text:
Q: shortcode for recent custom type post I'm not a coder so I may have done some "coding" crimes, apologies. I've bought a theme who has shortcodes for last posts but not for last "custom post type". So I tried to create last "custom post type" function modifying the Last post function provided.
Last post funtion:
function get_custom_posts( $params ) {
extract( shortcode_atts( array (
    'number' => '1',
    'excerpt' => 290,
    'readmore' => 'no',
    'readmoretext' => 'Read more'
), $params ) );

//$latest_posts = get_posts( 'category=0&numberposts=' . $number . '&suppress_filters=false');     OLD
$latest_posts = query_posts( 'cat=0&posts_per_page=' . $number );
wp_reset_query();

$result = '
';
$count = count($latest_posts);
foreach ($latest_posts as $key => $latest_post) {
  $author = get_the_author_meta('nickname', $latest_post->post_author );
  $post_link = get_permalink( $latest_post->ID );
  $date = mysql2date(get_option('date_format'), $latest_post->post_date);
  $category = get_the_category_list( ', ', $parents = '', $latest_post->ID );

  $result .= '

';
  // POST THUMBNAIL
  if (get_the_post_thumbnail( $latest_post->ID, 'thumbnail' )) {
      $result .= '
';
      $result .= '';
      $result .= get_the_post_thumbnail( $latest_post->ID, 'thumbnail' );
      $result .= '';
      $result .= '
';
  }

  // POST BODY
  $result .= '
';
  $result .= '

' . $latest_post->post_title . '

';
  if ( $latest_post->post_excerpt ) {
      $result .= '
' . $latest_post->post_excerpt . '
';
  }
  else {
      $limit = $excerpt;
      $my_text = substr($latest_post->post_content, 0, $limit);
      $pos = strrpos($my_text, " ");
      $my_post_text = substr($my_text, 0, ($pos ? $pos : -1)) . "...";
      $read = "";
  if($readmore == 'yes'){
    $read = ' '.$readmoretext.'';
  }
      $result .= '
' . strip_tags($my_post_text) . $read . '
';
      //$result .= '
' . substr_replace( $latest_post['0']->post_content, '...', 350 ) . '
';
  }

  $result .= '
';

  if ($count - 1 != $key)
  $result .= do_shortcode('[rule]');

  $result .= '

';
}
$result .= '
';

return $result;
}
add_shortcode( "get_posts", "get_custom_posts" );

Then I tried to modify it like this, (my custom post type is ait-dir-item):
function get_custom_items( $params ) {
extract( shortcode_atts( array (
    'number' => '1',
    'excerpt' => 290,
    'readmore' => 'no',
    'readmoretext' => 'Read more'
), $params ) );

//$latest_posts = get_posts( 'category=0&numberposts=' . $number . '&suppress_filters=false');     OLD
$latest_items = WP_query( 'post_type' = 'ait-dir-item', 'cat=0&posts_per_page=' . $number );
wp_reset_query();

$result = '
';
$count = count($latest_items);
foreach ($latest_items as $key => $latest_item) {
  $author = get_the_author_meta('nickname', $latest_item->post_author );
  $post_link = get_permalink( $latest_item->ID );
  $date = mysql2date(get_option('date_format'), $latest_item->post_date);
  $category = get_the_category_list( ', ', $parents = '', $latest_item->ID );

  $result .= '

';
  // POST THUMBNAIL
  if (get_the_post_thumbnail( $latest_item->ID, 'thumbnail' )) {
      $result .= '
';
      $result .= '';
      $result .= get_the_post_thumbnail( $latest_post->ID, 'thumbnail' );
      $result .= '';
      $result .= '
';
  }

  // POST BODY
  $result .= '
';
  $result .= '

' . $latest_item->post_title . '

';
  if ( $latest_item->post_excerpt ) {
      $result .= '
' . $latest_item->post_excerpt . '
';
  }
  else {
      $limit = $excerpt;
      $my_text = substr($latest_item->post_content, 0, $limit);
      $pos = strrpos($my_text, " ");
      $my_post_text = substr($my_text, 0, ($pos ? $pos : -1)) . "...";
      $read = "";
  if($readmore == 'yes'){
    $read = ' '.$readmoretext.'';
  }
      $result .= '
' . strip_tags($my_post_text) . $read . '
';
      //$result .= '
' . substr_replace( $latest_item['0']->post_content, '...', 350 ) . '
';
  }

  $result .= '
';

  if ($count - 1 != $key)
  $result .= do_shortcode('[rule]');

  $result .= '

';
}
$result .= '
';

return $result;
}
add_shortcode( "get_items", "get_custom_items" );

Of course, this function doesn't work... Can someone help me? I want to modify this function instead of installing a plugin because I want to use the same classes so I will have the same design for both shortcodes.
Thanks in advance!

A: The problem looks to me to be this line:
$latest_items = WP_query( 'post_type' = 'ait-dir-item', 'cat=0&posts_per_page=' . $number );

That is very odd. First, you are missing the new keyword. Without it you get an "undefined function" error.
Second, with WP_Query, and a number of other functions, you can use a query-string-like function argument, or an array. You have (broken) elements of both in there, which isn't going to work.
For a query-string-like parameter, you'd need:
$latest_items = new WP_query('post_type=ait-dir-item&cat=0&posts_per_page='.$number);

See how that is all one string, and yours isn't? You have three distinct strings, an = floating loose, and a comma that is going to indicate that you are actually using two parameters and not just one. If you have debugging enabled you'd see errors.
But use an array. Those query-string-like parameters are hard to read, hard to edit, and error prone because of it. At least that is my take.
$latest_items = new WP_query(
  array(
    'post_type' => 'ait-dir-item',
    'cat' => 0,
    'posts_per_page' => $number,
  )
);

Also, the cat=0 is unnecessary. As near as I can tell, the query is identical with or without it.

A: This seems to work with custom post type :
function get_custom_posts( $params ) {
extract( shortcode_atts( array (
'number' => '1',
'excerpt' => 290,
'readmore' => 'no',
'cpt' => 'post',
'readmoretext' => 'Read more'
), $params ) );

//$latest_posts = get_posts( 'category=0&numberposts=' . $number .      '&suppress_filters=false');     OLD
$latest_posts = query_posts( 'post_type='.$cpt.'&posts_per_page=' . $number );
wp_reset_query();

$result = '
';
$count = count($latest_posts);
foreach ($latest_posts as $key => $latest_post) {
  $author = get_the_author_meta('nickname', $latest_post->post_author );
  $post_link = get_permalink( $latest_post->ID );
  $date = mysql2date(get_option('date_format'), $latest_post->post_date);
  $category = get_the_category_list( ', ', $parents = '', $latest_post->ID );

  $result .= '

';
 // POST THUMBNAIL
  if (get_the_post_thumbnail( $latest_post->ID, 'thumbnail' )) {
      $result .= '
';
      $result .= '';
      $result .= get_the_post_thumbnail( $latest_post->ID, 'thumbnail' );
      $result .= '';
      $result .= '
';
  }

  // POST BODY
  $result .= '
';
  $result .= '

' . $latest_post->post_title . '

';
  if ( $latest_post->post_excerpt ) {
      $result .= '
' . $latest_post->post_excerpt . '
';
  }
  else {
      $limit = $excerpt;
      $my_text = substr($latest_post->post_content, 0, $limit);
      $pos = strrpos($my_text, " ");
      $my_post_text = substr($my_text, 0, ($pos ? $pos : -1)) . "...";
      $read = "";
  if($readmore == 'yes'){
    $read = ' '.$readmoretext.'';
  }
      $result .= '
' . strip_tags($my_post_text) . $read . '
';
      //$result .= '
' . substr_replace( $latest_post['0']->post_content, '...', 350 ) . '
';
  }

  $result .= '
';

  if ($count - 1 != $key)
  $result .= do_shortcode('[rule]');

  $result .= '

';
}
$result .= '
';

return $result;
}
add_shortcode( "get_posts", "get_custom_posts" );

To use it just write [get_posts cpt="ait-dir-item"] in your posts
EDIT: it's bad practise to use query_post() in this context

A: I'm review your code throughly but the problem is in the wp_query parameter.so please replace this line to my new one
$latest_items = WP_query( 'post_type=ait-dir-item&cat=0&posts_per_page=' . $number )

this will help.I'm sure about that.

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A computer stores bits, and 8 bits make a byte.
Some bytes are not like other bytes -- bytes can mean different things.
Some bytes mean an integer, some bytes mean a list; we say that they have different types.
An integer is a group of integers, like 1, 2, 3, and so on, and so forth.
A boolean is a group that has 2 members, True and False.
But we're getting ahead of ourselves, so let's start from 0.
A void type has no members. It is void.
A unit type has only 1 member, and the member is called unit. You see it as () in many places.
What type has 2 members?
True and False are both members of boolean. True is not False, and False is not True. But they are both boolean.
This reads as: "The type boolean has 2 members, it can either be True or False".
Suppose you're deciding if you should continue reading.
be Undecided (there is no try).
We observe that the type decision has 3 members.
In the Decided case, there are 2 members.
The number of members is the same as the number of members in a boolean, because the Decided case is made up entirely of boolean.
In the Undecided case, there is 1 member, similar to Unit.
How did we get 3? A decision can be Decided (2) or Undecided (1). 2 + 1 = 3.
See how or and + works in similar ways.
That's why they call it a sum type!
Let's make a Pair of booleans.
How many members are in this type? 4!
How did we get 4?
For the first boolean, there are 2 members, True and False.
And for each of these 2 members, the second boolean can be True or False.
And that's where the name product comes from.
You've made a decision, and depending on the decision you will either continue reading, or not.
Here's one particular way to encode this decision-to-action process.
If you have decided, you follow your decision. If you are undecided, you continue reading and see what happens.
continue is a function, its type is decision -> boolean.
Let's ask the same question: how many members are there in the decision -> boolean type?
A function of this type has to, for each member of the input type, decision with 3 members, give a member of the output type, boolean with 2 members.
So the total number of unique functions of this type is 2 * 2 * 2 = 8, or equivalently, 2 ^ 3!
So you see, function types are also called exponential types.
A Pair boolean integer is a type, but a Pair boolean a', where a' depends on what boolean actually is, True or False, is called a dependent sum type.
It says that that the pair depends on the first item in the pair, if it is True, we have a Pair of boolean * boolean, but if it is False, we have a Pair of boolean * integer.
To count how many members there are in this type pair, we can treat it like a sum type.
when True, the second item has to be a boolean, which has 2 possible value.
when False, the second item has to be a integer, which for simplicity sake, can have 256 values.
Therefore, the total number of members is 2 + 256 = 258.
Why is this called sigma? Because you are taking the sum of all possible types across all values of the type being depended on (first item in the pair).
A decision -> boolean is a function type, with an argument of type decision, and a return type boolean.
But a function decision -> a', where a' depends on what decision actually is, True or False, is called a dependent product type.
This says that when the type of continue depends on the value of the argument.
When decision is Decided True, continue returns an integer, when Decided False or Undecided, it returns a boolean.
Let's count how many members type has. Recall how we counted the number of members a function type has previously, we multiplied the number of members in the output type n times, where n is the number of member in the input type.
In this case, since the number of members in the output type changes, we have to multiply them one by one instead of taking an exponent, to get 256 * 2 * 2 = 1024.
Why is this called pi? Because you are taking the product of all possible types across all values of the type being depended on (argument of the function).
And actually, the function space is a special case of the pi type, where the return type is constant, i.e. does not depend on the value of the argument.
We can think of types as a set of things. We say that some value is of that type when it is a member in that set.
For example, boolean is the set of things, which contains True and False.
A sum type is a disjoint union, or tagged union.
A product type is a cartesian product.
A sigma is the symbol to do a sum over a sequence.
A pi is the symbol to do a product over a sequence.
A lot of this comes from, or is related, to category theory.
I chose to say that a type has 2 members to invoke the idea of thinking about types as sets. Other words that can be used include value (rejected because it means too many things).
The real reason why the words sum, product, etc are used probably has less to do with counting members, and more to do with category theory. But it works well enough if you are trying to get an intuition about the meaning.
Manis Goregaokar. What Are Sum, Product, and Pi Types?
Idris, which has dependent types, for tips on the made up syntax.


Your visit to St. Petersburg can be complete with a stop at one of the souvenir markets. There are several types and locations to choose from. Some of the best markets are hidden off Nevsky Prospect and can be found with my help as your personal tour guide in St. Petersburg. At various markets you'll find the traditional stacking dolls, amber and other types of memorabilia. Almost all of the vendors will be able to speak English and negotiating is a large part of the purchase in these markets. So come prepared to negotiate for bargains!


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Our Tactical Communications Training Promotes the Goal of Law Enforcement: To Gain Voluntary Compliance.
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text:
Q: Force WWW to use HTTPS How do I force www to use https and not just http?
For example, when visiting http://www.example.com it redirects to https://www.example.com but if you don't include the www, you don't get redirected to the SSL version.
RewriteEngine on
RewriteBase /

RewriteCond %{HTTP_HOST} !^www\.(.*)$
RewriteCond %{REQUEST_URI} !^/admin
RewriteCond %{REQUEST_URI} !^/crossdomain.xml
RewriteRule ^(.*)$ https://www.%{HTTP_HOST}/$1 [R=301,L]

A: Below condition should work for both www and without it.
RewriteCond %{HTTP_HOST} ^(www\.)?example.com [NC]


text:
Wildlife Linkages
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What You Should Know About the Pacific Flyway
December 11, 20172017-12-11
By Matt Dolkas,
It's December and it's getting properly cold outside. With winter on the horizon, birds across the continent are moving south and on these cold late-fall mornings I sometimes wish I could do the same.
The lack of an abundant food source is what, for most, demands a move south. And for some species, this means flying as far as Central and South America.
It's always amazed me how birds find their way during these annual migrations. Some scientists believe migratory birds take their navigational cues from the sun, moon and stars. Others think they simply follow geographic landmarks like rivers and mountains. Nobody knows for sure and maybe never will.
What we do know is that migratory birds consistently follow the same flight paths, converging every year on "flyways." Think of them like nature's interstate system.
The Pacific Flyway (roughly illustrated above) is one of four flyways in North America used by migratory birds for their annual migrations. The San Francisco Bay, and the diversity of habitat surrounding the Bay, create one of the most important stopovers for migratory birds on this flyway.
There are four flyways that span across North America–the Pacific, Central, Mississippi and Atlantic Flyways (don't worry, there won't be a quiz on any of this).
California lies within the Pacific Flyway, an area that stretches from the Arctic to the coast of Mexico, and from the Rocky Mountains to the Pacific. North to south it's over 4,000 miles long and, in places, over 1,000 miles wide.
The San Francisco Bay is a critically important stopover for birds moving along the Pacific Flyway. As the largest estuary (tidal mouth of a large river) on the Pacific coasts of both North and South America, the Bay offers an abundance of habitat for tired migrant birds. And it's not just wetlands that make the Bay Area so attractive. There's a diversity of habitat types here offering refuge for birds of all kinds – it's an avian paradise.
The importance of Bay Area open spaces to the Pacific Flyway is clearest when considering the concentration of the region's Important Bird Areas (IBAs). Yea, what's an IBA? The National Audubon Society is leading an effort to identify, monitor and protect the planet's most important places for birds. These areas are determined using internationally agreed criteria and are called, quite simply, "Important Bird Areas."
The interactive map below illustrates our region's IBAs and the overlap between these areas and the land we've helped protect. Explore more below and learn about one of the Pacific Flyways' greatest stopovers:
Want to learn more about birds in the Bay Area? Download our Natural Guide to Bay Area Birds—an illustrated guide to the most iconic birds in our open spaces!
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Monarch Butterfly: Speak, Monarch. Tell Us How to Save You.
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"What the article never considers is whether charter schools are needed and whether the state would be wiser to invest the same hundreds of millions millions in improving the public schools that most students attend."
It is a scam folks. First the GOP defunds everthing, forcing cities to cut essential services. Public schools are the first to g, and when they fail because there his no money to support firing professional practitioners, or ensure that class size supports learning, or provide materials and programs the support the teacher, and the student... they use what taxpayer money remains to enrich the charter school owners.
So WHY? do you support Diane Ravitch, who supports the NEA?
You too were screwed by the unions, as you have explained before. So I simply ask "Why?" as you have advocated doing.
Thanks in advance for your hopefully edited response.


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Witness a Coyote and Badger Become Best Buddies Thanks to This Gift of a Video
By Megan McCluskey
February 7, 2020 11:08 AM EST
First, there was the fox and the hound. Now, there's the coyote and the badger.
In footage that was captured by a wildlife camera set up by environmental conservation organization Peninsula Open Space Trust (POST), a coyote and badger who appear to be palling around can be seen traveling together through a culvert under a Bay Area highway.
Neal Sharma, POST's wildlife linkages program manager, told CNN that POST believes this is the first time that this type of behavior has been observed in the Bay Area.
"Having that interaction on film and seeing how these two different animals that lead different lives, how they interact, it's just so exciting," Sharma said.
The video took off after it was shared on Twitter on Monday by user Russ McSpadden, earning over 771,000 likes and 183,000 retweets as of Friday morning. It even got a shoutout from Captain America himself, Chris Evans.
"I'm choosing to believe that the coyote sounds like Michael J. Fox and the badger sounds like Sam Elliot and they're best friends on an adventure," Evans wrote.
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text:
Home :: Forums :: CHRONIC CONDITIONS :: Bronchitis and Pneumonia
Acute Bronchitis: Two Cases
Bronchitis and Pneumonia
Abstract: Two cases of acute, epidemic bronchitis are presented along with discussion of the characteristic symptoms in each case and the case analyses. Some of the author's confirmed characteristic symptoms of Carcinosin are identified; e.g., waking at 4 a.m., amelioration from music, sleep position on the back with one or both hands above the head, craving for sweets or fats. Suggested guidelines for the taking of the acute homeopathic case (which may be considered controversial) are provided; i.e., focus upon only those symptoms which have arisen in association with the acute disturbance, ignoring all preceding chronic symptomatology. The common misconception of dividing remedies into acute and chronic disease categories is debunked, the author emphasizing that any remedy may act curatively in either setting.
Keywords: bronchitis, acute case taking, characteristic symptoms, acute remedies, chronic remedies, Carcinosin.
Two cases of acute, epidemic bronchitis are reported below. The first of these patients had seen me once two years ago for another acute, epidemic illness; the second sees me regularly for ongoing treatment and well-child care.
Both cases were taken in a very specific manner: focusing in on those changes that have occurred since the patient became ill. Only symptoms of the acute episode are included, and all of the patient's recent changes are included in these reports. None of the symptoms in either case comes from the patient's chronic state.
Hering tells us, "In all...cases the symptoms appearing last...are always the most important."( 1) Hahnemann teaches, "In [acute disease] the principal symptoms become prominent and recognizable to the senses more quickly, so the taking of the case requires far less time and there are far fewer questions to ask..."( 2) I ask only one question when I take an acute case, but I ask it over and over again: "What has changed since...?"
When taking an acute case focus your patient on his or her state since the illness began. Don't allow any divergence. Keep asking what else has changed since they've become ill. Interrupt whenever they move off track and bring them back to the time since they've been sick. Consider only these symptoms in your case analysis. If you haven't tried acute case taking using this approach I'd encourage you to do so. The accuracy and effectiveness of your prescribing for acute cases will improve dramatically.
HC is a 10 year old who'd had a cough and a sore throat for 3 days when she saw me 12/22/95. She was much worse after 7 p.m. She described a pain behind her ears when she coughed. Her throat was better while warm drinks were being swallowed. She had a tickle in her throat pit after eating ice cream or drinking milk. She described a light green expectoration that tasted a bit salty. She coughed when her throat hurt and when her throat tickled. Exertion made her throat tickle, which made her cough. Her right ear hurt when she coughed. She had decreased energy. She couldn't sleep after a party last night (she was up till midnight). She woke at 4 a.m. to blow her nose. She felt warmer than usual. She wanted to listen to music the night she got sick, which was not only unusual for her but also helped her feel better. She had a grilled cheese sandwich for lunch today, which didn't hurt to swallow.
Exam: Afebrile. Tympani: R -- cerumen, L -- clear. Oropharynx red. Shotty cervical nodes. Clear lungs. A throat swab sent to the lab was negative for Strep.
Impression: Bronchitis with pharyngitis.
Case Analysis
The characteristic symptoms in this case were: 1) waking at 4 a.m. (why should a 10 year old who was up till midnight wake up at 4 a.m. for any reason?); 2) ameliorated by music (she not only wanted to listen to music, but she also felt better by doing so!); 3) desired fatty food which ameliorated (a grilled cheese sandwich is nothing if it isn't greasy -- cheese between buttered bread that's warmed till it oozes); and 4) warmer than usual.
The following chart represents the MacRepertory(R) repertorization of the above symptoms with the "Analysis" options set to "Strange, Rare & Peculiar" and to "Keynotes" with the "Analysis Preferences" set to "Enhance Analyses" and to "Double Underline Value" for both choices. These settings make the program rank the remedies according to what's most characteristic about the remedies in relation to the symptoms I've chosen to repertorize.
Sulphur, on first pass, seems an appropriate prescription for this patient who is hot, wakes at 4 a.m., and craves fatty food. Natrum muriaticum also covers all these symptoms and is sensitive to music as well. However the best match, the most similar remedy, is Carcinosin.
Prescription and Follow-Up
Carcinosin 1M once.
Mom reported complete disappearance of all acute symptoms within 36 hours. HC has been well since.
KS (19 months old). Acute cough. She awoke at 11:30 p.m. and 4:30 a.m. She had circumscribed red cheeks. Her tongue was bright red in the center. Her ears itched. It looked like she was scratching in them. She wanted music on a lot, in the kitchen, in the bathroom, in the car, etc. She sang along and danced. It made her more lively. She slept on her back with one or both arms above her head. She craved sweets.
Exam: Afebrile; tympani clear: oropharynx clear; no cervical nodes; lungs clear except for coarse central sounds posteriorly with no dullness or fremitus; regular heartbeat with no murmur.
Impression: Bronchitis.
The characteristic symptoms in this case were: 1) waking at 4 a.m.; 2) music ameliorates; 3) dancing; 4) sleeping on her back with her arms above her head; 5) desires sweets. Remember that all of these were changes from her chronic state.
The following chart represents the MacRepertory(R) repertorization of the above symptoms with the analysis options set as above.
Tarentula hispanica needed to be considered for this patient because of the combination of music and dancing in the characteristic symptoms. Sulphur covered the early waking and the craving for sweets. Pulsatilla covered the sleep position. Natrum muriaticum and Lycopodium also had to be considered. However, the best match for the totality of these characteristic symptoms was again Carcinosin.
Carcinosin 200c once.
Three day follow-up: She had an emotional aggravation for two days, then she became better. She was coughing less. Her cheeks were not as red. Her cheeks began to get redder again five days later, so Mom gave Pulsatilla without contacting me. (Don't you love those moms?) Her cough started to return three days later and a second dose of Carcinosin, was given at that point. She has been well since.
I want to make two points as I discuss these cases. The first is about how I prescribe Carcinosin. The second is about the distinction many homeopaths make between "acute" and "chronic" remedies.
Carcinosin is one of our newest remedies. Foubister's ground breaking work was published only about 40 years ago. Our collective literature is very sketchy when it comes to this remedy. It is not well proven. Some of the symptoms I find most characteristic of Carcinosin are not well described anywhere. A few of these symptoms are found in these cases: waking at 4 a.m., ameliorated by music (persons that respond well to Carcinosin may be so very sensitive to music that they weep at its inherent beauty), sleep position on the back with one or both hands above the head, and craving for sweets or, even more characteristically, fats.
Patients of mine who have responded well to Carcinosin have exquisite sensitivities to beauty, dancing, travel, reprimand, and world affairs, as well as music. They have intense cravings for fats, salt, ice cream, chocolate, bacon, and sweets. They are ameliorated by the ocean. There are many other Carcinosin symptoms; 996 are listed in the Complete Repertory, v. 3.01. But these exquisite sensitivities and strong food cravings form a core group of very characteristic symptoms, any one of which should lead you to investigate the possibility of prescribing Carcinosin.
The second point I want to discuss is perhaps the more important of the two: Carcinosin, a nosode, is generally considered a chronic remedy yet was prescribed quite successfully in these acute cases of epidemic bronchitis. Many homeopaths in America today, and perhaps in the world, seem to group remedies into "acute" and "chronic" categories, certain remedies like Arnica, Drosera, Arundo, and Sarsaparilla being labeled "acute" remedies and certain others like Sulphur, Natrum muriaticum, Lycopodium, and the nosodes being labeled "chronic" remedies. I believe this distinction is misleading. If one categorizes remedies into "acute" and "chronic" groups one will tend to miss prescriptions because one would never imagine Rumex, for instance, would fit a constitutional case or that Carcinosin would fit an acute case. Any remedy can act acutely under certain circumstances and act constitutionally under certain other circumstances, the certain circumstances being the characteristic symptoms in the case.
Please remember Hahnemann's often repeated directive to bring a pure mind to the practice of homeopathy. In a prefatory chapter to the Materia Medic Pura Hahnemann said the following about the pure mind: "In order to be able to observe well, the medical practitioner requires to possess...the capacity and habit of noticing carefully and correctly....Poetic fancy, fantastic wit, and speculation must for the time be suspended, and all overstrained reasoning, forced interpretation, and tendency to explain away things must be suppressed. The duty of the observer is only to take notice of the phenomena and their course....This capability of observing accurately is never quite an innate faculty; it must be chiefly acquired by practice, by refining and regulating the perception of the senses, that is to say, by exercising a severe criticism in regard to the rapid impressions we obtain of external objects, and at the same time the necessary coolness, calmness, and firmness of judgment must be preserved, together with a constant distrust of our own powers of apprehension."( 3)
The symptoms which characterize any case, be it acute or chronic, will lead to the proper prescription, be it an "acute" or a "chronic" remedy. I think that's worth repeating: the characteristic symptoms in each case necessarily lead to the proper, homeopathic prescription.( 4)
(1.) Hering, Constantine. Analytical Repertory of the Symptoms of the Mind, preface.
(2.) Hahnemann, Samuel. Organon of Medicine, S82.
(3.) Hahnemann, Samuel. "The medical observer: A fragment." Materia Medical Pura, vol. 2.
(4.) Hahnemann, Samuel. Organon of Medicine, S153.
American Institute of Homeopathy.
By Rodney Schaffer
‹ Breakthrough for breathing Antibiotics Not Warranted For Acute Bronchitis ›


text:
Frontiers in Surgery
Zongbing You
Tulane University, United States
Daotai Nie
Southern Illinois University School of Medicine, United States
Benyi Li
University of Kansas Medical Center, United States
Front. Surg., 07 June 2018 | https://doi.org/10.3389/fsurg.2018.00040
Expression and Localization of Cathepsins B, D and G in Cancer Stem Cells in Liver Metastasis From Colon Adenocarcinoma
Shreeja Mehrotra1, Susrutha K. Wickremesekera1,2, Helen D. Brasch1, Bede Van Schaijik1, Reginald W. Marsh1,3, Swee T. Tan1,4* and Tinte Itinteang1
1Gillies McIndoe Research Institute, Wellington, New Zealand
2Department of General Surgery, Upper Gastrointestinal, Hepatobiliary & Pancreatic Section, Wellington Regional Hospital, Wellington, New Zealand
3University of Auckland, Auckland, New Zealand
4Wellington Regional Plastic, Maxillofacial and Burns Unit, Hutt Hospital, Wellington, New Zealand
Aim: We have previously identified and characterized cancer stem cell (CSC) subpopulations in liver metastasis from colon adenocarcinoma (LMCA). In this study we investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs.
Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for cathepsins B, D and G was performed on 4μm-thick formalin-fixed paraffin-embedded LMCA sections from nine patients. Immunofluorescence (IF) IHC staining was performed on three representative samples of LMCA from the original cohort of nine patients, to determine the localization of these cathepsins in relation to the CSC subpopulations. NanoString mRNA analysis and Western Blotting (WB) were used to examine the transcript and protein expression of these cathepsins, respectively. Enzyme activity assays were utilized to determine their functional activity. Data acquired from counting of cells staining positively of the cathepsins on the DAB IHC-stained slides and from Nanostring mRNA analysis were subjected to statistical analyses to determine significance.
Results: DAB IHC staining demonstrated expression of cathepsins B, D and G within LMCA. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4− cells within the tumor nests and the OCT4+ CSC subpopulation within the peritumoral stroma. NanoString mRNA analysis showed significantly greater transcript expression of cathepsin B and cathepsin D, compared to cathepsin G. WB confirmed expression of cathepsin B and cathepsin D proteins, while cathepsin G was below detectable levels. Enzyme activity assays showed functional activity of cathepsin B and cathepsin D.
Conclusion: Our study demonstrated novel finding of the expression of cathepsin B, cathepsin D, and possibly cathepsin G by the putative CSC subpopulations within LMCA.
Colorectal cancer (CRC) is the third most common cancer worldwide, and the second major cause of cancer-related deaths in developed countries (1). In New Zealand, there are approximately 2,800 new cases of CRC annually with approximately a 50% mortality resulting from the disease (2).
The etiology and pathogenesis of CRC is complex (3) with environmental and lifestyle factors playing a significant role in colorectal carcinogenesis. An increase in the incidence of CRC paralleling economic development and adoption of a Western lifestyle, has been reported in several countries (4). Dietary and lifestyle risk factors include a high intake of unsaturated fats and red meat, total energy intake, excessive alcohol use and sedentary lifestyle (3). Somatic and inherited mutations have also been implicated (3).
Over 90% of CRC are adenocarcinoma (5). The liver is the most frequent site of CRC metastasis, via the portal venous system (1). Liver metastasis is a significant contributor to mortality in patients with CRC, with 60–70% of patients who die from advanced CRC demonstrating liver metastasis (6). Over half of patients with CRC will develop liver metastasis during their lifetime. In patients with metastatic CRC, survival is seldom more than three years despite advancements in chemotherapy and biological agents. Liver resection provides the greatest likelihood of long-term survival in these cases, with five-year survival rates of up to 58%. However, only around 20% of cases are resectable at the time of diagnosis (7, 8).
The cancer stem cell (CSC) concept of cancer proposes that a small subset of cells within the tumor - the CSCs, possess innate biological characteristics for self-renewal (9). CSCs are thought to be responsible for tumorigenesis, tumor differentiation, maintenance, spread and relapse (9, 10). Recent reports have demonstrated the expression pattern of specific CSC markers, such as CD133, CD44, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) and epithelial cell adhesion molecule (EpCAM), in CRC (11). There is also evidence indicating the presence of CSC subpopulations within CRC, with unique expression patterns of embryonic stem cell (ESC) markers (11).
We have previously identified and characterized three subpopulations of CSCs within liver metastasis from colon adenocarcinoma (LMCA): a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4+ subpopulation and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4− subpopulation confined to the peritumoral stroma (PTS), and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4− subpopulation within the tumor nests (TNs) (12). We speculate the existence of a hierarchy of CSCs within LMCA, with the OCT4+ cells within the PTS putatively representing the most primitive CSC subpopulation (12).
The renin-angiotensin system (RAS) is known classically for its role in regulating blood pressure and fluid and electrolyte balance. There is increasing evidence indicating dysregulation of the RAS in malignancy and is associated with poor patient outcomes (13). The paracrine mechanisms of local RAS may affect tissue angiogenesis, cellular proliferation, apoptosis and inflammation, thereby playing an important role in tumorigenesis (14). Inhibition of the RAS restrains tumor growth, metastasis and angiogenesis (13). Components of the RAS are expressed by CSC subpopulations in a number of cancer types including isocitrate dehydrogenase-wildtype glioblastoma (IDHWGB) (15) and oral cavity squamous cell carcinoma (OCSCC) affecting different subsites (16–18). These CSCs have been proposed as a potential therapeutic target by manipulation of the RAS. Cathepsins B, D and G are proteases, which provide bypass loops for the RAS (19) are also expressed by GB (20) and oral tongue SCC (21), suggesting potential treatment targeting these cathepsins.
Cathepsin B, a cysteine protease, converts inactive pro-renin into active renin (20, 22). Cathepsin D, an aspartyl protease, functionally similar to renin, converts angiotensinogen to angiotensin I (20, 23). Cathepsin G, a serine protease, produces angiotensin II from angiotensin I and directly from angiotensinogen (20, 24).
Cathepsins are produced as inactive proenzymes that undergo processing to become active enzymes. Cathepsin B is initially generated as an N-glycosylated proenzyme of 39 kDa and is subsequently modified under acidic conditions to the single-chain form of mature enzyme of 29 kDa (25). This process is strongly inhibited by pepstatin, an aspartic protease inhibitor, suggesting that an aspartic protease plays a role in propeptide-processing of the proenzyme (25). Cathepsin D is synthesized as a preproenzyme comprised of 412 amino acid residues (26). Following removal of the signal peptide within the endoplasmic reticulum, the proezyme is transported to an acidic compartment, where it is subsequently activated by proteolytic removal of a 44-amino acid residue prodomain. The mature form of cathepsin D can exist as either a single or two-chain form, with both forms displaying equal activity (26). Cathepsin G is a serine protease of the chymotrypsin family. It undergoes proteolytic activation by cathepsin C and is stored within neutrophil azurophilic granules as an active protease (27, 28).
Cathepsins have been implicated in invasion and metastasis in CRC, due to their ability to degrade extracellular matrix (ECM) in response to inflammatory and oncogenic stimuli (6, 29).
In this study, we investigated the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations within LMCA we have previously identified (12) using immunohistochemical (IHC) staining, immunofluorescence (IF) IHC staining, Western blotting (WB), and NanoString mRNA expression analysis. Enzymatic activity assays were performed to determine functional activity of these cathepsins. The presence of these cathepsins may underscore potential treatment target in the treatment of LMCA.
Tissue Samples
The same LMCA samples from nine male patients, aged 50–80 (mean, 65) years (Table 1) included in our previous study (12), were sourced from the Gillies McIndoe Research Institute Tissue Bank for this study which was approved by the Central Regional Health and Disability Ethics Committee (ref. no. 15/CEN/106). Written informed consent was obtained from all patients.
Table 1. Characteristics of Liver Metastasis from Colon Adenocarcinoma Samples.
Histochemical and Immunohistochemical Staining
Hematoxylin and eosin (H&E) staining was performed on 4μm-thick formalin-fixed paraffin-embedded LMCA sections from nine patients to confirm the presence and areas of TNs and the PTS, by an anatomical pathologist (HDB). These LMCA sections then underwent DAB IHC staining, as previously described (12), using primary antibodies for cathepsin B (1:1,000; cat# sc-6490-R, Santa Cruz, CA, USA), cathepsin D (1:200; cat# NCL-CDm, Leica, Newcastle-upon-Tyne, UK), cathepsin G (1:200; cat# sc-33206, Santa Cruz, CA, USA), EMA (ready-to-use, cat# PA0035, Leica), and OCT4 (1:30, cat# MRQ-10, Cell Marque, Rocklin, CA, USA). All DAB IHC-stained slides were mounted in Surgipath Micromount (Leica).
IF IHC staining was performed to determine co-expression of two proteins on three samples of LMCA from the original cohort of nine patients used for DAB IHC staining. Vectafluor Excel anti-mouse 488 (ready-to-use; cat# VEDK2488, Vector Laboratories, Burlingame, CA, USA) and Alexa Fluor anti-rabbit 594 (1:500; cat# A21207, Life Technologies, Carlsbad, CA, USA) were utilized to detect the combinations. All IF IHC-stained slides were mounted in Vecta Shield Hardset mounting medium with 4′,6′-diamino-2-phenylindole (Vector Laboratories).
Positive control tissues used for the primary antibodies were human placenta for cathepsin B; human breast carcinoma for cathepsin D; mouse bone marrow for cathepsin G; and human seminoma for OCT4, as previously described (21). A negative LMCA control sample was prepared for DAB IHC staining by using an IgG isotype control (ready-to-use; cat# IR600, Dako, Santa Clara, CA, USA). For IF IHC staining, a negative control was performed using a section of LMCA tissue with the combined use of primary isotype mouse (ready-to-use; cat# IR750, Dako, Copenhagen, Denmark) and rabbit (read-to-use; cat# IR600, Dako) antibodies. All antibodies were diluted with Bond primary antibody diluent (cat# AR9352, Leica), and DAB and IF IHC staining was carried out on the Leica Bond Rx autostainer, as previously described (12).
DAB IHC-stained slides were viewed using an Olympus BX53 microscope and photographed with an Olympus DP21 digital camera (Olympus, Tokyo, Japan). IF IHC-stained slides were viewed and imaged using an Olympus FV1200 biological confocal laser-scanning microscope and processed with cellSens Dimension 1.11 software using 2D deconvolution algorithm (Olympus).
Cell Counting and Statistical Analyses
Using the Olympus BX53 light microscope fitted with an Olympus DP21 digital camera, cell counting was performed using ImageJ (https://imagej.net/Welcome) on six fields of view of each DAB IHC-stained slide of the nine LMCA samples at 400x magnification. Fields of view were selected from regions that exhibited the highest density of staining. The proportion of cells within the TNs and the PTS stained positively in each field of view was calculated and the results were subjected to t-tests for related samples, using SPSS v.22 statistical package.
NanoString mRNA Expression Analysis
RNA extracted from six snap-frozen LMCA samples from the same cohort of nine patients used for DAB IHC staining, was analyzed using NanoString nCounter™ Gene Expression Assay (NanoString Technologies, Seattle, WA, USA), as previously described (12). Probes for the genes encoding for cathepsin B (NM_001908.2), cathepsin D (NM_001909.3), cathepsin G (NM_001911.2), and the housekeeping gene GUSB (NM_000181.1) were used in the analysis. Raw data was analyzed by nSolverTM software (NanoString Technologies) using standard settings and results were normalized against the housekeeping gene, GUSB, and subjected to t-tests for related samples, using SPSS v.22 statistical package, to determine the relative abundance of each cathepsin to each other.
Total protein was extracted and precipitated from three snap-frozen LMCA samples of the original cohort of nine patients included for DAB IHC staining, separated by SDS-PAGE and transferred to a PVDF membrane using previously described methods (30). Detection of the proteins was performed on the iBind Flex (cat# SLF2000, Thermo Fisher Scientific) using the primary antibodies cathepsin B (1:250; cat# SC-6490-R, Santa Cruz), cathepsin D (1:250; cat# SC-6486, Santa Cruz), cathepsin G (1:250; cat# ab197354, Abcam, Cambridge, UK), and β-actin (1:500; cat# ab8226, Abcam). Appropriate secondary antibodies were goat anti-rabbit Alexa Fluor 647 (1:2000; cat# A21244, Life Technologies) for cathepsin B, chicken anti-goat Alexa Fluor 647 (1:2000; cat# A21469, Life Technologies) for cathepsin D, goat anti-mouse Alexa Fluor 488 (1:2000; cat# A21202, Life Technologies) for β-actin, and goat anti-rabbit HRP (1:2000; cat# ab6721, Abcam) for cathepsin G. Clarity Western ECL (cat# 1705061, Bio-Rad) was used as the substrate for visualizing HRP detected protein bands and the Chemi Doc MP Imaging System (Bio-Rad) and Image Lab 5.0 software (Bio-Rad) were used for band detection and analysis. All experiments were performed in triplicates. Snap-frozen tonsillar tissue was used as control tissue for cathepsin B and cathepsin D with a recombinant cathepsin G protein (cat# H00001511-Q01, Novus Biologicals, Littleton, CO, USA) as the control for cathepsin G. Matched mouse (1:500; cat# ab18443, Abcam) and rabbit (1:500; cat# ab171870, Abcam) isotype controls were used as appropriate negative controls.
Enzymatic Activity Assays
Enzymatic activities of cathepsin B and cathepsin D were determined in snap-frozen LMCA samples from the same three patients used in WB, using enzymatic activity assay kits for cathepsin B (cat# ab65300; Abcam) and cathepsin D (cat# ab65302; Abcam), as previously described (21). Fluorescence was measured in a NuncTM F96 MicroWellTM black polystyrene plate (cat# 136101, Thermo Fisher Scientific) using the Varioskan Flash plate reader (cat# MIB5250030, Thermo Fisher Scientific). Tonsil and denatured tonsil tissue lysates were used as appropriate positive and negative controls, respectively. All experiments were performed in duplicates with averages taken.
Histochemical and DAB IHC Staining
H&E staining of 4 μm-thick formalin-fixed paraffin-embedded sections of all nine LMCA samples confirmed the presence of the tumor on the slide by an anatomical pathologist (HDB). DAB IHC staining showed cytoplasmic expression of cathepsin B (Figure 1A, brown) by cells predominantly within the TNs (arrows) and some cells within the PTS (arrowheads). Granular cytoplasmic staining of cathepsin D (Figure 1B, brown) was also localized to cells within the TNs (arrows) and some cells within the PTS (arrowheads). Cytoplasmic expression of cathepsin G (Figure 1C, brown) was localized to a few cells within the PTS (arrowheads). Interestingly there was minimal staining for cathepsin B (Figure S1A, brown), cathepsin D (Figure S1B, brown) and cathepsin G (Figure S1C, brown) on the adjacent normal hepatocytes.
Figure 1. Representative 3,3-diaminobenzidine immunohistochemical-stained sections of liver metastasis from colon adenocarcinoma demonstrating cytoplasmic expression of cathepsin B [(A), brown] within the tumor nests (TNs, arrows) and the peritumoral stroma (PTS, arrowheads). Granular cytoplasmic staining of cathepsin D [(B), brown] was localized to cells within the TNs (arrows) and those within the PTS. Cytoplasmic expression of cathepsin G [(C), brown] was demonstrated in cells within the PTS (arrowheads). Nuclei were counter-stained with hematoxylin [(A-C), blue]. Original magnification: 400×.
Positive controls for cathepsin B (Figure S1D, brown), cathepsin D (Figure S1E, brown) and cathepsin G (Figure S1F, brown) demonstrated the expected staining patterns in human placenta, breast carcinoma and mouse bone marrow, respectively. The negative control demonstrated no staining (Figure S1G).
IF IHC Staining
IF IHC staining was performed on three representative LMCA samples from the original cohort of nine patients used for DAB IHC staining, to determine the expression of cathepsins B, D and G in relation to the CSC subpopulations, previously identified. Interestingly, cathepsin B was expressed by cells within the TNs (Figure 2A, red, arrows) and the OCT4+ cells within the PTS (Figure 2A, green, arrowheads). Similarly, cathepsin D was expressed by the OCT4− cells within the TNs (Figure 2B, red, arrows) and the OCT4+ CSCs within the PTS (Figure 2B, green, arrowheads). This was in contrast to cathepsin G which was localized to a few cells within the PTS (Figure 2C, red, arrows), separate from the OCT4+ subpopulation (Figure 2C, green, arrowheads). Images of the individual stains are presented in Figure S2. Minimal staining was present on the negative control (Figure S2G), confirming the specificity of the primary antibodies used.
Figure 2. Representative immunofluorescence immunohistochemical-stained sections of liver metastasis from colon adenocarcinoma demonstrating expression of cathepsin B [(A, D), red] by cells within the TNs (arrows) and the OCT4+ cells within the peritumoral stroma [(A, D), green, arrowheads]. Cathepsin D [(B, E), red] was similarly expressed by cells within the TNs (arrows) and the OCT4+ CSC subpopulation [(B, E), green, arrowheads]. Cathepsin G [(C, F), red] was localized to a few cells within the PTS (arrows), separate from the OCT4+ subpopulation (C, F, green, arrowheads). All slides were counter-stained with 4',6'-diamino-2-phenylindole. A-C: scale bars: 20 µm. D, E, F: magnified view of A, B, C, respectively.
Cell counting on DAB IHC-stained slides for cathepsins B, D and G (Figure 3A) in the nine samples of LMCA demonstrated significantly greater expression of cathepsin B by the cells within the TNs compared to those within the PTS (t = 4.384, p = 0.007). There was no significant difference between cells stained positively within the TNs and those within the PTS for cathepsin D (p = 0.589) and cathepsin G (p = 0.927).
Figure 3. Cell counting of 3,3-diaminobenzidine immunohistochemical-stained slides of liver metastasis from colon adenocarcinoma demonstrating cells within the tumor nests (TNs) and peri-tumoral stroma (PTS) stained positively for cathepsins B, D and G. There was statistical difference in the abundance between the cells within the TNs and those within the PTS (**p < 0.001) (A). Average relative expression of cathepsins B, D, and G mRNA transcripts in samples of colorectal adenocarcinoma metastasis to the liver from six patients, normalized over housekeeping gene GUSB and presented as relative units (*p < 0.05) (B).
NanoString mRNA expression analysis confirmed the presence of mRNA transcripts for cathepsin B, D and G relative to the housekeeping gene GUSB in all six snap-frozen LMCA samples (Figure 3B). Statistical analysis of the NanoString data demonstrated that there was no significant difference between the mean level of expression of cathepsin B compared to that of cathepsin D (p = 0.345). However, the mean level of expression of cathepsin B (p = 0.028) and the mean level of expression of cathepsin D (p = 0.028) were significant greater than that of cathepsin G.
WB of the three snap-frozen LMCA samples demonstrated the presence of bands at the expected molecular weight for cathepsin B (Figure 4A) and cathepsin D (Figure 4B). Cathepsin B was detected at the appropriate molecular weight of 25 kDa in all three LMCA samples. Cathepsin D was detected at the corresponding molecular weight of 28 kDa in all three LMCA samples. Cathepsin G was not detected in any of the LMCA tissue samples at the expected molecular weight of 29 kDa (Figure 4C), with specificity of the antibody confirmed in the positive control, which demonstrated detection of a band at the expected molecular weight of 37 kDa, consistent with the manufacturer's product information. β-actin (Figure 4D) confirmed approximate equivalent protein loading for all LMCA samples examined. The tonsillar tissue confirmed specificity for cathepsin B and cathepsin D. The recombinant cathepsin G protein confirmed specificity for cathepsin G. The rabbit and mouse IgG isotype negative controls demonstrated minimal staining, as expected (Figure S3A,B ). The complete WB for cathepsins B, D and G are provided (Figure S3C-D).
Figure 4. Representative Western blot images of total protein extracted from samples of liver metastasis from colon adenocarcinoma from three patients demonstrating the presence of cathepsin B (A) and cathepsin D (B), but not cathepsin G (C). β-actin was used as a housekeeping protein (D).
To determine the functional activity of both cathepsin B and cathepsin D, which were detected by WB, we performed enzymatic activity analysis on the same three snap-frozen LMCA tissue samples, for both cathepsins. All the LMCA samples demonstrated enzymatic activity of cathepsin B and cathepsin D relative to that of the positive and negative control tissues (Figure 5). This provides evidence that these cathepsins are functional.
Figure 5. Enzymatic activity assays showed both cathepsin B and cathepsin D were active in all three samples of liver metastasis from colon adenocarcinoma relative to the positive and negative controls. Their activity is expressed as relative fluorescent unit (RFU).
The expression of ESC markers in CRC has been well documented and various combinations of markers have been reported. OCT4, SOX2, NANOG, Krüppel-like factor 4 (KLF4) and c-MYC play a significant role in triggering pluripotency in somatic cells, and they have been used to identify CSC subpopulations within various types of cancer (31). CD44, LGR5 and EpCAM are markers that are of particular relevance to CRC (11). Furthermore, CD133+ cells that have been isolated from primary colon cancer and liver metastases, have been shown to be capable of initiating tumor growth in immunodeficient mice (32).
The self-renewing capacity of CSCs and their persistence despite conventional therapy offers a possible explanation for tumor recurrence and metastasis (33). CSCs are able to invade and metastasize by acquiring an epithelial-mesenchymal transition (EMT) phenotype, which can be determined by examining the expression of E-cadherin and vimentin (34). Several signaling pathways are known to promote stem cell maintenance and induce EMT (33). In particular, Wnt/β-catenin signaling has been reported to significantly influence the regulation of growth and maintenance of CRC CSCs (34). Similarly, the Notch signaling pathway is thought to contribute to colon CSC viability, tumorigenicity and self-renewal (33).
Our previous characterization of three putative CSC subpopulations within LMCA led us to propose the existence of a hierarchy of CSCs within this tumor (12). Interestingly, IF IHC staining showed the expression of cathepsin B and cathepsin D by the OCT4+ CSC subpopulation within the PTS of the LMCA samples we have examined. However, cathepsin G was localized to a separate OCT4− subpopulation.
In vitro and in vivo studies have demonstrated the involvement of the RAS in proliferative signaling, evasion of growth suppressors, resistance of cell death, angiogenesis, reprogramming of energy metabolism, inflammation, cell migration, invasion and metastasis (35). The expression of components of the RAS by these CSCs, suggests the presence of a paracrine RAS involved in the regulation of CSCs (19). Furthermore, ACE inhibitors and ATIIR1 antagonists, which inhibit ATII production and action respectively, appear to exert inhibitory effects on cancer progression, vascularization and metastasis (36).
We have previously reported the expression of cathepsin B and cathepsin D by CSC subpopulations within oral tongue SCC (21) and IDHWGB (20), suggesting the presence of bypass loops for the RAS. To the best of our knowledge, this is the first report demonstrating the expression of cathepsin B and cathepsin D, and possibly cathepsin G in LMCA, suggesting the presence of RAS bypass loops.
DAB IHC staining and WB confirmed protein expression of cathepsin B and cathepsin D and the enzyme activity assays demonstrated the functional activity of these cathepsins. IF IHC staining showed the expression of cathepsin B and cathepsin D to the CSCs within the TNs and the PTS. Cathepsin G was localized to only a few cells within the PTS and it is not surprising that it was below detectable levels on WB. The expression of cathepsins B, D and G was confirmed at the transcriptional level with NanoString mRNA expression analysis. This indicates more abundant transcript expression of cathepsin B and cathepsin D, compare to that of cathepsin G, which was detected at low levels.
Cathepsin B can directly or indirectly degrade ECM in CRC by stimulating other proteases or by blocking their inhibitors. In vivo studies have demonstrated inhibition of cathepsin B by selective compounds leads to in reduced liver metastases by up to 60% and reduced liver metastases burden up to 80% (6).
Overexpression of cathepsin D has been associated with many types of cancers such as gastric carcinoma (37), melanoma (38), ovarian cancer (39) and breast cancer (40). It has been postulated that cathepsin D directly promotes tumor growth by degrading and remodeling the basement membrane and the stroma surrounding the tumor, and also acts indirectly by stimulating other enzymes or in conjunction with other cathepsins (2). Kirana et al (2). have demonstrated increased cathepsin D expression in cells from the main tumor body in late stage CRC to be significantly associated with subsequent distant metastasis and reduced cancer-specific survival.
Both cathepsin B and cathepsin D have been shown to be involved in ECM degradation in CRC, and levels of these cathepsins and their activity have been reported to be increased in the invasion edge of CRC (6). The expression of cathepsin B and cathepsin D by the CSC subpopulations within LMCA suggest these CSC subpopulations may be a therapeutic target. Further investigation into the expression of the RAS by these CSC subpopulations may underscore an effective targeted treatment strategy for this cancer.
This report presents novel finding of the expression of cathepsin B, cathepsin D, and possibly cathepsin G by the putative CSC subpopulations within LMCA.
Data from this study consists of a relatively small sample size, although it provides the foundation for future larger studies.
Further in vitro and in vivo data is required to validate the functional role of these cathepsins within LMCA.
Detection and functional analysis of components of the RAS in relation to CSCs in LMCA is required to underscore how these CSCs can be most effectively targeted.
This study was carried out with the approval of the Central Health and Disability Ethics Committee (ref. no. 13/CEN/130) with written informed consent from all subjects in accordance with the Declaration of Helsinki.
TI and ST formulated the study hypothesis. TI, SW and ST designed the study. SW, SM, HB, ST and TI interpreted the DAB and IF IHC data. SW, ST and TI interpreted the NanoString mRNA data. SM performed cell counting on DAB IHC stained slides. BVS performed WB and enzymatic activity assays. RM conducted statistical analysis and interpreted the results. SM, ST and TI drafted the manuscript. All authors commented on and approved the manuscript.
TI and STT are inventors of the PCT patent applications Cancer Diagnosis and Therapy (No. PCT/NZ2015/050108), and Cancer Therapeutic (PCT/NZ2018/050006). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Ms. Liz Jones of the Gillies McIndoe Research Institute for her assistance in IHC staining. This work is supported by the Lloyd Morrison Colon Cancer Research Programme.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fsurg.2018.00040/full#supplementary-material
Figure S1. 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining of normal liver adjacent to liver metastasis from colon adenocarcinoma (LMCA) showing minimal staining of hepatocytes for cathepsin B (A, brown), cathepsin D (B, brown) and cathepsin G (C, brown). Positive controls of DAB IHC staining for cathepsin B (D, brown), cathepsin D (E, brown) and cathepsin G (F, brown) demonstrating the expected staining patterns in human placenta, breast cancer and bone marrow, respectively. The negative control (G) of a LMCA section demonstrated no staining. Nuclei were counter-stained with hematoxylin (A-G, blue). Original magnification: 400x.
Figure S2. Split images of immunofluorescence immunohistochemical-stained sections of colon adenocarcinoma metastasis to the liver (LMCA) shown in Figure 2 demonstrating co-expression of cathepsin B (A, red) with OCT4 (B, green), cathepsin D (C, red) and OCT4 (D, green), cathepsin G (E, red) and OCT4 (F, green). A negative control (G) to test the specificity of the fluorescent secondary antibodies is performed on a section of LMCA. Cell nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (A-G, blue). Scale bars: 20 µm.
Figure S3. Western blots of matched IgG isotype negative controls with a fluorescent secondary antibody confirming specificity for the cathepsin B and cathepsin D bands with β-actin as a housekeeping protein (A), and with a HRP-conjugated secondary confirming non-specific binding for the 50 kDa bands present in the cathepsin G blot (B). Complete western blot images for cathepsin B (blue) and β-actin (green) (C), cathepsin D (D) and cathepsin G (E).
1. Zarour LR, Anand S, Billingsley KG, Bisson WH, Cercek A, Clarke MF, et al. Colorectal cancer liver metastasis: evolving paradigms and future directions. Cell Mol Gastroenterol Hepatol (2017) 3(2):163–73. doi: 10.1016/j.jcmgh.2017.01.006
2. Kirana C, Shi H, Laing E, Hood K, Miller R, Bethwaite P, et al. Cathepsin D Expression in Colorectal Cancer: From Proteomic Discovery through Validation Using Western Blotting, Immunohistochemistry, and Tissue Microarrays. Int J Proteomics (2012) 2012:245819:1–10. doi: 10.1155/2012/245819
3. Fearon ER. Molecular genetics of colorectal cancer. Annu Rev Pathol (2011) 6:479–507. doi: 10.1146/annurev-pathol-011110-130235
4. Fanali C, Lucchetti D, Farina M, Corbi M, Cufino V, Cittadini A, et al. Cancer stem cells in colorectal cancer from pathogenesis to therapy: controversies and perspectives. World J Gastroenterol (2014) 20(4):923–42. doi: 10.3748/wjg.v20.i4.923
5. Fleming M, Ravula S, Tatishchev SF, Wang HL. Colorectal carcinoma: Pathologic aspects. J Gastrointestinal Oncol (2012) 3(3):153–73.
6. Nadal C, Maurel J, Gascon P. Is there a genetic signature for liver metastasis in colorectal cancer? World J Gastroenterol (2007) 13(44):5832–44. doi: 10.3748/wjg.v13.i44.5832
7. Liu LX, Zhang WH, Jiang HC. Current treatment for liver metastases from colorectal cancer. World J Gastroenterol (2003) 9(2):193–200. doi: 10.3748/wjg.v9.i2.193
8. Misiakos EP, Karidis NP, Kouraklis G. Current treatment for colorectal liver metastases. World J Gastroenterol (2011) 17(36):4067–75. doi: 10.3748/wjg.v17.i36.4067
9. Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, et al. Cancer stem cells-perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res (2006) 66(19):9339–44. doi: 10.1158/0008-5472.CAN-06-3126
10. Allegra A, Alonci A, Penna G, Innao V, Gerace D, Rotondo F, et al. The cancer stem cell hypothesis: a guide to potential molecular targets. Cancer Invest (2014) 32(9):470–95. doi: 10.3109/07357907.2014.958231
11. Munro MJ, Wickremesekera SK, Peng L, Tan ST, Itinteang T. Cancer stem cells in colorectal cancer: a review. J Clin Pathol (2018) 71(2):110–6. doi: 10.1136/jclinpath-2017-204739
12. Humphries HN, Wickremesekera SK, Marsh RW, Brasch HD, Mehrotra S, Tan ST, et al. Characterization of cancer stem cells in colon adenocarcinoma metastasis to the liver. Front Surg (2017) 4:76. doi: 10.3389/fsurg.2017.00076
13. George AJ, Thomas WG, Hannan RD. The renin-angiotensin system and cancer: old dog, new tricks. Nat Rev Cancer (2010) 10(11):745–59. doi: 10.1038/nrc2945
14. Ager EI, Neo J, Christophi C. The renin-angiotensin system and malignancy. Carcinogenesis (2008) 29(9):1675–84. doi: 10.1093/carcin/bgn171
15. Bradshaw AR, Wickremesekera AC, Brasch HD, Chibnall AM, Davis PF, Tan ST, et al. Glioblastoma multiforme cancer stem cells express components of the renin-angiotensin system. Front Surg (2016) 3:51. doi: 10.3389/fsurg.2016.00051
16. Itinteang T, Dunne JC, Chibnall AM, Brasch HD, Davis PF, Tan ST. Cancer stem cells in moderately differentiated oral tongue squamous cell carcinoma express components of the renin-angiotensin system. J Clin Pathol (2016) 69(10):942–5. doi: 10.1136/jclinpath-2016-203736
17. Featherston T, Yu HH, Dunne JC, Chibnall AM, Brasch HD, Davis PF, et al. Cancer stem cells in moderately differentiated buccal mucosal squamous cell carcinoma express components of the renin-angiotensin system. Front Surg (2016) 3:52. doi: 10.3389/fsurg.2016.00052
18. Ram RS, Brasch HD, Dunne JC, Davis PF, Tan ST, Itinteang T. Cancer stem cells in moderately differentiated lip squamous cell carcinoma express components of the renin-angiotensin system. Front Surg (2017) 4:30. doi: 10.3389/fsurg.2017.00030
19. Munro MJ, Wickremesekera AC, Davis PF, Marsh R, Tan ST, Itinteang T. Renin-angiotensin system and cancer: A review. Integr Cancer Sci Therap (2017) 4(2):1–6.
20. Koh SP, Wickremesekera AC, Brasch HD, Marsh R, Tan ST, Itinteang T. Expression of cathepsins B, D. and G in isocitrate dehydrogenase-wildtype glioblastoma. frontiers in surgery (2017) 4:28.
21. Featherston T, Marsh RW, van Schaijik B, Brasch HD, Tan ST, Itinteang T. Expression and localization of cathepsins B, D, and G in Two cancer stem cell subpopulations in moderately differentiated oral tongue squamous cell carcinoma. Front Med (2017) 4:100. doi: 10.3389/fmed.2017.00100
22. Neves FA, Duncan KG, Baxter JD. Cathepsin B is a prorenin processing enzyme. Hypertension (1996) 27(3 Pt 2):514–7. doi: 10.1161/01.HYP.27.3.514
23. Naseem RH, Hedegard W, Henry TD, Lessard J, Sutter K, Katz SA. Plasma cathepsin D isoforms and their active metabolites increase after myocardial infarction and contribute to plasma renin activity. Basic Res Cardiol (2005) 100(2):139–46. doi: 10.1007/s00395-004-0499-3
24. Rykl J, Thiemann J, Kurzawski S, Pohl T, Gobom J, Zidek W, et al. Renal cathepsin G and angiotensin II generation. J Hypertens (2006) 24(9):1797–807. doi: 10.1097/01.hjh.0000242404.91332.be
25. Kawabata T, Nishimura Y, Higaki M, Kato K. Purification and processing of rat liver procathepsin B. J Biochem (1993) 113(3):389–94. doi: 10.1093/oxfordjournals.jbchem.a124056
26. Beckman M, Freeman C, Parish CR, Small DH. Activation of cathepsin D by glycosaminoglycans. Febs J (2009) 276(24):7343–52. doi: 10.1111/j.1742-4658.2009.07444.x
27. Korkmaz B, Moreau T, Gauthier F, Elastase N. Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions. Biochimie (2008) 90(2):227–42. doi: 10.1016/j.biochi.2007.10.009
28. Korkmaz B, Horwitz MS, Jenne DE, Gauthier F, Elastase N. Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacol Rev (2010) 62(4):726–59. doi: 10.1124/pr.110.002733
29. Kuester D, Lippert H, Roessner A, Krueger S. The cathepsin family and their role in colorectal cancer. Pathol Res Pract (2008) 204(7):491–500. doi: 10.1016/j.prp.2008.04.010
30. Tan K, Brasch HD, van Schaijik B, Armstrong JR, Marsh RW, Davis PF, et al. Expression and localization of cathepsins B, D, and G in dupuytren's disease. Plast Reconstr Surg Glob Open (2018) 6(2):e1686. doi: 10.1097/GOX.0000000000001686
31. van Schaijik B, Davis PF, Wickremesekera AC, Tan ST, Itinteang T. Subcellular localisation of the stem cell markers OCT4, SOX2, NANOG, KLF4 and c-MYC in cancer: a review. J Clin Pathol (2018) 71(1):88–91. doi: 10.1136/jclinpath-2017-204815
32. O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature (2007) 445(7123):106–10. doi: 10.1038/nature05372
33. Puglisi MA, Tesori V, Lattanzi W, Gasbarrini GB, Gasbarrini A. Colon cancer stem cells: controversies and perspectives. World J Gastroenterol (2013) 19(20):2997–3006. doi: 10.3748/wjg.v19.i20.2997
34. Roy S, Majumdar AP. Cancer Stem Cells in Colorectal Cancer: Genetic and Epigenetic Changes. J Stem Cell Res Ther (2012) Suppl 7(6):1–6. doi: 10.4172/2157-7633.S7-006
35. Wegman-Ostrosky T, Soto-Reyes E, Vidal-Millán S, Sánchez-Corona J. The renin-angiotensin system meets the hallmarks of cancer. J Renin Angiotensin Aldosterone Syst (2015) 16(2):227–33. doi: 10.1177/1470320313496858
36. Deshayes F, Nahmias C. Angiotensin receptors: a new role in cancer? Trends Endocrinol Metab (2005) 16(7):293–9. doi: 10.1016/j.tem.2005.07.009
37. Allgayer H, Babic R, Grützner KU, Beyer BC, Tarabichi A, Wilhelm Schildberg F, et al. An immunohistochemical assessment of cathepsin D in gastric carcinoma: its impact on clinical prognosis. Cancer (1997) 80(2):179–87.
PubMed Abstract | Google Scholar
38. Bartenjev I, Rudolf Z, Stabuc B, Vrhovec I, Perkovic T, Kansky A. Cathepsin D expression in early cutaneous malignant melanoma. Int J Dermatol (2000) 39(8):599–602. doi: 10.1046/j.1365-4362.2000.00025.x
39. Lösch A, Schindl M, Kohlberger P, Lahodny J, Breitenecker G, Horvat R, et al. Cathepsin D in ovarian cancer: prognostic value and correlation with p53 expression and microvessel density. Gynecol Oncol (2004) 92(2):545–52. doi: 10.1016/j.ygyno.2003.11.016
40. Abbott DE, Margaryan NV, Jeruss JS, Khan S, Kaklamani V, Winchester DJ, et al. Reevaluating cathepsin D as a biomarker for breast cancer: serum activity levels versus histopathology. Cancer Biol Ther (2010) 9(1):23–30. doi: 10.4161/cbt.9.1.10378
Keywords: colorectal cancer, cathepsin, cancer stem cells, liver, metastasis, renin-angiotensin system, bypass loops
Citation: Mehrotra S, Wickremesekera SK, Brasch HD, Van Schaijik B, Marsh RW, Tan ST and Itinteang T (2018). Expression and Localization of Cathepsins B, D and G in Cancer Stem Cells in Liver Metastasis From Colon Adenocarcinoma. Front. Surg. 5:40. doi: 10.3389/fsurg.2018.00040
Received: 30 January 2018; Accepted: 30 April 2018;
Zongbing You, Tulane University, United States
Daotai Nie, Southern Illinois University School of Medicine, United States
Benyi Li, University of Kansas Medical Center, United States
Copyright © 2018 Mehrotra, Wickremesekera, Brasch, Van Schaijik, Marsh, Tan and Itinteang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Swee T. Tan, swee.tan@gmri.org.nz


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Ghostly Theorosa Bridge
Valley Center, Kansas
About 12 miles north of Wichita, Kansas sits the small town of Valley Center. Now having a population of a little more than 5,000 residents, the settlement was born in 1872 on the banks of the Little Arkansas River. Located along the ever important railroad, the town grew to include a post office, three stores, two blacksmith shops, a grain elevator, and several homes by the early 1880's.
Though now a bedroom community to nearby Wichita commuters, one of biggest attractions in this quiet town is that of the haunted Theorosa Bridge, also known as the 109th Street Bridge and the Crybaby Bridge.
On an old country road three miles north of town on Meridian Street, this old bridge spans Jester Creek at the intersection with 109thstreet.
There are several legends about the haunting of this bridge all based on a baby drowned in the creek below. Today, the bridge is a simple concrete bridge that normally no one would pay any attention to, except for the legends.
The first legend tells of settlers who were passing through the area in the late 19th century when they were attacked by Indians and a baby named Theorosa was kidnapped. Her grief-stricken mother was said to have left the wagon train to search for her missing daughter and reportedly continues to look today as her mournful cry can still be heard calling out for her child.
Another legend has it that a skirmish between the cavalry and an Indian tribe living by the creek occurred about this time. In this version, an Indian woman is stabbed and her baby is dropped into the creek and drowned.
Theorosa's Bridge. Photo by Beth Golay/KMUW
Yet another tale suggests that a woman named Theorosa is a young woman who has an illegitimate baby and drowns it in Jester Creek to hide her shame. Later, overcome by guilt, she drowns herself in the creek as well.
Finally, another tells of an engaged woman who fell in love with another man and bore his child. Jealous with rage, her fiancé reportedly through the baby over the bridge into the creek and Theorosa jumped off the bridge to save her baby, but drowned herself, instead.
In any event, the bridge has reported to have been haunted for years and years. Many have reported seeing floating balls of light, eerie shapes, and the apparition of a woman in the area around the bridge. Cars are said to mysteriously stall as they cross, or if they should stop, will feel the entire vehicle begin to shake. Others report cold breezes which seemingly come from nowhere, and the sounds of mournful voices or the chilling cries of a baby.
Yet others say that the weather is consistently different at the bridge than it is in the rest of the area. Rumor has it that if you call out to Theorosa, telling her that you have her baby, she'll come out of the water and attack you.
The original iron and wood bridge that first stood at Jester Creek for decades burned down in 1974, was rebuilt, and was destroyed by fire again in 1976. Afterwards, it was closed for the next fifteen years. However, in 1991, the road was reopened and the current concrete bridge was built that continues to serve travelers across the creek.
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By Hiring an Attorney With More Than Three Decades of Experience in Family Law, You Will Get Qualified, Aggressive and Reliable Representation in Court!
The primary things people are concerned about when they get divorced are how they are going to afford to live and what will happen to their children. As a lawyer, I work with my clients to determine their goals and expectations, working diligently to make sure they have the best possible outcome from their divorce. Going through a divorce is one of the most difficult times in a person's life. If you are entering into a divorce or any other family law dispute, I will provide you the professional and compassionate guidance and representation you will need.
I have seen many people through this tough time, and I understand what you are going through. I am not only an experienced attorney, but also a certified mediator. Couples can come to me before matters escalate to a divorce trial, and mediate their differences in a more informal setting, where they have more input and control of their outcome. This can be accomplished through various methods of alternate dispute resolution, including attending independent mediation, utilizing collaborative lawyers, or attending a structured settlement conference after your dissolution case has been filed.
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I was born in Guadalajara Mexico and raised in Oakland California. My seminary formation took me across borders from Tijuana Mexico to San Antonio Texas to Rome Italy. Ministry to people in prison and ministry to those struggling with addictions are very life giving to me as well as ministering to Spanish speaking communities. MY ministry has included parish work, shrine ministry, preaching workshops and retreat ministry. Fr. Salvador Gonzalez, OMI is the current director of the national Shrine of Our Lady of the Snows and has been ministering in the Diocese of Belleville for nine years.
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Raul Salas, OMI, born in Brownsville, TX. I am 69 years old and ordained 42 yrs. I am the oldest of 5 children. My ministry has been primarily formation, high school minor seminary for 16 yrs, 11 years Shrine ministry at the Shrine of Our Lady of the Snows, 12 years in formation of our Scholastic seminarians (those seminarians in the final years of theology and preparation for final vows or ordination), and 3 years as a parochial vicar in a small parish in Texas. I am presently director of Hispanic Ministry at the Shrine of Our Lady of the Snows plus availability for other ministerial duties. While it is a scary part of ministry for me, it is the task of preaching. I feel it is a great responsibility to announce the Good News of God's saving action and to witness the hope and joy it brings to people.
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Mark Dean, OMI, a Missionary Oblate of Mary Immaculate, is currently on staff at King's House Retreat and Renewal Center in Belleville, IL, and will soon be moving to the National Shrine of our Lady of the Snows to continue work in retreat ministry. Previously he has (among a variety of assignments) served as a formator to Oblate seminarians, pastor to Sweden's northern-most parish, and chaplain to both university students and men incarcerated. Being the third born of five sons, Mark enjoys the privileges of being a middle child, and standing in the middle, along with Virtue.
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Fr. Elvis Ngosa Mwamba, I am an Oblate originally from Africa – delegation of Zambia. I was ordained in 2018, July the 7th. My first assignment was ministry at Our Lady of the Snows Shrine in Belleville a ministry I never thought I will do. I have found so much joy in the ministry I do as a Pilgrims Director. One thing that stands out in my ministry is journeying with people of different faith and listening to their stories, with a compassionate and listening heart and finding answers in their situation and courageously help them to go out and be carries of the good news to all.
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Most of his years as an Oblate missionary have involved him in parish ministry, serving together both Anglo and Hispanic parishioners in locations such as Lowell, MA, Los Angeles, CA, Eagle Pass, TX among others. He values the call to recognize and collaborate with the charisms and gifts of the laity in today's church and pursue the struggles for justice in our society. Prioritizing God's poor with their many faces is both a challenging and fulfilling dimension in Fr. Jim's Oblate ministry.
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Eva Cassidy: The Story at The Edinburgh Festival Fringe
The Amphitheatre of theSpace @ Symposium Hall features a magical combination of the music of Eva Cassidy with the voice of Elsa Jean McTaggart as she pays a heartfelt musical homage to her favourite artist.
Elsa Jean McTaggart
Many Fringe audiences will already know Elsa Jean McTaggart from her popular traditional Scottish themed shows, and also perhaps from last year's launch of this show at Fringe 2017. This show, although still featuring many of Eva Cassidy's iconic songs, is a new show with some changes to the set list based on audience requests from last year's show. Of course, there are some Eva Cassidy songs/arrangements that simply have to be in a show like this, and 'Fields of Gold' and 'Over The Rainbow' are two of them. There are a few surprises in this set list too, and as I know Elsa has been working hard on these new songs, I am not spoiling the surprise for you; go and see the show.
Eva Cassidy was a unique and distinctive voice in music, and Elsa Jean McTaggart is also a unique and distinctive voice, and although the two are definitely not the same, I can think of few singers better vocally suited than Elsa to be doing an Eva Cassidy themed show. I am deliberately avoiding labelling this as a 'tribute show' as it is definitely not one in the normal sense of the genre. Elsa Jean McTaggart is making no attempt to be 'Eva Cassidy', but like so often Eva herself, Elsa is singing songs that she loves to sing. This show is a tribute to Eva Cassidy in the best and most honest way – through her musical legacy. (Tom King)
Tickets: https://www.thespaceuk.com/shows/2018/
Festiva Fringe


With an automatic gate, you have higher security and greater convenience when entering and exiting your property. Our job at "Gate Repair Brea" is to ensure that your gate performs reliably and safely. That is why we offer a full array of services for such systems from new gate installation to maintenance and repair. Our solutions cover each and every component of the system including the opener and the intercom. When required, count on us to provide full emergency assistance in the fastest possible way. Our company works with all types of gates from swing and sliding units to overhead models. The make, brand and year of manufacturing are irrelevant. Expect to receive servicing of the highest quality.
When an issue occurs, entrust us with its resolution. You can have complete confidence in our expert knowledge and skills and in our swift response as well. You will receive the required service within the shortest time. Over the years, we have developed a highly effective approach to the repair of gates and it is implemented strictly by all of our technicians in California. It begins with thorough inspection and testing for identifying the cause of the problem. Once this is done, the best repair technique is used to eliminate the particular problem. Both our techniques and our equipment are technologically advanced. This enables us to achieve the perfect result quickly. Count on us to repair gate motors and hardware components such as tracks, wheels, and hinges equally well.
One of the major factors behind the popularity of our service is that we pay close attention to details. When swing gate hinges are fixed, for instance, our technician will clean them and lubricate them to ensure that they work smoothly and quietly at all times. It is our commitment to take all required steps for achieving perfection. When a component is beyond repair, it is replaced with a new one of the same specifications. Our company is always fully stocked on replacement components including gate remotes. They are hand-picked to ensure that each one is of the highest quality.
Since you most certainly use your electric gate on a daily basis, it should be easy for you to notice any troubling signs. When this happens, you should get in touch with our gate repair company in Brea right away to ensure that small malfunction will not turn into a huge problem. We are readily available to answer all of your questions and to help you without delay.
* Why is my sliding gate making popping sounds? This shows that the rollers have become extensively worn out. Our technician will inspect the system to confirm the cause of the problem and will replace the old components with new matching ones which are strong and durable.
* How come the electric gate is not closing? This issue is usually due to faulty safety sensors. Less often, the problem is will the opener. In any case, the malfunctioning device will be repaired promptly. If it is beyond repair, it will be replaced.
Hire us at Gate Repair Brea for installing a new gate and opener for you when the time comes. Our service includes full assistance throughout the comparison shopping process. The gate will be installed with precision. The opener will be set securely in place and all of its settings will be adjusted. Count on us for the intercom programming as well.
Do you need assistance with your gate today? Our company offers same day emergency gate repair services in zip code 92821. With expertise in the newest openers for residences in California, our technicians utilize their knowledge to fix electric gates and guarantee safety.
Looking for a reliable gate service provider in Orange County? Look no further! Our company offers a wide selection of services from driveway gate installation to welding hinge repair. Count on us for same day services, and for fast response in emergencies.