reasonably certain he won't pull another boomerang on us and have to go right back in via the godawful Local Teaching Hospital ER).
* If today's test results are all good, Hotter is hoping they'll send him home tomorrow. He sounded crabby and rather put out about being at Club Med today, which is always a good sign.
* I haven't been able to find it in myself to care about this even a little bit until today, but right before Hotter went back in-patient my lodging arrangements for BlogHer kind of fell apart, and apparently that trusting the universe thing is still crap if you're me. The universe is still a cunt. So please let me know if you need a roommate (I'm fine with sharing a bed or hell, I'll sleep on the floor!), and also let me know if you're trying to acquire a full conference pass, because I'm starting to think my getting to BlogHer this year is a lost cause (dammit).
* My mother-in-law. Lord, y'all. The very least I can say about her right now is that she promised Hotter she'd come down here to see him, but then remembered that it's been 6,000 miles since her last oil change, refused to let Loud and Crazy (my brother-in-law) change her oil (which he is perfectly capable of doing, mind you, it's not that he isn't), and then refused to let Loud and Crazy take her car to Big and Scary (my other brother-in-law, who is a professional mechanic) and let HIM do it, and that is her official reason for not coming to visit her son in ICU: she is overdue for an oil change. Discuss!
ETA: Well, shit y'all. I just spoke with one of the surgeons and the chief radiologist reviewed Hotter's MRI from last night and determined that they ARE concerned about the fluid around the liver, it has NOT decreased (although it has also not INCREASED dramatically, which is kind of the best possible worst-case scenario), and it is, in fact, bile. Which is undesirable to have leaking in your abdominal cavity. They're leaving him NPO and doing an ERCP this afternoon to figure out whether the bile is coming from one of the ducts they clipped during surgery or from the liver itself (which is what they currently suspect). If that is in fact the case they're going to have to open his incision back up, wash him out, possibly do some minor repairs, and place a drain. He is NOT coming home tomorrow but at least they're closer to figuring this out?

subset: commoncrawl


text:
SILVIS, Ill. (AP) Brian Harman won his first PGA Tour title on Sunday, using three straight birdie putts in the final five holes to pull away from veteran Zach Johnson to win the John Deere Classic.
Harman had a 5-under 66 in the final round for a 22-under-262 total and a one-stroke victory over Johnson to win $846,000, along with the last exemption for next week's British Open.
Harman, a 27-year-old Georgian in his third full season on the PGA Tour, withstood Johnson's challenge by making three birdies beginning at No. 14 to get to 23 under with two holes to play. Johnson, playing four pairs ahead of Harman, birdied the 17th to get within a stroke, but ran out of holes.
Jhonattan Vegas raced into a tie for third with a closing 65, ending up tied with Jerry Kelly at 265. Scott Brown and Tim Clark tied for fifth at 266.


text:
Background Info
We Go Into More Detail About
- Overtraining Syndrome (OTS) and how it affects the Ultra-marathon world.
- Where the ultra-marathon sport is going.
- The importance of rest during your training.
- What is the ultra-marathon world like?
- How should you train for an ultra-marathon versus training for a marathon?
- How Meaghen went from not running at all to running her first ultra-marathon.
Links and Resources
- Meaghen Brown
- Running on Empty (Outside Magazine)
- Mike Foote - Ultramarathoner
- Scott Jurek sets record for Appalachian Trail
- Non functional overtraining
- Geoff Rowes - Ultramarathoner
- Michael Wardian - Ultramarathoner
- Hardrock 100 Ultra-marathoner
- Kilian Jornet - Ultramarathoner


text:
Outdoor Rug by Treasure Garden - Charleston Honey
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- 1 - year Fade Warranty
- Made in Belgium
Tags: outdoor, porch, rug, treasure, garden, charleston, honey, patio, rugs


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text: Shooting: Gear Gear Posted 06-30-2012 Magnum Research, Inc.® Introduces .22LR Semi-Automatic Rifle Contact Information: Sheryl Gallup Gallup Media Marketing, Inc. 763-300-1132 firstname.lastname@example.com Date: June 26, 2012 PEARL RIVER, NY – USA based, Magnum Research, Inc. (MRI) introduces the latest in their Magnum Lite® line of .22 LR semi-automatic rifles. The exciting new features of the MLR22AT will quickly become a favorite among .22 fans. Features of the new MLR22AT include an ambidextrous lightweight thumbhole 10/22® stock made from polypropylene with fiber additives for maximum strength. The new stock design features a comb height designed for comfort, with a semi-palm swell on both sides of the pistol grip, a molded-to-fit rubber butt plate, and the comb height easily accommodates optics. The new rifle will be equipped with the Magnum Lite® revolutionary patented graphite bull barrel, exclusive to Magnum Research. The patented uni-directional graphite fibers, parallel to the bore axis, results in a barrel with 6x the stiffness of the equivalent weight of steel. The lighter barrel weights (from 13-16 oz. depending on barrel length) are easier to handle and dissipate heat up to 43% faster than steel. The result is extremely accurate rimfire performance. Additional features of the MLR22AT include: .22LR Benz target chamber designed specifically for semi-automatics; full floating barrel for maximum accuracy, CNC-machined receiver from 6061-T6 aircraft aluminum forging with an integral weaver-type rail for optics, and a black anodized finish. The MLR22AT weighs only 4.25 lbs., has a barrel length of 17 inches, and an overall length of 35.5 inches. This rifle utilizes the 10/22® trigger group and 10/22® rotary magazines. MRI bolts are manufactured and machined from heat-treated and thoroughly hardened 4140 steel with minimum headspace for better accuracy. MSRP for the MLR22AT will be $562. For more information about Magnum Research products, visit www.magnumresearch.com. To learn more about Kahr Arms, visit www.kahr.com, or contact Sheryl Gallup at email@example.com 10/22® is a registered trademark of Sturm, Ruger® & Co. Inc. Share This Story Next From Gear Practical Rifle Scope Zeroing Related Comments Categories News Stories Tips Gear Videos Photo Galleries Viewer Photo Galleries Features Concealed Carry & Personal Defense Down Range TV Short Stories by Larry Potterfield AllOutdoor.com Outdoor Blog Sponsored Content Streamlight Shooting Check Out All The Photos Explore the United States. Find information about and activities within your state. Get Started


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text:
I hope everyone will enjoy making this Happy Father's Day To A Classic card. You can give it to your dad or your kids' dad, and it makes a fun Birthday card if you don't need either of those too. I love how easy it is to position the sentiments on the clear blocks just by using the grid paper.
Wednesday, May 13, 2015
Guy Greetings Antique Car
Posted by RhondaG -the stampin' bunny at 6:43 AM
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text:
How To: Fix Safari 6 crashing on Mountain Lion
It's been a couple days since Apple released Mountain Lion, and overall, I hope you're enjoying it as much as we are. After some reports from users, though, we believe there is a bug in Mountain Lion that could cause Safari 6 to crash for some users. Here's what's going on and how to fix it.
With the help of our excellent users and Daniel Jalkut over at Red Sweater, we've looked into this bug and it appears to be caused by a problem with proxy settings under Mountain Lion. This could be due to changes you made to your proxy configuration, or due to some apps that make these changes for you.
If you are experiencing Safari 6 crashing under Mountain Lion with our 1Password extension installed, a quick fix is to adjust your proxy configuration settings under System Preferences > Networking. Here is our document that outlines more details and another that shows you how to adjust your proxy settings.
We'll bring you more details as soon as we get them.


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GEO343 Remote Sensing
Principles, methods, techniques of remote sensing - including air photo interpretation and photogrammetry; their use in identification, analysis, and management of physical, cultural, and economic resources, application to geography and related physical and social sciences. Computer applications are included.
- 4
- Community Service
- Field Study
- Liberal Arts
- Research
- Must be enrolled in the following level: Undergraduate
- May not be enrolled in the following class: Freshman
- GEO273 Minimum Grade of D- or GEO202 Minimum Grade of D- or GLG201 Minimum Grade of D- or ANT211 Minimum Grade of D-


text: One of Martin County's most famous residents is profiled in December's Vanity Fair: "He's lost a step or two. 'Or five,' he says. At times, his hands tremble. He leans his spindly frame on a black cane. He never once rises from the sofa…Reynolds still can't believe he blew it with Sally Field, whom he terms the 'love of my life'… He's got a girlfriend, Rhonda Stearns, a local woman he's known for years… … Click here to read more


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Choice Tracks – Back For More, Perfectly Imperfect, Fearless, Genetic Process, When Black Turns To White
It's been seven long years since new Pyramaze music has been released, and after three very solid albums which featured a couple of different vocalists between them, it was time for this Danish melodic metal band to give us something new to digest. Along with new vocalist Terje Heroy in the mix, we have some very well done melopower! Pyramaze has a short, yet significant history of making some very dark and thought-provoking metal and Disciples Of The Sun is a definite addition to their arsenal of albums. There are bits on this album that remind me a bit of a more modern sound (I think it has to do with the vocal styles of the vocalist and not a direction change that the band is consciously taking). I think there is still more this band can do, and maybe now they have finally found that voice that they need to continue to build momentum upon. It's not that their music hasn't been good, as I have enjoyed each of their releases (they all have something to offer the listener), but I have just been waiting for this band to reach that next glorious level and they have fallen just a bit short each time. I think Disciples Of The Sun is the next step on the evolutionary ladder and after a seven year stretch, it's a big step, but there are still a few more rungs left on the ladder that need to be taken. If the band can stick together and stay out of the transitional stages, they are well within reach!
Choice Tracks – Back For More, Perfectly Imperfect, Fearless, Genetic Process, When Black Turns To White
True Metal Lives
The Voice Of The Underground
October 2018


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Birthday boy Kenny not expecting more gifts in Russia
The locals had done their homework. When Stephen Kenny checked into his hotel room in St Petersburg late on Tuesday night, after a trip from Dublin which was delayed by issues with Dundalk's chartered flight, there was some cake and a message awaiting him from hotel management, congratulating him on the occasion of his 45th birthday over the weekend.
Another gift, of sorts, came his way yesterday morning when Martin O'Neill confirmed that two Dundalk players - Andy Boyle and Daryl Horgan - would be named in the Republic of Ireland squad for next week's World Cup tie in Austria.
Kenny is not expecting any presents when he takes his side to a snow-kissed pitch in a chilly St Petersburg this evening, the 9pm local time kick-off ensuring that temperatures will be a real test for the recently crowned Airtricity League champions.
Dundalk winning tonight is unlikely, as classy European outfits like Lyon, Benfica, PSV and Sevilla have all tried, and failed, to win in St Petersburg in recent times.
"I am led to believe that Zenit are the highest-ranked team in the competition and we are the lowest, so for us to get a victory or a result would be a real shock throughout Europe," Kenny said.
"If we got that it would put us in a very strong position in the group.
"Listen, they're a brilliant team and we'll have to be brilliant to get something extraordinary. But we are capable of that."
Belfast Telegraph


text:
Product Reviews Write A Review
Sort Reviews by: Date | Rating Comments
1 5 stars by Gig'em6 out of on July 28th 2015
Billboard Hamburger
Length of ownership: 1 week-1 month
Pros: These are the best looking invitations ever. Our guests are excited about the party just because the invitations make it look like so much fun!
Cons: None


text:
RK Swamy BBDO gets communication mandate for PM?s Jan Dhan Yojana
MUMBAI: The financial inclusion program of the government of India labeled ?Prime Minister?s Jan Dhan Yojana? is being formally launched by the PM Narendra Modi on 28 Aug in New Delhi.
The launch communication mandate has been assigned to RK Swamy BBDO, who is charged with developing a fully integrated creative and media communication package. A committee comprising the department of financial services, ministry of finance, several leading banks and the Indian banks association led the agency selection process.
The program encompasses all media including TV, radio, newspapers, outdoor, internet and social media. The launch communication for this program commenced on 23 August in newspapers and on 25 August on television across the country. A superbly animated TV commercial shows a lot of people carrying their money into the bank and easily opening their bank accounts. In the end, the people join hands and morph into the logo.
RK Swamy BBDO CEO Shekar Swamy said, ?The Prime Minister?s Jan Dhan Yojana is so crucial for the welfare of millions. As an organisation, we place the top most priority to support such nation-building projects. We are privileged to be associated with it, and we are sparing no effort to ensure that the program is successful.?


text:
Frequently Asked Questions
Is Chamber membership a good investment?
It's a great investment. Ask any Lake County Chamber member — they'll tell you they recoup their membership costs many times over in benefits and discounts. The discounts on Workers Comp rates alone can save over what you're probably paying now.
Which Lake County Chamber of Commerce should I join?
It depends. If your company is in, Madison, for instance, you should probably start there. But if your company does business in other parts of Lake County, you might also consider membership in other Lake County Chambers.
How much does membership cost?
Membership costs vary from Chamber to Chamber. The logical place to start is by becoming a member of the Chamber where your business is actually located.
Of course you may decide, as many of our members have, to join additional Chambers in the places where you do business as well. Multiple Chamber memberships mean an increased networking base!
Are membership benefits the same with each Lake County Chamber?
More or less. All of our Chambers offer the "biggies" — affordable group health plans through Anthem Blue Cross/Blue Shield, Discounted Worker's Compensation rates from CompManagement, networking/business building opportunities, speakers and programs. But each Chamber also has its own special membership perks and pluses!
I'm already spread pretty thin. Is Chamber of Commerce membership time consuming?
It's your call entirely. Some of our members pop in for a meeting or an event whenever they can, others attend functions religiously, and/or volunteer to serve on Chamber boards and advisory committees. Naturally, the more you attend the greater your networking opportunities, but we fully understand that our members are busy folks.
Are there any other reasons to join?
Sure! First, it's fun. When you're a Chamber member you meet new friends, and get to join in some cool after-hours activities. Second, your support of your local Chamber helps us mount lots of important behind-the-scenes efforts to foster a good business climate in your market, and act as an assertive pro-business voice in Columbus and Washington.


text:
Pope's South Korea visit brings China opportunity
Pope Francis' greeting to Chinese President Xi Jinping as he flew to South Korea early Thursday was a rare cordial exchange between the sides that have no diplomatic relations and are embroiled in a sometimes bitter contest for authority.world Updated: Aug 14, 2014 11:54 IST
Pope Francis' greeting to Chinese President Xi Jinping as he flew to South Korea early Thursday was a rare cordial exchange between the sides that have no diplomatic relations and are embroiled in a sometimes bitter contest for authority.
Vatican protocol calls for Francis to send telegrams to heads of state whenever he flies through their airspace. Usually they pass unnoticed, but Thursday's telegram was unique because the last time a pope wanted to fly over China, in 1989, Beijing refused.
"Upon entering Chinese air space, I extend best wishes to your excellency and your fellow citizens, and I invoke the divine blessings of peace and well-being upon the nation," the pope's message said.
China's Foreign Ministry did not immediately reply to faxed questions about the pope's greeting and any Chinese response. While no Chinese officials are taking part in the visit, individual Chinese Catholics, including the many studying in South Korean seminaries and universities, were expected to attend events.
Vatican officials say there is a dialogue with Chinese authorities. But the core issue dividing them - Rome's insistence on naming bishops - remains.
"These are friendly gestures, but I wouldn't expect any breakthroughs," said Anthony Lam of the Holy Spirit Study Center in Hong Kong that closely monitors the church in mainland China.
Relations between Beijing and Rome have been tense since 1951, when China severed ties with the Holy See after the officially atheistic Communist Party took power and set up its own church outside the pope's authority. China persecuted the church for years until restoring a degree of religious freedom and freeing imprisoned priests in the late 1970s.
While China's state media largely ignored the pope's visit, brief reports about the first-ever papal flight over China and extending of greetings were posted to the popular Twitter-like Weibo microblog site - an indication they weren't a censored topic.
The popular Global Times tabloid also carried a round-up of foreign media reports on the issue, along with comments from Chinese expert on Vatican diplomacy Kong Chenyan calling it a "positive development" in China-Vatican relations.
However, Kong said China remained unhappy with what it sees as the Vatican's lack of respect for Chinese sovereignty, its relations with Taiwan, and lingering resentment over the canonization of saints viewed by the ruling party as enemies of the Chinese people.
First Published: Aug 14, 2014 11:50 IST


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Jealousy inside a relationship can trigger numerous problems but some people just have no idea how you can cope with jealousy. Some people can become irrationally jealous of their partner towards the extent that they don't wish to let them out of their site. They don't like their partner to even speak to or look at another woman or man or they become extremely jealous. The partner on the receiving finish of this jealousy might turn out to be tired of it
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Has he cheated on you prior to? Do you know for a reality that he has cheated or do you just suspect it? Has he given you reason to suspect he is cheating? You need to discover whether you've a valid reason for not trusting him.
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If you have been trying for a lengthy time to rebuild the trust but just can't get past his cheating then perhaps this relationship has run its course and you should move on.
Whether or not you trust your partner or not, if he flirts with other individuals do you think he is going to cheat? If not then why are you currently jealous?
Jealousy is truly only a response to a threat but if that threat becomes real as well as your partner flirts with someone after which cheats on you with that person, then the threat goes beyond jealousy and into betrayal.
The second method to help you deal with jealousy is to discover for your partner to flirt all they want and not let it get to you. Try to view flirting as an all-natural response that people have toward other individuals that they discover appealing. Even when your partner flirts with other ladies he still chooses to become with you so you really have no cause to become jealous of flirting knowing that you are the individual he chooses.
The third method to deal with jealousy would be to use it for your advantage inside a relationship. In the event you do this though you do need to be careful that it isn't taken too far. Showing a little possessiveness is actually enjoyable for many people and can add a spark to a relationship.
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Mar. Drugs 2012, 10(6), 1225-1243; doi:10.3390/md10061225
Abstract: Gorgonian corals possess many novel natural products that could potentially mediate coral-bacterial interactions. Since many bacteria use quorum sensing (QS) signals to facilitate colonization of host organisms, regulation of prokaryotic cell-to-cell communication may represent an important bacterial control mechanism. In the present study, we examined extracts of twelve species of Caribbean gorgonian corals, for mechanisms that regulate microbial colonization, such as antibacterial activity and QS regulatory activity. Ethanol extracts of gorgonians collected from Puerto Rico and the Florida Keys showed a range of both antibacterial and QS activities using a specific Pseudomonas aeruginosa QS reporter, sensitive to long chain AHLs and a short chain N-acylhomoserine lactones (AHL) biosensor, Chromobacterium violaceium. Overall, the gorgonian corals had higher antimicrobial activity against non-marine strains when compared to marine strains. Pseudopterogorgia americana, Pseusopterogorgia acerosa, and Pseudoplexuara flexuosa had the highest QS inhibitory effect. Interestingly, Pseudoplexuara porosa extracts stimulated QS activity with a striking 17-fold increase in signal. The stimulation of QS by P. porosa or other elements of the holobiont may encourage colonization or recruitment of specific microbial species. Overall, these results suggest the presence of novel stimulatory QS, inhibitory QS and bactericidal compounds in gorgonian corals. A better understanding of these compounds may reveal insight into coral-microbial ecology and whether a therapeutic potential exists.
1. Introduction
Gorgonian corals are found in coral reefs worldwide, but are most abundant and diverse in Caribbean reefs [1]. The Caribbean gorgonians are dominant over scleractinian corals—both in size and number [1,2], are a rich source of natural products [3], and are important ecological components in Caribbean reef habitats [4,5]. Their success can be attributed partly to their diverse array of natural products that act as feeding deterrents against fish and invertebrate predators [3,6,7]. In addition, gorgonian natural products may also play a role in antifouling [8] and in mediating coral-microbe interactions [9,10,11]. Microbes are abundant in marine systems and natural constituents of healthy corals including a surface mucus layer [12,13,14,15]. It appears that many corals host rich bacterial diversity that is distinct from the water column, species specific, and play protective roles in the coral "holobiont" (symbiotic organism that includes the coral animal polyp, endosymbiotic dinoflagellate, and bacteria [16,17,18,19]). However, when coral bacteria communities are disrupted, this may render corals more susceptible to disease and mortality from pathogenic bacteria [11,20,21]. Thus, keeping microbial populations under control is a very important mechanism to maintaining coral health.
Corals may control microbe populations with immune defenses [22,23], self-cleaning of mucus [24], or secrete chemicals that inhibit microbial growth, attachment, and behavior [8,11,25]. Such chemicals could include compounds that regulate bacteria communication or have antimicrobial activity. Bacteria communicate with one another using signaling molecules which accumulate in the environment with increasing bacterial density in a process termed quorum sensing (QS). When the concentration of signaling molecules accrues beyond a certain threshold, they interact with their cognate receptor and upregulate target gene expression. The best characterized QS molecules are N-acylhomoserine lactones (AHL) [26,27,28] produced by Gram-negative bacteria. Due to the prevalence of Gram-negative bacteria in the marine environment, we focused on AHL-based QS signaling for this study. Gram-negative bacteria use QS to facilitate and coordinate a range of cellular processes such as motility [29], luminescence [30], biofilm formation [31], expression of virulence factors [32], and is an important mechanism in the colonization of eukaryotic organisms [33,34]. Growing documentation of QS activity in corals, suggest that QS is also important in coral-bacteria associations. Extracts of various stony and soft corals from the Great Barrier Reef have QS regulatory properties [35,36] and recently, production of QS signals have been reported in cultures of coral associated bacteria [37,38]. Most notable, Alagely et al. [39] demonstrated that compounds isolated from the surface of healthy corals could modulate QS regulated behavior in the coral pathogen Serratia marcescens.
In the Caribbean, different studies have produced conflicting conclusions as to whether gorgonians possess compounds with antibacterial activity against marine bacteria and whether this is a significant component of microbial defense [9,10]. The differences between studies are mostly attributed to different interpretations of what constitutes significant antimicrobial activity based on the disc diffusion assay method. Furthermore, Jensen et al. [9] suggest that the maintenance of broad-spectrum antimicrobial activity is not a leading factor in the evolution of chemical defenses in gorgonians. In addition to revisiting the scope of antibacterial activity in corals, it is also necessary to address other mechanisms for microbial regulation in gorgonians. The intricacy of how gorgonians regulate microbial populations remains largely unknown. It is not clear whether gorgonians have intrinsic chemical defenses against potentially pathogenic marine bacteria or other mechanisms for microbial regulation, such as quorum sensing (QS). Caribbean gorgonians have properties that modulate bacterial physiology and quorum sensing (QS) has been suggested as a possible mechanism [11]. Yet, to date, Caribbean gorgonians have not been surveyed for QS activity. The overall objective of this study was to determine whether or not Caribbean gorgonians have detectable levels of antimicrobial and QS activity. We surveyed dominant Caribbean gorgonians for the presence of QS activity and bactericidal activity against a suite of model bacterial strains, including ecologically relevant marine bacteria known to cause coral diseases. The gorgonians studied represent dominant genera from two locations in the Caribbean, Puerto Rico and the Florida Keys. We hypothesized that antimicrobial and QS activity would be detectable but would vary among the dominant gorgonian genera in the Caribbean.
2. Results
2.1. Antimicrobial Activity
Ethanol extracts from all eight gorgonians showed significant but variable antibacterial activity against Gram-negative and Gram-positive bacterial strains (Table 1, Table 2 and Figure 1). Overall the extracts from Pseudoplexaura porosa and Pseudopterogorgia acerosa demonstrated the highest inhibitory activity against all the bacteria strains, and Eunicea laciniata had the lowest bactericidal activity. Gorgonian extracts had greater inhibitory activity against Gram-positive than Gram-negative bacteria (Table 2). P. porosa, Plexaura homomalla, Pseudopterogorgia americana and P. acerosa all demonstrated significantly higher activity against Gram-positive strains. The gorgonian coral extracts also exhibited significantly higher bactericidal activity against non-marine compared to marine strains (Figure 2a). P. porosa, P. Americana, P. acerosa and E. laciniata had higher activity against non-marine bacterial strains, while Briareum sp. had the same activity against all bacteria regardless of the Gram staining or environmental source.
|Bacteria species
|Gram Stain
|Methicillin resistant Staphylococcus aureus (MRSA)
|Non marine
|Methicillin sensitive Staphylococcus aureus (MSSA)
|Non marine
|Vancomycin resistant Enterococcus faecium (VRE)
|Non marine
|Enterococcus faecalis
|Non marine
|Bacillus subtilis
|Non marine
|Escherichia coli
|Non marine
|Pseudomonas aeruginosa (PAO1)
|Non marine
|Serratia marcescens (MG1)
|Non marine
|M. Teplitski
|Serratia marcescens
|Serratia marcescens (PDL100)
|Vibrio alginolyticus
|GenBank # X744690
|Vibrio parahaemolyticus (PP-A2)
|Genbank # FJ892748
|ANOVA data
|Coral species
|Bacteria species
|Coral species × Bacteria species
|Gram stain (+) or (−)
|Coral species × Gram stain
|Environment (non-marine or marine)
|Coral species × Environment
Among the bacteria strains, Bacillus subtilis was most sensitive to gorgonian extracts followed by vancomycin resistant Enterococcus (VRE) and methicillin-sensitive Staphylcoccus aureus (MSSA), Escherichia coli and Pseudomonas aeruginosa strain PAO1 (Table 2 and Figure 2b). The coral extracts had higher inhibitory activity against the vacomycin resistant Enterococcus than the susceptible strain, while there was no difference in inhibitory activity between the drug resistant Staphylcoccus aureus and the resistant strain. In contrast, very little inhibitory activity was observed against many of the marine bacteria, in fact there was negligible activity against all three S. marcescens strains (not different from the control). Of the Vibrio species, there was higher susceptibility to the coral extracts in V. alginolyticus than V. parahaemolyticus.
2.2. Quorum Sensing (QS) Inhibitory Activity in Gorgonian Extracts
The potential of gorgonian extracts to antagonize bacteria cell-cell communication systems was investigated using two reporter bioassays, Pseudomonas aerugionosa 3-oxo-C12-HSL (long chain AHL) and Chromobacterium violaceum CV026 (short chain AHL). Differences in antagonistic effects in the long chain AHL assay were observed among the corals tested (ANOVA F = 53.39, p < 0.0001, Figure 3). The gorgonians Pseudopterogorgia americana, Pseudopterogorgia acerosa, and Plexuara flexuosa and Gorgonia ventalina had the highest inhibitory effect on QS, while the other coral species did not have significant inhibitory activity above the controls. To exclude the possibility that reduced fluorescence was a result of growth inhibition rather than antagonism of QS activity, growth curves of P. aeruginosa PAO1-JP2 (pKR-C12) with and without extracts were determined. At a concentration of 250 µg/mL, extracts did not significantly affect bacterial growth of the las/rhl1 double mutant PAO1-JP2 reporter strain with pKR-C12 biosensor during the incubation time.
A decrease in violacein was expected in the Chromobacterium violaceum CV026 assay if coral extracts contained anti-QS compounds that interfered with short chain AHLs or their production. Violacein production was reduced by 50% in the presence of Pseudopterogorgia acerosa, Pseudopterogorgia americana, and Pseudoplexaura porosa extracts, while Gorgonia ventalina inhibited violacein by 30% (ANOVA F = 29.03, p < 0.0001, Figure 4). All other gorgonians tested did not affect violacein production in the Chromobacterium violaceum CV026 bioassay. Growth inhibition was also tested for C. violaceum, but none of the extracts affected bacterial growth (data not shown).
2.3. Quorum Sensing (QS) Stimulatory Activity in Gorgonian Extracts
With the exception of Pseudoplexaura porosa and Gorgonia ventalina, none of the gorgonian extracts induced QS activity in the long chain AHL bioassay. P. porosa extract stimulated QS activity with a maximum 17-fold increase in signal over negative control (ANOVA F = 7314.64, p < 0.0001, Figure 5). G. ventalina had low, but appreciable, pro-QS activity with a 2-fold induction over negative control. For the Chromobacterium violaceum CV026 assay, none of the gorgonian extracts induced violacein production.
3. Discussion
Antimicrobial and quorum sensing (QS) compounds have been proposed as potential mechanisms for microbial regulation in Caribbean corals [9,10,11,37,38,39,40]. Overall, our study is in agreement with Kim 1994 [10] and Jensen et al. 1996 [9] in that the incidence of antibiotic activity against non-marine bacteria was significantly greater than marine strains. Specifically, we detected little activity against all three S. marcescens strains and Vibrio parahaemolyticus PP-A2, a strain isolated from the surface of healthy Pseudoplexaura porosa. S. marcescens PDL100 and V. alginolyticus are both coral pathogens, it is possible that these and other marine bacteria are successful pathogens due to the low bactericidal activity or resistance of the corals and their ethanol extracts.
In addition, little activity was observed against Gram-negative strains relative to Gram-positive strains of bacteria, which supports the notion that gorgonians lack broad-spectrum antimicrobial activity. Jensen et al. [9] has suggested that antimicrobial activity might not be a crucial factor in the evolution of secondary metabolites. This may be a characteristic that is specific to Caribbean gorgonians, because broad-spectrum activity is found in corals from other locations around the globe [41]. Caribbean gorgonians may rely on other mechanisms, such as inducible immune defenses [22,23,42,43] or QS, for microbial regulation. Nonetheless, the presence of strong antimicrobial activity against human pathogens is notable for the potential of development of novel antimicrobial therapies, especially against antibiotic resistant bacterial strains [44].
There is growing evidence that bacteria cooperative consortia (known as biofilms) with higher organisms may involve regulation of quorum sensing (QS) [31,45,46,47]. We investigated QS antagonism and inductive activity in Caribbean gorgonian corals as a possible method for microbial control. Considering the widespread occurrence of Gram-negative bacteria in the marine environment [48,49,50] and the prevalence of N-acyl-homoserine lactones in marine bacteria [36,51,52], our investigation targeted LuxI/LuxR-type quorum sensing systems of Gram-negative bacteria. We used two different QS reporters for detecting long and short chain AHL signaling molecules in the gorgonian extracts. Our results show that Caribbean gorgonian corals possess compounds that both antagonize and stimulate or induce QS activity (summarized in Table 3). A third of the gorgonians screened had activity that antagonized QS activity, while only two induced QS activity. QS activity seemed to be genus specific, as activity was restricted to corals from the genera Pseudoplexaura, Plexaura, Pseudopterogorgia and Gorgonia. The three species from the genus Eunicea, along with Briareum and Muriceopsis lacked any QS inhibitory or inductive activity entirely.
|Collection Location
|Coral Species
|Inhibitory QS Biossays
|Inductive QS Biossays
|Pseudomonas aeruginosa Long chain AHL
|Chromobacterium violaceum CV026 Short chain AHL
|Pseudomonas aeruginosa Long chain AHL
|Chromobacterium violaceum CV026 Short chain AHL
|Puerto Rico
|Pseudoplexaura porosa
|Puerto Rico
|Plexaura flexuosa
|Puerto Rico
|Plexaura homomalla
|Puerto Rico
|Pseudopterogorgia americana
|Puerto Rico
|Pseudopterogorgia acerosa
|Florida Keys
|Plexaurella sp.
|Puerto Rico
|Eunicea laciniata
|Puerto Rico
|Eunicea tourneforti
|Florida Keys
|Eunicea mammosa
|Puerto Rico
|Briareum sp.
|Puerto Rico
|Gorgonia ventalina
|Florida Keys
|Muriceopsis flavida
Moderate quorum sensing (QS) inhibition was detected in Pseudopterogorgia acerosa, Plexuara flexuosa, Pseudopterogorgia americana and Gorgonia ventalina, using a specific Pseudomonas aeruginosa screen (long chain AHLs), while more potent inhibition was found in P. americana, P. acerosa, P. porosa, and G. ventalina using the Chromobacterium violaceum screen (short chain AHLs). Interestingly, P. acerosa and P. americana were antagonistic in both bioassays, which may indicate a mechanism conserved within the genus Pseudopterogorgia. The genus Pseudopterogorgia is known for having a rich diversity of bioactive natural products with diverse in vitro activities, such as anti-inflammatory [53], antimicrobial components [9,11] and ichthyodeterrents [6,54]. In addition, P. americana has copious amounts of mucus with properties that regulate metabolic and antibiotic activity in coral-associated bacteria and was suggested to be under QS control [11]. Skindersoe et al. [35] reported that true soft corals (Order Alcyonacea) from the Great Barrier Reef exhibited the highest levels of quorum sensing (QS) inhibitory activity in proportion to other marine organisms examined. The occurrence of QS antagonism in the Great Barrier Reef and Caribbean coral extracts suggests QS activity is a widespread phenomenon in Anthozoan-microbial interactions.
Eukaryotes and prokaryotes can both activate or inhibit QS, which can elicit a range of functional responses in either group [55,56,57]. For example, bacteria isolated from marine sponges produce AHLs that are hypothesized to promote bacterial colonization of sponge surfaces [36,52]. In contrast, the red alga Delisea pulchra, synthesizes AHL mimics, known as furanones, that disrupt QS signaling and inhibit bacterial colonization of the alga surfaces [58,59]. Moreover, resident coral bacteria produce compounds that regulate the growth of other microbes [11] and may also be a source of QS antagonistic compounds.
It is important to note the limitations of the assays used in this study. The gorgonian extracts may have AHL concentrations below the threshold of sensitivity to the biosensors or nonspecific AHLs to the biosensor. In addition, the long chain AHL Pseudomonas aeruginosa biosensor has a limited range of detection for the acyl moiety and is most sensitive to 3-oxo-C12-HSL and 3-oxo-C10-HSL AHLs.
Strong quorum sensing (QS) induction was detected in Pseudoplexuara porosa while very moderate levels of QS stimulation were observed in Gorgonia ventalina in the Pseudomonas aeruginosa long chain AHL assay. In contrast, no QS induction was detected with the Chromobacterium violaceum CV026 assay. The ability of G. ventalina to have both very moderate anti-QS activity as well as moderate pro-QS activity is very interesting. Since no exogenous AHLs are added in the QS induction assay, it is entirely possible that extracts from G. ventalina have several different types of compounds in their crude extracts. Some compounds may mimic AHLs to stimulate QS, and some inhibit the QS activation pathway, or bind to the exogenous AHL to inhibit QS. Since this was not a phenomenon that occurred with any of the other 11 gorgonian corals examined, it is possible this is a phenomenon unique to G. ventalina. Of the corals we examined, G. ventalina is one of the most studied mostly due to its susceptibility to fungal and parasitic diseases [60]. As such, many different types of defense mechanisms from antifungal to antibacterial defenses have been identified [61,62]. The ability to moderately inhibit and stimulate QS may be a direct or indirect result of the diversity of defensive strategies employed by this coral.
Crude extracts from Pseudoplexaura porosa, on the other hand, very definitively stimulated QS and produced a signal that was comparable to approximately 40% of the inducing synthetic AHL (50 nM, 3-oxo-C12-HSL). Furthermore, P. porosa extracts showed high levels of inhibition in the Chromobacterium violaceum CV026 bioassay. Long chain AHLs (N-acyl side chains from C 10 to C 14), in the presence of N-hexanoylhomoserine lactones (HHL) are known to inhibit violacein pigment production in the Chromobacterium violaceum CV026 bioassay [63,64]. In our studies, there was a negative correlation between anti-QS activity in the Chromobacterium violaceum CV026 assay and pro-QS activity in the long chain AHL Pseudomonas aerugionosa 3-oxo-C12-HSL assay. Collectively, these results suggest the presence of long chain AHLs or homologs in P. porosa extracts. In marine systems, long chain AHLs have been detected in sponges [36,65], marine snow [51], and in cultures of coral-associated bacteria [38]. Our results add to the growing evidence that long chain AHLs may be prevalent in marine microbial niches [36,51,52] and could indicate high bacterial densities within or on P. porosa tissues. Further study is needed to better understand what role QS stimulatory molecules have in gorgonian microbial ecology.
4. Experimental Section
4.1. Gorgonian Coral Collection
Fragments of gorgonian corals were collected using SCUBA from La Parguera, Puerto Rico in July of 2007 and June of 2008 at the Looe Key Reef research site (24 33.78′N, 8124.05′W) in the Florida Keys, USA. Gorgonian fragments (5 cm each from 5 individual coral colonies) were collected at depths of 5–10 m. Divers wore gloves to collect the corals and all coral fragments were kept in separate bags. Five colonies were sampled for the following species: Briareum sp., Eunicea laciniata, Eunicea tourneforti, Plexaura flexuosa, Plexaura homomalla, Pseudoplexaura porosa, Pseudopterogorgia americana, and Pseudopterogorgia acerosa, for Puerto Rico, and Gorgonia ventalina, Plexaurella sp., Muriceopsis flavida, and Eunicea mammosa for the Florida Keys. All specimens were identified by L.R. Hunt, L.D. Mydlarz, E. Weil (Puerto Rico), and E. Bartells (Florida). Coral fragments were flash frozen in liquid nitrogen and shipped on dry ice to the University of Texas at Arlington, and stored at −80 °C until use.
4.2. Extract Preparation
Gorgonian fragments (5 colonies) were pooled to provide sufficient amount of extract for bioassays, then lyophilized on a VirTis Benchtop K lyophilizer (The VirTis Company, Gardiner, NY, USA) and ground in a mortar with a pestle to a fine powder, and subjected to an overnight ethanol extraction at room temperature. 100% ethanol (Decon Labs, Inc., King of Prussia, PA, USA) was added at a ratio of 10 mL to every 0.2 g of homogenized coral. Extracts were then transferred to pre-weighed vials, evaporated to dryness under N2, and final weights determined. All samples were diluted in 100% ethanol to a stock concentration of 100 mg/mL and stored at −20 °C until further analysis.
4.3. Antibacterial Assays
Gorgonian ethanol extracts were tested for antimicrobial activity against a suite of Gram-negative and Gram-positive non-marine and marine bacteria (Table 1). The selection of marine bacteria included recognized microbial pathogens of coral such as Serratia marcescens PDL100 (courtesy Kim Ritchie), which has been identified as the causative agent of white pox disease in elkhorn coral Acropora palmata [66], and Vibrio alginolyticus potential pathogen of Caribbean Yellow Band Disease (courtesy Kim Ritchie) [67]. Other marine bacteria included S. marcescens from channel water, Cheesequake Salt Marsh, NJ, V. parahemolyticus PP-A2 isolated from the mucus of healthy Pseudoplexaura porosa,. In addition to marine bacteria, both Gram-positive and Gram-negative tester strains representing many human bacterial pathogens (Table 1) were used to address the range of bacterial inhibition by the gorgonian extracts. Marine bacteria were grown in diluted in nutrient media Difco marine broth (Becton, Dikinson and Co., Le Pont de Claix, France) and non-marine strains in Luria Broth (LB, Miller, Novagen, Merck KGaA, Darmstadt, Germany).
Antimicrobial activity was tested using a bacteria turbidity assay and conducted in 96-well flat-bottom plastic microplates (Greiner bio-one, Monroe, NC, USA). This assay method requires less sample volume than the disc-diffusion assay and has been previously utilized to analyze antimicrobial activity in corals [68]. Assays were run in triplicate wells and experiments contained bacteria with extract, along with the following controls: bacteria only, bacteria + antibiotic, or bacteria + ethanol (to control for ethanol effects). Assays were run at appropriate temperatures for the select bacteria (37 °C for non-marine bacteria and human pathogenic strains, and 29 °C for marine and environmental strains). Ethanol extracts of gorgonians were diluted in nutrient media and 100 µL was added into each well for a final concentration of 250 µg/mL per well (and less than 2.5% ethanol final concentration). Overnight bacteria cultures, in exponential growth, were diluted to an optical density (OD600) of 0.2 and 100 µL added to wells containing extracts for a final assay volume of 200 µL per well. The plates were gently mixed and an initial (time 0) OD600 reading was recorded with a Biotek Synergy 2 spectrophotometer (Bio-Tek Inc., Winooski VT). Plates were then placed in a shaking incubator and hourly readings were recorded for 5 h with a final reading at 24 h. All assays were run in triplicate or quadruplet.
Growth inhibition of bacteria cultures was measured by comparing the growth rate (GR) of bacteria with coral extracts to bacteria-ethanol controls. The GR was calculated with the following formula: 3.3 × log ((tf/ti)/n), where, tf = final OD600, ti = initial OD600, and n = tf − ti. The linear portion of logarithmic growth was used to select the initial and final hour and was kept standard between runs. Percent growth inhibition was determined by calculating the difference in GR of wells with gorgonian extract to ethanol control values (without the addition of extracts).
4.4. Isolation and Identification of Vibrio parahaemolyticus
To isolate culturable bacteria, mucus samples were collected from Psuedoplexaura porosa and diluted in sterile seawater. Diluted mucus was then spread onto glycerol artificial seawater agar and incubated at 24 °C. Bacteria that grew on the agar were subcultured into marine broth and identified to species. For species identification of culturable bacteria isolates, bacterial genomic DNA was extracted using a Ultra Clean Microbial DNA isolation kit (Mo BIO Carlsbad, CA, USA) per manufacturer's instructions. PCR amplification was performed on genomic DNA using Rln (forward primer GCTCAGATTGAACGCTGGCG) and U2 (reverse primer ACATTTCACAACACGAGCAGT) oligonucleotides, which correspond to positions within the Escherichia coli 16S rRNA gene [69]. A ~1100 bp product, from PCR amplification, was identified with agarose gel electrophoresis and purified with a PCR purification kit (Qiagen Valencia, CA, USA). PCR products were sequenced via BigDye™ terminator cycling and automated sequencing (ABI Prism 3100xl) using Rln and U2 for forward and reverse strand synthesis respectively. Consensus sequences were then analyzed via GenBank BLAST searches and a percent identity match was made to known bacteria. Thus far, only one bacteria species, Vibrio parahaemolyticus, was identified from P. porosa mucus and the DNA sequence corresponding to this V. parahaemolyticus isolate has been deposited into GenBank (Accession number FJ892748).
4.5. Pseudomonas aeruginosa 3-oxo-C12-HSL Bioassay
Long chain N-acylhomoserine lactones (AHLs) (i.e., 3-oxo-C12-HSL) were detected in gorgonian ethanol extracts using a sensitive QS reporter strain, Pseudomonas aeruginosa PAO1-JP2 (pKR-C12), previously developed [70,71]. The bioassay procedure for detecting long chain AHLs and pro and antiquorum sensing activity with PAO1-JP2 (pKR-C12) is well established [71] and was utilized in the present study with little modification. P. aeruginosa was obtained from Professor B. Iglewski from University of Rochester Medical Center, New York, USA. Pseudomonas aeruginosa PAO1-JP2 (a las/rhl1 double mutant unable to produce AHL signals) [70] harbors the biosensor plasmid pKR-C12 [72] and was used for testing inhibition or stimulation of QS. The reporter relies on the well-characterized plasmid pKR-C12, which contains a translational fusion of the lasB elastase gene (a virulence gene) of P. aeruginosa to green fluorescent protein (gfp) gene, encoding an unstable version of the Gfpmut3 protein [72]. Notably, the unstable variant of gfp is useful in that it allows detection of transient gene expression [73] and enables sensitive QS detection in real time. Moreover, pKR-C12 contains the lasR gene, under control of a lac-type promoter, which encodes the cognate receptor for the long chain AHL, 3-oxo-C12-HSL [72]. Thus, the reporter strain PAO1-JP2 (pKR-C12) used in this study was specific to the las QS system and most sensitive to long chain AHLs [72].
Overnight cultures of the reporter strain grown at 37 °C in LB broth were diluted four-fold into fresh LB media, and grown for an additional 1 h. Crude extracts were added to microtiter plates to a final concentration of 250 µg/mL. After addition of 50 nM of 3-oxododecanoyl-L-homoserine lactone (a synthetic signal molecule, 3-oxo-C12-HSL, Cayman Chemical, Ann Arbor, MI, USA) to reporter strain cultures, 100 µL aliquots of culture were loaded onto plates containing the extracts. For controls, wells were loaded with the same volume of ethanol as the extracts for every experiment. Thereafter, microtiter plates were incubated at 37 °C for 4 h with agitation. Fluorescent signals (excitation = 485 nm; emission = 528 nm) and absorbance (OD600) were quantified with a Synergy 2 spectrophotometer (Bio-Tek Instruments, Gen 5 Software, Winooski, VT, USA). Values were reported as % inhibition fluorescence relative to control values (minor fluorescence due to ethanol without the addition of extracts). To test for stimulation of QS, assay conditions were the same as above except the AHL signaling molecule (3-oxo-C12-HSL) was not added to the reporter strain culture. Stimulatory values were reported as relative fluorescent units (RFU) and were compared to negative control cultures (no AHL added).
4.6. Chromobacterium violaceum CV026 Bioassay
The N-acylhomoserine lactones (AHL) biosensor strain Chromobacterium violaceum CV026 served as an indicator organism by quantifying violacein (pigment) synthesis, a phenotype under QS control. Violacein production is dependent upon external addition of N-hexanoylhomoserine lactone (C6-HHL, Cayman Chemical, Ann Arbor, MI, USA) [64] in C. violaceum CV026, QS induction was tested by adding coral extracts to a 96-well plates containing overnight (18 h) cultures of C.violaceum CV026 following methods previously described [63]. Plates were incubated for 16 h at 27 °C on a plate shaker. Violacein pigment production was detected by drying the plates at 60 °C overnight, resolubilizing the violacein pigment in 100 µL of DMSO, and determining A590. HHL was added to assay wells at an optimal induction concentration (3.7 × 10−8 M HHL) to test for QS inhibition. The percentage inhibition of violacein production was correlated to ethanol control values without the addition of extracts and values were reported as percent inhibition relative to control values. For induction of QS, HHL was not added to assay wells and the presence of violacein in the wells indicated QS induction. For controls, wells were loaded with the same volume of ethanol as extracts for every experiment.
4.7. Statistical Analysis
Data were tested for normality (Shapiro-Wilk) and homogeneity of variances (Levene) prior to analysis. Data sets met parametric analyses constraints and were analyzed using ANOVA with Tukey post-hoc tests with JMP Statistical Discovery Software version 9.0 (SAS Institute Inc., Cary, NC, USA).
5. Conclusions
In this study, we report evidence for both inhibition and stimulation of quorum sensing (QS) activity and strong antimicrobial activity against select bacteria in Caribbean gorgonian extracts. These data indicate that microbial regulation in corals may occur by several different mechanisms, such as controlling bacteria growth directly or indirectly through modulating QS. Since the coral extracts were not very effective against several representative marine bacteria, controlling bacteria through direct inhibition or induction of the QS signals may be an alternative strategy. Although not a strategy employed equally by all genera of corals examined in this study. To our knowledge, this is the first demonstration of QS stimulation in Caribbean gorgonian corals. Testing of chemical extracts can suggest the presence of AHLs (or compounds with similar activity) in coral tissues, but cannot determine which component of the holobiont are the source of the AHL-like molecules. Identification of the AHL source is a crucial next step, as well as the bacterial phenotypes under AHL control. Our study also found that Caribbean gorgonians have strong antimicrobial activity against pathogenic bacteria of human concern and little antimicrobial activity against certain marine bacteria. Gorgonians may need to rely on other mechanisms, such as QS or inducible immune defenses, to control these relevant bacteria. Further work is needed to better understand microbial regulation in the coral environment and further characterize gorgonian antimicrobials with potential benefit to humans.
This work was funded by the National Science Foundation Award # OCE-0849799, the University of Texas at Arlington startup funds (LDM and KRD) and the Research Enhancement Program (LDM). Corals were collected under the specifications of the Florida Fish and Wildlife Conservation Commission nonresident saltwater fishing licenses as well as research permit number FKNMS-2007-050, for Gorgonia ventalina. We are most grateful to B.H. Iglewski for Pseudomonas aeruginosa PAO1 and PAO1-JP2 strains, L. Eberl for plasmid pKR-C12, and R. McLean for Chromobacterium violaceum strain CV026, K. Ritchie for Vibrio alginolyticus and Serratia marcscens PDL100 (ATCC # 39018), and M. Teplitski for Serratia marcescens MG1 and Serratia marcescens (ATCC # 39006). We would like to thank three anonymous reviewers for comments that improved the manuscript.
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Liquid: The VRBO of Bikes
Bicycling is the best way to see a city.
If you agree with that statement, you'll want to sign up with liquid.com. It's a new worldwide network of bike owners and would-be bike renters that seeks to connect the two groups with an ease never before attained. Through Liquid, bike owners rent their bikes to travelers, cyclists, and even bike-less locals that are just looking to get in a bit of exercise or exploration time in over 400 cities in 80 countries.
The initiative launched in New York City and San Francisco last April, and in September it started a national beta program.
How it works: Kind of like traveler-favorite Vacation Rentals by Owner (VRBO). Bike owners fill out a few details on the bike they are willing to rent out, submit photos of the sweet chariot, and in 24 to 48 hours, after Liquid checks for quality, price, and accuracy, the bike is ready for rent. You earn a little cash (bike rentals seem to run anywhere from $10 to $50 on the site) and help out one of your tribe. Bike renters search online by location, bike size, and type to get a list of available rides nearby. From there, a renter makes a request from the lister and asks any questions he or she might have. Once the lister approves the request, it's a match made in bicycle heaven.
A recent search for Hartford, Connecticut, and San Francisco, California, yielded lots to choose from, though bike rental choices in Burlington, Vermont, brought back just one result. Like couchsurfing.org, which connects travelers with a free bed and possibly a tour guide, or VRBO, the options are only as good as the participants.
So add your bike to the virtual fleet, and when you're on the road, take someone else's wheels for a spin.
—Berne Broudy


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Update. This is the part where you think I'm going to tell you this particular one has been removed, right? Well, since I'm not part of this club and so I can't see their super secret list. Oh wait, yes I can, it's on Tumblr. Regardless, judging by the update on the GISHWHES website, I would very much doubt it's been removed.
Commandment 4 – Harassment – It's come to our attention that a number of teams have been verbally harassing authors and politicians to achieve items. If someone doesn't want to help you, this is no reason to verbally attack them. We are trying to create art and change lives, not hurt people. Your team will be docked points if we determine that you are breaking Commandment 4. Perhaps more importantly, you are dinging your karma credit. Be nice to people. Remember, you are representing the global community of GISHWHES and that community believes that through art we can change lives – not hurt them.
Wow. Self-aggrandizement much? I say it hasn't been removed because I notice below, they make reference to #173 being removed. In case you're curious as to what that would be, it's this:
#173. Some things should be broken. Let's see if we can crash williamshatner.com: get all your friends to visit the site at precisely 9:02 AM PST, Monday August 4th. You must show a screencap of the ensuing error message including the URL in the URL bar. (Hint: Don't visit the site before that exact time, as it will give the servers time to take on the load.) 65 POINTS
Ho Ho Ho. Random acts of kindness. Crash someone's website. Yeah. Lovely people all around.
Also, an upberfan has pointed out that some of the things on the list were user submitted. Unless Misha Collins and Co. don't bother approving them, it's a moot point.
Are you familiar with Misha Collins? I wasn't until today. I don't watch much TV. But apparently he's on a CW show called Supernatural. He also has a yearly charity event called GISHWHES, or the Greatest International Scavenger Hunt The World Has Ever Seen. This event, by the way, started as a publicity stunt to help Collins try to win the People's Choice Award.
The scavenger hunts in previous years have had some great feats of accomplishment, helping Hurricane Sandy victims, entertaining dialysis patients, and giving blood. And every year they donate money to Collins' charity Random Acts.
A visit to the GISHWHES website also lists such accomplishments as "Recycling Parties," "Teaching people they can dance anywhere," "Helping adults reclaim their childhood," and other such inane projects that can only be dreamed up by wealthy elite narcissists designed to make people do their bidding. Hidden Cash, anyone? Participants also can win grand prize trips, because what's the point of charity if there isn't something in it for you too, right?
Calm down. I'm not "hating." Okay, yes I am. But so what. If you have money to give to the needy, just do it for fuck's sake. Don't make people jump through hoops. Most of the items on this years GISHWISH scavenger hunt list have nothing to do with any participant really benefitting from the activities. Unless you count the proverbial helping someone "step out of their shell," which I don't. The majority of the list though has little to do with even helping anyone-- unless you count promoting GISHWISH as helping someone.
Some choice examples form the list of 185 things :
19. Stand in front of a recognizable landmark or monument, wear something magnificent, and in whatever your native language is, complete the following sentence: Gishwhes makes me feel____
22. Because of our subversive influence, most governments have declared GISHWHES to be a national security threat. Have a uniformed officer explain to a unit of servicemen how to neutralize a hostile GISHER.
185. Get "GISHWHES" spelled out at night by using the effect of lit and unlit office windows in a skyscraper. We must be able to clearly see the word and it must span at least 30 stories. 291 POINTS
183. Last year, NASA used their official twitter account to politely ask us to stop bothering the astronauts on the space station. NASA has undoubtedly spent the past 12 months regretting this hastily-made decision, so we are giving them a second chance to get in on the fun… Get "GISHWHES conquers space" or "GISHWHES " written on or in something orbiting in space. 400 points if an astronaut poses with the inscription. No photoshopping allowed! A couple of teams cheated last year on the space item and were disqualified. 289 POINTS
The one that has people upset though, is #78: Get a previously published Sci-Fi author to write an original story (140 words max) about Misha, the Queen of England and an Elopus. -59 POINTS
You see, these well-meaning but very dumb participants got it into their brain to bombard a lot of published science fiction writers with a plea to write a story about Misha, the Queen of England, and an Elopus. No doubt they forgot about the part that it was for charity and so weren't in the know but instead opened email after email from people who came across as the breathless scavenger hunt nerds they are. You see, it never occurred to these well meaning sheeple that writers get paid to write and charity or not, it's their job. And the job requires a great deal of effort.
Just as it never occurred to Misha Collins to, I don't know, pool a sizable list of science-fiction authors who might be willing to participate, so the entire community wouldn't be inundated? Guess that would've taken effort, huh? Maybe it should have gone on the scavenger hunt list.
At least one writer, (who isn't even considered a science fiction writer, mind you) Lauren DeStefano sounded off very loudly on it.
Here's an excerpt from her Facebook page, emphasis mine:
This year, Misha Collins got the fun idea to ask his fans to contact published sci-fi authors to write a story about him.
And by fun I mean rude. Whether or not he gave this any thought, Misha Collins has just subjected authors to dozens of emails from HIS fans asking us to write a story. About him. For free. Maybe it didn't occur to Misha that we are actual people with a job to do. Maybe it didn't occur to him that we are not his employees. Maybe, and this is the most likely, he just thought it would be fun.
But, like anyone else, authors are just people with a job to do. We have our own fans to interact with (and we do so without encouraging them to solicit free work from others), and we have our own stories to write, and our own bills to pay. I also have my own emails from readers to read and respond to, and I am so not appreciating this flood of boilerplate "Write this story for Misha" requests which I seem to have received even though my books are not traditionally classified as sci-fi.
So, because Misha didn't think of us authors as real human beings with a job of our own to do, I am appealing to all of you to see that we are. Please share and pass this along, and please, please, please do not ask me to work for Misha for free so he can have a good laugh. I am right now working on my own books, which I am writing for all of you. And frankly you guys are more important to me than being an item on this scavenger hunt.
Good for Lauren! She was of course painted by the same sheeple who bombarded her with emails as being "insensitive." Either that or some commenters implored her to do it for charity (because blackmail always works) or worse, just insulted her with thinly disguised threats like this comment underneath her post. I didn't include the person's name here which is more tact that that person demonstrated Ms. DeStefano.
You basically wrote a story about Misha Collins right here already, I don't know why it's such a big deal to write a story for your fans in the name of charity. Rather than start all this drama it may have been better (and less time consuming) had you just wrote a short story and sent the same one to your fans or even posted that story on facebook for everyone to use. If I was participating in this contest and had sent you an email I'd be half tempted to send this post as a reply. You may want to rethink topics like this in the future as many of your fans are loyal to supernatural and it's a fandom that really sticks together and due to this post it may result in a loss of fans. I'm sorry, but this was handled poorly.
I need to remember this tactic.
"You should really bake three dozen cupcakes for me instead of complaining that I want you to work for free, otherwise you might lose me as a customer."
"You need to remember that those of us who are fans of your music might not like your comments, and it'd be a real shame if you lost our support, now wouldn't it."
Hell, even science fiction writer Neil Gaiman, whose wife Amanda Palmer was infamous for trying to get musicians to work for free, opted out, albeit in a more aloof way. I imagine he was tired of fielding the comments and emails, too.
It isn't just the tone of the so-called fan responses who already complain the loudest when content creators dare suggest they be paid for their work that really irks me. Nor is it that, charitable giving aside, Misha Collins' self-serving narcissism which reaches Zach Braff levels of annoyance hatched an ill-conceived scheme to get his participants to hound people. Although that is annoying, too.
No, what really bothers me is that in this list of things to do, Misha Collins is also asking you the scavenger hunt participant, to create content as well. Even he sees content the same way as the Facebook commenters: It's no big deal. Just shut up and do it.
Again from the scavenger hunt list:
152. Make a children's Pop-Up book about the CROATOAN Virus ending the world
167. If GISHWHES were a destination vacation, what would the brochure look like?
Oh PLEASE let me design a brochure for you, and write and make a pop-up book, too. PLEASE let's devalue our talent and content just for YOU, Misha! Pretty please! That's not even mentioning the filmed clips of you doing something stupid that no doubt are a part of that list, and will no doubt, be uploaded to Youtube where ads will live and money will be made, except of course from the person who created the content.
I understand this is the new normal now. People don't value content. And when someone who creates content dares to stand up and say "I'm not working for free," people shit on them. I get it. What I don't get is the very same person who makes a living creating content asks other people to do it for free, in the hopes that the entrants might win a grand prize trip to wherever, the odds being slim. Or worse, the same person puts established authors in a position where, if they say no to a scavenger hunt, they are made to feel like a horrible person because they aren't helping a charity.
How exactly is that a random act of kindness?


text: PlayMG Corp. is launching its MG Pocketable Wi-Fi Android-App Gaming System for holiday shoppers, making it available for Black Friday and Cyber Monday shoppers starting as early as 9:00 p.m. Eastern on Thanksgiving evening. The three bundles available include an MG system, case and combo charger for $159, and an MG system and two cases for $169. $40 in games, credit and an 8GB Micro SD card is included with every MG.


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(1933– ),
born Bendigo, grew up on a Riverina sheep property and was educated at Melbourne Grammar and at the University of Melbourne. He has worked as a teacher and administrator ...
Subjects: Literature.
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Sri Lanka captain Kumar Sangakkara elected to bat after winning the toss against the West Indies in the second Test on Tuesday.
The hosts made three changes from the side that drew the opening Test in Galle, bringing in pacemen Nuwan Kulasekara and Suranga Lakmal, and spinner Rangana Herath in place of Dammika Prasad, Thilan Thushara and Suraj Randiv.
Lakmal made his Test debut.
The West Indies replaced Andre Russell with left-arm spinner Sulieman Benn for the only change in their team.
No play was possible in the morning session due to a wet outfield.
Sri Lanka: Kumar Sangakkara (capt), Mahela Jayawardene, Tillakaratne Dilshan, Tharanga Paranavitana, Thilan Samaraweera, Angelo Mathews, Prasanna Jayawardene, Nuwan Kulasekara, Ajantha Mendis, Suranga Lakmal, Rangana Herath.
West Indies: Darren Sammy (capt), Brendan Nash, Chris Gayle, Adrian Barath, Carlton Baugh, Sulieman Benn, Darren Bravo, Dwayne Bravo, Shivnarine Chanderpaul, Kemar Roach, Shane Shillingford.
Umpires: Richard Kettleborough (ENG) and Asad Rauf (PAK)
TV umpire: Steve Davis (AUS)
Match referee: Alan Hurst (AUS)
Sri Lanka elect to bat in second Windies Test
Sri Lanka captain Kumar Sangakkara elected to bat after winning the toss against the West Indies in the second Test on Tuesday.


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