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My app will be using slightly thicker separator lines in tableViews - about 3x the thickness. I have already done this in some areas by setting separatorStyle to none and customizing cell backgroundViews with custom backgrounds that include my thick separator.
The issue with this method is that it only applies to real rows. Thus if I have a tableView that fills the whole screen but only has two rows, then only the two rows will have the custom separators. The workaround I've been using is to create blank cells (with the custom background) whenever the actual rows don't fill the entire table. However, this workaround is not feeling very practical.
Things would be much simpler if I could either modify the default separator's thickness...is there any way to do this, or something similar?


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There is always something going on at the Columbus Arts Council located in the Rosenzweig Arts Center! Check out the side panel for more event information or go to our "Calendar" page for a quick glance at this months events.
Will Kimbrough is a singer, songwriter, award-winning guitarist, multi-instrumentalist, producer, performer and bandleader with a voracious appetite for every aspect of music. Born and raised in the deep south of the Alabama Gulf Coast, he ingested eclectic FM radio sounds and the music of nearby New Orleans. In his quest to develop his musical capacities to their fullest, Will Kimbrough has become a modern-day Renaissance Man.
Kimbrough's body of work reflects a lifetime of performing, writing and collaborating from more angles than Da Vinci's protractor. True to his search for universal truths, his profound musical knowledge and expert creations reflect years of learning the intricacies of folk, blues, gospel, country, rock'n'roll, punk rock and jazz.

subset: commoncrawl


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$util.date("MMMM d, yyyy",$topLevel.timeZone)
SHERMAN, Tex. -- A Sherman firefighter was arrested Thursday morning for possession of meth. Firefighter Zach Cornell was taken into custody around nine o'clock Thursday morning after Sherman police searched his vehicle and found meth.
Zach Cornell, 31, was arrested Thursday morning after an anonymous caller notified the Sherman Police Department of a suspicious man passed out behind the wheel of a car on Sunset Boulevard.
Police say Cornell was off duty, but still in uniform. When officers searched Cornell's car, they found a pipe and less than one gram of methamphetamine.
"At that point in time, a search warrant obtained for the residence at this point in time, nothing has been recovered from the residence," Sgt. Dawsey said.
Police say Cornell was arrested and charged with possession of methamphetamine less than one gram in a drug free zone. They say Cornell was arrested within 1,000 feet of Wakefield Elementary School, which enhances the charge to a third degree felony.
Sherman Fire Chief Jeff Jones says Cornell has been employed with the City of Sherman as a firefighter since 2000. No word yet if he will be suspended or put on administrative leave with the Sherman Fire Department. Jones says that decision will be made after results from Cornell's drug tests are analyzed.
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Khokababu Movie ReviewRoshini Mukherjee, TNN, Jan 19, 2012, 11.57AM IST
Our advise, keep that logic behind, dig into a tub of corn and let Khokababu do the rest.
Filmein sirf teen cheezon ki wajah se chalti hain... Entertainment, entertainment, entertainment... So claimed "The Dirty Picture". Going by that picture then, Khokababu too sums up about D Sankaraaiya's directorial debut: "No gyan, no bigyan, only entertainment."
And so, Khoka Roy, (Dev) BA fail, ensures that he keeps his audience entertained. If not through the entire span then at least for most part of it. The "markatari" Khokababu is one "chalu cheez" and ably matches his "brain power" against Shankar's (Ferdous) muscle power. From sweeping the possessive brother's beautiful sister, Puja (Subhashree), off her feet, to marrying her in private, from keeping the union a secret to winning over suitor, Ajay (Dibyendu) to his side, Khoka is a master at his game. Need we also mention that the hero finally gets the girl, beats up the baddies and also extracts jamai ador from the brother?
The story hardly breathes of originality though. It takes one back to the times of rivalry between two families, of blood, gore and revenge. If the first half is more about Khoka's display of wit and romanticism, the latter is sprinkled with action and dance sequences, where songs keep popping up a bit too frequently for your comfort.
But what makes this ride one hell of an entertainer is apna Khokababu's action, emotion and execution. Dev, the actor, or the "non-actor" as he claims to be, has performed to the best of his ability. But it would be nice to see him test different waters now that he has already made a mark as a commercial hero. And yes, Ferdous is a welcome change. He brings to the fore the different shades of Shankar. Subhashree's wafer thin figure looks ravishing and rocking alongside Dev's machismo. Ashish, Locket, Partha Sarathi as Peto and Subhashish as Dhananjoy Khara provide able support to the plot and the characters.
Rishi Chanda scores with the title track as much as he does with the "Khoka 420" number. Then there are, "Je deshe chena jana manush kono nai", "Soniye tu", "Pyar ka jhatka", to name a few. The picturesque foreign locales and high-flying action (quite literally so) keep the audience engaged.
Our advise, keep that logic behind, dig into a tub of corn and let Khokababu do the rest.


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Lego Cake
Buttercream iced. Fondant accents. Large legos are Rice Crispy Treats covered in fondant. Small legos are handcut fondant pieces. (note to self - next time find a mold :) Lego man is fondant. Inside cake was rainbow colors to match the outside!
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1. e-Penis
Always wanted to know how big your e-Penis is ?
2. Twumped
Twumped is basically a website that shows the twitter users that have recently broken up or been dumped by their girlfriend/boyfriend, and then allows you to message the twitter account owner.
3. Twitter Friends Network Browser
Twitter Friends Network Browser lets you type in a user name, and you get a map of their Twitter picture, their name and their latest tweet, and the same goes for their friends. Click or drag the icon to see more info.
4. textgasm
Textgasm allows you to share your secrets in 140 characters or less and other users are able to post comments or cast votes.
5. Post like a Pirate
Post like a Pirate is pretty self explanatory Matey.
6. Cursebird
Cursebird shows you a real-time feed of swearing on Twitter.
7. Twitter Tussle
Enter the two terms you want to see compete and Twitter Tussle will create a bird fight to the death to see which of the words is more popular on Twitter.
8. TwitterLoo
TwitterLoo has been dubbed as "The weirdest Twitter application you'll ever use!"
This post was created by a user and has not been vetted or endorsed by BuzzFeed's editorial staff. BuzzFeed Community is a place where anyone can post awesome lists and creations. Learn more or post your buzz!
- DNC Day 2: Bill Clinton took the crowd down memory lane, and then Hillary broke through a virtual glass ceiling 💥
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The lawyer for Sandra Bland's mother said a former Waller County Jail guard told him under oath that he falsified entries in a jail log connected… Read More
The Republicans wrapped up their 2016 convention last night and Donald Trump had a "message" for Black America: He is the Law & Order… Read More
Charles Kinsey went to retrieve a 23-year-old autistic man who wandered from a mental health center out into the street Monday, and he was shot by… Read More
We are not "old Black" nor "new Black" we are ALL Black.
"Old Time D.C.," a nostalgia-filled Facebook page, recently stirred a distant memory that struck a chord, given current events. "Who… Read More
Airbnb has selected former Attorney General Eric Holder to help "craft a world-class anti-discrimination policy," according to a blog… Read More
"For the serious ones, the real ones, the alpha ones, we know what it's gonna take….revenue and blood," so said Gavin Long… Read More
It started with an emotional cry from rapper Killer Mike in the wake of the police shooting deaths of Alton Sterling and Philando Castile: "We… Read More
A Black Lives Matter group based in Wichita, Kansas originally planned a protest Sunday, but decided to have a cookout with police instead, Read More
As his historic presidency draws to a close, there's much to reflect on the legacy of President Obama. And just as importantly, there's… Read More
In 1968, the world watched as America imploded. Our descent into the Vietnam War and the continuing struggles for civil rights drew stark contrasts… Read More
We are not "old Black" nor "new Black" we are ALL Black.
Vernon Reid Talks 30 Yrs of the Black Rock Coalition
Justin Adu Explains African Amedia
Beautifully Brown Grace Jones
Why Did Ben Carson Mock Police Brutality
Detroit's New Activist Generation


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The Defense Department is planning to expand its defense industrial base cybersecurity pilot program, and expects to get the green light from the White House within the next two months, DOD CIO Teri Takai said April 24.
"We've been working on this for two years now," Takai told a cybersecurity forum organized by Rep. Jim Moran (D-Va.) in Arlington, Va., according to Federal News Radio. "Our plan this year is to expand this, and I think it'll give a lot more companies the opportunity to share not only with us, but with each other."
The program currently has 37 participants; once new rules are approved by the Office for Management and Budget, that number would grow to 200.
In January it was announced that the Homeland Security Department would take an active role in the project, but it would still be overseen by DOD, where the project was originally rolled out in July 2010 by then-Deputy Defense Secretary William Lynn. With DHS on board, the project was designated as the Joint Cybersecurity Services Pilot (JCSP), according to a DHS privacy assessment released in January. According to published reports, Takai indicated that DHS may launch its own version of a public-private cybersecurity initiative.
Related story
DHS takes key role in cybersecurity program
DOD's voluntary pilot project is designed to facilitate open communication on cyber attacks and malicious cyber activity among private-sector companies, internet service providers and government agencies.
"We provide information to them as it relates to threats, and they also share in a confidential manner the threats they face and the actions they're taking," Takai said of defense contractors, according to BusinessWeek. "We all get hit by the same kinds of things," but also different threats from time to time.
As the DIB pilot prepares for expansion, it's unclear whether DOD and the participants have addressed issues that were criticized by some members of Congress and in a Washington Post report on an internal study of the program, conducted by Carnegie Mellon University researchers earlier this year.
The study showed that the information-sharing plan is viable and ISPs are capable of handling classified intelligence from the National Security Agency, but it also revealed the program wasn't effective in stopping cyber attacks that participants could have prevented themselves without the shared data.
"Unfortunately, the report on the DIB Pilot Program highlights one of my continuing points, that there is no silver bullet in cybersecurity," Rep. James Langevin (D-R.I.), co-founder of the Congressional Cybersecurity Caucus, said in a Jan. 17 statement. "Signature-based defenses alone will never be enough to secure our defense contractors, our classified networks or our critical infrastructure. We need a comprehensive approach to cybersecurity that incorporates innovative information-sharing arrangements with industry, while also boosting our capabilities for our own defenses and those who manage our critical infrastructure."
About the Author
Amber Corrin is a former staff writer for FCW and Defense Systems.
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In the latest issue: grid security, agile development, BYOD and more!
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The latest version of the Defense Department-wide Enterprise Service Management Framework places more emphasis on managing IT risk.
Despite GSA's efforts to accelerate the FedRAMP approval process, the lack of agency reciprocity puts the program's central goals at risk.
Got some early-career colleagues who are doing great things in federal IT? Nominate them for FCW's 2016 Rising Star awards.
The agency software inventories required under the draft category management policy will be treasure maps that lead to efficient, effective, streamlined buying and unprecedented savings.
As experts warned of the "dire" threats posed by outdated federal technology, lawmakers grilled top feds, debated workforce issues and inched closer to backing a $3.1 billion fix-it fund.
The agency is making strides in developing and deploying border technologies but still has some weaknesses.
Agencies have spent almost $23 billion on legacy IT over the past three years, according to reports to Congress. Is a revolving fund the answer?
The United Kingdom will go live with its governmentwide digital identity platform, GOV.UK Verify, in the coming days. The U.S. government will need a little more time.
What contracting officer representatives really think is needed to fix contract management.
Please do not tie down the national infrastructure with the way DoD does its networks!
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text: Non-Game Software Is Now Available On SteamJason Schreier10/02/12 2:59pmFiled to: SteamValveSoftwaretweetKotakucore3dmarkGamemaker143EditInvite manuallyPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink Programs like GameMaker and 3DMark are now available on Steam.Valve sent over a press release today to announce the news. Here's what they have to offer: ArtRage Studio Pro CameraBag 2 GameMaker: Studio 3D-Coat 3DMark Vantage 3DMark 11 Valve adds: Advertisement Many of the launch titles will take advantage of popular Steamworks features, such as easy installation, automatic updating, and the ability to save your work to your personal Steam Cloud space so your files may travel with you. GameMaker Studio, for example, features integration with Steam Workshop that allows GameMaker users to share their work via Steam. You can browse through Steam's software offerings—all 10% off during the launch—on their website.Reply143 repliesLeave a reply


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I'm really curious.
I'm really curious.
I don't know who, but I can guess why.
Multi user refers to 'users'. But the functionality is Multi Sites and makes a lot more sense.
@elfin Well, WordPress already supported multiple users and multiple sites, before WordPress MU. So the naming is clear as mu-d to me.
I'm assuming MU "merged" with WP because the core code grew and became more adaptable, ultimately superseding MU's raison d'etre. Did this factor into the name change? In the process, did "MU" morph into something else, chang direction, change focus?
Maybe calling people "users" is no longer fashionable?
There was a blog post written about this topic. You can read about it on Andrew Nacin's blog.
The dev team decided. The old name really wasn't descriptive of what it actually did.
With the merge, the codesbase was rolled in,but it's still pretty much the same. It didn't morph into something else, just changed some names.
And "multisite" is easier to pronounce. I never knew if I should say "MU" or "em-yoo" with WordPress MU. :)
Right, I had to answer a number of people who were trying MU for multi-author blogs, which single WordPress supported anyway.
This topic has been closed to new replies.


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This entire park is very interesting: His boyhood home, Ebenzier Baptist Church, the museum & the grave. My only surprise was how dirty the reflecting pool was. There was someone cleaning it but it was a bit surprising to see it was not pristine. That seemed a bit disrespectful to the memory of the man.
But, overall, it is worth the time to visit this park and it should not be missed.
Own or manage this property? Claim your listing for free to respond to reviews, update your profile and much more.


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Letters: Not surprisedJune 17,2014
It was inevitable that the grocers association and such would sue Vermont over the new GMO labeling law they are lobbyists who make money by fighting these battles. Now is the opportunity for those who feel strongly about labeling GMOs to donate money to the Vermont fund to fight this legal battle.
Other countries have GMO labeling laws. Consumers in the United States have the right to know too. If the GMO companies are so proud of what they produce, then they should be voluntarily labeling instead of fighting. In the long run this is a battle that the GMO companies are going to lose. Labeling will become the default. The question is how much money will the grocers association and the GMO companies waste fighting rather than doing the right thing, which is to produce a safe product and marketing it well.
Walter Jeffries
West TopshamMORE IN LettersIn an editorial in The Times Argus, Peter Galbraith is aiming to quash special interests. Full StoryThere was never any real doubt about the fact that the DNC stacked the deck against Senator... Full StoryTo say that this years political season has been weird is probably an understatement. Full Story
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Rapid, Transient Fluconazole Resistance in Candida albicans Is Associated with Increased mRNA Levels of CDR
Marr, Kieren A.
Lyons, Christopher N.
Rustad, Tiger
Bowden, Raleigh A.
White, Theodore C.
Antimicrobial Agents and Chemotherapy
Fluconazole-resistant Candida albicans, a cause of recurrent oropharyngeal candidiasis in patients with human immunodeficiency virus infection, has recently emerged as a cause of candidiasis in patients receiving cancer chemotherapy and marrow transplantation (MT). In this study, we performed detailed molecular analyses of a series of C. albicans isolates from an MT patient who developed disseminated candidiasis caused by an azole-resistant strain 2 weeks after initiation of fluconazole prophylaxis (K. A. Marr, T. C. White, J. A. H. vanBurik, and R. A. Bowden, Clin. Infect. Dis. 25:908–910, 1997). DNA sequence analysis of the gene (ERG11) for the azole target enzyme, lanosterol demethylase, revealed no difference between sensitive and resistant isolates. A sterol biosynthesis assay revealed no difference in sterol intermediates between the sensitive and resistant isolates. Northern blotting, performed to quantify mRNA levels of genes encoding enzymes in the ergosterol biosynthesis pathway (ERG7, ERG9, and ERG11) and genes encoding efflux pumps (MDR1, ABC1, YCF, and CDR), revealed that azole resistance in this series is associated with increased mRNA levels for members of the ATP binding cassette (ABC) transporter superfamily, CDR genes. Serial growth of resistant isolates in azole-free media resulted in an increased susceptibility to azole drugs and corresponding decreased mRNA levels for the CDR genes. These results suggest that C. albicans can become transiently resistant to azole drugs rapidly after exposure to fluconazole, in association with increased expression of ABC transporter efflux pumps.
Related Articles
Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors.
Sanglard, D
Ischer, F
Monod, M
Bille, J
Antimicrobial Agents and Chemotherapy
Some Candida albicans isolates from AIDS patients with oropharyngeal candidiasis are becoming resistant to the azole antifungal agent fluconazole after prolonged treatment with this compound. Most of the C. albicans isolates resistant to fluconazole fail to accumulate this antifungal agent, and this has been considered a cause of resistance. This phenomenon was shown to be linked to an increase in the amounts of mRNA of a C. albicans ABC (ATP-binding cassette) transporter gene called CDR1 and of a gene conferring benomyl resistance (BENr), the product of which belongs to the class of major facilitator multidrug efflux transporters (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378-2386, 1995). To analyze the roles of these multidrug transporters in the efflux of azole antifungal agents, we constructed C. albicans mutants with single and double deletion mutations of the corresponding genes. The mutants were tested for their susceptibilities to these antifungal agents. Our results indicated that the delta cdr1 C. albicans mutant was hypersusceptible to the azole derivatives fluconazole, itraconazole, and ketoconazole, thus showing that the ABC transporter Cdr1 can use these compounds as substrates. The delta cdr1 mutant was also hypersusceptible to other antifungal agents (terbinafine and amorolfine) and to different metabolic inhibitors (cycloheximide, brefeldin A, and fluphenazine). The same mutant was slightly more susceptible than the wild type to nocodazole, cerulenin, and crystal violet but not to amphotericin B, nikkomycin Z, flucytosine, or pradimicin. In contrast, the delta ben mutant was rendered more susceptible only to the mutagen 4-nitroquinoline-N-oxide. However, this mutation increased the susceptibilities of the cells to cycloheximide and cerulenin when the mutation was constructed in a delta cdr1 background. The assay used in the present study could be implemented with new antifungal agents and is a powerful tool for assigning these substances as putative substrates of multidrug transporters.
Related Articles
Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing
Wang, Huiping
Kong, Fanrong
Sorrell, Tania C
Wang, Bin
McNicholas, Paul
Pantarat, Namfon
Ellis, David
Xiao, Meng
Widmer, Fred
Chen, Sharon CA
BMC Microbiology
Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.
The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.
The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.
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In Vitro Low-Level Resistance to Azoles in Candida albicans Is Associated with Changes in Membrane Lipid Fluidity and Asymmetry
Kohli, Avmeet
Mukhopadhyay, Kasturi
Rattan, Ashok
Prasad, Rajendra
Antimicrobial Agents and Chemotherapy
The present study tracks the development of low-level azole resistance in in vitro fluconazole-adapted strains of Candida albicans, which were obtained by serially passaging a fluconazole-susceptible dose-dependent strain, YO1-16 (fluconazole MIC, 16 μg ml−1) in increasing concentrations of fluconazole, resulting in strains YO1-32 (fluconazole MIC, 32 μg ml−1) and YO1-64 (MIC, 64 μg ml−1). We show that acquired resistance to fluconazole in this series of isolates is not a random process but is a gradually evolved complex phenomenon that involves multiple changes, which included the overexpression of ABC transporter genes, e.g., CDR1 and CDR2, and the azole target enzyme, ERG11. The sequential rise in fluconazole MICs in these isolates was also accompanied by cross-resistance to other azoles and terbinafine. Interestingly, fluorescent polarization measurements performed by using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene revealed that there was a gradual increase in membrane fluidity of adapted strains. The increase in fluidity was reflected by observed change in membrane order, which was considerably decreased (decrease in fluorescence polarization values, P value) in the adapted strain (P value of 0.1 in YO1-64, compared to 0.19 in the YO1-16 strain). The phospholipid composition of the adapted strain was not significantly altered; however, ergosterol content was reduced in YO1-64 from that in the YO1-16 strain. The asymmetrical distribution of phosphatidylethanolamine (PE) between two monolayers of plasma membrane was also changed, with PE becoming more exposed to the outer monolayer in the YO1-64 strain. The results of the present study suggest for the first time that changes in the status of membrane lipid phase and asymmetry could contribute to azole resistance in C. albicans.
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Reverse Genetics in Candida albicans Predicts ARF Cycling Is Essential for Drug Resistance and Virulence
Epp, Elias
Vanier, Ghyslaine
Harcus, Doreen
Lee, Anna Y.
Jansen, Gregor
Hallett, Michael
Sheppard, Don C.
Thomas, David Y.
Munro, Carol A.
Mullick, Alaka
Whiteway, Malcolm
PLoS Pathogens
Candida albicans, the major fungal pathogen of humans, causes life-threatening infections in immunocompromised individuals. Due to limited available therapy options, this can frequently lead to therapy failure and emergence of drug resistance. To improve current treatment strategies, we have combined comprehensive chemical-genomic screening in Saccharomyces cerevisiae and validation in C. albicans with the goal of identifying compounds that can couple with the fungistatic drug fluconazole to make it fungicidal. Among the genes identified in the yeast screen, we found that only AGE3, which codes for an ADP-ribosylation factor GTPase activating effector protein, abrogates fluconazole tolerance in C. albicans. The age3 mutant was more sensitive to other sterols and cell wall inhibitors, including caspofungin. The deletion of AGE3 in drug resistant clinical isolates and in constitutively active calcineurin signaling mutants restored fluconazole sensitivity. We confirmed chemically the AGE3-dependent drug sensitivity by showing a potent fungicidal synergy between fluconazole and brefeldin A (an inhibitor of the guanine nucleotide exchange factor for ADP ribosylation factors) in wild type C. albicans as well as in drug resistant clinical isolates. Addition of calcineurin inhibitors to the fluconazole/brefeldin A combination only initially improved pathogen killing. Brefeldin A synergized with different drugs in non-albicans Candida species as well as Aspergillus fumigatus. Microarray studies showed that core transcriptional responses to two different drug classes are not significantly altered in age3 mutants. The therapeutic potential of inhibiting ARF activities was demonstrated by in vivo studies that showed age3 mutants are avirulent in wild type mice, attenuated in virulence in immunocompromised mice and that fluconazole treatment was significantly more efficacious when ARF signaling was genetically compromised. This work describes a new, widely conserved, broad-spectrum mechanism involved in fungal drug resistance and virulence and offers a potential route for single or improved combination therapies.
Author Summary
Candida albicans is a fungus that normally resides as part of the microflora in the human gut. Candida species can cause superficial infections like thrush in the healthy human population and life-threatening invasive infections in immunocompromised patients. Fungal infections are often treated with azole drugs, but due to the fungistatic nature of these agents, C. albicans can develop drug resistance, leading to therapy failure. To improve the action of azoles and convert them into fungicidal drugs, we first systematically analyzed the genetic requirements for tolerance to one such azole drug, fluconazole. We show, both genetically and pharmacologically, that components of the ARF cycling machinery are critical in mediating both azole and echinocandin tolerance in C. albicans as well as several other pathogenic Candida species and in the pathogenic mold Aspergillus fumigatus. We highlight the importance of ARF cycling in drug resistance by showing that genetic compromise of ARF functions overrides common drug resistance mechanisms in clinical samples and other key regulators of azole/echinocandin tolerance. We validated the therapeutic potential of ARF cycling in two mouse models and provide evidence that drug treatment is more efficacious when ARF activities are genetically compromised. Our study demonstrates a new mechanism involved in two important aspects of the biology of human fungal pathogens and provides a potential route for improved antifungal therapies.
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Role of ATP-Binding-Cassette Transporter Genes in High-Frequency Acquisition of Resistance to Azole Antifungals in Candida glabrata
Sanglard, Dominique
Ischer, Francoise
Bille, Jacques
Antimicrobial Agents and Chemotherapy
Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753–2765, 1999). Deletion of CgCDR1 in C. glabrata rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type C. glabrata exposed to fluconazole in a medium containing the drug at 50 μg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 × 10−4 to 4 × 10−4). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation of CgCDR1. The CgCDR1 deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 μg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to CgCDR1, called CgCDR2, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both CgCDR1 and CgCDR2 suppressed the development of HFAR in a medium containing fluconazole at 5 μg/ml, showing that both genes are important mediators of resistance to azole derivatives in C. glabrata. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in C. glabrata. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both CgCDR1 and CgCDR2.
Related Articles
Prevalence of Molecular Mechanisms of Resistance to Azole Antifungal Agents in Candida albicans Strains Displaying High-Level Fluconazole Resistance Isolated from Human Immunodeficiency Virus-Infected Patients
Perea, Sofia
López-Ribot, José L.
Kirkpatrick, William R.
McAtee, Robert K.
Santillán, Rebecca A.
Martínez, Marcos
Calabrese, David
Sanglard, Dominique
Patterson, Thomas F.
Antimicrobial Agents and Chemotherapy
Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles in C. albicans strains displaying high-level fluconazole resistance (MICs, ≥64 μg/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14α-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.
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A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole
Harrison, Benjamin D.
Hashemi, Jordan
Bibi, Maayan
Pulver, Rebecca
Bavli, Danny
Nahmias, Yaakov
Wellington, Melanie
Sapiro, Guillermo
Berman, Judith
PLoS Biology
When exposed to the antifungal drug fluconazole, Candida albicans undergoes abnormal growth, forming three-lobed "trimeras." These aneuploid trimeras turn out genetically variable progeny with varying numbers of chromosomes, increasing the odds of creating a drug-resistant strain.
Candida albicans, the most prevalent human fungal pathogen, is generally diploid. However, 50% of isolates that are resistant to fluconazole (FLC), the most widely used antifungal, are aneuploid and some aneuploidies can confer FLC resistance. To ask if FLC exposure causes or only selects for aneuploidy, we analyzed diploid strains during exposure to FLC using flow cytometry and epifluorescence microscopy. FLC exposure caused a consistent deviation from normal cell cycle regulation: nuclear and spindle cycles initiated prior to bud emergence, leading to "trimeras," three connected cells composed of a mother, daughter, and granddaughter bud. Initially binucleate, trimeras underwent coordinated nuclear division yielding four daughter nuclei, two of which underwent mitotic collapse to form a tetraploid cell with extra spindle components. In subsequent cell cycles, the abnormal number of spindles resulted in unequal DNA segregation and viable aneuploid progeny. The process of aneuploid formation in C. albicans is highly reminiscent of early stages in human tumorigenesis in that aneuploidy arises through a tetraploid intermediate and subsequent unequal DNA segregation driven by multiple spindles coupled with a subsequent selective advantage conferred by at least some aneuploidies during growth under stress. Finally, trimera formation was detected in response to other azole antifungals, in related Candida species, and in an in vivo model for Candida infection, suggesting that aneuploids arise due to azole treatment of several pathogenic yeasts and that this can occur during the infection process.
Author Summary
Fungal infections are a particularly challenging problem in medicine due to the small number of effective antifungal drugs available. Fluconazole, the most commonly prescribed antifungal, prevents cells from growing but does not kill them, giving the fungal population a window of opportunity to become drug resistant. Candida albicans is the most prevalent fungal pathogen, and many fluconazole-resistant strains of this microbe have been isolated in the clinic. Fluconazole-resistant isolates often contain an abnormal number of chromosomes (a state called aneuploidy), and the additional copies of drug resistance genes on those chromosomes enable the cells to circumvent the drug. How Candida cells acquire abnormal chromosome numbers is a very important medical question—is aneuploidy merely passively selected for, or is it actively induced by the drug treatment? In this study, we found that fluconazole and other related azole antifungals induce abnormal cell cycle progression in which mother and daughter cells fail to separate after chromosome segregation. Following a further growth cycle, these cells form an unusual cell type that we have termed "trimeras"—three-lobed cells with two nuclei. The aberrant chromosome segregation dynamics in trimeras produce progeny with double the normal number of chromosomes. Unequal chromosome segregation in these progeny leads to an increase in the prevalence of aneuploidy in the population. We postulate that the increase in aneuploidy greatly increases the odds of developing drug resistance.
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Amino Acid Substitutions in the Cytochrome P-450 Lanosterol 14α-Demethylase (CYP51A1) from Azole-Resistant Candida albicans Clinical Isolates Contribute to Resistance to Azole Antifungal Agents
Sanglard, Dominique
Ischer, Françoise
Koymans, Luc
Bille, Jacques
Antimicrobial Agents and Chemotherapy
The cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance of C. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378–2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression in Saccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different cloned CYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1 genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1 gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.
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The ATP Binding Cassette Transporter Gene CgCDR1 from Candida glabrata Is Involved in the Resistance of Clinical Isolates to Azole Antifungal Agents
Sanglard, Dominique
Ischer, Françoise
Calabrese, David
Majcherczyk, Paul A.
Bille, Jacques
Antimicrobial Agents and Chemotherapy
The resistance mechanisms to azole antifungal agents were investigated in this study with two pairs of Candida glabrata clinical isolates recovered from two separate AIDS patients. The two pairs each contained a fluconazole-susceptible isolate and a fluconazole-resistant isolate, the latter with cross-resistance to itraconazole and ketoconazole. Since the accumulation of fluconazole and of another unrelated substance, rhodamine 6G, was reduced in the azole-resistant isolates, enhanced drug efflux was considered as a possible resistance mechanism. The expression of multidrug efflux transporter genes was therefore examined in the azole-susceptible and azole-resistant yeast isolates. For this purpose, C. glabrata genes conferring resistance to azole antifungals were cloned in a Saccharomyces cerevisiae strain in which the ATP binding cassette (ABC) transporter gene PDR5 was deleted. Three different genes were recovered, and among them, only C. glabrata CDR1 (CgCDR1), a gene similar to the Candida albicans ABC transporter CDR genes, was upregulated by a factor of 5 to 8 in the azole-resistant isolates. A correlation between upregulation of this gene and azole resistance was thus established. The deletion of CgCDR1 in an azole-resistant C. glabrata clinical isolate rendered the resulting mutant (DSY1041) susceptible to azole derivatives as the azole-susceptible clinical parent, thus providing genetic evidence that a specific mechanism was involved in the azole resistance of a clinical isolate. When CgCDR1 obtained from an azole-susceptible isolate was reintroduced with the help of a centromeric vector in DSY1041, azole resistance was restored and thus suggested that a trans-acting mutation(s) could be made responsible for the increased expression of this ABC transporter gene in the azole-resistant strain. This study demonstrates for the first time the determinant role of an ABC transporter gene in the acquisition of resistance to azole antifungals by C. glabrata clinical isolates.
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Evaluation of Differential Gene Expression in Fluconazole-Susceptible and -Resistant Isolates of Candida albicans by cDNA Microarray Analysis
Rogers, P. David
Barker, Katherine S.
Antimicrobial Agents and Chemotherapy
The opportunistic fungal pathogen Candida albicans is the major causative agent of oropharyngeal candidiasis (OPC) in AIDS. The development of azoles, such as fluconazole, for the treatment of OPC has proven effective except in cases where C. albicans develops resistance to fluconazole during the course of treatment. In the present study, we used microarray technology to examine differences in gene expression from a fluconazole-susceptible and a fluconazole-resistant well-characterized, clinically obtained matched set of C. albicans isolates to identify genes which are differentially expressed in association with azole resistance. Among genes found to be differentially expressed were those involved in amino acid and carbohydrate metabolism; cell stress, cell wall maintenance; lipid, fatty acid, and sterol metabolism; and small molecule transport. In addition to CDR1, which has previously been demonstrated to be associated with azole resistance, the drug resistance gene RTA3, the ergosterol biosynthesis gene ERG2, and the cell stress genes CRD2, GPX1, and IFD5 were found to be upregulated. Several genes, such as the mitochondrial aldehyde dehydrogenase gene ALD5, the glycosylphosphatidylinositol synthesis gene GPI1, and the iron transport genes FET34 and FTR2 were found to be downregulated. Further study of these differentially regulated genes is warranted to evaluate how they may be involved in azole resistance. In addition to these novel findings, we demonstrate the utility of microarray analysis for studying the molecular mechanisms of drug resistance in pathogenic organisms.
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Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole
Spitzer, Michaela
Griffiths, Emma
Blakely, Kim M
Wildenhain, Jan
Ejim, Linda
Rossi, Laura
De Pascale, Gianfranco
Curak, Jasna
Brown, Eric
Tyers, Mike
Wright, Gerard D
Molecular Systems Biology
The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.
The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.
Chemical screens with a library enriched for known drugs identified a diverse set of 148 compounds that potentiated the action of the antifungal drug fluconazole against the fungal pathogens Cryptococcus neoformans, Cryptococcus gattii and Candida albicans, and the model yeast Saccharomyces cerevisiae, often in a species-specific manner.Chemogenomic profiles of six confirmed hits in S. cerevisiae revealed different modes of action and enabled the prediction of additional synergistic combinations; three-way synergistic interactions exhibited even stronger synergies at low doses of fluconazole.The synergistic combination of fluconazole and the antidepressant sertraline was active against fluconazole-resistant clinical fungal isolates and in an in vivo model of Cryptococcal infection.
Rising fungal infection rates, especially among immune-suppressed individuals, represent a serious clinical challenge (Gullo, 2009). Cancer, organ transplant and HIV patients, for example, often succumb to opportunistic fungal pathogens. The limited repertoire of approved antifungal agents and emerging drug resistance in the clinic further complicate the effective treatment of systemic fungal infections. At the molecular level, the paucity of fungal-specific essential targets arises from the conserved nature of cellular functions from yeast to humans, as well as from the fact that many essential yeast genes can confer viability at a fraction of wild-type dosage (Yan et al, 2009). Although only ∼1100 of the ∼6000 genes in yeast are essential, almost all genes become essential in specific genetic backgrounds in which another non-essential gene has been deleted or otherwise attenuated, an effect termed synthetic lethality (Tong et al, 2001). Genome-scale surveys suggest that over 200 000 binary synthetic lethal gene combinations dominate the yeast genetic landscape (Costanzo et al, 2010). The genetic buffering phenomenon is also manifest as a plethora of differential chemical–genetic interactions in the presence of sublethal doses of bioactive compounds (Hillenmeyer et al, 2008). These observations frame the difficulty of interdicting network functions in eukaryotic pathogens with single agent therapeutics. At the same time, however, this genetic network organization suggests that judicious combinations of small molecule inhibitors of both essential and non-essential targets may elicit additive or synergistic effects on cell growth (Sharom et al, 2004; Lehar et al, 2008). Unbiased screens for drugs that synergistically enhance a specific bioactive effect, but which are not themselves individually active—termed a syncretic combination—are one means to substantially elaborate chemical space (Keith et al, 2005). Indeed, compounds that enhance the activity of known agents in model yeast and cancer cell line systems have been identified both by focused small molecule library screens and by computational methods (Borisy et al, 2003; Lehar et al, 2007; Nelander et al, 2008; Jansen et al, 2009; Zinner et al, 2009).
To extend the stratagem of chemical synthetic lethality to clinically relevant fungal pathogens, we screened a bioactive library of known drugs for synergistic enhancers of the widely used fungistatic drug fluconazole against the clinically relevant pathogens C. albicans, C. neoformans and C. gattii, as well as the genetically tractable budding yeast S. cerevisiae. Fluconazole is an azole drug that inhibits lanosterol 14α-demethylase, the gene product of ERG11, an essential cytochrome P450 enzyme in the ergosterol biosynthetic pathway (Groll et al, 1998). We identified 148 drugs that potentiate the antifungal action of fluconazole against the four species. These syncretic compounds had not been previously recognized in the clinic as antifungal agents, and many acted in a species-specific manner, often in a potent fungicidal manner.
To understand the mechanisms of synergism, we interrogated six syncretic drugs—trifluoperazine, tamoxifen, clomiphene, sertraline, suloctidil and L-cycloserine—in genome-wide chemogenomic profiles of the S. cerevisiae deletion strain collection (Giaever et al, 1999). These profiles revealed that membrane, vesicle trafficking and lipid biosynthesis pathways are targeted by five of the synergizers, whereas the sphingolipid biosynthesis pathway is targeted by L-cycloserine. Cell biological assays confirmed the predicted membrane disruption effects of the former group of compounds, which may perturb ergosterol metabolism, impair fluconazole export by drug efflux pumps and/or affect active import of fluconazole (Kuo et al, 2010; Mansfield et al, 2010). Based on the integration of chemical–genetic and genetic interaction space, a signature set of deletion strains that are sensitive to the membrane active synergizers correctly predicted additional drug synergies with fluconazole. Similarly, the L-cycloserine chemogenomic profile correctly predicted a synergistic interaction between fluconazole and myriocin, another inhibitor of sphingolipid biosynthesis. The structure of genetic networks suggests that it should be possible to devise higher order drug combinations with even greater selectivity and potency (Sharom et al, 2004). In an initial test of this concept, we found that the combination of a non-synergistic pair drawn from the membrane active and sphingolipid target classes exhibited potent three-way synergism with a low dose of fluconazole. Finally, the combination of sertraline and fluconazole was active in a G. mellonella model of Cryptococcal infection, and was also efficacious against fluconazole-resistant clinical isolates of C. albicans and C. glabrata.
Collectively, these results demonstrate that the combinatorial redeployment of known drugs defines a powerful antifungal strategy and establish a number of potential lead combinations for future clinical assessment.
Resistance to widely used fungistatic drugs, particularly to the ergosterol biosynthesis inhibitor fluconazole, threatens millions of immunocompromised patients susceptible to invasive fungal infections. The dense network structure of synthetic lethal genetic interactions in yeast suggests that combinatorial network inhibition may afford increased drug efficacy and specificity. We carried out systematic screens with a bioactive library enriched for off-patent drugs to identify compounds that potentiate fluconazole action in pathogenic Candida and Cryptococcus strains and the model yeast Saccharomyces. Many compounds exhibited species- or genus-specific synergism, and often improved fluconazole from fungistatic to fungicidal activity. Mode of action studies revealed two classes of synergistic compound, which either perturbed membrane permeability or inhibited sphingolipid biosynthesis. Synergistic drug interactions were rationalized by global genetic interaction networks and, notably, higher order drug combinations further potentiated the activity of fluconazole. Synergistic combinations were active against fluconazole-resistant clinical isolates and an in vivo model of Cryptococcus infection. The systematic repurposing of approved drugs against a spectrum of pathogens thus identifies network vulnerabilities that may be exploited to increase the activity and repertoire of antifungal agents.
antifungal; combination; pathogen; resistance; synergism
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Replacement of Candida albicans with C. dubliniensis in Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis Treated with Fluconazole
Martinez, Marcos
López-Ribot, José L.
Kirkpatrick, William R.
Coco, Brent J.
Bachmann, Stefano P.
Patterson, Thomas F.
Journal of Clinical Microbiology
Candida dubliniensis is an opportunistic yeast that has been increasingly implicated in oropharyngeal candidiasis (OPC) in human immunodeficiency virus (HIV)-infected patients but may be underreported due to its similarity with Candida albicans. Although most C. dubliniensis isolates are susceptible to fluconazole, the inducibility of azole resistance in vitro has been reported. Thus, the use of fluconazole prophylaxis in the treatment of these patients may have contributed to the increasing rates of isolation of C. dubliniensis. In this study, yeast strains were collected from the oral cavities of HIV-infected patients enrolled in a longitudinal study of OPC. Patients received fluconazole for the suppression or treatment of OPC, and isolates collected at both study entry and end of study were chosen for analysis. Samples were plated on CHROMagar Candida medium for initial isolation and further identified by Southern blot analysis with the species-specific probes Ca3 (for C. albicans) and Cd25 (for C. dubliniensis). Fluconazole MICs were determined by using NCCLS methods. At study entry, susceptible C. albicans isolates were recovered from oral samples in 42 patients who were followed longitudinally (1 to 36 months). C. albicans strains from 12 of these patients developed fluconazole resistance (fluconazole MIC, ≥64 μg/ml). C. dubliniensis was not detected at end of study in any of these patients. Of the remaining 30 patients, eight (27%) demonstrated a replacement of C. albicans by C. dubliniensis when a comparison of isolates obtained at baseline and those from the last culture was done. For the 22 of these 30 patients in whom no switch in species was detected, the fluconazole MICs for initial and end-of-study C. albicans isolates ranged from 0.125 to 2.0 μg/ml. For the eight patients in whom a switch to C. dubliniensis was detected, the fluconazole MICs for C. dubliniensis isolates at end of study ranged from 0.25 to 64 μg/ml: the fluconazole MICs for isolates from six patients were 0.25 to 2.0 μg/ml and those for the other two were 32 and 64 μg/ml, respectively. In conclusion, a considerable number of patients initially infected with C. albicans strains that failed to develop fluconazole resistance demonstrated a switch to C. dubliniensis. C. dubliniensis in this setting may be underestimated due to lack of identification and may occur due to the impact of fluconazole on the ecology of oral yeast species.
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Correlation between In Vitro and In Vivo Antifungal Activities in Experimental Fluconazole-Resistant Oropharyngeal and Esophageal Candidiasis
Walsh, Thomas J.
Gonzalez, Corina E.
Piscitelli, Steven
Bacher, John D.
Peter, Joanne
Torres, Richard
Shetti, Daiva
Katsov, Victoria
Kligys, Kristina
Lyman, Caron A.
Journal of Clinical Microbiology
Oropharyngeal and esophageal candidiasis (OPEC) is a frequent opportunistic mycosis in immunocompromised patients. Azole-resistant OPEC is a refractory form of this infection occurring particularly in human immunodeficiency virus (HIV)-infected patients. The procedures developed by the Antifungal Subcommittee of the National Committee for Clinical Laboratory Standards (NCCLS) are an important advance in standardization of in vitro antifungal susceptibility methodology. In order to further understand the relationship between NCCLS methodology and antifungal therapeutic response, we studied the potential correlation between in vitro susceptibility to fluconazole and in vivo response in a rabbit model of fluconazole-resistant OPEC. MICs of fluconazole were determined by NCCLS methods. Three fluconazole-susceptible (FS) (MIC, ≤0.125 μg/ml) and three fluconazole-resistant (FR) (MIC, ≥64 μg/ml) isolates of Candida albicans from prospectively monitored HIV-infected children with OPEC were studied. FR isolates were recovered from children with severe OPEC refractory to fluconazole, and FS isolates were recovered from those with mucosal candidiasis responsive to fluconazole. Fluconazole at 2 mg/kg of body weight/day was administered to infected animals for 7 days. The concentrations of fluconazole in plasma were maintained above the MICs for FS isolates throughout the dosing interval. Fluconazole concentrations in the esophagus were greater than or equal to those in plasma. Rabbits infected with FS isolates and treated with fluconazole had significant reductions in oral mucosal quantitative cultures (P < 0.001) and tissue burden of C. albicans in tongue, soft palate, and esophagus (P < 0.001). In comparison, rabbits infected with FR isolates were unresponsive to fluconazole and had no reduction in oral mucosal quantitative cultures or tissue burden of C. albicans versus untreated controls. We conclude that there is a strong correlation between in vitro fluconazole susceptibility by NCCLS methods and in vivo response to fluconazole therapy of OPEC due to C. albicans.
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Experimental Induction of Fluconazole Resistance in Candida tropicalis ATCC 750
Barchiesi, Francesco
Calabrese, David
Sanglard, Dominique
Falconi Di Francesco, Luigi
Caselli, Francesca
Giannini, Daniele
Giacometti, Andrea
Gavaudan, Stefano
Scalise, Giorgio
Antimicrobial Agents and Chemotherapy
Candida tropicalis is less commonly isolated from clinical specimens than Candida albicans. Unlike C. albicans, which can be occasionally found as a commensal, C. tropicalis is almost always associated with the development of fungal infections. In addition, C. tropicalis has been reported to be resistant to fluconazole (FLC). To analyze the development of FLC resistance in C. tropicalis, an FLC-susceptible strain (ATCC 750) (MIC = 1.0 μg/ml) was cultured in liquid medium containing increasing FLC concentrations from 8.0 to 128 μg/ml. The strain developed variable degrees of FLC resistance which paralleled the concentrations of FLC used in the medium. The highest MICs of FLC were 16, 256, and 512 μg/ml for strains grown in medium with 8.0, 32, and 128 μg of FLC per ml, respectively. Development of resistance was rapid and could be observed already after a single subculture in azole-containing medium. The resistant strains were cross-resistant to itraconazole (MIC > 1.0 μg/ml) and terbinafine (MIC > 512 μg/ml) but not to amphotericin B. Isolates grown in FLC at concentrations of 8.0 and 32 μg/ml reverted to low MICs (1.0 μg/ml) after 12 and 11 passages in FLC-free medium, respectively. The MIC for one isolate grown in FLC (128 μg/ml) (128 R) reverted to 16 μg/ml but remained stable over 60 passages in FLC-free medium. Azole-resistant isolates revealed upregulation of two different multidrug efflux transporter genes: the major facilitators gene MDR1 and the ATP-binding cassette transporter CDR1. The development of FLC resistance in vitro correlated well with the results obtained in an experimental model of disseminated candidiasis. While FLC given at 10 mg/kg of body weight/day was effective in reducing the fungal burden of mice infected with the parent strain, the same dosing regimen was ineffective in mice infected with strain 128 R. Finally, the acquisition of in vitro FLC resistance in strain 128 R was related to a loss of virulence. The results of our study elucidate important characteristics and potential mechanisms of FLC resistance in C. tropicalis.
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Azole Resistance by Loss of Function of the Sterol Δ5,6-Desaturase Gene (ERG3) in Candida albicans Does Not Necessarily Decrease Virulence
Vale-Silva, L. A.
Coste, A. T.
Ischer, F.
Parker, J. E.
Kelly, S. L.
Pinto, E.
Sanglard, D.
Antimicrobial Agents and Chemotherapy
The inactivation of ERG3, a gene encoding sterol Δ5,6-desaturase (essential for ergosterol biosynthesis), is a known mechanism of in vitro resistance to azole antifungal drugs in the human pathogen Candida albicans. ERG3 inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified a C. albicans clinical isolate (VSY2) with high-level resistance to azole drugs in vitro and an absence of ergosterol but normal filamentation. Sequencing of ERG3 in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (erg3-1) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming that ERG3 inactivation was the mechanism of azole resistance. Additionally, the replacement of both ERG3 alleles by erg3-1 in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinical ERG3 mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole both in vitro and in vivo, the ERG3-derived mutant of SC5314 was resistant only in vitro and was less virulent than the wild type. This suggests that VSY2 compensated for the in vivo fitness defect of ERG3 inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation of ERG3 does not necessarily affect filamentation and virulence.
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Patterns of in vitro activity of itraconazole and imidazole antifungal agents against Candida albicans with decreased susceptibility to fluconazole from Spain.
Martinez-Suarez, J V
Rodriguez-Tudela, J L
Antimicrobial Agents and Chemotherapy
Two groups of recent clinical isolates of Candida albicans consisting of 101 isolates for which fluconazole MICs were < or = 0.5 microgram/ml (n = 50) and > or = 4.0 micrograms/ml (n = 51), respectively, were compared for their susceptibilities to fluconazole, clotrimazole, miconazole, ketoconazole, and itraconazole. Susceptibility tests were performed by a photometer-read broth microdilution method with an improved RPMI 1640 medium supplemented with 18 g of glucose per liter (RPMI-2% glucose; J. L. Rodríguez-Tudela and J. V. Martínez-Suárez, Antimicrob. Agents Chemother. 38:45-48, 1994). Preparation of drugs, basal medium, and inocula was done by the recommendations of the National Committee for Clinical Laboratory Standards. The MIC endpoint was calculated objectively from the turbidimetric data read at 24 h as the lowest drug concentration at which growth was just equal to or less than 20% of that in the positive control well (MIC 80%). In vitro susceptibility testing separated azole-susceptible strains from the strains with decreased susceptibilities to azoles if wide ranges of concentrations (20 doubling dilutions) were used for ketoconazole, miconazole, and clotrimazole. By comparison with isolates for which fluconazole MICs were < or = 0.5 microgram/ml, those isolates for which fluconazole MICs were > or = 4.0 micrograms/ml were in general less susceptible to other azole drugs, but different patterns of decreased susceptibility were found, including uniform increases in the MICs of all azole derivatives, higher MICs of several azoles but not others, and elevated MICs of fluconazole only. On the other hand, decreased susceptibility to any other azole drug was never found among strains for which MICs of fluconazole were lower.
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Multiple Resistant Phenotypes of Candida albicans Coexist during Episodes of Oropharyngeal Candidiasis in Human Immunodeficiency Virus-Infected Patients
Lopez-Ribot, Jose L.
McAtee, Robert K.
Perea, Sofia
Kirkpatrick, William R.
Rinaldi, Michael G.
Patterson, Thomas F.
Antimicrobial Agents and Chemotherapy
Mechanisms of resistance to azoles in Candida albicans, the main etiologic agent of oropharyngeal candidiasis (OPC), include alterations in the target enzyme (lanosterol demethylase) and increased efflux of drug. Previous studies on mechanisms of resistance have been limited by the fact that only a single isolate from each OPC episode was available for study. Multiple isolates from each OPC episode were evaluated with oral samples plated in CHROMagar Candida with and without fluconazole to maximize detection of resistant yeasts. A total of 101 isolates from each of three serial episodes of OPC from four different patients were evaluated. Decreasing geometric means of fluconazole MICs with serial episodes of infection were detected in the four patients. However, 8-fold or larger (up to 32-fold) differences in fluconazole MICs were detected within isolates recovered at the same time point in 7 of 12 episodes. Strain identity was analyzed by DNA typing techniques and indicated that isolates from each patient represented mainly isogenic strains, but differed among patients. A Northern blot technique was used to monitor expression of ERG11 (encoding lanosterol demethylase) and genes coding for efflux pumps. This analysis revealed that clinical isolates obtained from the same patient and episode were phenotypically heterogeneous in their patterns of expression of these genes involved in fluconazole resistance. These results demonstrate the complexity of the distribution of the molecular mechanisms of antifungal drug resistance and indicate that different subpopulations of yeasts may coexist at a given time in the same patient and may develop resistance through different mechanisms.
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Amino Acid Substitutions at the Major Insertion Loop of Candida albicans Sterol 14alpha-Demethylase Are Involved in Fluconazole Resistance
Alvarez-Rueda, Nidia
Fleury, Audrey
Morio, Florent
Pagniez, Fabrice
Gastinel, Louis
Le Pape, Patrice
PLoS ONE
In the fungal pathogen Candida albicans, amino acid substitutions of 14alpha-demethylase (CaErg11p, CaCYP51) are associated with azole antifungals resistance. This is an area of research which is very dynamic, since the stakes concern the screening of new antifungals which circumvent resistance. The impact of amino acid substitutions on azole interaction has been postulated by homology modeling in comparison to the crystal structure of Mycobacterium tuberculosis (MT-CYP51). Modeling of amino acid residues situated between positions 428 to 459 remains difficult to explain to date, because they are in a major insertion loop specifically present in fungal species.
Methodology/Principal Finding
Fluconazole resistance of clinical isolates displaying Y447H and V456I novel CaErg11p substitutions confirmed in vivo in a murine model of disseminated candidiasis. Y447H and V456I implication into fluconazole resistance was then studied by site-directed mutagenesis of wild-type CaErg11p and by heterogeneously expression into the Pichia pastoris model. CLSI modified tests showed that V447H and V456I are responsible for an 8-fold increase in fluconazole MICs of P. pastoris mutants compared to the wild-type controls. Moreover, mutants showed a sustained capacity for producing ergosterol, even in the presence of fluconazole. Based on these biological results, we are the first to propose a hybrid homology structure-function model of Ca-CYP51 using 3 different homology modeling programs. The variable position of the protein insertion loop, using different liganded or non-liganded templates of recently solved CYP51 structures, suggests its inherent flexibility. Mapping of recognized azole-resistant substitutions indicated that the flexibility of this region is probably enhanced by the relatively high glycine content of the consensus.
The results highlight the potential role of the insertion loop in azole resistance in the human pathogen C. albicans. This new data should be taken into consideration for future studies aimed at designing new antifungal agents, which circumvent azole resistance.
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Heteroresistance to Fluconazole and Voriconazole in Cryptococcus neoformans
Mondon, P.
Petter, R.
Amalfitano, G.
Luzzati, R.
Concia, E.
Polacheck, I.
Kwon-Chung, K. J.
Antimicrobial Agents and Chemotherapy
Cryptococcus neoformans isolates that exhibited unusual patterns of resistance to fluconazole and voriconazole were isolated from seven isolates from two different geographical regions: one isolate from an Israeli non-AIDS patient and six serial isolates from an Italian AIDS patient who had suffered six recurrent episodes of cryptococcal meningitis. Each isolate produced cultures with heterogeneous compositions in which most of the cells were susceptible, but cells highly resistant to fluconazole (MICs, ≥64 μg/ml) were recovered at a variable frequency (7 × 10−3 to 4.6 × 10−2). Evidence showed that this type of resistance is innate and is unrelated to drug exposure since the Israeli patient had never been treated with azoles or any other antimycotic agents. Analysis of clonal subpopulations of these two strains showed that they exhibited heterogeneous patterns of resistance. The number of subpopulations which grew on fluconazole or voriconazole agar declined progressively with increasing azole concentration without a sharp cutoff point. For the Italian serial isolates, the number of clonal populations resistant to fluconazole (64 μg/ml) and voriconazole (1 μg/ml) increased steadily, yielding the highest number for the isolate from the last episode. Attempts to purify a sensitive subpopulation failed, but clones highly resistant to fluconazole (100 μg/ml) and moderately resistant to voriconazole (1 μg/ml) always produced a homogeneous population of resistant cells. Upon maintenance on drug-free medium, however, the majority of the homogeneously resistant cells of these subclones lost their resistance and returned to the stable initial heteroresistant phenotype. The pattern of heteroresistance was not affected by the pH or osmolarity of the medium but was influenced by temperature. The resistance appeared to be suppressed at 35°C and was completely abolished at 40°C. Although heterogeneity in azole resistance among subpopulations of single isolates has been reported for Candida species, the transient changes in expression of resistance under different growth conditions reported here have not been observed in fungal pathogens.
Related Articles
Clinical Significance of Azole Antifungal Drug Cross-Resistance in Candida glabrata
Panackal, Anil A.
Gribskov, Jennifer L.
Staab, Janet F.
Kirby, Katherine A.
Rinaldi, Michael
Marr, Kieren A.
Journal of Clinical Microbiology
Candida glabrata, which can become resistant to fluconazole, is a common cause of bloodstream infection. This study was performed to determine the significance of cross-resistance to new azole drugs among C. glabrata isolates recovered as a cause of infection in azole-treated hematopoietic stem cell transplant (HSCT) recipients. Seven cases of invasive candidiasis caused by C. glabrata occurred in HSCT recipients who were receiving azole therapy between January 2000 and December 2004 in our institution. Case characteristics were ascertained. Sequential colonizing and invasive isolates were examined to determine susceptibilities to fluconazole, itraconazole, and voriconazole, and molecular relatedness by restriction fragment length polymorphism (RFLP) analysis. Twenty-three C. glabrata isolates were recovered from 4 patients who developed candidemia while receiving fluconazole and three patients who developed candidemia while receiving voriconazole. The mode MICs of fluconazole, itraconazole, and voriconazole for these isolates were ≥64 μg/ml (range, 4 to ≥64 μg/ml), 2 μg/ml (range, 0.25 to ≥16 μg/ml), and 1 μg/ml (range, 0.03 to ≥16 μg/ml), respectively. Kendall tau b correlation coefficients demonstrated significant associations between the MICs of voriconazole with fluconazole (P = 0.005) and itraconazole (P = 0.008). Colonizing and invasive isolates exhibiting variable susceptibilities had similar RFLP patterns. These observations suggest that C. glabrata exhibits considerable clinically significant cross-resistance between older azole drugs (fluconazole and itraconazole) and voriconazole. Caution is advised when considering voriconazole therapy for C. glabrata candidemia that occurs in patients with extensive prior azole drug exposure.
Related Articles
Microbiological Screening of Irish Patients with Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Reveals Persistence of Candida albicans Strains, Gradual Reduction in Susceptibility to Azoles, and Incidences of Clinical Signs of Oral Candidiasis without Culture Evidence▿†
McManus, Brenda A.
McGovern, Eleanor
Moran, Gary P.
Healy, Claire M.
Nunn, June
Fleming, Pádraig
Costigan, Colm
Sullivan, Derek J.
Coleman, David C.
Journal of Clinical Microbiology
Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) are prone to chronic mucocutaneous candidiasis, which is often treated with azoles. The purpose of this study was to characterize the oral Candida populations from 16 Irish APECED patients, who comprise approximately half the total number identified in Ireland, and to examine the effect of intermittent antifungal therapy on the azole susceptibility patterns of Candida isolates. Patients attended between one and four clinical evaluations over a 5-year period, providing oral rinses and/or oral swab samples each time. Candida was recovered from 14/16 patients, and Candida albicans was the only Candida species identified. Interestingly, clinical diagnosis of candidiasis did not correlate with microbiological evidence of Candida infection at 7/22 (32%) clinical assessments. Multilocus sequence typing analysis of C. albicans isolates recovered from the same patients on separate occasions identified the same sequence type each time. Fluconazole resistance was detected in isolates from one patient, and isolates exhibiting a progressive reduction in itraconazole and/or fluconazole susceptibility were identified in a further 3/16 patients, in each case correlating with the upregulation of CDR- and MDR-encoded efflux pumps. Mutations were also identified in the ERG11 and the TAC1 genes of isolates from these four patients; some of these mutations have previously been associated with azole resistance. The findings suggest that alternative Candida treatment options, other than azoles such as chlorhexidine, should be considered in APECED patients and that clinical diagnosis of oral candidiasis should be confirmed by culture prior to the commencement of anti-Candida therapy.
Related Articles
Emergence of Resistance of Candida albicans to Clotrimazole in Human Immunodeficiency Virus-Infected Children: In Vitro and Clinical Correlations
Pelletier, René
Peter, Joanne
Antin, Cynthia
Gonzalez, Corina
Wood, Lauren
Walsh, Thomas J.
Journal of Clinical Microbiology
Oropharyngeal candidiasis (OPC) is a common opportunistic infection in human immunodeficiency virus (HIV)-infected patients and other immunocompromised hosts. Clotrimazole troches are widely used in the treatment of mucosal candidiasis. However, little is known about the potential contribution of clotrimazole resistance to the development of refractory mucosal candidiasis. We therefore investigated the potential emergence of resistance to clotrimazole in a prospectively monitored HIV-infected pediatric population receiving this azole. Adapting the National Committee for Clinical Laboratory Standards M27-A reference method for broth antifungal susceptibility testing of yeasts to clotrimazole, we compared MICs in macrodilution and microdilution assays. We further analyzed the correlation between these in vitro findings and the clinical response to antifungal therapy. One isolate from each of 87 HIV-infected children was studied by the macrodilution and microdilution methods. Two inoculum sizes were tested by the macrodilution method (103 and 104 CFU/ml) in order to assess the effect of inoculum size on clotrimazole MICs. The same isolates also were tested using a noncolorimetric microdilution method. Clotrimazole concentrations ranged from 0.03 to 16 μg/ml. Readings were performed after incubation for 24 and 48 h at 35°C. For 62 (71.2%) of 87 clinical isolates, the MICs were low (≤0.06 μg/ml). The MIC for 90% of the strains tested was 0.5 μg/ml, and the highest MIC was 8 μg/ml. There was no significant difference between MICs at the two inoculum sizes. There was 89% agreement (±1 tube) between the microdilution method at 24 h and the macrodilution method at 48 h. If the MIC of clotrimazole for an isolate of C. albicans was ≥0.5 μg/ml, there was a significant risk (P < 0.001) of cross-resistance to other azoles: fluconazole, ≥64 μg/ml (relative risk [RR] = 8.9); itraconazole, ≥1 μg/ml (RR = 10). Resistance to clotrimazole was highly associated with clinically overt failure of antifungal azole therapy. Six (40%) of 15 patients for whom the clotrimazole MIC was ≥0.5 μg/ml required amphotericin B for refractory mucosal candidiasis versus 4 (5.5%) of 72 for whom the MIC was <0.5 μg/ml (P = 0.001; 95% confidence interval = 2.3 to 22; RR = 7.2). These findings suggest that an interpretive breakpoint of 0.5 μg/ml may be useful in defining clotrimazole resistance in C. albicans. The clinical laboratory's ability to determine MICs of clotrimazole may help to distinguish microbiologic resistance from the other causes of refractory OPC, possibly reducing the usage of systemic antifungal agents. We conclude that resistance to clotrimazole develops in isolates of C. albicans from HIV-infected children, that cross-resistance to other azoles may develop concomitantly, and that this resistance correlates with refractory mucosal candidiasis.
Related Articles
Genome-Wide Expression Profile Analysis Reveals Coordinately Regulated Genes Associated with Stepwise Acquisition of Azole Resistance in Candida albicans Clinical Isolates
Rogers, P. David
Barker, Katherine S.
Antimicrobial Agents and Chemotherapy
Candida albicans is an opportunistic human fungal pathogen and a causative agent of oropharyngeal candidiasis (OPC), the most frequent opportunistic infection among patients with AIDS. Fluconazole and other azole antifungal agents have proven effective in the management of OPC; however, with increased use of these agents treatment failures have occurred. Such failures have been associated with the emergence of azole-resistant strains of C. albicans. In the present study we examined changes in the genome-wide gene expression profile of a series of C. albicans clinical isolates representing the stepwise acquisition of azole resistance. In addition to genes previously associated with azole resistance, we identified many genes whose differential expression was for the first time associated with this phenotype. Furthermore, the expression of these genes was correlated with that of the known resistance genes CDR1, CDR2, and CaMDR1. Genes coordinately regulated with the up-regulation of CDR1 and CDR2 included the up-regulation of GPX1 and RTA3 and the down-regulation of EBP1. Genes coordinately regulated with the up-regulation of CaMDR1 included the up-regulation of IFD1, IFD4, IFD5, IFD7, GRP2, DPP1, CRD2, and INO1 and the down-regulation of FET34, OPI3, and IPF1222. Several of these appeared to be coordinately regulated with both the CDR genes and CaMDR1. Many of these genes are involved in the oxidative stress response, suggesting that reduced susceptibility to oxidative damage may contribute to azole resistance. Further evaluation of the role these genes and their respective gene products play in azole antifungal resistance is warranted.
Related Articles
Fluconazole Susceptibility of Vaginal Isolates Obtained from Women with Complicated Candida Vaginitis: Clinical Implications
Sobel, J. D.
Zervos, M.
Reed, B. D.
Hooton, T.
Soper, D.
Nyirjesy, P.
Heine, M. W.
Willems, J.
Panzer, H.
Antimicrobial Agents and Chemotherapy
Despite considerable evidence of azole resistance in oral candidiasis due to Candida species, little is known about the azole susceptibilities of the genital tract isolates responsible for vaginitis. The fluconazole susceptibilities of vaginal isolates obtained during a multicenter study of 556 women with complicated Candida vaginitis were determined by evaluating two fluconazole treatment regimens. Of 393 baseline isolates of Candida albicans, 377 (96%) were highly susceptible to fluconazole (MICs, <8 μg/ml) and 14 (3.6%) were resistant (MICs, ≥64 μg/ml). Following fluconazole therapy, one case of in vitro resistance developed during 6 weeks of monitoring. In accordance with the NCCLS definition, in vitro fluconazole resistance correlated poorly with the clinical response, although a trend of a higher mycological failure rate was found (41 versus 19.6% on day 14). By using an alternative breakpoint of 1 μg/ml, based upon the concentrations of fluconazole achievable in vaginal tissue, no significant differences in the clinical and mycological responses were observed when isolates (n = 250) for which MICs were ≤1 μg/ml were compared with isolates (n = 30) for which MICs were >1 μg/ml, although a trend toward an improved clinical outcome was noted on day 14 (odds ratio, >2.7; 95% confidence interval, 0.91, 8.30). Although clinical failure was uncommon, symptomatic recurrence or mycological relapse almost invariably occurred with highly sensitive strains (MICs, <1.0 μg/ml). In vitro fluconazole resistance developed in 2 of 18 initially susceptible C. glabrata isolates following fluconazole exposure. Susceptibility testing for women with complicated Candida vaginitis appears to be unjustified.
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Researchers at UNC-Chapel Hill want to learn about your experience – to share your story, join the LECHE research survey here:
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For further information regarding this study please contact the study coordinator (Anitha Muddana) at firstname.lastname@example.com or 984-974-9008.]]>
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Compensation Available: Participants will receive up to $150 for taking part in this study.
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7/2012- 6/2017
Participation Details
2 visits (1 visit at the UNC Dental School and 1 visit at the Women's Hospital)
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Qualifiers for the study
Developed new symptoms or experienced exacerbation of existing symptoms of pelvic pain
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Under 21 and over 64, pregnant, hyperthyroidism, epilepsy, rheumatoid arthritis, lupus erhematosis, ulcerative colitis, Crohn's disease, bowel resection
Study Contact Person Name and Number Jasmine Lewis, 1-866-857-9394
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Cirque du Soleil: Journey of Man
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Track Listings
|1. Journey Of Man - Roxane Potvin
|2. Overture
|3. Taiko - The Tribe From Cirque Du Soleil's Mystere
|4. Birth
|5. The Forest - Toumany Kouyate
|6. In The Beginning
|7. Youth
|8. Flying
|9. Reveil
|10. Banquine
|11. Journey Of Man (Instrumental)
|12. Trip Hop
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I had the chance to see *Journey of Man* on an Imax screen and as wonderful as the movie itself was, the music was even more enchanting. It's one soundtrack that can truly stand apart and be appreciated on its own.
From listening to this cd you will be invited to feel a beautiful kind of melancholy, revitalizing energy, and subtle strains of menace. For a writer, or a dreamer, it's a must. You will forget yourself within minutes of first turning it on.
If there were no sight, only sound, *Journey of Man* would be the music to live by.
But such is not the case. The music is wonderful--lush, rich, pictoral in and of its own right. The CD is totally self contained and musically enriching to the listener. The composition of Benoit Jutras is stunning, and the musicianship is flawless. Invest in this recording, even if you never have a chance to view the film.
Man - what a bunch of babble. Anyway, this is a great cd - some really wonderful orchestrations and the most classical of any Cirque soundtrack.
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Exciting, energetic, and powerful are just a few wors I can use to describe the music on this disk. If ever there was a sound track that actually put the movie to shame, this is... Read morePublished on Feb. 13 2004 by Len Manning
enough said! The music is great... far better than the half asked movie that Cirque created (no offense to those who liked it... so do i he he).Published on July 31 2002
The soundtrack of this IMAX film is incredible. If you have never listened to a cirque CD you have to get this one. Read morePublished on March 31 2002
Hi! If you have ever seen this movie, especially on the big IMAX screen, you know what I'm talking about when I say it's the BEST! Read morePublished on Feb. 18 2002
I went to Chicago for my birthday on last January, I thought that the travel was my brithday present, but when i came out of the IMAX theather on Navy Pier after seeing this movie,... Read morePublished on May 14 2001 by ruben tejeda navarrete
Just saw the IMAX production at Metrion in SF today, absolutely beautiful. Brings their art to a new dimension. Read morePublished on Feb. 5 2001 by Aaron Christeaan
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Book your room at Hotel Coral Suites
Get an overview of this hotel3.5-star Panama City hotel in El Cangrejo with free breakfast
Located in El Cangrejo, this hotel is within 2 mi (3 km) of Via Espana, Multicentro Panama, and Multiplaza Pacific Mall. National Bank House Museum and Museum of Natural Science are also within 3 mi (5 km).
Hotel Features
An outdoor pool, a 24-hour fitness center, and a bar/lounge are available at this smoke-free hotel. Free buffet breakfast is provided, as well as free WiFi in public areas, free self parking, and a free shopping center shuttle. Additionally, a rooftop terrace, a 24-hour business center, and dry cleaning are onsite.
Room Amenities
All 62 rooms boast private pools and offer free WiFi and kitchens. Sofa beds and refrigerators are standard, as are LCD TVs with cable channels.Would you like to give us feedback? Share it! Opens in a new window
Hotel Coral SuitesCalle D, El Cangrejo Panama City Panama 083000377
Hotel Amenities
Hotel Amenities
Hotel Coral Suites features an outdoor pool and a 24-hour fitness center. A bar/lounge is on site where guests can unwind with a drink. A complimentary breakfast is offered each morning. High-speed wireless Internet access is complimentary.
A 24-hour business center is on site at this 3.5-star property. This Panama City hotel also offers a rooftop terrace, tour/ticket assistance, and laundry facilities. For a surcharge, the property provides a shuttle from the hotel to the airport (available on request) and a beach shuttle. Onsite self parking is complimentary.
Hotel Coral Suites is a smoke-free property.
- 24-hour business center
- 24-hour fitness facilities
- 24-hour front desk
- Airport transportation - drop-off (surcharge)
- Bar/lounge
- Beach shuttle (surcharge)
- Cruise terminal shuttle (surcharge)
- Dry cleaning/laundry service
- Elevator/lift
- Free breakfast
- Free self parking
- Free WiFi
- Laundry facilities
- Number of buildings/towers - 1
- Number of floors - 6
- Outdoor pool
- Porter/bellhop
- Rooftop terrace
- Free shopping center shuttle
- Smoke-free property
- Total number of rooms - 62
- Tours/ticket assistance
Family Friendly Amenities
- Cribs/infant beds (complimentary)
- Free Wi-Fi
- Full-sized refrigerator/freezer
- Kitchen
- Laundry facilities
- Microwave
- Outdoor pool
- Sofa bed
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free self parking
Room Amenities
- Air conditioning
- Cable TV service
- In-room climate control (air conditioning)
- Coffee/tea maker
- Free newspaper
- Free toiletries
- Cookware, dishware, and utensils
- Free cribs/infant beds
- Daily housekeeping
- Designer toiletries
- Desk
- Free WiFi
- Full-sized refrigerator/freezer
- Hair dryer
- In-room safe
- Iron/ironing board
- Kitchen
- Microwave
- Phone
- Premium bedding
- Premium TV channels
- Private bathroom
- Private pool
- Shower/tub combination
- Sofa bed
- Sofa bed size
Where to Eat
Guests are offered a complimentary buffet breakfast each morning.
Nearby Things to Do
Recreational amenities at the hotel include an outdoor pool and a 24-hour fitness center.
- Golfing nearby
To make requests for specific accessibility needs, complete the "Special or Accessibility Requests" section of "Trip Preferences" when you book your hotel.
Hotel Policies
Check-in time starts at 3 PM
Check-in time ends at 12:30 AM
Special check-in instructions:
Check-out time is 1 PM
Payment types
- No pets or service animals allowed
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
No rollaway/extra beds available
No pets or service animals allowed
Optional extras
- Airport shuttle fee: PAB 30.00 per person
- Airport shuttle fee per child: PAB 10.00 (from 12 to 16 years old)
Hotel Name
- Coral Suites
- Coral Suites Hotel
- Coral Suites Panama City
- Hotel Coral Suites
- Hotel Coral Suites Panama City
- Hotel Coral Suites Panama/Panama City
We should mention
This property offers transfers from the cruise terminal and airport (surcharges may apply). Guests must contact the property with arrival details 24 hours prior to arrival, using the contact information on the booking confirmation. No pets and no service animals are allowed at this property.


text:
Colonels to appear on SLC-TV
Published: Thursday, July 19, 2012 at 8:00 a.m.
Last Modified: Thursday, July 19, 2012 at 8:56 a.m.
The Nicholls State football team will appear in at least two games on the Southland Conference Television Network in 2012.
Nicholls' first SLC TV game will be on Oct. 20 against Stephen F. Austin in Nacogdoches, Texas. On Nov. 3, the Colonels open up a doubleheader on the Southland Television Network when they host McNeese State. Both games start at 3 p.m.
Local television affiliates include KWBJ in Morgan City, WHNO in New Orleans and WBTR in Baton Rouge on WBTR.
Reader comments posted to this article may be published in our print edition. All rights reserved. This copyrighted material may not be re-published without permission. Links are encouraged.


text:
Imagine that a user request page.htm in its browser. It travels through the internet and one of proxy server (P1) serves a cached version. Then, the user refreshes the page, another request travels through the internet and reaches proxy server P2, which serves a document that was cached at a different moment. The user sees 2 different contents.
Question: Could this happen ?
Assuming that the expiry policy is set to an absolute date, that would not happen. (Well in theory, because the server clocks are not necessarily synchronised). But what about relative time window cache policies ?
If it can happen, how do you get around the potential issue ?


text: What a great find ... The premises are well kept the staff Excellent and very accommodating ... This was just a turn around trip. However when I come to Sacramento this will be my place of choice. The view is so peaceful make sure to book your room on the inside so you take advantage of the romantic setting ...... More


text:
Virginia state police say they believe the human remains discovered on a southern Albermarle County farm are those of Morgan Harrington, the Virginia Tech student who has been missing since Oct. 17.
State Police Superintendent Steve Flaherty said Tuesday an autopsy will be conducted to confirm that the remains are Morgan Harrington. Authorities say they found significant items and evidence at the scene.
Farmer David Bass says he was surveying his property Tuesday morning looking at fences when he spotted the remains. The farm is about 10 miles from where 20-year-old Harrington was last seen.
Harrington became separated from friends after she left a Metallica concert at the John Paul Jones Arena in Charlottesville arena and was denied re-entry.
The Roanoke woman's parents were seen at the Charlottesville state police barracks Tuesday afternoon.
Cops: Remains of Missing Va. Student Found
© 2010 CBS Interactive Inc. All Rights Reserved. This material may not be published, broadcast, rewritten, or redistributed. The Associated Press contributed to this report.


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Samsung exhibit, E3, Los Angeles, Calif.
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The Violent Femmes perform on the first of three days of concerts in Newport, R.I.
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