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Q: Error building Intellij Scala Plugin I am trying to follow the instructions at
https://confluence.jetbrains.com/display/SCA/Setting+up+Scala+plugin+project+in+IntelliJ+IDEA
I am using Idea 14.1.14.
Have downloaded the sources and downloadIdea.
And after importing the project set the Project SDK to 1.7.
Not sure if the above is enough info to go on; but on make project I get the below error; is it possible to guess at what I've got incorrect?
Error:scalac: error while loading Object, Missing dependency 'object scala in compiler mirror', required by /usr/lib/jvm/java-7-oracle/jre/lib/rt.jar(java/lang/Object.class)
Error:scalac: Error: object scala in compiler mirror not found.
scala.reflect.internal.MissingRequirementError: object scala in compiler mirror not found.
    at scala.reflect.internal.MissingRequirementError$.signal(MissingRequirementError.scala:17)
    at scala.reflect.internal.MissingRequirementError$.notFound(MissingRequirementError.scala:18)
    at scala.reflect.internal.Mirrors$RootsBase.getModuleOrClass(Mirrors.scala:53)
    at scala.reflect.internal.Mirrors$RootsBase.getModuleOrClass(Mirrors.scala:66)
    at scala.reflect.internal.Mirrors$RootsBase.getPackage(Mirrors.scala:173)
    at scala.reflect.internal.Definitions$DefinitionsClass.ScalaPackage$lzycompute(Definitions.scala:161)
    at scala.reflect.internal.Definitions$DefinitionsClass.ScalaPackage(Definitions.scala:161)
    at scala.reflect.internal.Definitions$DefinitionsClass.ScalaPackageClass$lzycompute(Definitions.scala:162)
    at scala.reflect.internal.Definitions$DefinitionsClass.ScalaPackageClass(Definitions.scala:162)
    at scala.reflect.internal.Definitions$DefinitionsClass.init(Definitions.scala:1377)
    at scala.tools.nsc.Global$Run.<init>(Global.scala:1229)
    at xsbt.CachedCompiler0$$anon$2.<init>(CompilerInterface.scala:116)
    at xsbt.CachedCompiler0.run(CompilerInterface.scala:116)
    at xsbt.CachedCompiler0.run(CompilerInterface.scala:102)
    at xsbt.CompilerInterface.run(CompilerInterface.scala:27)
    at sun.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
    at sun.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:57)
    at sun.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
    at java.lang.reflect.Method.invoke(Method.java:606)
    at sbt.compiler.AnalyzingCompiler.call(AnalyzingCompiler.scala:102)
    at sbt.compiler.AnalyzingCompiler.compile(AnalyzingCompiler.scala:48)
    at sbt.compiler.AnalyzingCompiler.compile(AnalyzingCompiler.scala:41)
    at org.jetbrains.jps.incremental.scala.local.IdeaIncrementalCompiler.compile(IdeaIncrementalCompiler.scala:29)
    at org.jetbrains.jps.incremental.scala.local.LocalServer.compile(LocalServer.scala:26)
    at org.jetbrains.jps.incremental.scala.remote.Main$.make(Main.scala:62)
    at org.jetbrains.jps.incremental.scala.remote.Main$.nailMain(Main.scala:20)
    at org.jetbrains.jps.incremental.scala.remote.Main.nailMain(Main.scala)
    at sun.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
    at sun.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:57)
    at sun.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
    at java.lang.reflect.Method.invoke(Method.java:606)
    at com.martiansoftware.nailgun.NGSession.run(NGSession.java:319)
Warning:scalac: No 'scala-library*.jar' in module dependencies [compiler_settings]

A: I have faced a similar issue recently even after having the compatible versions of java,sbt and scala SDK but found few reasons why we get this error when we importing sbt project into Intellij.

*

*Check the scala-SDK is present in the global libraries under File->Project Structure -> Global Libraries.

Please find the below screenshot.

*Even after fixing the first issue you still face this issue. Then look for the external dependencies which you have placed under /lib folder check the checksum of that jar if possible are make sure it has the right size. This should solve the issue.

Apart from the above issue, you might end up with dependencies source linking issues in the code when you open the project in IntelliJ. This is because of the additional root modules which are present under File -> Project modules -> modules. In the modules panel, you will see projectxxx-src and projectxx-test additional to projexctxx-process and projectxx-process-build. So remove the projectxx-src and projectxx-test.

A: Go to File -> Project structure -> Global libraries and add Scala SDK there. In my case, two SDKs were already present (in ivy folder) and offered for selection.

A: You have to have Java 8 JDK for Scala to work
see the scala site for further info

A: Only Java 8 JDK version should be used along side scala. Not even higher JDK versions.

A:
better also check here. It solved my own building errors

A: In my case, I had to specify the correct JDK home path in the Project structure -> SDKs.
Since my JAVA_HOME variable was not set yet, Intellij was taking a default value which doesn't exist.
Replace the JDK Home Path by the the right path of the JDK (in my case it was the following : /Library/Java/JavaVirtualMachines/adoptopenjdk-8.jdk/Contents/Home)
Replace 'adoptopenjdk-8.jdk' by your folders' name (might be jdk_1.8.0_xxx.jdk)
I hope it helps.

A: Double-Check JDK Compatibility
I encountered this issue when the Java SDK being used by my IntelliJ project was too "new" for the version of Scala I was using. For example, IntelliJ defaulted to using Java 12 for my project when my project was using Scala version 2.11.8. However, according to the documentation, the minimum required Scala version for JDK 11 is 2.11.12; therefore, I resolved this problem by changing the JDK used in IntelliJ to Java 8 by going to File -> Project Structure -> Project -> Project SDK


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HomeCommunityChurchGreek Orthodox churches in Sydney deliver hampers and toys to those in...
Greek Orthodox churches in Sydney deliver hampers and toys to those in need this Christmas
Photos: Facebook.
CommunityChurchInitiatives
With just two weeks until Christmas Day, Greek Orthodox churches across Greater Sydney have started getting into the festive spirit by organising hamper and toy drives for people in need.
Christmas toys for the Sydney Children's Hospital:
Volunteers from the Saint Catherine Greek Orthodox Church in Mascot have worked hard over the last few weeks gathering Christmas toys for the Sydney Children's Hospital in Randwick.
Two of these volunteers, Eirini and Sebastian, visited Big W at Eastgardens to purchase a variety of children's toys for the initiative, such as fluffy elephants, LEGO, tennis balls and among many other things guaranteed to bring joy to a child's life.
Eirini and Sebastian, visited Big W at Eastgardens to purchase children's toys.
According to a Facebook post, the purchase of the toys was made possible with the help of "wonderful donors from our parish, especially the family of Aris Koutsougeras who donated a substantial amount."
Once bought, the toys were delivered by Eirini and Sebastian, as well as Father Athanasios Giatsios, to the children's hospital. Although the group weren't able to hand out the toys individually this year due to COVID-19 restrictions, they still brought smiles to the faces of the children and their families.
The toys were delivered to the Sydney Children's Hospital in Randwick.
Elsewhere, the church was also holding its annual Christmas Food Collection to help other vulnerable people in the community as well.
Hundreds of non-perishable items were donated and these were then packaged by a number of young volunteers from SOFIA UNSW, Sydney University Greek Society (SUGS), the Hellenic Society of the University of NSW (Hellsoc UNSW) and the St Catherine's Youth Fellowship.
The packages were then delivered to six charities in the local community including: Pantry4ThePeople, St Elesa Food Initiative, South East Neighbourhood Pantry, Street Buffet, Bayside Women's Shelter and Ronald McDonald House in Randwick.
Mascot's annual Christmas Food Collection.
Christmas hampers for refugee families:
This year, St Nectarios Parish and Community in Burwood are continuing their tradition of preparing 120 Christmas hampers for refugee families through Metro Assist.
The parish's Youth In Action group held a virtual Trivia Night during lockdown which was attended by 100 people and raised over $4,000 for the hampers. Parishioners also donate groceries and gifts to be included in the Christmas hampers.
Hampers delivered to refugee families last year. Photo: Facebook.
"This is a project that our youth are so passionate about and is one of their key community service activities for the year…" Christina Efthymiades, a Youth In Action mentor, tells The Greek Herald.
"Seeing the smiles of the refugee families we support and sending them some Christmas joy and our love is our blessing and a wonderful team building exercise for our youth."
At the same time, the Parish is also holding its annual Christmas carols on Sunday, December 19 in the courtyard of the church. The event involves the singing of Christmas carols, a visit from Santa Claus and gifts are presented to children, chanters, caretakers and key volunteers.
St Nectarios Burwood's Christmas carols last year. Photo: Facebook.
"Our annual Christmas Carols is one of the highlights of our year and we are so happy this event can proceed this year. It is a beautiful gathering of our community and we eagerly await the smiling faces of our children as they receive their gifts from Santa," Penelopy Kioussis, President of the Board, says.
2021 Christmas Hamper Appeal:
GO Family and Friends Ministry, together with Mother Maria of Paris Mission, the St Elesa Food Initiative, Feed the People and Ark of Love, have launched their 2021 Christmas Hamper Appeal.
As part of the appeal, the Greek Orthodox organisations are calling on people across Greater Sydney to fill a box with non-perishable items such as canned food, bottled water, cake mix and pasta. To get into the Christmas spirit, treats such as pudding and chocolates, as well as Christmas decorations, are also welcome.
These boxes can then be dropped off on either Saturday, December 11 between 9am and 11am or Sunday, December 12 between 8am and 11am, at the Greek Orthodox churches in Bankstown, Belmore, Parramatta, Blacktown, Thornleigh, Kogarah, Liverpool, Gymea, Crows Nest and Surry Hills.
In the meantime, the Mother Maria of Paris Mission is also holding its annual Toy Drive and will be collecting donations up until December 15 at All Saints Greek Orthodox Church in Belmore.
Mother Maria of Paris Mission have teamed up with the Canterbury Bankstown Bulldogs.
This year, the Mission has partnered with local rugby league team, the Canterbury Bankstown Bulldogs, who have already donated $5000 worth of toys to the initiative.
Team players will also help members from the mission distribute the toys to children in hospital as well as the Riverwood Community Centre.
"It's really all about bringing some joy and cheer to families and children that have been without it for a while," program co-ordinator, Chris Constanti, tells The Greek Herald.
*Note: This is not an extensive list of all church initiatives available across Greater Sydney.
The Mission's toy drive.
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The Fliptime Machine is a fully immersible device using, visual, audible, motion and participatory elements that when mixed together, open pathways in the space time continuum and allow the traveler to see events as they took place in the past. Join us for a ride to the past and beyond!!!!
The Chalkboard Art Car will be back at Utopiafest this year for your scribbling pleasure. Find the 1981 Chevy El Camino on the festival grounds throughout the weekend!
The DRUNKLUCK Motel is designed as a surrealist homage to one of the most quintessential American traditions: the budget motel. By merging physical, visual and theatrical elements - the DRUNKLUCK MOTEL will provide festival-goers with a familiar space - reimagined in a surrealist way that encourages connection and creation.
Sacred Tribe will be bringing out their large multicolored sculpture consisting of individual triangular polygons framed out of wood that stack upwards while uniformly twisting in a helical path. Come climb and experience this multi-layered art piece. Sacred tribe is an Austin-based collective that produces art, sculpture, and jewelry.
String Theory Art Installation - Amazing yarn-based fence murals and multi layered sculptures by the amazing folks at Start Productions.
Because of the Lotus symbiotically fuses fine art with digital art, while creating a space where the art can be viewed as a new medium with the viewer being fully immersed in the piece. Paintings, murals and other inanimate objects are brought to life using the arts of Projection Mapping, 3D and 2D animation, Laser Projection, and now Virtual Reality. The use of biofeedback equipment, specifically a wireless EEG headset, allows participants to control the digital art based on brainwave pattern.
Two or more telephones that only talk only to each other. These telephones are simple to set up simply connected by wire and requiring no power. The US Navy uses these and they can clearly communicate up to 5 miles apart.
Live Art - by the amazing Ami of GonzoViz.com.
Hand-sewn quilts with color changing led lights in the frame.. as the lights change color the fabric changes and warps!
Come one, Come all! But not all at once. Bring your instruments and step right up to experience the Magick Solar Suitcases! The Magick Solar Suitcases are a completely solar powered, live, interactive experience where anyone can help create the next song on the spot! You can help make the next song by playing one of the Suitcases, grabbing the microphone, plugging in an instrument of your own or asking a question. You can find the Suitcases throughout the festival grounds. All are children of the immortal bliss, get your instruments!
The Mesmerize Lounge by IECreatives is an outdoor pop-up art gallery constructed out of up-cycled materials from corporate event waste. Treat the art as photo backdrops and to take a load-off in the lounge at anytime during the festival. The wood and plastic crates have been salvaged and re-used. IECreatives is a team of artists based in Austin, Texas who produce multi-sensory & immersive installations for entertainers, brands, artists, agencies, and community organizations.
Spunshiine - Artist Haley Ashley is bringing her trippy laser cut fabric installations to UTOPiAfest!
Witness radiant fire performances Friday and Saturday nights brought to you by Cherry Cola Dog.
Bask in the beauty and relax in the beehive Honeycomb Dome by Ruthie Monroe!
We are proud to offer a variety of workshops on art, massage, permaculture, acroyoga and so much more!


We at Little Bean not only love coffee, we love art as well. Currently, we're featuring two local artists' work on our walls, and will be periodically rotating throughout the year. All work is for sale directly from the artists.
my style, if it must be labeled is, Abstract Expressionism; it's my own individual perspective, emotional angst that only I fully understand. It may shed light on some, and it may not. I paint big, I paint as big as possible, there is far too much inside of me to contain my emotions on a small canvas. I use latex & acrylic house paint mainly, they are more fluid and forgiving, my true emotions end up coming out exactly how they should, not how I planned.
my art is expressing on canvas what words will never a do, a chaotic past that created a mold of beauty.
my art is me, my truth, my fear, my insecurities, my defects, ALL of me.
I express to you on canvas what you may not ever be able to see, but you will understand that I am here, I am me, because of my past.


text:
Dieudonné Cardinal Nzapalainga, Spiritan, receives…
Central AfricaDemocratic Republic of CongoGeneralate
On 16th April 2022, Dieudonné Cardinal Nzapalainga, Spiritan, was invited by the National Episcopal Conference of Congo to an international colloquium marking the 65th anniversary of the Catholic University of Congo (UCC), in which he received the Golden Medal of Honour. Cardinal Nzapalainga who is the Archbishop of Bangui, Central Africa Republic (CAR) is well known, among others, for being outspoken and acting on the importance of interreligious dialogue in peace building.
Delivering the inaugural lecture on 26th April, Cardinal Nzapalainga stressed that many conflicts facing some countries today can be resolved through interreligious dialogue: "Interreligious dialogue is an indispensable path to peace building in the world today and deserves to be considered an absolute necessity and the deepest aspiration of the human race."
In his presentation titled, "Interreligious Dialogue at the Service of Peace for a New Humanism", the Cardinal added that with the health crisis and the war in Ukraine and the various armed conflicts in several African countries, "the call for the construction of a school of peace is of vital importance."
To this end, Cardinal Nzapalainga said, "It is necessary and inevitable to open up and dialogue with other religions."
Cardinal Nzapalainga added, "Religions must be present in all initiatives that can contribute to the advent of a lasting peace, for all religions have a mission to work for peace."
"It is therefore impossible to talk about peace if one is not yet ready to enter into a relationship with people who do not share my religion," he further added.
He went on to highlight the importance of hospitality, which he described as "the welcome and recognition of the other in his or her difference without this difference being the cause of disputes."
We congratulate our Confrere, Cardinal Nzapalainga for his remarkable commitment to our Spiritan ministry in interreligious dialogue and peace building.
"We try in dialogue to co-operate honestly with the leaders and the faithful of other religions as well as with those who do not believe in God. We put our trust in the Holy Spirit, leading both us and them "to the complete truth" (Jn 16,13). [SRL 16.3].
"Each circumscription and each Spiritan carries the concern for interreligious dialogue. Let commitment for interreligious dialogue be integrated into the strategic plan of the circumscription, according to the reality and concrete challenges of each circumscription." [Decisions of the General Chapter of Bagamoyo II].
This article is also available in: Français Português
Categories: Central Africa, Democratic Republic of Congo, Generalate 2022-05-03 1 Comment
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Tragedies and 'miracles' in the Central African Republic (CAR)
Information on the presentation of the 'Positio' regarding the virtues of our Founder, the Servant of God Claude François Poullart des Places
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Ty is Co-director of Light Bearers Ministry and teaches on a variety of topics, emphasizing God's unfailing love as the central theme of the Bible. He has authored eight best-selling books, including his newest, A God Named Desire, which paints a compelling picture of God's character.
Ty can be reached via www.digma.com and other places on the web. Light Bearers used to have their ministry not far from where I live over the border in Washington State. I first went to their camp meetings probably 25 years ago and have always enjoyed Ty's messages.


Texas voters will decide the fate of nine proposed amendments to the state constitution in the coming election.
Early voting began Oct. 21 and runs through Nov. 1. Numbers reported Oct. 25 showed that voting is on track to outpace turnout in recent elections.
The Secretary of State�s office reports daily voting totals for the 15 most populous counties in the state, and by the end of Oct. 24, the fourth day of early voting, about 95,000 Texans had cast a ballot at the polls in those counties. That was more than double the 45,379 voters who had voted at the same point in 2011 constitutional amendment election, the Secretary of State�s office said.
The Nov. 5 constitutional amendment election is the first statewide election since photo ID requirements for voting in person went into effect. Computer users who have an Internet connection can see daily voter turnout numbers for early voting in this and past elections by visiting the elections tab at sos.state.tx.us.
On Oct. 21, the Texas Education Agency and Texas School Safety Center at Texas State University jointly pointed school districts and charters toward resources available to help address and combat bullying and cyber-bullying on campuses.
Texas School Safety Center�s inaugural Texas Bully Prevention Summit in San Marcos is set for Oct. 30.
On location in Israel on Oct. 23, Gov. Perry and Israeli President Shimon Peres, joined by Texas A&M University Chancellor John Sharp and other TAMU System executives, announced the creation of an international branch of Texas A&M University in Israel.
Funding for development and construction of the campus, to be located in the predominantly Arab city of Nazareth, will come from private donations.
In early October, state Sen. Tommy Williams, R-The Woodlands, announced he would not remain in office for the 2015 session of the Texas Legislature.
Last week, Williams, an accountant by trade, said he would resign from office, effective at midnight, Oct. 25.
Williams chaired the budget-writing Senate Finance Committee in 2013. In 2011, he chaired the Senate Transportation Committee. He began is legislative career in 1997 as a House member. In 2003 he was elected to the Senate.
Targeting the Rio Grande Valley, this state, local and federal multi-agency effort brought about an increase patrols Sept. 15 through Oct. 4�to address three public safety issues: criminal activity; commercial vehicles traffic on roadways and unsafe driving practices.
�These 24/7 saturation patrols were sustained until the cartels were forced to curtail their drug and human smuggling operations in the targeted area,� the DPS reported. Beefed up law enforcement in the area resulted in decreases in the illegal trafficking of marijuana, cocaine, methamphetamines, U.S. currency and stolen vehicles, plus a list of other positive effects.
Ed Sterling is the director of member service for Texas Press Association. Capital Highlights is provided to member publications and their readers.


The super talented fast rising music act Mark Owi is out with another mind blowing single titled "Gwumolo" (Song of appreciation). The song which has the great African vibes was produced by Kezzy Kleff, mixed by Suka and the guitar by Fiokee. Mark is currently signed to Ballership Ents.
You better get ready to dance to this one I swear. This dude just know how to thrill his audience. The beat is out of this world, this is completely African. It reminds me so much of us not trying so hard to be someone else in anyway. And vocally, Mark did justice to the song.
It is actually a song of praise, ainya lo'woicho ko da no ya lum ma (Praise be to God for all he has done for me). Trust me, you would want to keep this one.


bpm'online | Technology Advisors, Inc.
Bpm'online is a global provider of process-driven CRM for marketing, sales, and service. The core value of bpm'online products is the agility to change processes faster than ever and align marketing, sales and service on a single platform. Users love bpm'online's engaging interface with a social look and feel, free from redundant information to keep them focused on what's relevant. Today, the company employs 600+ experts and serves over 6,500 customers worldwide. Check out this video to learn more.
bpm'online's unique functionality serves as a simultaneous marketing, sales, and service platform, so you gain the ability to switch between processes with ease.
Enjoy working with the modern, streamlined user interface that is free from redundant information, keeping you focused only on what's relevant.
Bpm'online is three products on one platform, so it connects the dots between marketing, sales, and service for total alignment between departments.
In 2015 Gartner included bpm'online in the Gartner Magic Quadrant for the CRM Customer Engagement Center. Forrester also gave bpm'online the highest overall score among strong performers for its product in The Forrester Wave.
Bpm'online sales is a cutting-edge end-to-end sales cycle management software that drives customer acquisition, development, and retention.
Bpm'online marketing is a robust marketing automation software that fosters demand generation excellence by identifying customers' needs and nurturing their interests until prospects are successfully transformed into opportunities.
Bpm'online service provides effortless access to features and information regardless the device being used to access it. Bpm'online service has two versions, Customer Center and Service Enterprise. Both include: omnichannel communications, a contact center, BPM, service catalog, knowledge management, synchronization & integration, and a system designer.
What are the differences between the two? How do you know which version is right for you? See below for additional details about Customer Center and Service Enterprise.
Best for companies looking to manage omnichannel communications.
Best for medium to large companies looking for high-level service across multiple channels.
BpmSchemaTools is an application developed by TAI to simplify the analysis and documentation of a bpm'online database. If you are an implementor or end user and need a quick way to extract and document all of your bpm'online objects(tables/views) and their properties(fields), this is the tool for you!
Let's take a look at the most crucial steps in the CRM selection process and identify possible pitfalls and solutions that can help you avoid costly mistakes and achieve the best outcome of your CRM initiative.
CRM technology underpins nearly every customer facing process today. Sales, marketing, and service cycles can suffer when CRM software makes it difficult to customize and automate processes. Cumbersome manual CRM processes can lead users to neglect key steps, resulting in fewer sales and decreased customer loyalty.


The Huygens Software is named after Christiaan Huygens, who studied and described the diffraction of light waves. The Software includes many image processing options, for brightfield and all types of fluorescence Microscope images. With Huygens it is possible to perform image deconvolution and restoration, interactive analysis and volume visualization in 2D, 3D, multi-channel and time. Additional Server and GPU-based Options ensure maximization of hardware performance. Please click here for more information about Huygens deconvolution. Feel also free to download the software and request a free trail license.
The Huygens software comes in different flavors offering solutions for both single users and multi-user facilities. We also offer a Huygens web-based interface, Huygens Remote Manager, available for submitting deconvolution tasks via your internet browser to the compute engine Huygens Core. It is possible to get Huygens and additional options as a node-locked license, leased license or in a floating license configuration.
Huygens Essential: For wizard guided deconvolution and visualization.
Huygens Professional: For a complete set of image restoration and analysis tools.
Huygens Core: A complete Huygens Compute Engine to program scripts and process a large number of images automatically.
Huygens Scripting: A reduced version of Huygens Core.
Huygens Titan: A microscopic images indexing tool.
Results of the Huygens deconvolution of a widefield image.
You can download the Huygens software from our website for free. After installing and starting the Huygens software, you can request a temporary test license to explore the Huygens deconvolution, visualization and restoration options. Feel free to try out all our products for free. There are also many FreeWare tools in the software that can run without any license.
Scientific references to SVI software.


Mel Ott, Willie Mays, Willie McCovey, and Barry Bonds combined to hit 2,212 home runs for the Giants in regular season contests. And while this quartet added another 16 homers in postseason play for the franchise, none of them hit a walk-off homer in a Division Series, League Championship Series or World Series; but Dusty Rhodes did.
In Game 1 of the 1954 World Series, after Mays drew a one-out walk and stole second to open up a base for an intentional walk, Rhodes came to the plate pinch-hitting for Monte Irvin, took a measurement of Bob Lemon on the mound, and electrified the 52,000-plus at the Polo Grounds with a three-run shot to right.
Sixty years later -- and with an expanse of water rather than a city skyline helping to frame the ballpark -- Travis Ishikawa added his name alongside Rhodes', and in the process gave the Giants an opportunity to collect the franchise's eighth World Series title.
With San Francisco and St. Louis tied at 3-3 in the ninth inning of Game 5, left fielder Ishikawa sent the Giants to the World Series with a three-run, series-ending homer.
Ishikawa joined Rhodes as the second Giants player to hit a walk-off homer in postseason play and the third, along with Rhodes and Kenny Lofton (Game 5 of the 2002 NLCS) to own a walk-off hit.
• The Giants, in addition to becoming the fourth team since 1969 to clinch a pennant on a homer, became the 10th team overall to clinch the LCS in walk-off fashion, joining the '72 Reds (NLCS Game 5, George Foster scored on a wild pitch as the Reds defeated the Pirates, '76 Reds (NLCS Game 3, Ken Griffey delivers a single as the Reds defeat the Phillies), '78 Dodgers (NLCS Game 4, Bill Russell produces a single as the Dodgers defeat the Phillies), '92 Braves (NLCS Game 7, Francisco Cabrera delivers a two-run single as the Braves defeat the Pirates), '99 Braves (NLCS Game 6, Andruw Jones works a bases-loaded walk as the Braves defeat the Mets) and 2002 Giants on Lofton's walk-off single vs. the Cardinals. Of these nine teams to win the LCS in walk-off fashion, only one -- the 1976 Reds -- went on to win the World Series.
• The Giants have captured 21 pennants in the World Series era: 1904-05, '11-'13, '17, '21-24, '33, '36-37, '51, '54, '62, '89, 2002, '10, '12 and '14.
• One of the Giants' three homers Thursday came from Joe Panik, who gave San Francisco an early 2-1 lead with his two-run shot in the third. He has played 10 career postseason games, collecting 11 total hits and hitting safely in seven of them.
Panik is the 18th player in Giants history to hit safely in at least seven of his first 10 postseason games; he's the first Giants player to be shy of his 24th birthday (he turns 24 on Oct. 30) and hit safely in at least seven of his first 10 postseason games; and his 11 hits through his first 10 postseason games tie him for the 10th most in Giants history. Will Clark, with 16, had the most.
• In the win, third baseman Pablo Sandoval doubled and singled and extended his playoff on-base streak to 23 games in a row. The switch-hitter is now tied with Lou Gehrig and Lance Berkman for the sixth-longest streak in postseason history. Reaching safely in his next contest would tie Sandoval with Carlos Beltran and Carlos Ruiz . Miguel Cabrera, with 31 straight, holds the all-time mark.
• In a no-decision, San Francisco starter Madison Bumgarner allowed three runs in eight innings. This was his 10th career postseason start and 11th overall postseason appearance, with this Game 5 line giving him a career 2.67 ERA in 67 1/3 innings. He is one of six Giants pitchers with at least 50 postseason innings. Christy Mathewson had the most (101 2/3, with a 0.97 ERA), followed by Art Nehf (78 IP, 1.96 ERA), Bumgarner, Tim Lincecum (54 2/3 IP, 2.47 ERA) and Matt Cain (51 1/3 IP, 2.10 ERA).
• With the Giants on their way to World Series to face the Royals, the 110th Fall Classic will be just the second to feature two Wild Card teams. In 2002, the Giants lost to the Angels in seven games.
• In the eighth inning, San Francisco's Michael Morse delivered a pinch-hit, game-tying home run.
Before Morse's shot there had been only one other game-tying, pinch-hit homer in a series-clinching affair. In Game 4 of the 1978 NLCS, Philadelphia's Bake McBride hit a solo homer off the Dodgers' Rick Rhoden in the seventh inning to tie the game at 3-3. Russell then won the game in the bottom of the 10th when he singled off Philadelphia's Tug McGraw to drive in Ron Cey and send the Dodgers to the Fall Classic.
• With this win, Bruce Bochy has four pennants on his managerial resume (1998 with the Padres; 2010, '12 and '14 with the Giants). Including 19th century managers, he is one of 23 to have at least four. The other 22 are in the Hall of Fame.


Indian intelligence agencies have warned that a Delhi-Kabul flight of India's carrier Air India may be hijacked by terrorists, said local daily The Times of India online on Sunday.
A high alert has been sounded after the intelligence input and the Indira Gandhi International airport in the Indian capital has been alerted and stringent checks of even the staff members have been instructed, said the report.
The authorities have put in place "multi-level" checking and have issued strictures to ensure they are followed judiciously, with additional ladder-point check put in place and the ground staff asked to thoroughly check all cabin baggage once the passengers boarded, said the report.
Commandos will also patrol the adjoining areas of the airport with machine gun-mounted vehicles. The number of flight marshals have also been increased and cabin crew have been asked to look out for problem elements, it added.
In 1999, terrorists hijacked an Air India passenger plane in southern Afghanistan'sKandahar city.


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About | Nick Young
Nick Young writes and performs indie rock with a touch of twang, sounding like a cross between vintage Wilco, Old 97s & Steve Earle.
Nick Young's twang infused new album "South Lima" is a stripped-down affair, heavy on acoustic and pedal steel guitars but retaining Young's signature hooks and exquisite knack for melody. The Rochester, NY based artist recorded the album in nearby Buffalo with his long time bass player and friend Tim Mroz and the record was named after the small (very small) town where Young wrote almost all of the songs that appear on the release. Young's sophomore effort comes almost 3 years after his solo debut "Truth Is" and after nearly 50,000 miles of touring in support of that album. Embracing the troubadour within, Young will once again bring his "twang-folk" (or whatever the hell you want to call it) to the masses, armed with an acoustic guitar, his songs and an authentic passion for performing.
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Sermon given by Rev. Doug Bunnell on Sunday May 15, 2016, at First Presbyterian Church of Bellingham. Isaiah 43:1-5 & John 6:16-21.
Sermon given by Rev. Doug Bunnell on Easter Sunday, March 27, 2016, at First Presbyterian Church of Bellingham. He is risen!


This fall we ran one of our favorite contests- #WinterParkPups. Who doesn't love looking at puppy pictures? Our Winter Park fans really went all out to show us their pups! Picking the winners was quite the task, but we can just not be done with this contest before giving some of these 4 legged models a call out for honorable mention. Without further ado, please spend hours "awwing" over our ten honorable mention pups. Thank you to everyone who entered, and follow us on social to see if we reprise this contest!


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Home > Vol 8, No 16 >	Weber
Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer
Helga Weber, José R. Valbuena, Mustafa A. Barbhuiya, Stefan Stein, Hana Kunkel, Patricia García, Carolina Bizama, Ismael Riquelme, Jaime A. Espinoza, Stephen E. Kurtz, Jeffrey W. Tyner, Juan Francisco Calderon, Alejandro H. Corvalán, Manuel Grez, Akhilesh Pandey, Pamela Leal-Rojas and Juan C. Roa _
Helga Weber1,2,*, José R. Valbuena3,*, Mustafa A. Barbhuiya4, Stefan Stein5, Hana Kunkel5, Patricia García3,6, Carolina Bizama3,6, Ismael Riquelme1,2, Jaime A. Espinoza7, Stephen E. Kurtz8, Jeffrey W. Tyner8, Juan Francisco Calderon9, Alejandro H. Corvalán6,10, Manuel Grez5, Akhilesh Pandey4,11, Pamela Leal-Rojas1, Juan C. Roa3,6
1Center of Excellence in Traslational Medicine (CEMT) and Scientific and Technological Bioresource Nucleus (BIOREN), Universidad de La Frontera, Temuco, Chile
2Department of Pathology, Faculty of Medicine, Temuco, Chile
3Department of Pathology, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
4McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
5Gene Therapy Unit, Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Germany
6Center for Investigation in Translational Oncology (CITO), Advanced Center for Chronic Diseases (ACCDiS), Millennium Institute on Immunology and Immunotherapy, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
7SciLifeLab, Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Solna, Stockholm, Sweden
8Division of Hematology and Medical Oncology, Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA
9Center for Genetics and Genomics, School of Medicine, Clínica Alemana de Santiago - Universidad del Desarrollo, Santiago, Chile
10Department of Hematology Oncology, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
11Department of Biological Chemistry, Department of Oncology, Department of Pathology, The Johns Hopkins University School of Medicine Baltimore, MD, USA
*These authors contributed equally to this work
Juan C. Roa, email: jcroa@med.puc.cl
Pamela Leal-Rojas, email: pamela.leal@ufrontera.cl
Keywords: gallbladder cancer, HSP90 inhibitors, geldanamycin, 17-AAG, gallbladder cancer xenografts
Received: October 13, 2016 Accepted: February 01, 2017 Published: February 16, 2017
Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors.
In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies.
In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05).
The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%).
Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.
Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer | Weber | Oncotarget
Gallbladder cancer (GBC) is the most common type of biliary-tract carcinomas. It has a high morbidity and poor prognosis as it is often associated with early metastasis and invasiveness [1]. GBC tumors affect thousands of individuals worldwide (annual incidence of 2.2/100,000), but has a much higher impact and mortality in Chile, China, Japan and India [2]. Complete surgical resection is the only potentially curative approach in early stages of the disease. However, most patients cannot undergo surgery since GBC is usually diagnosed at much more advanced stages [3]. Chemotherapy has been the main treatment option in circumscribed, advanced or metastatic biliary-tract carcinoma. A combination of chemotherapy with gemcitabine and a platinum-based agent is regarded as the standard treatment for patients with advanced biliary-tract cancer. However, the response to standard chemotherapy and radiotherapy is extremely poor, with modest impact on overall survival [4]. Therefore, new therapeutic options are needed to improve survival and response rates. Understanding the signaling pathways in GBC, such as Hedgehog, PI3K/AKT/mTOR, Notch, ErbB, MAPK and angiogenesis, holds great promise for the development of new effective treatment strategies [2].
High-troughput, rapid small molecule inhibitor screenings to identify therapeutic targets for cancer become tractable with the development of new technologies [5]. This method has been used so far to screen compounds for treating a number of heamatological malignancies [6]. We employed the same method to screen 130 potential treatment drugs in gallbladder cancer cell lines and identified a set of small molecule inhibitors including 17AAG, Velcade, Volasartib and YM-155 as the most potent growth inhibitors of these cell lines. We subsequently tested the HSP90 inhibitor 17AAG in pre-clinical cancer models.
HSP90 is a ubiquitous ATP-dependent molecular chaperone that facilitates the maturation of more than 200 downstream proteins, including key mediators of signal transduction, cell-cycle control and transcriptional regulation [7]. HSP90 also stabilizes numerous oncoproteins that facilitate malignant progression and maintain transformed cellular phenotypes as well as enables tumor cells to escape apoptotic death [8]. Its deregulation has been associated with the pathogenesis of various human cancers [9–11].The use of inhibitors against HSP90 has been proposed as an attractive therapeutic strategy in numerous tumors based on the fact that HSP90 clients comprise proteins that contribute to all of the six hallmarks of cancer Therefore, its inhibition could simultaneously interfere with key pathophysiological processes of tumor cells, leading to more effective clinical results [9].
Geldanamycin (GA), a benzoquinone ansamycin antibiotic isolated from Streptomyces hygroscopicus, is a first generation HSP90 inhibitor with potent anti-tumor effects in vitro. However, it was never evaluated in clinical trials mainly due to metabolic instability, limited solubility, and induction of severe hepatotoxicity in animals [12]. 17-AAG (Tanespimycin), an analog molecule chemically derived from GA, was the first HSP90 inhibitor to enter clinical trials [13]. In preclinicals models it has shown antitumor activity in various types of cancer, such as colon, breast, ovarian, and melanoma tumors [12, 14].
In this study we systematically screened 130 small molecule for gallbladder cancer inhibition. We evaluated antitumoral activity of HSP90 inhibitors, 17-AAG and geldanamycin, in vitro and on a preclinical subcutaneous tumor model and showed the potential of the 17-AAG for further clinical investigations.
Small molecule inhibitors with therapeutic potential for GBC
Based on previous publications by the co-authors of this study about the methodology high-throughput rapid small molecule inhibitor screening [5, 6], we pre-selected drugs from the FDA approved list of anti-cancer kinase and other small molecule inhibitors that were computationally and genetically (siRNA screening) tested in series of cancer cell lines. We adopted 130 drugs taking cue from those previous studies (Supplementary Table 1). In the rapid screen of these 130 drugs, we identified small molecules inhibitors including 17-AAG (Tanespimycin), Eleslomol, Velcade, Volasartib and YM-155 as the five most potent drugs across GBC cell lines. (Figure 1). Most of these drugs are either in clinical trials or have been determined to be effective against a wide range of cancers in preclinical tests. However, these molecules have not been investigated for their efficacy in GBC. It is important to note that all the seven GBC cell lines showed resistance against a series of widely used antitumoral drugs included in the screen. The IC50 values for the seven GBC cell lines of the drugs tested is provided in Figure 1 and Supplementary Table 1. These drugs are known to target many different kinases and receptors and have proved effective in other types of cancer. The results corroborate with the lack of effective chemotherapy-based treatment for GBC. Notably, five of them proved to be potent against the seven GBC cell lines investigated. Among these five candidates, we selected the HSP90 inhibitor 17-AAG (Tanespimycin) for pre-clinical validation as a potential therapeutic molecule for GBC.
Figure 1: Top five most potent drugs for gallbladder cancer. Seven human gallbladder cancer cell lines GB-d1, G415, SNU308, NOZ, TGBC1TKB, TGBC2TKB and TGBC24TKB were used for the rapid small molecule inhibitor screen including a panel of 130 small molecule inhibitors. Cell viability testing was carried out on the small-molecule inhibitor screening plates and synergy plates using a cell proliferation assay.
17-AAG and GA reduce cell viability and cell migration in GBC cell lines in vitro
Due to HSP90 expression was highly upregulated in G-415 and GB-d1, compared with the other studied cell lines (data not shown), these cells lines were selected for further assays.
Cell viability was analyzed by MTS assay after treatment with increasing concentrations of GA or 17-AAG for 24, 48, and 72 hours. As shown in Figure 2A–2D, 17-AAG and GA significantly reduced cell viability in both cell lines as early as 24 hours after exposure to the inhibitors (P < 0.001).
Figure 2: In vitro effects of 17-AAG and GA on cell growth and cell migration in two GBC cell lines. (A, B) G-415 and (C, D) GB-d1 cells were treated with increasing concentrations of 17-AAG or GA. Cell viability was determined after 24, 48, and 72 hours of treatment. Data are shown as mean ± SD of at least three independent experiments in quintuplicate (***P < 0.001; ns: not significant). (E, F) Cell migration was evaluated in G-415 and GB-d1 cells treated with 17-AAG or GA (12 μM and 15 μM, respectively) for 24 hours. Control cells received an equivalent amount of solvent only. Data are shown as mean ± SD (***P < 0.001).
To establish the effect of 17-AAG and GA on cell migration, GBC cell lines were exposed to 17-AAG (12 μM), GA (15 μM), or 0.01% DMSOfor 24 hours. After this time, migration rates were significantly lower in treated versus untreated cells. Relative migration rates observed in G-415 were 18.3% (17-AAG) and 11.7% (GA) (P < 0.001) compared to the DMSO control, while GB-d1 showed 3.4% (17-AAG) and 7.4% (GA) (P < 0.001). (Figure 2E and 2F).
Exposure to 17-AAG and GA reduces in vitro expression of HSP90 target proteins in GBC cells in vitro
To further investigate the in vitro effects of 17-AAG and GA in GBC cell lines, we evaluated the expression of HSP90 and target proteins by immunoblotting. Cells were exposed to 17-AAG (12 μM), GA (15 μM) or DMSO for 24 hours and were lysed and analyzed by western blot using commercial antibodies. As shown in Figure 3, increased levels of HSP90 were observed upon HSP90 inhibition in G-415 and GB-d1 cells lines. On the other hand, HSP90 target proteins, EGFR, AKT, phospho-AKT, phospho-ERK and Cyclin D1 were strongly inhibited by 17-AAG or GA treatments in both cell lines. Treatment with either HSP90 inhibitors markedly decreased Cyclin B1 expression in Gb-d1 cells, but enhanced its expression in G-415 cells. No significant changes were observed in total ERK or survivin protein expression under the treatment conditions assayed.
Figure 3: In vitro effects of 17-AAG and GA on HSP90 expression and on HSP90 client protein expression in two GBC cell lines. G-415 and GB-d1 cells were treated with 17-AAG or GA (12 μM and 15 μM, respectively) for 24 hours. Control cells received an equivalent amount of solvent only. Western blot analysis was carried out using antibodies against HSP90 and HSP90 clients proteins. Protein loading was normalized using an antibody recognizing α-tubulin.
17-AAG and GA treatment lead to G2/M cell cycle arrest and induces apotosis in GBC cells in vitro
To determine whether the antiproliferative effect of 17-AAG and GA was due to cell cycle arrest, G-415 and GB-d1 cells were treated either with 17-AAG (12 μM), GA (15 μM), or DMSO for 24 hours. Cell cycle fractions were determined by PI-DNA staining and FACS analysis. After 24 hours of exposure, 17-AAG and GA treatments resulted in a statistically significant increase of G2/M phase cells compared with the controls, both in G-415 and in GB-d1 cells (P < 0.001). These results were accompanied by a decrease in G0/G1 and S phase populations. (Figure 4).
Figure 4: Effect of 17-AAG and GA on cell cycle on human GBC cells. (A and B) G-415 and (C and D) GB-d1 cells were treated with dimethylsulfoxide or 12 μM and 20 μM of 17-AAG or with 15 μM and 20 μM of GA for 24 h. Cells were processed for analysis of cell cycle distribution by flow cytometry. Data are shown as mean ± SD of three independent samples and are representative of two independent experiments. (***P < 0.001; ns: not significant).
Cell cycle arrest was followed by induction of apoptosis, as determined by FACs analysis of annexin V staining and caspase 3/7 activity. Apoptosis was evaluated after 24, 48 and 72 hours of treatment with 12 uM and 20 uM of 17-AAG and GA. For 17AAG-treated-G-415 cells, the percentage of apoptotic cells was notably higher after 72 hours of treatment, 18.7% (12 μM) and 20.7% (20 μM) compared to control cells that showed 2.2% (P < 0.001) (Figure 5A). Similar findings were observed after treatment with GA, where the percentage of apoptosis cells increased after 72 hours of treatment to 12.7% (12 μM) and 26.0% (20 μM) compared to control cells that showed 2.2% (P < 0.001) (Figure 5B). GB-d1 cells treated with 17-AAG exhibited more susceptibility to apoptosis than G-415, with 33.8% (12 μM) and 66.2% (20 μM) of apoptotic cells after 24 hours of treatment compared to untreated control (16%; P < 0.001). After 72 hours of treatment these cells showed 69,9% (12 μM) and 97,4% (20 μM) of apoptotic cells whereas control cells showed 7,5% of positive cells (P < 0.001) (Figure 5C). Using the same concentrations of GA, the amount of apoptotic cells increased to 43.2% (12 μM) and 39.7% (20 μM) after 72 hours, whereas control cells showed 10.5% (P < 0.001) (Figure 5D).
Figure 5: Effect of 17-AAG and GA on apoptosis in human GBC cells. (A and B) G-415 and (C and D) GB-d1 cells were treated with 17-AAG or GA and harvested after 24, 48, and 72 hours for annexin V /propidium iodide (PI) staining and analized by flow cytometry. Data are shown as mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant). Activity of Caspase-3/7 in G-415 cells (E and F) and in GB-d1 cells (G and H) treated with 17-AAG or GA for 72 h was assessed by Flow Cytometry using a fluorogenic substrate for detection of activated caspases 3 and 7 in apoptotic cells. Data are shown as mean ± SD (**P < 0.01; ***P < 0.001; ns: not significant).
We confirmed these results using a CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit at 72 hours after treatment. We observed that the amount of activated caspases-3 and -7 significantly increased after 17-AAG or GA treatments in both GBC cell lines. G-415 cells treated with 17-AAG showed 16.2% (12 μM) and 19.3% (20 μM) of activated caspases-3 and-7 whereas control cells showed 2,0% (P < 0.01 and P < 0.001, respectively) (Figure 5E). G-415 cells treated with GA showed 25.7%(12 μM) and 65.7% (20 μM) of activated caspases-3 and-7 whereas control cells showed 2.5% (P < 0.001) (Figure 5F). GB-d1 cells treated with 17-AAG showed 68.5% (12 μM) and 85.1% (20 μM) of activated caspases-3 and-7 whereas control cells showed 3.5% (P < 0.001) (Figure 5G). Finally, GB-d1 cells treated with GA showed 55.4% (12 μM) and 50.8% (20 μM) of activated caspases-3 and-7 whereas control cells showed 17.5% (P < 0.01) (Figure 5H).
17-AAG inhibits tumor growth on xenograft gallbladder cancer model
Based on the in vitro evidence, we decided to study further whether HSP90 inhibitors can be therapeutically effective on established s.c. human gallbladder tumors in vivo. The effect of 17-AAG on tumor growth was evaluated in xenograft GBC tumor models. Briefly, 2 × 106 G-415 cells were injected subcutaneously into NOD-SCID mice. When tumors reached an average volume of 50 mm3, mice were treated with 17-AAG at a concentration of 25 mg/kg administered i.p., daily for 5 days per week for 4 weeks. Mice were sacrificed 34 days after initiation of the treatment and a necropsy was performed that included removal of the entire tumor area. Mice bearing G-415 tumors and treated with 17-AAG exhibited 69.6% reduction in average tumor size (P < 0.05), as well as 64.9% in tumor weight (P < 0.05) compared to the control (Figure 6A and 6B). In addition, a decrease of the Ki67 index was observed in treated mice compared to controls, although it was not statistically significant (Supplementary Figure 1). Expression of HSP90 and its target proteins in tumor tissues obtained at the end of the in vivo studies were assessed by western blot analysis. As shown in Figure 6C, treatment with 17-AAG inhibitor significantly decreased the expression of phospho-AKT in G-415 tumors (p value). However, no significant changes were observed in total AKT, HSP90, HSP70, Cyclin B1 and Cyclin D1 protein expression under the treatment conditions assayed.
Figure 6: In vivo efficacy of 17-AAG on human GBC xenografts. (A and B) Tumor growth of mice harboring G-415 tumors. G-415 cells were injected subcutaneously into NOD-SCID mice. When tumors reached a volume of approximately 50 mm3, they were treated with 17-AAG or vehicle, as described in the materials and methods section. Animals were sacrificed at day 34 after treatment initiation. Data are expressed as mean ± SD. 17-AAG exerted a statistically significant antitumor effect compared to the groups treated with vehicle (*P < 0.05 at day 34). (C) Western blot analysis of HSP90 and HSP90 clients proteins of tumor tissue from mice treated with vehicle or 17-AAG.
Expression levels of HSP90 in GBC tissues
In order to establish whether HSP90 is frequently expressed in GBC, we examined the expression of HSP90 by IHC on TMAs. Examples of staining intensity are illustrated in Figure 7, showing weak, moderate and strong staining to HSP90 in the cytoplasm of GBC cells (Figure 7A). In most cases, HSP90 showed a diffuse pattern of staining. HSP90 expression was seen in 182/209 cases of GBC (87%). HSP90 was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26% ) (Figure 7B). There were no significant differences in HSP90 expression between dysplasia and early and advanced carcinoma lesions.
Figure 7: Immunohistochemical staining of HSP90 in gallbladder cancer tissue. (A) Representative images from gallbladder lesions showing positive immunoreactivity for HSP90 in three cases of GBC: a) weak intensity b) moderate intensity c) strong intensity. (B) Frequency distribution of HSP90 immunohistochemical staining in gallbladder cancer.
High throughput rapid small molecule inhibitor screening has been successfully used to screen drugs for a series of heamatological malignancies [6]. Through the same approach we screened 130 small molecule inhibitors for GBC, which has allowed us to identify many promising compounds/drugs for using against GBC, such as, 17-AAG, Velcade, Volasertib and YM-155. As 17-AAG is a multi-target drug that acts on a diverse set of regulatory pathways [15], we decided to evaluate the therapeutic efficacy of this HSP90 inhibitor and its analog geldanamycin, in vitro and in preclinical models of human GBC in NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in GBC tumors.
HSP90 is a chaperone protein involved in the stabilization and conformational maturation of numerous oncoproteins [8]. Many studies have demonstrated that HSP90 is associated with the pathogenesis, poor prognosis and resistance to therapy of various human cancers [10, 11, 16, 17]. HSP90 target proteins are known to contribute to nearly every aspect of the oncogenic process including immortality, survival, anti-apoptosis, genomic instability, neoangiogenesis and metastasis [18–20]. Inhibition of HSP90 through blockade of ATP-binding sites of the HSP90-partner complex results in the dephosphorylation and/or the proteasomal degradation of these target proteins and leads to potent antitumor activity [7].
Several studies have demonstrated the therapeutic efficacy of HSP90 inhibitors on in vitro models and its suppression of tumor growth in numerous solid tumors in human subcutaneous growing xenografts [21, 22]. However, no studies have reported the use of HSP90 inhibitors on in vitro or in vivo GBC models. Although currently no HSP90-targeting agents have been approved for human use, several clinical trials have shown their antitumor activity, alone or in combination with other cytotoxic agents, in multiple tumor types [23]. 17-AAG (Tanespimycin) is the first HSP90 inhibitor that has been investigated in phase II clinical trials in patients with thyroid, pancreatic or kidney cancer [24], and with promising results in patients with metastatic breast cancer [25].
New water-soluble analogs of 17-AAG, such as 17-DMAG (Alvespimycin) and IPI-504 (Retaspimycin) have proved to have a longer half-life in plasma, greater oral bioavailability, superior antitumor activity in preclinical studies and lower toxicity compared with 17-AAG [12]. In the past years there has been a considerable increase in the discovery of HSP90 inhibitors, progressing from first-generation derivatives of natural products to second and third-generation fully synthetic small molecules with improved pharmacokinetic profiles [13].
Even though HSP90 is widely expressed in normal tissues, HSP90 inhibitors selectively kill cancer cells but not normal ones due to the fact that HSP90 in tumor cells is mostly in its active conformation as part of multi-chaperone complexes that have 100-fold greater binding affinity for 17-AAG compared to the uncomplexed, latent form of HSP90 that is present in normal cells [7, 26].
Currently 17-AAG is not under clinical study, but other HSP90 inhibitors, with even better pharmacological and toxicological properties, such as retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, are currently under investigation [27]. STA-9090 (Ganetespib), having improved pharmacologic properties in terms of potency, safety, and tolerability compared with other first- and second generation HSP90 inhibitors, has reached phase III clinical trial and currently 12 clinical trials are underway (https://clinicaltrials.gov).
The induction of cell cycle arrest and apoptosis are common mechanisms proposed for the antiproliferative effect of 17-AAG on cancer cells [28, 29]. Similar to what has been observed in other tumoral cells treated with HSP90 inhibitors [30, 31], our studies showed that 17-AAG and GA decreased cell viability and cell migration in GBC cells and these effects were strongly associated with a marked decrease of HSP90 target proteins such as EGFR, AKT, phospho-AKT, phospho-ERK, cyclin D1 and cyclin B1. Also, in concordance to other studies, we observed a marked increase in expression of HSP90 after 17-AAG or GA treatment, likely through activation of the heat shock transcription factor 1 (HSF1), the master stress-inducible regulator, in the cytoplasm and the nucleus [32–36], or by disrupting nuclear HSP90/multichaperone complexes that inhibit activation of DNA-bound HSF1 [37].
Molecular changes observed in our study were consistent with the increased G2/M cell cycle arrest and apoptosis observed in our in vitro studies. It is widely known that ERK1/2, Cyclin B1 and Cyclin D1 are implicated in the regulation of the G2/M phase transition and are required for mitosis to occur normally [38]. It has also been demonstrated that blockade of EGFR results in a significant inhibition of the in vitro and in vivo growth of several cell lines derived from human carcinomas of various histological types [39, 40]. In a cohort of 49 samples from patients with advanced biliary tract carcinomas (BTC), 13 of which corresponded to GBC, Pignochino et al., showed that EGFR was expressed in 38.5% of GBC cases, and phospho-MAPK and phospho-AKT were expressed in < 46% of these cases, indicating the activation of the EGFR pathway [40]. They also demonstrated that blocking EGFR/HER2 signaling resulted in considerable antiproliferative effects on in vitro models of BTC. It was also shown that targeting EGFR/HER2 pathways enhances the antiproliferative effect of gemcitabine in biliary tract and gallbladder carcinomas [40]. Inhibition of HSP90 target proteins such as AKT and phospho-AKT, which are frequently active or overexpressed in many cancer cells, including GBC, affect the PI3K/AKT/mTOR signaling pathway which plays an important role in regulating cell cycle progression, apoptosis and cell survival [41, 42].
Considering HSP90 interactions with a number of oncogenic proteins, the observed overall effect of GA and 17-AAG on the studied GBC cell lines depends on the additive effect of all these proteins [29]. This could explain that although we observed an increased expression of Cyclin B1 in G-415 cells after treatments either with GA or 17-AAG, this did not affect the inhibitory effect on proliferation and cell migration in this cell line. Interestingly, Gazitt et al., also reported that treatment with 17-AAG caused an upregulation of Cyclin B1 in osteosarcoma cell lines, and association of this phenomenon with mitotic blockage [43].
Our in vivo study demonstrated for the first time the therapeutic efficacy of a HSP90 inhibitor in GBC xenografts. The significant tumor mass reduction observed in 17-AAG-treated mice was associated with a marked decrease in phosphorylation of AKT. This is especially relevant, considering that previous studies by our group have shown upregulation of the PI3K/AKT/mTOR signaling pathway in advanced GBC [44, 45]. It is widely known that AKT protects cells from apoptosis by phosphorylating and inactivating several key apoptotic molecules [46]. Dephosphorylation and inactivation of AKT increases sensitivity of the cells to apoptosis-inducing stimulus [47]. Several studies have shown that 17-AAG sensitizes tumor cells to taxol-induced apoptosis [48] and synergizes with cisplatin inducing apoptosis in cisplatin-resistant esophageal squamous cell carcinoma cell lines [22]. 17-AAG also sensitizes radioresistant cells to radiation by inhibiting the PI3K-Akt pathway [49]. Arlander et al., reported that 17-AAG-mediated disruption of Chk1 activation drastically sensitized various tumor cells to gemcitabine, which is, in combination with platinum-based agents, the standard treatment for patients with advanced biliary-tract cancer [50].
Previously, our group have demonstrated that rapamycin exert strong therapeutic efficacy on subcutaneous xenograft tumors of human GBC [51]. Also, Ding et al., showed that the combination of rapamycin, Cl-1040 and 17-AAG reduced circulating prostate cancer cells in a mouse model for circulating prostate cancer DNA quantification [52]. These findings suggest that the combination of rapamycin and 17-AAG could be a strategy that should be considered for future studies.
Unlike in vitro assays, no expression changes in other proteins were observed in vivo. This could be explained by the dose regimen used in the treated mice, which was the dose tolerated for the animals, but clearly was not sufficient to affect most of the evaluated HSP90 target proteins from tumor samples. To optimize the in vivo dosing regimen and to reach a suitable efficacy-tolerability balance may require the use of other HSP90 inhibitors with even better pharmacological and toxicological properties, such as 17-DMAG, which is a water soluble analog of 17-AAG, or second or third generation HSP90 inhibitors [53, 54]. Also, more studies should be conducted to determine whether other proteins or pathways are affected by 17-AAG in GBC models.
In conclusion, our findings provide a rationale for the potential use of HSP90 inhibitors as a novel, highly effective therapeutic strategy for GBC treatment.
Seven human GBC cell lines GB-d1, G-415, SNU308, NOZ, TGBC1TKB, TGBC2TKB and TGBC24TKB were used for the rapid small molecule inhibitor screening. These cell lines exhibit different invasive and differentiation properties. Detalis are provide in Supplementary Table 2. G-415, NOZ, TGBC1TKB, TGBC2TKB and TGBC24TKB were purchased from Riken BioResource Center (Ibaraki, Japan), GB-d1 and SNU308 were provided by Anirban Maitra (Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA). GB-d1 was authenticated by short tandem repeat analysis.
G-415 and GB-d1 were grown in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and maintained in a 37°C atmosphere containing 5% CO2. The other cell lines SNU308, NOZ, TGBC1TKB, TGBC2TKB and TGBC24TKB were cultured in DMEM- high glucose with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and maintained in a 37°C atmosphere containing 5% CO2.
Clinical samples
A total of 209 gallbladder specimens from patients who underwent curative surgical resection upon GBC diagnosis at Hospital Clinico de la Pontificia Universidad Católica (Santiago, Chile) between 1989 and 2007 were included in this study. None of the included patients with advanced GBC received any neoadjuvant or coadjuvant therapy, or radical surgery after the cholecystectomy. The clinicopathologic features of these patients were obtained from medical records and are summarized as follows: Histologically, cases were classified according to lesion type as dysplasia (n = 18), early carcinoma (n = 38), or advanced carcinoma (n = 171). Early and advanced carcinoma were further classified according to grade of differentiation: well-differentiated (n = 22), moderately differentiated (n = 81), and poorly differentiated (n = 106).
HSP90 inhibitors
17-Demethoxy-17-allylaminogeldanamycin (17-AAG) inhibitor (A6880) was purchased from LC Laboratories (Woburn, MA, USA) and geldanamycin (GA) (ant-agl-5) was purchased from InvivoGen (San Diego, CA, USA). For in vitro assays the inhibitors were resuspended to 10 mM in dimethylsulfoxide (DMSO) as stock solutions and stored at −20°C. Inhibitors were diluted in culture medium before each in vitro experiment and 0.01% DMSO in culture medium was used as a vehicle control. For in vivo studies 17-AAG was dissolved in DMSO at 25 mg/ml and stored at –20°C.
Mouse monoclonal antibody anti-HSP90 (NCL-HSP90) was purchased from Novocastra Laboratories (Newcastle-upon-Tyne, UK) and used for both western blot and immunohistochemical analyses. Mouse monoclonal antibody anti-EGFR (#280005) was purchased from Invitrogen Life Technologies (Grand Island, NY, USA), anti-Cyclin B1 (SC-245) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal antibodies anti-AKT, clone 11E7 (#4685), anti-phospho-AKT, clone D9E (Ser473, #4060), anti-p44/42 MAPK (ERK1/2) (#4695), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204, #4370), anti-survivin (#2808) were purchased from Cell Signaling Technology (Danvers, MA, USA), anti-α-tubulin (32–2500) was purchased from Invitrogen Life Technologies and Cyclin D1, clone EP12 (M364201) was purchased from Dako North America Inc. (Carpinteria, CA, USA) and used for Western blot analysis. The secondary antibodies anti-rabbit IgG, goat HRP-linked (#7074) and anti-mouse IgG, goat HRP-linked (#7076) were purchased from Cell Signaling Technology. For Ki67 index, automated staining was performed using an antibody against Ki67 antigen (Clone MIB1 DAKO) at a dilution of 1:100. Ki67 index was measured using the open access web application ImmunoRatio [55] for automated image analysis using 4–10 images per sample, depending on tumor size and excluding necrotic areas. Statistical analyses were performed using an unpaired Mann Whitney test using GraphPad Prism v. 6.
Small molecule inhibitor screening on gallbladder cancer cell lines
Cells were seeded into 384 well assay plates at 400 cells/well in their respective media. Inhibitors were obtained from LC Laboratories (Woburn, MA, USA) and Selleck Chemicals (Houston, TX, USA) were dissolved in DMSO and added at a concentration series ranging from 10 mM to 0.01 mM as previously described [5]. The final concentration of DMSO used was 0.01% throughout the screening methods. After three days of culture at 37oC, 5% CO2, CellTiter96 (Promega Madison, WI, USA) was added and optical density was measured at 490 nm and used to determine cell viability. The concentration of inhibitor required to inhibit cell growth by 50% (IC50) was calculated using a non-linear regression analysis.
Cell viability assay
G-415 and GB-d1 cell lines were plated into 96 well plates at a density of 3 x 103 cells per well. After an overnight attachment period cells were treated either with 17-AAG, GA or 0.01% dimethylsulfoxide (as control). The number of viable cells was determined at 24, 48, and 72 hours using CellTiter 96 Aqueous One Solution Cell Proliferation assay (Promega Corp., Madison, WI). Briefly, 20 μl CellTiter 96 solution was added to each well and the plates were incubated for 1 hour after which the absorbance of each well was read at a wavelength of 490 nm using a multiwell plate reader (Autobio Labtec Instruments, Zhengzhou City, China). All assays were performed in five technical replicates, and each assay was repeated three times.
Transwell cell migration assay
Migration assays were performed using 24-well Transwell™ plates containing polycarbonate filters with an 8 μm pore size (BD Biosciences, Bedford, MA, USA). Complete medium was placed in the lower chamber to act as a chemoattractant and 5 × 104 cells of either G-415 or GB-d1 cells were seeded with serum-free medium into the upper chamber. Cells were exposed to 17-AAG (12 μM), GA (15 μM) or 0.01% DMSO (as control) for 24 hours at 37°C. After 24 hours, cells were fixed in methanol for 15 minutes and stained with 0.05% crystal violet in 25% methanol/PBS for 15 minutes. Cells on top of the membrane were removed using a cotton swab, and filters were washed with PBS. Cells on the underside of filters were viewed and counted under a microscope in 10 randomly selected fields.
Western blot analysis
For in vitro assays, cells were seeded on 75 cm2 culture plates. Cells at 60–70% were treated with 17-AAG (12 μM), GA (15 μM), or 0.01% dimethylsulfoxide (as control) for 24 hours. Cell lysates were prepared using RIPA buffer (Sigma Aldrich, St Louis, MO, USA). Briefly, cells were washed three times with cold PBS and lysed on ice with RIPA buffer containing a phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitor cocktail (Sigma Aldrich, St Louis, MO, USA). For in vivo experiments, tumor lysates were prepared on ice using cell lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 2 mM EDTA) containing a protease and phosphatase inhibitor cocktail (Sigma Aldrich, St Louis, MO, USA). Whole lysates were collected after centrifugation at 14.000 rpm for 10 minutes at 4°C. Protein concentrations were determined using a BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific Inc, Rockford, IL, USA) according to the manufacturer's instructions. Equal amounts of total cellular protein (40 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 4%–20% Mini-PROTEAN TGX Gels (Bio-Rad Laboratories Inc, Hercules, CA, USA) and electrotransferred to PVDF membranes (Immobilon®-P membrane Millipore, Bedford, MA, USA). Membranes were blocked with 1X Tris-buffered saline containing 0.05% Tween (TBST) and 5% fat-free milk for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies. After washing with TBST, membranes were further incubated with the secondary antibodies for 1 hour at room temperature. Antibody-bound protein bands were detected using SuperSignal™ West kit (Pierce, Thermo Scientific Inc, Rockford, IL, USA). Images were acquired with the ImageQuant LAS 500 system (GE Healthcare Life Sciences, Pittsburgh, PA, USA ). Alpha-tubulin expression was used as a loading control.
G-415 and GB-d1 cell lines were seeded into 6 well plates at a density of 1x105 cells per well. After overnight incubation, cells were treated either with 17-AAG (12 μM), GA (15 μM) or 0.01% dimethylsulfoxide (as control) for 24 hours. Cells were trypsinized, washed and fixed in 70% ethanol at −20°C overnight. Cells were then washed and incubated for 30 minutes at room temperature and in darkness in a solution containing 20 μg/ml Propidium Iodide (PI) (Sigma Aldrich, St. Louis, MO, USA), 100 μg/ml RNase (US Biological, Salem, MA) and 0,1% Triton X-100). Cells were evaluated on a FACSCanto II (Becton Dickinson, Franklin Lake, NJ) and data was analyzed with BD FACSDiva software, version 6.0. Experiments were performed in three technical replicates, and data was expressed as mean ± S.D.
Apoptosis analysis
G-415 and GB-d1 cell lines were plated into 6 well plates at a density of 1 × 105 and 6 × 104cells per well, respectively. After an overnight attachment period cells were treated either with 17-AAG, GA or 0.01% dimethylsulfoxide for 24, 48 and 72 hours. Cells were harvested by trypsinization and apoptosis was determined using two different methods, Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen, Eugene, OR) and CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Life Technology, Carlsbad, CA), according to the manufacturer's protocol. Data were collected and analysed on FACSCanto II (Becton Dickinson, Franklin Lake, NJ) using BD FACSDiva software, version 6.0. Annexin V (+)/PI (-) and annexin V (+)/PI (+) cells were considered early and late apoptotic cells, respectively, and both were counted as total apoptotic cells. Caspase 3/7 (+)/Sytox (−) and caspase 3/7 (+)/Sytox (+) also were counted and considered as total apoptotic cells. Experiments were performed in three technical replicates and a total of 10,000 cells were analyzed in each individual experiment.
In vivo tumor xenograft assays
8 to 12-week-old NOD-SCID mice (obtained from Jackson Laboratory, Bar Harbor, USA) were subcutaneously injected in one flank with 2 × 106 cells of G-415 resuspended in 200 μl of PBS with 30% of Matrigel (Matrigel™ Basement Membrane Matrix, BD Biosciences). When the average tumor reached 50 mm3, mice were randomly separated into two groups and treated with 17-AAG and its respective vehicle. 17-AAG was administered at a daily intraperitoneal (i.p) dose of 25 mg/kg for 5 days per week for 4 weeks. Tumor volumes were estimated twice a week from caliper measurements (volume = 0.52 × (width)2 × length).
HSP90 immunohistochemical staining
Whole tissue sections (WTSs) and tissue microarrays (TMAs) were used for this study. Three different representatives areas were sampled for TMAs construction according to previously published methods [56]. None of these patients had received preoperative chemotherapy. HSP90 expression was evaluated using a previously described immunohistochemistry protocol [56]. Briefly, heat induced antigen retrieval was performed prior to immunohistochemical staining. Sections were incubated with a HSP90 monoclonal antibody (1:200 dilution, Novocastra Laboratories) at 4°C overnight. Detection of signal was achieved using the LSAB + kit (DAKO, Carpinteria, CA, USA) according to the manufacturer's recommendations. 3,3′-diaminobenzidine/H2O2 (DAKO) was used as chromogen and slides were counterstained with hematoxylin. We used gastric cancer tissue as positive control and omission of the primary antibody was used as a negative control. The immunohistochemical staining of HSP90 was examined by two independent and specialized pathologists (JRV, AHC) without any information about clinicopathological features or prognosis. Immunohistochemical staining was evaluated based on staining intensity and positive cells within the whole tissue section using a previously described scoring system [56]. Briefly, an arbitrary 20% cutoff was used to determine HSP90 positivity. Only cytoplasmic staining was considered positive for HSP90, and expression was graded visually as negative, weak or moderate/strong [56].
Use of gallbladder specimens from patients was approved by the IRB of the School of Medicine, Pontificia Universidad Católica de Chile. Mouse husbandry and animal experiments has been conducted in accordance with the ethical standards and according to the local animal protection law.
Statistical analyses of in vitro and in vivo experiments were performed by analysis of variance (one way ANOVA) followed by Tukey's multiple comparisons test. P values < 0.05 were considered significant. Data analysis was performed with the GraphPad Prism 5 (GraphPad Software, Inc. San Diego CA).
We would like to thank Dr. Sebastian Newrzela (Senckenberg Institute of Pathology; Goethe-University of Frankfurt Medical School; Frankfurt am Main, Germany) and Tanya Syzonenko (Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus. Frankfurt. Germany) for technical support.
The authors report no conflicts of interest in this work.
FONDECYT (National Fund for Scientific and Technological Development) grants 1130204, 11130515 and 1151008; Fondap-Conicyt 15130011, CEGIN (Center of Genetic and Immunological Studies) grant 09CN14-5960. Millennium Institute on Immunology and Immunotherapy P09/016-F.
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Every established brand knows the pain of growing old. All the things that have served them so well in the past start to lose their lustre, becoming less and less effective by the day. But the prospect of an overhaul can be daunting. Here's a few things the £13 billion Bond franchise has taught us about keeping a brand exciting.
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Abraham Lincoln once said "The best way to predict your future is to create it". With the introduction of every new Bond actor, comes a new era for the franchise. So first consideration should be given to what you are trying to build towards. Think about where you want to be, then support it with a plan of what you need to do to get there – not the other way around!
The man portraying Bond is significant, but would we consider the move from Sean Connery to Roger Moore, or Pierce Brosnan to Daniel Craig a change or evolution?
When it comes to brands, change can be a scary word. Change can represent a departure from your brand's direction to pursue another. On the other hand, evolution is about remaining true to your original vision and values, while continually adjusting and developing to support endurance and growth. In the case of 007, we've seen 24 films with six lead actors over more than 56 years. How long has it been since your brand last evolved?
003. Your brand is defined by what's around it as much as it's defined by itself.
Staying relevant to the world's evolution should spur how your brand evolves. No matter what film you watch, Bond remains unmistakeably Bond, but notice how the suave and sophisticated 60's were quickly replaced by the attitude-driven, entertaining 70's?
Fast-forward to Daniel Craig's gritty, action-packed Casino Royale in 2006 as a response to the Jason Bourne reboot with Matt Damon in 2002 which re-established the spy genre for the modern era. A new era for Bond, influenced greatly by context. Remember, your brand doesn't live in a vacuum – it's defined by what is around it as much as it's defined by itself.
Daniel Craig's Bond rediscovered the rawness of Ian Fleming's original 007 novel characterisation that had been forgotten by his predecessors. For brands, looking back to their roots is a great way of finding a way forward with authenticity.
005. Invest in your equities.
"Bond. James Bond." From the mesmerising opening titles to the Aston Martin, gorgeous women and "Shaken, not stirred", brand 007 boasts a load of signatures inseparable from the franchise.
It didn't happen overnight – in fact it's something Bond has had to work hard at. These little trinkets of recognition and association have been carefully placed in people's minds over decades. Religion and football teams are other great examples of 'brands' that invest in long-term equities.
006. Always think of your fans.
At the end of the day, your success will be defined by your fans and many brands would do well to be reminded of this. Your fans should be the reason that you do what you do. Brand Bond does this amazingly well with the way it launches its films and continually gives fans what they want while at the same time introducing them to something new, every time.
007. Never complete the story.
Bond has a formula that could see it continue for generations. With storytelling being at the heart of branding today, one of the most important lessons is to never complete your brands' narrative. Always leave it open ended, because you will need to add the next chapter before you know it.


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Q: Dynamic regexp_extract based on column value in spark I'm trying to know if there is a way to implement dynamic regexp_extract in my dataset based on the column value.
For example:
if my column A value is "N06" I need to use regex as "(?<=2020:).?\\n"
else if my column A value is "N02" I need to use regex as "(?<=2026:).?\\n"
ds.withColumn("extracted",functions.regexp_extract(functions.col("A"),regex,0))

A: Try to use when and otherwise like this:
when(col("C") === "N06", regexp_extract(col("A"), regex1, 0))
  .otherwise(regexp_extract(col("A"), regex2, 0))

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