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In the final analysis, there are three reasons why people go to war; self-defense, greed, and religion. Of the three, self-defense is the only morally justified reason for engaging in war because, obviously, if you are defending yourself against a foreign aggressor you did not start the hostilities and are only fighting to save yourself and your fellow citizens from the wrongful actions of an attacking nation. War truly is a curse upon mankind not only because innocent people are killed, but also because of the senseless destruction and the needless squandering of natural resources which inevitably occurs. In today's world this last consequence is especially poignant because population levels have put a strain on our natural resources and environment. Clearly, any nation which wages war out of greed or religion is a pox on mankind and its leaders are war criminals who should be treated accordingly.
The United States of America was the greatest nation in all of history and the reasons for this are no accident. We were a nation conceived in liberty and born of revolution in keeping with the governing principles subscribe to by our Founding Fathers. It was adherence to these principles which forged the nation we once had. The past tense is appropriate to describe our situation because the nation we used to have no longer exists. It has gone the way of the dodo bird because not one governing principle subscribed to and considered inviolate by our Founding Fathers is functional or operative. And this conclusion can be reached with conviction and absolute certainty. We are a nation who has lost its way and the best illustration of this is that we use war as tool of diplomacy and a means to spread a non-existent democracy throughout the world. How has all this come to pass?
The founding fathers feared the uneducated and ignorant masses. Because of this fear they gave us a republic instead of a democracy. In a democracy, the masses rule and mob psychology prevails. In a republic established by a written constitution, the rule of law is paramount and the rights of individuals trump the predilections and wiles of the majority. A republic is the government given to us by the Founders and it is this kind of government which we have lost. And this came to pass because somewhere along the line because our leaders stopped believing we should be governed according to the ideals and principles embodied within our constitution and instead, be governed for their personal benefit while keeping us ignorant of what is really going on. This is where we are today. Our leaders lie to us all the time and no longer fear the uneducated and ignorant masses because they can control and manipulate our perceptions of the world through the media. What our Founding Fathers feared most is now a valued tool used by those who rule us. How has all this come to pass?
Capitalism is the goose that laid the golden egg for Americans. It provided us with a bounty envied by the rest of the world and all of us take great pride in being American primarily because of our economic success. However, while capitalism is an ideal economic system for a nation to have to provide for the distribution of goods and services, it is the worst possible system to have when capitalists control government. The purpose of government is to level the playing field for all concerned. The goal of a capitalist is to seek advantage in the market place. These two goals are at cross purposes to each other and when capitalist get the upper hand over government it becomes corrupt and serves the needs of corporations instead of the needs of society. This is exactly what happened in the United States and this is why everything that could possible go wrong with America has come to pass. Our government is now so corrupt that we are no longer citizens but instead, subjects where we have no rights and corporate interests rule the day. How did all of this come to pass?
In 1913, while no one was paying attention, Congress created the Federal Reserve System and gave it the responsibility of printing money and regulating our economy. At first glance, this sounds well and good, but nothing could be further from the truth because the Federal Reserve System is controlled by the Federal Reserve Bank; a private corporation whose owners and principles are a mystery to us. In other words, by creating the Federal Reserve System, Congress turned over our economic well-being to a group of unknown insiders and because these people were empowered to loan us our own money at interest, they became so rich and powerful that they now dominate and control our government. Therefore, upon passage of the Federal Reserve Act, our republic died and our government became an oligarchy run by behind the scenes power brokers who have the final say in everything. The Republic died, democracy died, freedom died and Americans would never again have a say in how we are governed even though we are still allowed to vote for either one of the two puppets who we wrongfully think of as being our President.
In conclusion, the only solution to our problem is to rid ourselves of the Federal Reserve Bank and this can be accomplished by repealing the Federal Reserve Act. Repealing this despicable and ignoble act is simply a matter of getting enough members of Congress to support this action. However, in consideration of how corrupt and self-serving Congress has become, our present situation is such that it is far easier for an elephant to go through the eye of a needle than it is to get a Congressman to vote for something of which would be so beneficial to us. This is especially true when you consider that such a vote constitutes a vote against the people who rule and control the world.

domain: sociology


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[Frontiers in Bioscience 3, a11-15, February 1, 1998]|
TARGETED INHIBITION OF HEPATITIS B VIRUS GENE EXPRESSION: A GENE THERAPY APPROACH
Frank Y.-T. Tung and Steven W. Bowen
Department of Infectious Disease and Microbiology, University of Pittsburgh, Pittsburgh PA 15261
Received 1/21/98 Accepted 1/28/98
In this report, we describe a potential gene therapy approach for persistent HBV infection. Antisense RNAs of the HBV surface antigen gene expressed from a retroviral vector can effectively inhibit HBsAg expression by 50-75%. The advantage of using long antisense RNA (0.8 kilobases in this case) to inhibit the target sequence is that variant viruses should also be inhibited by antisense RNA due to complementation to the target sequence. Many HBV variants have been identified and shown to contribute to differences in severity of disease (33). Recently, Yu et al, (34) reported a ribozyme-mediated inhibition of HBsAg by co-transfection assay which is similar to this paper. In that report, high molar ratio (at least >5) of ribozyme construct to HBV expression construct was used to achieve 80% inhibition by measuring endogenous polymerase activity. Due to a different HBV expression construct being used, the efficiency of inhibition can not be compared directly. However, it would be difficult to deliver high copy number of ribozyme to liver cells in practice.
The unique features of retroviral vectors for gene transfer are that they can provide long-term gene expression and are non-immunogenic to the host immune system. To achieve long lasting anti-HBV effect, the antisense transduced cells have to stably express antisesnse RNA in chronic HBV patients. Using current ex vivo gene transfer protocol, only a small percentage of hepatocytes can be transduced with a retroviral vector. However, this small percentage of antisense transduced liver cells may have a selective advantage over unprotected HBV infected cells as the result of immune-mediated damage. With the regenerating ability of normal hepatocytes, the antisense RNA protected cells may dominate the cell population in the liver.
In our assay system, the multiple copies of HBV genome still can enter the antisense transduced cells by co-transfection. Therefore, the anti-viral effect may be more significant in natural HBV infection. In that case, fewer copies of replicative RNA intermediates and/or viral mRNA are present in the infected cells as the targets of antisense RNA.

domain: literature


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Although, technically the movie Stand By Me was released in 1986, it wasn’t until March 1987 that it was released here in the UK. So despite being a year too late to celebrate its actual 25th Anniversary, I still wanted to celebrate it by doing a blog post of twenty-five years since its release here.
I was only 9 years old when this movie was released but still remember the first time I saw it. It was and still is one of my all time favorite movies to watch, it is one of those movies that has become timeless over the years, and is one of those movies that I grew up with, and still enjoy watching it even today. I have been a Stephen King fan for a number of years and have read the short-story ‘The Body’, that this movie was based upon.
If you haven’t seen this movie (where have you been the last 25 Years?) – The movie has now been released on a Special Edition Blu-Ray that can be purchased for £7.49 from Amazon.co.uk
The disc includes the following features:
- Blu-ray Exclusive: A video reunion with Director Rob Reiner and Actors Wil Wheaton & Corey Feldman for the first time in 25 years
- An audio commentary with Reiner
- Documentary featurette
- Stand By Me music video featuring River Phoenix and Wil Wheaton.
The Movie’s Synopsis
The story follows four 12-year old boys Gordie Lachance (Wil Wheaton), Chris Chambers (River Phoenix), Vern Tessio (Jerry O’Connell) and Teddy Duchamp (Corey Feldman), as they travel on a journey through the woods to find the dead body of the kid Ray Brower (Kent W. Luttrell). It is narrated by Richard Dreyfuss, who is supposed to be Gordie Lachance telling of his childhood story after he reads a newspaper article about the death of his friend.
Based in the fictional town of Castle Rock, Oregon over Labor Day weekend in September of 1959, young Gordie, a quiet boy renowned for telling stories and writing, is rejected by his father, following the death of his older brother Denny, who was like his best friend and who paid him much more attention than his parents ever did.
Gordie spends time with his school friends Chris Chambers and Teddy Duchamp in their tree-house when one of his other friends Vern Tessio tells each of them about a conversation he overheard, his brother Billy telling his friend about finding the body of Ray Brower while dumping a stolen car. The disappearance of the boy had mounted the largest police search that Castle Rock had ever seen and had been big news on the radio. Gordie, Chris, Teddy and Vern decide to embark upon a journey to see if they can find Ray’s body and become local heroes.
The boys set out, first encountering Milo Pressman and his dog Chopper when they pause to fill their canteens and take a break. Gordie is selected to go an get the food for the journey from the local store, but returns to find his friends have scarpered and realizes that Chopper is about to chase him. As he makes a run for it, his friends have already jumped the junkyard fence and are encouraging young Gordie. After which they begin to tease Chopper with the famous line from the story ‘Chopper Sick Balls’, Milo Pressman the junkyard owner is not best pleased.
They then walk along the train tracks and encounter a long train bridge, and decide to cross over here rather than going round. Gordie is anxious that a train might come around the corner at any moment. As Vern is concerned that he might fall decides to cross the bridge on his hands and knees, suddenly they see a train coming and begin to run for it. The boys have a close shave with the train.
The boys make camp and Gordie tells his friends a story of a boy who enters a pie eating contest to get back at the town folk who are always teasing him because of his size and weight, a story invented by Gordie. Later Chris reveals to Gordie he is tired of everyone singling him out because of his family always being involved in crime, and never making anything of himself. He confesses to Gordie that he did in fact take the milk money at school, but decided to give it back, but then it turns out one of the teachers stole the money and bought a new skirt with it.
They finally locate the body of Ray Brower and it reminds Gordie that his father liked his brother better than him. It is at this point the local bully “Ace” played by Keifer Sutherland and his gang consisting of Chris’s brother “Eyeball” Chambers, Vince Desjardins, Charlie Hogan, Billy Tessio and two other boys show up to take the body. Gordie threatens “Ace” with a handgun that Chris had brought with them for protection. Gordie decides no gang should get the credit for finding the kid Ray Brower and wants it to be reported anonymously to the authorities.
The boys return to town and say goodbye to each other. The narrator states that Vern later marries out of high school, had four children and became a forklift driver at the local lumber yard. Teddy tried to get in to the Army, but his eyes and ear kept him out. He eventually spent some time in jail and is now doing some odd jobs around Castle Rock. Chris was able to stick it out and took some advanced courses with Gordie, he later moved out of Castle Rock and became a lawyer. Chris who was always wanting to keep the best piece got in to a fight in a fast food restaurant, one of the men pulled a knife, and was stabbed, he died almost instantly.
The ending scene sees a father finishing off the memoirs and takes his son and his friend swimming.
I didn’t want this article to become one of my normal ‘Where are they now…’ posts. I wanted it to be a special post that focuses on a movie that I think everyone should see at least once in their lives.
I am going to focus on the four main characters of the movie, and only give a brief account of where they are now.
Gordie Lachance – Played by Wil Wheaton.
First of all, is Gordie the main character that this story is based around. It is supposed to be a memoir account of his childhood, so lets take a look at what Wil Wheaton has been up to since this movie finished filming.
Wil was born Richard William Wheaton III on July 29, 1972 in Burbank, California. He had a few movies prior to filming Stand By Me in 1986 and has go on to have a constant stream of acting and voice jobs since.
His most recent acting role is, as Dr Isaac Parrish in the television series ‘A Town Called Eureka’ and as Colin Mason in the television series ‘Leverage’.
In the recent reunion of the cast Wil gives an account of what it was like to see the cast members 25 years later.
He met with director Rob Reiner and co-stars Jerry O’Connell and Corey Feldman, and of course, there was an obvious absence in the room.
There were five chairs set up for us in a semi-cicle. Or names were on pieces of paper so we knew where to sit. I was between Rob and Corey, and Jerry and Richard sat to Corey’s left. When we all sat down, Rob looked down the row of seats and softly said to me, “it feels like there should be an empty seat here for River”.
People ask me about River all the time. He and I were close during filming, and for about a year or so after filming, but the sad truth is that he got sucked into a lifestyle that I just don’t have room in my life for, and we drifted apart. When he died, I was shocked and horrified, but I wasn’t completely surprised. I didn’t feel a real sense of loss at the time – but the River I knew and loved had been gone for a long time at that point – but I felt sad for his family, and angry at the people around him who didn’t do more to help him, to help himself. Since he died, when I’ve talked about him, I’ve felt like I’m talking about the idea of him, instead of the person I knew, if that makes sense.
But when Rob said that to me, with such sadness in his eyes, it was like I’d been punched in the stomach by eighteen years of suppressed grief. I knew that if I tried to say anything, all I would do was cry, and I didn’t know if I’d be able to stop. I took a deep breath, swallowed hard, and nodded. “Yeah” I whispered.
Born River Jude Phoenix, August 23, 1970 died at the age of 23 years old on October 31, 1993. He was the oldest brother of fellow actors Rain, Joaquin, Libtery and Summer Phoenix.
His real name was River Jude Bottom and was born in Madras, Oregon. The first child of Arlyn Sharon Dunetz and John Lee Bottom. He was named after the River of Life from the Hermann Hesse novel Siddhartha and received his middle name from The Beatles song ‘Hey Jude’.
It was after moving in with their maternal grandparents in Florida, that on April 02, 1979 the family officially changed their name to Phoenix, after the mythical bird that rises from its own ashes, symbolizing a new beginning.
River’s work covers some twenty-four films and television appearances, including that of the science fiction film Explorers and the coming-of-age film Stand By Me.
River died of drug-induced heart failure on the sidewalk outside the West Hollywood nightclub The Viper Room. He had been in the middle of filming the unreleased film Dark Blood at the time of his death.
Vern Tassio – Played by Jerry O’Connell
Born Jeremiah O’Connell, February 17, 1974 in New York City. Is best know for his roles in the television series Sliders (another of my favorites – that I haven’t seen in such a long time), My Secret Identity and of course as Vern Tessio in the film Stand By Me.
He began his acting career at a very young age. As a child he did commercial work for Duncan Hines Cookies. He was eleven years old when he landed his first feature film role as the character Vern Tessio in Rob Reiner’s Stand By Me.
Jerry has gone on to have acting roles in many television series including, The Defenders, Fish Hooks and most recently NTSF:SD:SUV.
Teddy Duchamp – Played by Corey Feldman
Born Corey Scott Feldman on July 16, 1971 in Los Angeles, California. The second of five children of Sheila Goldstein and Bob Feldman. He has an older sister Mindy, a yonger sister Brittnie and two younger brothers, Eden and Devin.
Corey was well known for his roles throughout the 1980’s in Hollywood films Friday the 13th: the Final Chapter, The Goonies, Stand by Me, The Lost Boys, License to Drive, Dream a Little Dream, Gremlins, Teenage Mutant Ninja Turtles and The Burbs.
Corey has gone on to star in television series, Psych and Easy to Assemble. He currently has several movies due for release in 2012, including Six Degrees of Hell (Completed), Zombex (Post-Production) and The Zombie King (Post-Production).
And finally… a couple of videos for you to enjoy:
The Original ‘Stand By Me’ Trailer
A Scene From the Movie
The Original ‘Stand By Me’Music Video- by Ben E. King
The ‘Stand By Me’ song – by John Lennon

domain: entertainment


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California’s San Mateo County is “seeking nominations for poet laureate, someone who can act as an ambassador for literary arts.” Do you have what it takes?
Slate has translated famous first lines of literature into emojis, and they’re surprisingly coherent. Pair with Jonathan Russell Clark‘s essay on opening sentences.
How many Millions readers reside in Miami? All of you should come check out this USpeak event hosted by the University of Miami’s Creative Writing Program. Students will read verse and short stories, and visiting professor Patricia Engel (author of Vida) will read as well. You can also pick up a copy of Mangrove, the university’s undergraduate literary journal.
Perhaps best known for her fiction, specifically her classic The Group, Mary McCarthy became a novelist almost by chance. “McCarthy was good at recycling – a term which she used herself – and good, also, as she admitted, at plagiarizing her own life. Nevertheless, her fiction lives, and some of it has been highly influential.” Margaret Drabble takes us through McCarthy’s major works of fiction, featured in Mary McCarthy: The Complete Fiction which was released this year in a deluxe collection for the very first time.

domain: library


The Lamentable Sonata
from album (The Red Jewel) by Tartaros
Let us empty the porcelain jars Light up the candelairs Orchestra! Play the lamentable sonata Scribe..
from album (Album Unknown) by Michael Nesmith
[Verse 1] Everybody see's it's you I'm the one that lost the view Everybody says we'..
When You're Old And Grey
from album (Tom Lehrer Revisited) by Tom Lehrer
The most po***r type of po***r song is of course the love song, and I'd like to illustrate sev..
Video not found
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With the first of their three album trilogy out into the world, Cold War Kids stopped by ‘The Late Show with Stephen Colbert’ to drop a song from New Age Norms 1 the other night. Performing the song “Complainer,” the band proved they haven’t lost their shine while giving their fans an idea of what to expect from their current tour.
Utilizing multiple screens with the imagery of the album, the band did what they do best on the show. The live performance felt well rehearsed and conveyed the song perfectly. Embracing a new era in the group, the live glimpse had plenty of energy and bespoke craftsmanship. Never ones to sit still, the band played with passion and weight.
The video can be viewed on YouTube or above. New Age Norms 1 is available on multiple formats directly from the band. Cold War Kids are on tour tonight in San Diego, CA at North Park Theatre until February 15 in Austin, TX at Stubb’s. The band’s complete tour routing can be accessed here.
Image Credits: Photo Courtesy of Big Hassle.


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Laguna Beach Books Meet The Author: Dr. Willie Parker Thursday January 18 2018.
Laguna Beach Books Meet The Author is at 6:00pm.
In Life’s Work, an outspoken, Christian reproductive justice advocate and abortion provider (one of the few doctors to provide such services to women in Mississippi and Alabama) pulls from his personal and professional journeys as well as the scientific training he received as a doctor to reveal how he came to believe, unequivocally, that helping women in need, without judgment, is precisely the Christian thing to do.
Laguna Beach Books Parking
Laguna Beach Books is located at 1200 South Coast Highway.
There is metered timed street parking in the area.
There is also garage token parking behind the book store.
Please ask Laguna Beach Books for a token before you leave.


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Members of the fibroblast growth factor (FGF) family and the FGF receptors (FGFRs) have been implicated in mediating various aspects of mammary gland development and transformation. To elucidate the molecular mechanisms of FGFR1 action in a context that mimics polarized epithelial cells, we have developed an in vitro three-dimensional HC11 mouse mammary epithelial cell culture model expressing a drug-inducible FGFR1 (iFGFR1). Using this conditional model, iFGFR1 activation in these growth-arrested and polarized mammary acini initially led to reinitiation of cell proliferation, increased survival of luminal cells, and loss of cell polarity, resulting in the disruption of acinar structures characterized by the absence of an empty lumen. iFGFR1 activation also resulted in a gain of invasive properties and the induction of matrix metalloproteinase 3 (MMP-3), causing the cleavage of E-cadherin and increased expression of smooth muscle actin and vimentin. The addition of a pan MMP inhibitor abolished these phenotypes but did not prevent the effects of iFGFR1 on cell proliferation or survival.
The pathways through which FGFs and their receptors signal appear to play important roles not only in normal development and wound healing but also in tumor formation and progression (Powers et al., 2000). To date, 22 distinct FGFs have been discovered that can act in an autocrine or paracrine fashion through a class of cell-surface tyrosine kinase receptors (RTKs; Grose and Dickson, 2005). The ligand binding capacity and tissue distribution of each receptor is highly variable (Dailey et al., 2005).
Several studies have now linked inappropriate expression of FGFs and their receptors to the development of human breast cancer (Dickson et al., 2000). For example, in some breast cancers, FGF receptors (FGFRs) 1, 2, and 4 are amplified (Penault-Llorca et al., 1995). Thus, a better understanding of the role of FGF signaling during breast cancer progression may facilitate the development of cancer therapies to target this signaling pathway.
The diversity and complexity of FGF ligand and receptor interactions makes it difficult to delineate the molecular mechanisms of action of individual FGF ligands or receptors, which determine a specific cellular response. Thus, an inducible FGFR (iFGFR) was developed as a ligand-independent dimerization and activation system to study the early events in FGFR signaling and transformation (Welm et al., 2002). In mouse mammary tumor virus–iFGFR1 transgenic mice, acute induction of iFGFR1 results in increased lateral budding of the mammary ductal epithelium and sustained activation induces alveolar hyperplasia and invasive lesions, which are associated with ECM remodeling and vascular branching in the stroma adjacent to these lesions. These data suggest a potential role for FGFs in initiating branching in the mammary gland and in restructuring the stroma to provide support for the developing ductal network. In addition, these results indicate that FGFR1 signaling may play a critical role in early mammary tumor progression.
To date, many of the studies of FGF signaling have been performed in 3T3 fibroblasts in two-dimensional (2D) culture systems, and it was not possible to study the effects of this pathway in a polarized architecture that mimics acinar structures of mammary epithelial cells in vivo. Unlike monolayer cultures, mammary epithelial cells grown in three dimensions recapitulate several features of mammary epithelium in vivo (Schmeichel and Bissell, 2003; Shaw et al., 2004). Therefore, the three-dimensional (3D) culture system has been used as a unique approach to investigate the signaling pathways involved in the early progression of cancer (Muthuswamy et al., 2001; Radisky et al., 2001; Debnath et al., 2003b).
Several human and mouse mammary epithelial cell lines have been shown to form acinar structures when grown in a basement membrane matrix (Shaw et al., 2004). In this paper, we show for the first time that another extensively used immortalized murine mammary epithelial cell line, HC11, can form acinus-like structures containing a single layer of polarized, growth-arrested cells when grown within a growth factor–reduced matrix. HC11 cells are a clonal mouse mammary epithelial cell line derived from a midpregnant mouse mammary gland. Upon hormonal stimulation, these cells differentiate and express milk proteins in vitro (Ball et al., 1988). We therefore combined the well-defined synthetic chemical ligand–induced dimerization strategy and HC11 3D cell cultures to establish a new model system to help dissect the molecular mechanisms through which iFGFR1 activation disrupts the polarized architecture of acinar units in the mammary gland and promotes an invasive phenotype.
Using this model system, we demonstrate that iFGFR1 activation rapidly disrupted cell polarity, induced proliferation, and promoted cell survival. In addition, matrix metalloproteinase 3 (MMP-3) expression was induced and appears to play a critical role during an epithelial-to-mesenchymal transition (EMT) characterized by cleavage of E-cadherin, expression of mesenchymal markers, and cell invasion. Importantly, our data suggest that increased MMP expression by iFGFR1 was critical for EMT but not for regulation of cell survival or proliferation. Furthermore, small interfering RNA (siRNA) targeted against MMP-3, but not -9, blocked cleavage of E-cadherin. These studies describe the first-time use of an inducible dimerization model to study FGFR1 signaling in an HC11 3D culture system.
culture of HC11 mammary epithelial cells
We generated 3D acinar structures by plating single HC11 cells on the matrix. At day 6, immunostaining and confocal imaging for phosphorylated histone H3 (p-histone H3), a marker of mitosis; α6 integrin, a basal surface marker (Fig. 1 A); and phospho-ezrin/radixin/moesin (p-ERM), an apical marker (Fig. 1 B), indicated that the structures consisted of unpolarized proliferative cells. We did not observe the staining of cleaved caspase 3, an apoptosis marker, at day 6 (unpublished data). At day 8, apical–basal polarization appeared within the cell clusters before lumen formation. The absence of p-histone H3 staining (Fig. 1 C) and the lack of increase in the size of the acini after day 8 suggested that the acini had reached a growth-arrested state. Immunostaining for α6 integrin and cleaved caspase 3 (Fig. 1 E) suggested that the acini consisted of two populations of cells: a polarized outer layer of cells that directly contacted the matrix and survived and an inner cluster of apoptotic and poorly polarized cells that did not contact the matrix. Additionally, HC11 acini displayed other aspects of polarity, such as basal deposition of laminin V, a basement membrane component that was detected by immunostaining with an anti–laminin V antibody (Fig. 1 F), and enrichment of p-ERM at the apical surface (Fig. 1 D). After 10–12 d in culture, the majority of the acini contained 15–40 polarized cells surrounding an empty lumen. Polarization of the cells was confirmed by immunostaining for α6 integrin and p-ERM (Fig. 1 G) and laminin V (Fig. 1 H). These results suggest that HC11 cells grown in three dimensions recapitulate several features of mammary epithelial cells in vivo, providing the structural and functional context necessary for examining the biological roles of genes during early-stage tumorigenesis.
iFGFR1 activation disrupts HC11 acinar morphology
To examine the downstream effects of iFGFR1 activation in HC11 3D cultures, we generated stable cell lines expressing iFGFR1. Two clones expressing different levels of iFGFR1 as determined by immunoblotting for expression of HA-tagged iFGFR1 (R1-high [R1-H] and R1-low [RI-L]) were chosen for further analysis (Fig. 2 A). To determine whether dimerization of iFGFR1 in the two clones activates downstream signaling pathways, we analyzed whether iFGFR1 induction could activate extracellular regulated kinase (ERK) in 2D cultures. On AP20187 induction, both clones showed sustained activation of ERK, which correlated with the level of iFGFR1 expression (Fig. 2, B and C). This result indicated that both clones transduce signals downstream of iFGFR1 to activate ERK. Cell cycle analysis was performed on the clones grown in two dimensions to determine whether the AP20187-induced iFGFR1 signaling pathway could also stimulate a biological response, such as proliferation. For this experiment, the clones were treated with AP20187 in serum-free medium, and cellular DNA content was determined by flow cytometry of propidium iodide–stained cells. Both clones showed a greater than twofold increase in the number of cells in S phase in response to AP20187 (unpublished data), which was consistent with previous studies using HC11 cells transiently transduced with iFGFR1 (Welm et al., 2002). These results suggested that dimerization of iFGFR1 in the two clones was sufficient to stimulate a biological response.
To examine the effects of iFGFR1 activation in preformed polarized HC11 acini, single cells of each clone were seeded on the matrix and allowed to grow for 10 d to form growth-arrested acini. When grown on the matrix in the absence of AP20187, the HC11 clones expressing iFGFR1 displayed the same growth and morphogenesis properties as nontransduced HC11 cells (unpublished data). After 10 d, growth-arrested polarized acini had formed and AP20187 were added to the medium for 5 d. Activation of iFGFR1 induced striking changes in the morphology of the acinar structures (Fig. 2 D). In contrast to the untreated iFGFR1 acini, >80% of the treated acini displayed dramatically increased acinar size (∼10-fold larger) and an invasive phenotype. Immunostaining with the anti–p-histone H3 antibody and confocal analyses of these structures demonstrated that untreated iFGFR1 acini remained growth arrested, whereas proliferation was reinitiated in the treated iFGFR1 acini that now contained lumens that were completely filled with cells (Fig. 2 E). Although the R1-H and -L clones had similar proliferation properties (not depicted), their invasive behaviors correlated with their iFGFR1 expression level. In a few acini (5% or less), cells dissociated and invaded the matrix after 24 h of AP20187 treatment. In 80–90% of the treated acini, a progressive architectural disruption occurred, and the acini displayed an invasive phenotype after 5 d of treatment. 5 d after stimulation, the R1-L clone had individual cells that dissociated from the acini and invaded the matrix, whereas the R1-H clone had long projections containing multiple cells that invaded the matrix (Fig. 2 D). The cells in these projections continued to proliferate (as determined by anti–p-histone H3 immunostaining) and invade the matrix, which was observed after 5 d of treatment (Fig. 2 E, arrow). Together, these results demonstrate that although proliferation in response to iFGFR activation was similar in the two clones, the severity of the invasive phenotype correlated with the level of FGFR1 expression.
iFGFR1 activation disrupts polarity, prevents luminal apoptosis, reinitiates proliferation, and promotes invasion in HC11 acini
Our previous study of iFGFR1 transgenic mice showed that iFGFR1 activation prevented apoptosis, induced proliferation, and altered cell polarity in mammary epithelial cells. Furthermore, iFGFR1-induced lesions have invasive characteristics (Welm et al., 2002). To examine whether iFGFR1 homodimers were able to activate downstream signaling and recapitulate some phenotypes of transgenic mice in polarized, growth-arrested 3D acini, iFGFR1 acini were stimulated with AP20187 for 24 h or 5 d, and cell polarity, proliferation, and apoptosis were monitored by immunostaining with the appropriate markers.
The serine/threonine protein kinase Akt (protein kinase B), a regulator of cellular proliferation and apoptosis (Scheid and Woodgett, 2001), is a downstream target of FGF signaling. To examine whether Akt signaling was activated on iFGFR1 induction in 3D cultures, we performed immunostaining on treated and untreated iFGFR1 acini using a phosphospecific Akt antibody that recognized an activation-specific phosphorylation site (anti–p-Akt Ser 473; Fig. 3 A, a and b). The untreated acini exhibited a random pattern of Akt activation only in the outer layer cells that directly contacted the matrix, whereas the treated structures displayed significantly increased levels of activated Akt present in both the luminal cells and the outer layer cells. This result suggests that the iFGFR1 homodimer is able to activate a physiological FGF signaling pathway in 3D cultures.
To determine whether iFGFR1 activation could reinitiate proliferation in 3D cultures, we stimulated day 10 iFGFR1 acini with AP20187 for 24 h or 5 d and then immunostained them with anti–p-histone H3 antibody to detect mitotic cells. The untreated iFGFR1 acini rarely displayed positive staining of p-histone H3 (Fig. 3, A [c] and B [c]). 24 h after stimulation, an approximately eightfold increase in the number of cells staining positive for p-histone H3 was observed in the treated iFGFR1 acini compared with untreated acini (Fig. 3, A [d] and C). This increase in proliferation was still detectable after 5 d of treatment, when we observed an approximately fourfold increase compared with untreated acini (Fig. 3, B [d] and C). These data suggest that iFGFR1 activation has the ability to reinitiate cell proliferation in the growth-arrested HC11 acini. Negative feedback mechanisms involving Sprouty, Sef, and Cbl (Hanafusa et al., 2002; Wong et al., 2002; Kovalenko et al., 2003) may attenuate iFGFR1 signaling after 5 d of treatment, leading to reduced cell proliferation.
To test whether iFGFR1 activation was able to disrupt cell polarity in the polarized acini, we performed immunostaining on iFGFR1 acini with antibodies against the polarity markers α6 integrin (Fig. 3, A [c and d] and B [c and d]) and p-ERM (Fig. 3 B, a and b). After 24 h of treatment, cell polarity within the acini was rapidly disrupted because α6 integrin staining was no longer limited to the basal-lateral region as seen in untreated acini. In addition, the enriched apical localization of p-ERM was also disrupted and was now detected around the entire periphery of the cells (unpublished data). This disruption of cell polarity persisted after 5 d of treatment. Thus, iFGFR1 activation resulted in a sustained loss of cell polarity.
To determine whether iFGFR1 activation could promote cell survival in the acini, apoptosis was analyzed by immunostaining with an anti–cleaved caspase 3 antibody. In comparison with the untreated acini, after 5 d of treatment, a dramatic decrease in the number of cells staining positive for cleaved caspase 3 was detected in the luminal cells (Fig. 3 B, e and f). The increased cell proliferation and inhibition of cell death appears to account for the increased size and filled lumens of treated iFGFR1 acini. Therefore, iFGFR1 activation has the ability to reinitiate cell proliferation and disrupt cell polarity in these growth-arrested polarized HC11 acini, suggesting that it can be used to investigate the early stages of tumor progression induced by iFGFR1 signaling.
iFGFR1 activation leads to dissociation of E-cadherin and β-catenin from the cell membrane and expression of mesenchymal markers
The invasive behavior of the cells upon iFGFR1 dimerization suggested that iFGFR1 signaling might affect molecules involved in cell–cell adhesion. To examine whether iFGFR1 activation disrupts membrane localization of E-cadherin and β-catenin, day 10 iFGFR1 acini were stimulated with AP20187 for 5 d and immunostained with anti–E-cadherin and anti–β-catenin antibodies (Fig. 4, A and B). Untreated iFGFR1 acini exhibited well-defined cell–cell junctions as indicated by membrane localization of E-cadherin and β-catenin, which were concentrated toward the basal-lateral region of the membrane. In the acini with iFGFR1 activation, diffusely localized E-cadherin and β-catenin were detected as indicated by diminished and punctate staining of both proteins. This finding demonstrated that localization of these adhesive molecules to the cell–cell junctions was lost in the architecturally disrupted acini.
FGFR1 has been suggested to be involved in EMT during mouse mesodermal development (Ciruna et al., 1997; Xu et al., 1999), and mislocalization and loss of E-cadherin at cell–cell junctions is an early event that is essential for cell invasion and EMT (Bracke et al., 1996; Bates and Mercurio, 2005). To investigate whether iFGFR1 activation can promote EMT in the HC11 3D culture system, day 10 iFGFR1 acini treated for 5 d with AP20187 were immunostained with antibodies to detect expression of mesenchymal markers such as smooth muscle actin (SMA) and vimentin (Fig. 4 C, b and c). In contrast to the untreated acini that did not express these markers (Fig. 4 C, a), SMA and vimentin expression were readily detectable in the iFGFR1-activated acini. This result, together with the acquisition of the invasive phenotype and loss of E-cadherin expression at cell–cell junctions, suggested that iFGFR1 activation results in EMT.
iFGFR1 activation alters the motility of monolayer HC11 cells
Disruption of cadherin-mediated adhesion has been suggested to affect the movement of epithelial monolayers (Owens et al., 2000; Wrobel et al., 2004). Because we also observed loss of membrane localization of E-cadherin upon iFGFR1 activation in HC11 cells in 2D cultures (unpublished data), we investigated whether activation of iFGFR1 affected the motility of HC11 monolayer cells. For this experiment, we examined the ability of iFGFR1-expressing HC11 cells in monolayer to break their intercellular contacts and move freely out of the epithelial sheet to repair a wound in the monolayer in response to AP20187 treatment (Fig. 5 A). After 20 h, cells expressing iFGFR1 did not migrate without AP20187 treatment. The R1-L cells displayed a slower level of migration into the wound space, whereas the R1-H cells migrated into the wound space rapidly after stimulation and nearly closed the wound in 20 h. To further confirm this result, a quantitative transwell migration assay was performed. Although both R1-L and -H cells migrated better than untreated cells, ∼2.5 times more migrating cells were detected in the R1-H clone than in the R1-L clone (Fig. 5 B). Therefore, the level of the motility of iFGFR1 stable clones correlated with the expression level of the iFGFR1 gene.
iFGFR1 activation induces MMP-3 expression and cleavage of E-cadherin
FGF signaling has been reported to induce MMP-3 expression (Pintucci et al., 2003), and MMP-3 overexpression has been reported to result in E-cadherin cleavage and EMT (Lochter et al., 1997). We therefore examined the expression level of MMP-3 after iFGFR1 activation by quantitative RT-PCR (Fig. 6 A). A significant increase in MMP-3 expression was observed after 24 h of iFGFR1 activation in HC11 monolayer cultures. A similar increase in MMP-9 expression was also detected (unpublished data). Interestingly, although induction of MMP-3 and -9 expression was observed in both the R1-H and -L clones, the level of the induction correlated with the iFGFR1 gene expression level (unpublished data).
To test whether iFGFR1 activation resulted in cleavage of E-cadherin, cell lysates and conditioned medium were collected from iFGFR1 clones in monolayer culture after 4 d of AP20187 treatment. Immunoblotting using antibodies against E-cadherin on the cell lysates revealed a reduction in the level of full-length E-cadherin (120 kD) expression after treatment (Fig. 6 B, a). A 40-kD fragment of E-cadherin was observed in the cell extract (Fig. 6 B, b), whereas an 80-kD cleaved product of E-cadherin was detected in the conditioned medium after treatment (Fig. 6 B, c). Total ERK was shown to indicate equal protein loading from the cell extracts (Fig. 6 B, d), and Ponceau S staining confirmed equal loading of the proteins from conditioned medium (not depicted). Because we did not detect down-regulation of E-cadherin expression in response to iFGFR1 activation by quantitative RT-PCR (unpublished data), rapid loss of full-length E-cadherin was probably attributable to proteolytic cleavage.
The iFGFR1-induced EMT phenotype can be blocked by a pan MMP inhibitor and is irreversible
MMPs are known to promote cellular invasion both in vitro and in vivo (Egeblad and Werb, 2002). To examine whether the invasive phenotypes induced by iFGFR1 activation are dependent on MMP activity, 10-d-old iFGFR1 acini were treated with AP20187 in the absence or presence of a pan MMP inhibitor, GM6001, for 5 d. In the AP20187-treated iFGFR1 acini, invasion of cells into the matrix was abolished in the presence of the MMP inhibitor (Fig. 7, A and B). Additionally, immunostaining for E-cadherin revealed that it was maintained at the cell–cell junctions in the GM6001-treated cultures but not in control cultures (Fig. 7 C). To determine whether the EMT phenotype was also prevented, AP20187-treated or AP20187/GM6001-treated iFGFR1 acini were immunostained with the anti-SMA antibody (Fig. 7 D). Inhibition of MMP activity abolished SMA expression in the HC11 acini with activated iFGFR1, suggesting that MMP activity was required for these cells to undergo EMT.
Interestingly, in the presence of AP20187 and GM6001, iFGFR1 acini still displayed increased acinar size and filled lumens. Additionally, immunostaining of AP20187/GM6001-treated iFGFR1 acini with antibodies to detect cell proliferation, apoptosis, and polarity showed results similar to those found in the iFGFR1 acini treated with only AP20187 (unpublished data). These data suggest that the induction of MMP activity is only required for iFGFR1 to induce cell invasion and EMT.
To test whether the EMT-like effect induced by iFGFR1 is reversible, the iFGFR1 acini were treated with AP20187 for 24 h and then AP20187 was removed and the phenotype was evaluated after 5 d. No reduction in the number of acini displaying the invasive phenotype was observed (Fig. 7 F). These acini also showed similar levels of vimentin and SMA staining (unpublished data). This result suggests that the EMT-like effect is not reversible. Interestingly, although dramatically reduced p-ERK was observed by immunoblotting after withdrawal of AP20187 for 24 h (Fig. 7 E), similar elevated levels of MMP-3 and -9 expression were observed by quantitative RT-PCR compared with the cells from which AP20187 was not withdrawn (not depicted). This suggests that there may be a positive feedback loop downstream of iFGFR1 signaling to maintain the expression of target genes even in the absence of continued iFGFR1 activation. The slightly elevated level of p-ERK observed after AP20187 withdrawal as compared with untreated cells may also be sufficient to maintain the induction of downstream target genes accounting for the lack of reversibility of the EMT phenotype.
MMP-3 is essential for E-cadherin cleavage induced by iFGFR1 activation
To determine whether MMP-3 plays an essential role during iFGFR1-induced EMT, we performed a test to see whether inhibition of MMP-3 expression in the iFGFR1 cells affects a critical step of EMT, loss of E-cadherin from cell–cell junctions. To do so, predesigned MMP-3 and -9 siRNA pools of four siRNA duplexes were used. Because a similar EMT effect was observed in both 2D and 3D cultures after iFGFR1 activation, the effects of MMP-3 and -9 siRNA pools were examined in 2D cultures. After siRNA transfection, a significant reduction in MMP-3 and -9 expression was detected in untreated and treated cells, respectively, by quantitative RT-PCR. After AP20187 treatment, MMP-3 siRNA–transfected cells displayed induction of MMP-9 expression but little MMP-3 induction (Fig. 8 A). Similarly, MMP-9 siRNA–transfected cells displayed induction of MMP-3 expression but little MMP-9 induction (unpublished data). This suggests that the MMP siRNAs are specific for their appropriate targets. siGLO, an RNA-induced silencing complex (RISC)–free siRNA, which was used as control, indicated that the transfection efficiency of the siRNA reached almost 90%. Additionally, siGLO-transfected cells displayed induction of both MMP-3 and -9 similar to nontransfected cells.
Interestingly, a significant reduction in the percentage of cells showing punctate staining of E-cadherin was observed in AP20187-treated MMP-3 siRNA–transfected iFGFR1 cells but not in siGLO control or MMP-9 siRNA–transfected R1 cells (Fig. 8, B and C). This suggests that MMP-3, but not -9, is involved in cleavage of E-cadherin downstream of iFGFR1 activation. These data are consistent with the reported function of MMP-3 in the other model systems (Lochter et al., 1997; Noe et al., 2001). More important, they suggest that MMP-3 is the critical downstream target of iFGFR1 activation involved in EMT.
We have previously described the AP20187-dependent iFGFR1 dimerization system that was developed and applied to a transgenic mouse model (Welm et al., 2002). Although a transgenic mouse model is critical for understanding certain aspects of epithelial tumorigenesis, such as the roles of the inflammatory response and angiogenesis, the complexity of the in vivo environment makes it difficult to delineate the molecular mechanisms involved in the early stages of oncogenesis. Therefore, we analyzed the molecular mechanisms of iFGFR1 in 3D cultures of HC11 mouse mammary epithelial cells to mimic the conditions under which iFGFR1 is activated in vivo.
There are two distinct stages of phenotypic progression observed in this 3D culture model: the first stage includes rapid loss of polarity, reinitiation of proliferation, and reduction of luminal cell apoptosis (observed 24 h after iFGFR1 activation). The second stage involves invasion of cells into the surrounding matrix and EMT (observed 4–5 d after iFGFR1 activation; Fig. 9 A).
Although several RTKs, including colony-stimulating factor receptor (CSF-1R), ErbB2, and Met, have been studied in MCF10A 3D cultures (Shaw et al., 2004), the present study is the first time that FGFR1 signaling has been examined in a 3D culture system. In the HC11 model system, iFGFR1 activation is inducible and allows us to study the consequences of iFGFR1 signaling with time on cell proliferation and invasion in the preformed and growth-arrested HC11 acinar structures. The only other example of using this AP20187-inducible system in a 3D culture system is the activation of ErbB2 in MCF10A mammary acini, which resulted in the formation of structures containing multiple acinar units with filled lumens but without invasive properties. Cooperation of the ErbB2 and TGFβ signaling pathways was required for the sustained, elevated activation of ERK and the induction of migration and invasion of MCF10A cells (Muthuswamy et al., 2001; Seton-Rogers et al., 2004). This result suggests that coordination of these two signal transduction pathways is required to promote cell invasion. Thus, it is surprising that FGFR1 signaling can promote both proliferation and invasion in the absence of other oncogenic events, and this may be a unique property of this RTK.
Another RTK, CSF-1R, when co-overexpressed with its ligand in MCF10A 3D cultures, resulted in a cell invasion phenotype similar to that observed after FGFR1 activation characterized by loss of E-cadherin from plasma membrane and release of individual cells into the matrix. However, in this case no reduction in total E-cadherin was observed. Thus, it was suggested that other mechanisms, such as an increased rate of endocytosis or a defect in the recycling of internalized molecules, may be involved in the loss of E-cadherin from the plasma membrane (Wrobel et al., 2004). Therefore, FGFR1 and CSF-1R appear to regulate cell invasion through distinct mechanisms. Although RTKs can activate a similar array of downstream signaling effectors, including ERK and phosphatidylinositol-3 kinase, their phenotypes as determined in 3D cultures appear to be quite distinct (Shaw et al., 2004). The current study of FGFR1 signaling in 3D cultures supports this observation.
To understand how iFGFR1 regulates cell invasion, we first demonstrated that iFGFR1 activation markedly disrupted cell–cell contacts characterized by the loss of E-cadherin and β-catenin at the cell membrane. Adherens junctions represent a powerful invasion suppressor complex in normal mammary as well as other epithelial cells (Herrenknecht et al., 1991; McCrea and Gumbiner, 1991).
Multiple mechanisms of inactivation of the E-cadherin–catenin complex in normal development and tumor formation have been reported, including genetic mutation, gene inactivation through methylation, repression of expression by Snail and other family members, and cleavage by MMPs (Berx et al., 1996; Bracke et al., 1996; Lochter et al., 1997; Ciruna and Rossant, 2001). Although it has been proposed that Snail expression downstream of FGFR1 is required for the normal repression of E-cadherin expression during early embryonic development (Ciruna and Rossant, 2001), we did not detect induction of Snail expression or inhibition of E-cadherin expression by quantitative RT-PCR in our system (unpublished data). This finding suggests that in the HC11 model during cell invasion, loss of E-cadherin is regulated through iFGFR1 by other mechanisms. Interestingly, after iFGFR1 activation, we observed a rapid increase in Twist expression, a known transcriptional repressor of E-cadherin during EMT (Yang et al., 2004). Because Twist has also been suggested to function as a transcriptional activator (Yang et al., 2004), it may be involved in inducing vimentin expression downstream of iFGFR1 signaling.
MMP-3 is one MMP that has been suggested to lead directly or indirectly to the cleavage of E-cadherin (Lochter et al., 1997; Noe et al., 2001) and is involved in promoting EMT and invasion in mammary tumor cells (Sternlicht et al., 1999). It has also been suggested to function through Rac1b and reactive oxygen species to induce expression of Snail and EMT (Radisky et al., 2005). That the effects of iFGFR1 signaling on cell invasion are dependent on MMP-3 activity is supported by several observations. First, quantitative RT-PCR revealed a rapid and dramatic increase in MMP-3 expression on FGFR1 activation, which was followed by E-cadherin cleavage, detected by immunoblotting. Therefore, in the HC11 model system, the decrease in E-cadherin after iFGFR1 activation is not regulated at the transcriptional level, which is dependent on increased Snail expression but is likely the result of cleavage by MMP-3. Second, the introduction of MMP-3 siRNA, not MMP-9 siRNA, into iFGFR1 cells inhibited the cleavage of E-cadherin, thus demonstrating the important role of MMP-3 during iFGFR1-induced EMT.
In iFGFR1-activated acini, the induction of SMA and vimentin expression was also detected. Expression of these mesenchymal markers together with the loss of E-cadherin and β-catenin from cell–cell contacts as well as the invasive behavior of the cells suggest that the HC11 mammary epithelial cells were converted from an epithelial to a mesenchymal phenotype after the activation of iFGFR1. Although it has been known that FGFR1 can stimulate EMT in mesoderm during gastrulation, it is intriguing to find that FGFR1 activation can also induce EMT in mammary epithelial cells. EMT is a hallmark of tumor progression associated with the acquisition of invasive and metastatic features of breast carcinomas (Thompson et al., 1994; Birchmeier and Birchmeier, 1995). Abolishment of these phenotypes by the pan MMP inhibitor GM6001 further suggests that MMP activity is required for iFGFR1 signaling to promote EMT and invasive ability of mammary epithelial cells.
In addition to MMP-3, we also observed induction of MMP-9 expression and activity after iFGFR1 activation (Welm et al., 2002). Thus, MMP-9 may function to activate local growth factors, stimulate angiogenesis in vivo, and degrade the ECM during cell invasion, whereas MMP-3 is critical for the cleavage of E-cadherin. This is another potential mechanism through which FGFR1 may act to promote cell invasion.
It is still not clear how cell polarity, proliferation, and survival are regulated by iFGFR1. Because the changes in these cellular processes upon iFGFR1 activation were not blocked by the addition of the MMP inhibitor (unpublished data), it is possible that they are regulated by independent signaling cascades downstream of iFGFR1. Therefore, these data suggest that MMP-3 induction may play an important role downstream of iFGFR1 during invasion and cell migration but was not required for iFGFR1-mediated effects on cell proliferation, cell polarity, and cell survival.
Based on these and previous studies that have reported the ability of MMP-3 to cleave E-cadherin and promote EMT (Lochter et al., 1997; Noe et al., 2001), we propose a model whereby iFGFR1 dimerization activates several downstream signaling pathways that lead to cell polarity disruption, acinar growth, and an invasive phenotype (Fig. 9 B). Multiple transcription factors can be activated and bind to the MMP-3 promoter to induce its expression, eventually resulting in increased MMP-3 activity in mammary epithelial cells. Subsequently, MMP-3 cleaves E-cadherin at the cell membrane, resulting in cell invasion and EMT. This may also provide one potential mechanism by which FGFR1 functions during ductal morphogenesis to regulate the invasion of mammary epithelium into the stromal fat pad. Aberrant regulation of this pathway may lead to early tumor progression. Thus, these results provide new insight into the possible mechanisms through which FGFR1 may contribute to breast cancer progression.
Although the iFGFR1 HC11 3D culture system has been proven to be an excellent system for studying some aspects of the FGFR1 signaling pathway (e.g., cell invasion and proliferation), inflammation and angiogenesis, two processes that are involved in tumor initiation and progression within the iFGFR1 transgenic mouse model, cannot be studied in this monotypic HC11 3D culture system. Although it may be possible for some of these processes to be modeled in organotypic 3D cultures, further analysis of iFGFR1 in transgenic mice will most likely be required to characterize the molecular mechanisms that regulate these processes.
Materials And Methods
Plasmids and cell culture
iFGFR1 was cloned into the EcoRI sites of the pBabe-puro using an MfeI fragment of pMMP-iFGFR1 including the FGFR1 intracellular kinase domain, two tandem FKBPv (FK506 binding protein) domains, the NH2-terminal myristoylation, and HA epitope sequences (Welm et al., 2002). HC11 cells were grown in growth media containing RPMI 1640 medium (JRH Biosciences) supplemented with 10% FBS (JRH Biosciences), 2 mM l-glutamine (JRH Biosciences), 10 ng/ml EGF (Life Technologies), 5 μg/ml insulin (Sigma-Aldrich), and 50 μg/ml gentamicin (Sigma-Aldrich). The 293T cell growth media contained DMEM (GIBCO BRL), 10% FBS, and 50 μg/ml gentamicin.
Generation of HC11s expressing iFGFR1
Cell transduction was done as described previously (Welm et al., 2002). Stable populations were obtained by selection with 2 μg/ml puromycin (Sigma-Aldrich). iFGFR1 expression was confirmed by immunoblotting. The selected clonal cell lines R1-H and -L were routinely grown in the medium with 2 μg/ml to maintain selection pressure. Serum starvation media for HC11 cells contained only RPMI supplemented with either 30 nM AP20187 (Ariad Pharmaceuticals) in ethanol or an equal volume of ethanol diluent alone as control.
HC11 cells were treated with trypsin and resuspended in growth medium. The cells were pelleted and resuspended in assay medium (RPMI supplemented with 2% FBS, 2 mM l-glutamine, 2.5 ng/ml EGF, 5 μg/ml insulin, and 50 μg/ml gentamicin) at a concentration of 25,000 cells/ml. Eight-chambered RS glass slides (Nalgene) were coated with 40 μl Matrigel basement membrane matrix (CLONTECH Laboratories, Inc.) per well and left to solidify for 15 min. The cells were mixed 1:1 with assay medium containing 4% Matrigel, and 400 μl was added to each chamber of the Matrigel-coated eight-chambered slide. Assay medium was replaced every 4 d. For stimulation with AP20187, the assay medium was replaced with the assay medium containing 100 μM AP20187 at day 10. For stimulation with AP20187 and GM6001 (Sigma-Aldrich), the assay medium was replaced with the assay medium containing 100 μM AP20187 and 10 μM GM6001 at day 10.
Wound healing assay
HC11 cells were seeded on glass coverslips at 5 × 104 cells per well and grown to confluence before overnight starvation in RPMI. A single wound was introduced in the monolayer using a micropipette tip, and the medium was replaced with RPMI supplemented with either 30 μM AP20187 in ethanol or an equal volume of ethanol diluent. Wound closure was monitored for 20 h.
0.5 ml of serum was added to the wells of a 12-well plate (Falcon). 5,000 R1-L and -H cells were plated in 0.5 ml of serum-free RPMI in 12-well format cell culture inserts containing 8-μm pores (Falcon). After a 20-h incubation, cells that had not migrated through the filters were removed using a cotton swab and the filters were stained with hematoxylin.
The following antibodies were used for immunoblot analysis: phospho-ERK and ERK (Cell Signaling); HA epitope (Santa Cruz Biotechnology, Inc.); DECMA-1, a rat monoclonal antibody that recognizes the extracellular domain of murine E-cadherin (Sigma-Aldrich); and E-cadherin, which recognizes the cytoplasmic tail of E-cadherin (Transduction Laboratories). Protein extracts from cultured cells were isolated by lysing the monolayer cell with RIPA buffer (1% Triton X-100, 1% NaDOC, 0.1% SDS, 20 mM Tris HCl, pH 7.5, 150 mM NaCl, and 1 mM EDTA) supplemented with Protease cocktail inhibitor (Roche) and phosphatase inhibitor (1 mM NaVO4 and 1 mM NaF) for 15–30 min at 4°C. Lysates were cleared for 15 min at 13,000 rpm, and supernatants were collected. Conditioned medium from monolayer cell cultures were harvested, centrifuged at 2,000 g for 15 min, and passed through a 0.22-μm filter, concentrated by Centricon (Millipore). Protein extracts and concentrated condition medium were quantitated by Bradford assay (Bio-Rad Laboratories). Proteins (30 μg) were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore). For immunostaining, blots were quenched with 5% nonfat dry milk in TBS containing 0.5% Tween 20. The membranes were incubated with primary antibody at a dilution of 1:1,000, followed by three 5-min washes and incubation with horseradish peroxidase–conjugated secondary antibodies at a dilution of 1:5,000. Chemiluminescence was developed based on the manufacturer's protocol (Pierce Chemical Co.).
Indirect immunofluorescence and image acquisition
The following primary antibodies were used for indirect immunofluorescent detection of specific antigens: anti–p-histone H3 (Ser10; Upstate Biotechnology); anti–cleaved caspase 3 (Asp175; Cell Signaling); anti–phospho-ezrin (Thr567)/radixin (Thr564)/moesin (Thr558) (Cell Signaling); anti-CD49f (Cell Signaling); anti–laminin V (Chemicon); anti–E-cadherin (Transduction Laboratories); anti–β-catenin (nonphosphorylated; Upstate Biotechnology); anti–phospho-Akt (Ser473; Cell Signaling). All first antibodies were used at a dilution of 1:200. Secondary antibodies were as follows: anti-mouse or anti-rabbit Texas red; anti-mouse or anti-rabbit coupled with Alexa Fluor dyes; anti-rat Texas red (Invitrogen). All secondary antibodies were used at a dilution of 1:500. Nuclei were stained with TOPRO-3 (Invitrogen). Immunostaining was performed as previously described (Debnath et al., 2003a). Confocal analyses were performed using a laser-scanning confocal microscope (model 510; Carl Zeiss MicroImaging, Inc.). The acquisition software used was LSM image browser (Carl Zeiss MicroImaging, Inc.). Phase-contrast images were captured using an inverted microscope (CK40-SLP; Olympus) and video camera (CCD-IRIS/RGB; Sony). The acquisition software used was Photoshop 5.0 (Adobe).
RNA was extracted from monolayer cells using Trizol as recommended by the manufacturer (Invitrogen). 5-μg samples of RNA were treated with DNase I as recommended by the manufacturer (Invitrogen) followed by generation of cDNA using Superscript II (Invitrogen). One tenth of the final reaction volume was used in quantitative PCR reactions as described previously (Yuen et al., 2002) containing 1 mM MgCl2, platinum Taq (Invitrogen), and 1× SYBR green (Invitrogen). PCR was performed using a thermocycler (ABI PRISM 7500; Applied Biosystems) with the following cycles: 94°C for 30 s, 55°C for 30 s, and 72°C for 34 s for 40 cycles. Relative quantitation of MMP-3 gene expression was calculated and normalized to cyclophilin expression levels using the 2−ΔΔCt method (Livak and Schmittgen, 2001). The following primer sequences were used: for MMP-3, 5′-TCCATGGAGCCAGGATTTC-3′ and 5′-TGCGAAGATCCACTGAAGAAGT-3′ (designed by F. Behbod, Baylor College of Medicine, Houston, TX), and for cyclophilin, 5′-TGAGCACTGGGGAGAAAGG-3′ and 5′-TTGCCATCCAGCCACTCAG-3′. MMP-9 primers were purchased from Superarray.
SMARTpool siRNA for MMP-3 and -9 and siGLO RISC-free siRNA as the control were introduced into subconfluent (75–80%) HC11 cells using DharmaFECT transfection reagent (Dharmacon) according to the manufacturer's instructions. After 24 h, the media were changed to RPMI, and the cells were starved overnight. The media were replaced with RPMI supplemented with either 30 μM AP20187 in ethanol or an equal volume of ethanol diluent the following day. After the induction for 48 h, the cells were collected for RNA analysis and immunostaining.
Online supplemental material
Fig. S1 shows untreated or treated R1-L and -H acini at low magnification.
We are grateful to Dr. F. Behbod for designing MMP-3 primers. We would also like to thank Dr. S. Muthuswamy, Dr. P.D. McCrea, and Dr. S. Grimm for helpful discussions.
W. Xian was supported by a postdoctoral fellowship from the Department of Defense Breast Cancer Research Program (DAMD 17-02-1-0285). K.L. Schwertfeger was supported by a Ruth L. Kirschstein National Research Service Awards fellowship (CA 97676-01). T.C. Vargo-Gogola was supported by a postdoctoral fellowship from the Department of Defense Breast Cancer Research Program (DAMD 17-03-1-0325). This research was supported by a National Institutes of Health grant (CA16303).
Abbreviations used in this paper: 2D, two-dimensional; 3D, three-dimensional; CSF-1R, colony-stimulating factor receptor; EMT, epithelial-to-mesenchymal transition; ERK, extracellular regulated kinase; FGFR, FGF receptor; iFGFR, inducible FGFR; MMP, matrix metalloproteinase; p-ERM, phospho-ezrin/radixin/moesin; p-histone H3, phosphorylated histone H3; RTK, tyrosine kinase receptor; siRNA, small interfering RNA; SMA, smooth muscle actin.

domain: medical


text:
Book by Todd Almond
Music and lyrics by Matthew Sweet
Choreographed by Joe Goode
Directed by Les Waters
Limited Season · Thrust Stage
April 9–May 16, 2010
Running time: 1 hour and 50 minutes, including one 15-minute intermission
Romance unfolds in a new musical wound around the tender love songs of Matthew Sweet’s landmark album, Girlfriend. Meeting in homeroom. Cruising through town. Holding hands. Cheering from the stands. That awkward first kiss. In the world premiere of Girlfriend at Berkeley Rep, boy meets boy. It’s an eternal story turned upside down, a dual-Romeo duet directed by Les Waters that’s innocent—and Sweet. “Girlfriend is the breathless testimony of a fool for love,” raves Rolling Stone, “a rock ‘n’ roll valentine that delivers subtle wisdom with an exhilarating kick.” Fall in love with the boy next door at Girlfriend.
Todd Almond · Book / Vocal Arrangements / Additional Orchestrations
Matthew Sweet · Music and Lyrics
Joe Goode · Choreographer
Les Waters · Director
David Zinn · Scenic and Costume Design
Japhy Weideman · Lighting Design
Jake Rodriguez · Sound Design
Julie Wolf · Music Director
Michael Suenkel · Stage Manager
Mina Morita · Assistant Director
Ryder Bach · Will
Jason Hite · Mike
Julie Wolf · Rhythm Guitar / Keyboards / Backing Vocals
Shelley Doty · Lead Guitar / Backing Vocals
Jean DuSablon · Bass
ieela Grant · Drums
“Love is in the air at Berkeley Rep’s Thrust Stage…A gentle, heartfelt, two-character chamber musical that celebrates the pain and joy of first love…Adolescent insecurity and the awkwardness of first romance inform every glance, gesture and warbled note in the performances of Ryder Bach and Jason Hite…The music, two attractive actors, and music director, keyboard player and vocalist Julie Wolf’s electrifying four-woman band make it an exhilarating joy.”—San Francisco Chronicle
“A rare treat—authentic, intimate and romantic, and, as the ‘I’ve Been Waiting’ lyric goes, ‘perfect in so many ways.’”—San Francisco Examiner
“A delicate gem of a musical that’s genuinely, well, sweet…The show soars during the musical interludes as Sweet’s pop anthems sweep us away with their irresistible exuberance…At its most powerful, Girlfriend takes you back to the time in your life when your favorite pop songs took on an almost-religious significance. It’s as if you turned up the dial on your own redemption.”—San Jose Mercury News / Bay Area News Group
Prologue: from the Artistic Director
I think humanity can be divided into two distinct categories: those who hated high school and those who loved it. (If you derived even mild pleasure from the experience, you fall into the latter group.) I hated it. At least that’s how I like to remember it. Spent most of my time feeling astonishingly weird, even in what should have been the most comfortable of circumstances. I was pretty sure that everyone was looking right through me, that my skin was not actually skin but some kind of translucent surface designed to give onlookers the best possible view of my quivering self. And what self were they looking at? There wasn’t much of a self there, as far as I could tell.
But there were things that could save me, even on the darkest of days. My friends, for one. Or this really tight pair of pants that I wore with a pair of really pointy shoes. And music. Always the music. Music that spoke to my secret self, a self that was fantastic and brilliant and irresistible. If only the world knew what the music was telling me. If only I had the courage to act on the music. Like a zillion other teenagers on the planet, I sat in my room playing music—some albums over and over again, some songs on an endless loop. I played music not just because I loved it, but because my life depended on it.
Girlfriend is a story that captures the eternal and impossible yearning of the adolescent heart. Two high school boys try to come to terms with their feelings for each other. At stake are their identities, their relationships with their families and their futures. Set to the rock music of Matthew Sweet (whose 1991 album of the same name was the inspiration for the story by Todd Almond), the play is an attempt to capture the sweetness that is buried within every teenage heart. It is a story without guile, without irony, without pretension. How oddly refreshing.
The intrepid Les Waters is at the helm again, wielding his directorial magic with a trusty team of sure-handed designers and two gifted young actors. It was Les’ inspiration to back the boys on stage with an all-girl band, giving the audience a full complement of interpretive choices. Because at the end of the play, it’s all about the choices. The ones you made or didn’t make. And the music you were listening to when you made them.
In it for the long haul: This Girlfriend is here to stay
By Madeleine Oldham
Matthew Sweet’s Girlfriend may not have been a multi-platinum bestseller, but it captured the heart of many a discerning listener. Legions of fans remain fiercely devoted to Sweet’s emotional songwriting and underdog persona. When the album first came out, no one expected it to become the sensation it did. We talked with playwright Todd Almond and other diehard Matthew Sweet fans to find out what’s so special about this unassuming yet captivating collection of songs.
I listened to Girlfriend every day when it came out in the early ‘90s. I dubbed copies (on cassette tape, of course) for my friends. I made my family listen to it on long road trips. I’ve bought it, misplaced it, replaced it, loaned it, uploaded it (these days), downloaded it and gifted it over the years. I wrote a musical about it, for heaven’s sake. Girlfriend is a thing on the planet that I love very much, and this musical is as much a love-letter to that album as it is anything else.
—Todd Almond, playwright
Can you articulate what drew you to the album?
I was unhappily living in Dullsville (Santa Fe), which is where my Dad lives. I hated it and was trying to move back to San Francisco. I was comforted by this music when I would play it while driving, and it would be one of the few things that I enjoyed doing. But this isn’t so much of a “memory” record for me. I can gladly listen to it nowadays and appreciate it on its own terms.
—Michelle, usher at the Warfield Theatre
If you listen to the album today, what does it make you think of?
I’m listening to it right now, and it makes me think of the girlfriend I just broke up with. These nice little complete and decorated sentiments put that relationship into a bit of perspective. It makes me feel better, actually. Matthew Sweet has a way of pounding my own significance into the ground with backing vocals and searing guitars.
Do you have a particular memory associated with Girlfriend?
Most of my memories of the album are of me driving (or, rather, sitting) in LA traffic singing along at the top of my lungs. “Nothing Lasts” is probably the most emotional song on the album, and I remember driving back to my apartment after breaking up with my girlfriend and playing that song. I didn’t sing along—I simply remember noticing the way the lights outside the car played across the dashboard as I drove. It wasn’t a heartbreaking moment, just a sober realization that it had to end eventually.
—Christian, story artist at Pixar
Do you have a favorite track?
I love all of them, but my top three are probably: “You Don’t Love Me,” “Winona” and “Thought I Knew You.” Oh wait…there’s “I’ve Been Waiting.” Oh yeah…“Looking at the Sun.” Oh forget it—I can’t answer the question. I love the whole thing.
I didn’t know nobody, then I saw you coming my way
By Mike Sablone
In 1992 (in suburban Massachusetts I wasn’t cutting-edge enough to have noticed that the album came out in 1991), I was 15 years old. My music taste, much like that of most suburban kids in a predominantly white suburb, started with comedy albums (Weird Al, Monty Python) and evolved into mostly rap and hip-hop (Public Enemy, Dr. Dre).
I had skipped bands like, say, the Rolling Stones, the Kinks, Led Zeppelin (I’m not too ashamed to admit that I just Googled their name to make sure I spelled it correctly), or that other band…the Beatles.
So when I first heard “Girlfriend” and “Evangeline” and “Divine Intervention,” it was like someone had plugged in my brain to this astonishing new series of sounds. Melody. Guitars. Drum fills. False endings. Vocal harmonies. I had no idea the history behind the album. All I knew was this record spoke to me.
Hearing the song “Girlfriend,” I finally understood what pop music was there for. It was there for me to understand that I wasn’t alone. It was there to tell me, “Don’t worry, I know you are a ball of terrified emotions right now, but this guy, this guy right here? He’s going to say everything you’re thinking. Everything you’re going to think. Everything that has been thought of before. And it’s all going to be OK.”
Popular music is popular because of this reason. This is why music works—it convinces teenagers that they’re not alone. It allows people to express everything they’re feeling, but in four minutes of perfect melody.
When I got to college two years later I met my best friend by talking to him about this album. We talked about how there was supposedly a college class that examined how perfect this album was. We talked about how pissed off we were that it wasn’t our college that offered this course. We then talked about the radio show we’d co-host for the next four years.
Girlfriend has a special place in my heart, but if I’m forced to say my favorite song it’s “Evangeline.” I remember MTV discussing the song, telling me that it was about a Japanese anime character. I remember trying to find out everything I could about this character. I remember then not caring, and making the song into a song that I wrote about whatever girl I had a hopeless crush on at the time.
You ask if the album still holds up for me? Ask me about the last girl I had a crush on. Ask me if I made her a mix CD. Do you need to ask me if I put “Evangeline” on there?
(Well, I might not have, unless I’m feeling bold. I usually wait ‘til the second or third mix CD. You can’t put “Girlfriend” on there—that’s just too obvious, and also? Kinda creepy. But I can kind of, sort of [but not really] get away with “Evangeline.” Note: this could be why I’m still single. I haven’t found the girl who appreciates this song as much as I do.)
I saw Matthew Sweet perform every time he came to Providence while I was in college. I drove down during the summer to see him in, like, 1995. WBRU, the terrible college radio station in Providence was playing “Good” by Better Than Ezra seemingly every five minutes. Driving around post-concert, I swore that if they played it twice while we hung out I’d buy the damn album to get them to stop playing it (the theory being that once I bought the record the radio station wouldn’t need to sell it so relentlessly to me). They, of course, played it four more times in the next 90 minutes. The next day I went and bought the record. I have no idea where that record is. I can still tell you exactly where my copy of Girlfriend is.
And that, ladies and gentlemen, is why I would take a bullet for Matthew Sweet.
Mike Sablone is a dramaturg working for Center Theatre Group in Los Angeles, where he develops new plays and musicals. He warns you not to get him started on anything music related as he won’t shut up. He is currently working on a 500-song Best of the Decade compilation that will rank songs, in reverse order of amazingness from 500 to 1. The CD will also include liner notes.
A conversation with Matthew Sweet
By Madeleine Oldham
Picture someone who has dedicated his life to rock ‘n’ roll: Matthew Sweet is hardly the first person who comes to mind. A polite, generous, hard-working Midwestern boy, Sweet comes across as, well…very sweet. Born in Lincoln, Nebraska in 1964, he has built a lengthy musical career through talent, perseverance and authenticity that defies traditional stereotypes of the raging, self-destructive musician. He has managed to remain his approachable and unpretentious self in the face of a world that revolves around flash and image. Berkeley Rep’s dramaturg and literary manager, Madeleine Oldham, had a chance to talk with Sweet last year during his Sunshine Lies tour, and the following are excerpts from that conversation.
You’ve been making music for a very long time now. Is it still fun?
You know, that’s a really good question. I think it is still fun for me. It’s a very hard business as far as trying to make it support you and your life and all that, and so I think there’s always this tendency when things aren’t going well to think, “Well, maybe I just have to do something else.” And I’ve gotten to a point a couple times where I felt that way. But I always tend to feel a lot better when I do music. It’s just what I do. I think there were times when I was afraid nobody cared, and I had to get used to the idea that no one else has to care about it but me.
But other than that, I think it’s always remained pretty fun for me. People will ask me, “Why did you get into music when you were really young?” And it’s the same kind of thing—it made me feel better than anything else, and I could lose myself in it and feel somehow freed a little bit from life, you know? And I think it’s still that way when it’s fun because it’s just something different; it’s a whole other way of feeling and thinking. It just kind of takes me away from real life.
And when you had those moments of “maybe nobody cares,” did you ever seriously think about doing something else?
I don’t think so, really. When I started out, I didn’t have any concept of what it would be like to have fans and all that. I really only cared about recording and writing songs and making a record. I didn’t really realize that you won’t get to keep doing that if you don’t sell some, you know? And my first couple records didn’t really sell, but I was lucky enough to follow the guy who had signed me around to a couple of labels. Then when Girlfriend took off, which was kind of a slow process, it was like this whole other dimension of what people thought about me. That sort of freaked me out and also added a lot of pressure, business-wise, to come up with something someone was gonna like, which wasn’t the kind of artist I was to begin with. So I think it put a damper sometimes on my spirit, just knowing there was that kind of pressure there, and in a weird way, even at the times I did the best, there was still this feeling it was not enough. And the music business was already heading the way it ended up at the end of the ‘90s, where it really started dissolving into people caring less and less about the music and more and more about actual numbers they could move.
So I think only when I was asking myself, “Will I get to make more records?” did I ever start to get worried and lose faith. But I like to go on my own MySpace page and see what everybody says about me and interact with people. Whenever I get it together to release something, I’m always really surprised that there’s anybody out there that cares. And it doesn’t need to be a large amount now, cause I’ve been around for a long time and the game has changed so much and I can sell small amounts of records. We’ll break even and make more records. And I think there’s some correlation between the less big business it is, the more I’m able to relax and get back to feeling like I did initially, which was just excited about music and doing it really mainly for me.
I also love playing live because you find out there are people out there who really do care about music, and they also care about seeing music live. And so I think that right now, people like me are trying to figure out how exactly you get to the people who do care if you release something.
And do you have thoughts about how you do that?
It’s really the same way we got going on Girlfriend in the beginning. I mean, it wasn’t like it suddenly happened. It started really small on a grassroots level, and lots of people worked hard to get other people turned on to it.
And that’s how we used to feel about so many artists—all those artists that we just revered from the ‘60s and ‘70s. Then records became less important as computers came in, and the Internet and all that, and there were just so many things to look at and be into. Back when we were into records, we would just sit in our rooms and it was all you had as your shield against your parents, or whatever.
How do you see the music business now?
Music itself is so unique, that I think it really can be independent of all those things. That’s why I think if you’re a musician who just does it, you can get in the mode of it, and it has its own life. And you hope it will find some people that like it so you can make a living. I mean, I wish somebody would just give me millions of dollars, and I would give my music away for free for the rest of my life—I’d rather do that! Before people were downloading music I would say to my manager, “Can’t we just, like, give it away free and just make money some other way?” ‘Cause I hated that feeling of pressure on the music to be successful.
Why do you think Girlfriend was such a successful album for you?
Well, I think basically people related to it. I was talking about feelings and relationships at a time when really it was an era of bands that were much less clear about what they were saying. The record didn’t sound like any other records right then either. So it was kind of radical sounding at the moment, which gave it this sort of grooviness thing. But I really think people just related to the feelings in the songs. It’s an interesting thing to see, and it’s really cool how much impact that can have. Like, that makes someone really never forget you, you know?
We’d like you to meet our wonderful new Girlfriend! Take a peek.
Les Waters and Todd Almond
Fall in love with the boy next door at Girlfriend, a Berkeley Rep world-premiere musical event inspired by songs from the 1990’s breakthrough album by Matthew Sweet—introduced by director Les Waters and writer Todd Almond.

domain: artistic


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That is an egregious offense to the subtitle gods but in some odd way I am proud of it. Coming in hot with another review is the next book in a series that is slowly creeping its way to the upper echelons of my heart. The True Bastards by Jonathan French is the second book of The Lot Lands series. It takes what made the first book such an interesting and fun read and improves upon it. The plot is much more expansive and original. Don’t just take my written word for it though, also listen to me verbally jerk the book off on another edition of the greatest fantasy, nay, the greatest literary podcast of all time: The Swords and Sports podcast featuring yours truly.
Click the link below to be transported to a magical place owned by a fruit (not a gay joke simply an apple joke) and have all your audio fantasies come true.
Now that is how you sell a fucking podcast. Where do I begin with this review. I already know this is going to be a clusterfuck but whatever. Jackal is no longer the POV character, instead we get our main homie Fetching. She is attempting to lead The True Bastards in the wake off having the Kiln destroyed by old sludgey but of course life ain’t that fucking easy. First she needs to contend with her own version of black lung, which is essentially that she is coughing up sludge like a frat boy throws up beer the morning after a sick rager. The town of Winsome has zero, nada, zilch food and motherfuckers are hungry. So she has to find out why none of the food is getting there. Turns out a pack of super hyenas controlled by a giant orc shaman are causing terror throughout The Lot Lands. Some frails have started setting up shop in an abandoned lot but none of the other hoofs no what to do. Hispertha is getting real mouthy in their quest to expand the empire. Basically she is going through a Lot. Get it, its because the series is called The Lot Lands. Let me end this monster paragraph.
That is quite the predicament to find oneself in. Fortunately for the lovely half-elf, half-orc super hybrid there is one simple answer. Violence. As the famous Jedi Mind Tricks song Violence begets Violence taught me, it’s that Violence begets Violence so that means madness ensues. We are talking hoof on hoof war, hoof on Tine war, hoof and hoof on magic orc shaman and hyena war, and all types of fun.
This fucking book is awesome. I love, love, love the use of all kinds of mythical races and that they can mix creating all different species that have to make their way in the world. I love that they ride hogs and shoot crossbows like they are a fucking motorcycle gang. I love the interplay between the clashing cultures and factions along The Lot Lands. The world Frenchy Lotana builds is absolutely brilliant. The characters are hilarious, believable and have realistic arcs. The curse words are great. The use of the word “cock” is without equal. Basically read this series if you like fun.
This book is literally the definition of chad. Easiest rating ever.


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Business Roundup: MO Hospitals Join Forces
Hospital and health system mergers, acquisitions and collaborations continue at a brisk pace during the week of October 21-26.
Missouri Hospitals Form Collaborative
Missouri's Saint Luke's Health System of Kansas City, BJC HealthCare of St. Louis, CoxHealth of Springfield, and Memorial Health System of Springfield, IL, have created a joint BJC Collaborative, LLC. The four health systems will remain independent under the collaborative, which will have a combined "footprint" of 4,821 hospital beds in Missouri, Illinois, and Kansas, and combined annual revenues of almost $7 billion.
The health systems forecast that the anticipated savings generated by the collaboration will allow them to deploy clinical programs and services to improve access and quality of care while lowering costs.
Saint Luke's is the largest non-profit healthcare provider in the Kansas City area and includes 11 hospitals and several primary and specialty care practices. BJC HealthCare is a 13 hospital system serving eastern Missouri and southern Illinois.
CoxHealth is Springfield, MO's only locally owned not-for-profit health system, and includes three hospitals and more than 65 clinics in 20 communities. Memorial Health System is a three-hospital, non-profit health care organization serving Illinois patients in a 40-county region.
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