text:
Habitat for Humanity of Bay County continues to make the American dream a reality
The non-profit is finishing up six houses in Callaway, one of them being their 100th home. Just down a few blocks, their first fundraiser house is underway.(WJHG)
By Katie Bente
Published: Nov. 28, 2022 at 6:08 PM CST
BAY COUNTY, Fla. (WJHG/WECP) - Trying to buy your first home in this market can seem unaffordable and unrealistic for many. But one local non-profit has spent the last three decades changing that reality for families across Bay County.
Homeownership is at the core of the American dream. But for many in Bay County, a dream is all it is. Habitat for Humanity has been making dreams come true for decades, building affordable homes for those in need.
"Oh, there's a tremendous need. We can't fulfill the complete need in Bay County as we all know since Michael, things are in pretty dire strains," Travis Herr, Habitat for Humanity of Bay County board member, said. "Somebody gets a house for the first time and they've been living in dilapidated, rat-infested roach-infested quarters for god knows how long it really is. It is moving."
But it's not a free pass for homebuyers.
"What we do is they actually end up with a mortgage on the home but it's for the cost to build a house so they'll end up with a $140,000 mortgage at 0%. So their payments, maybe $600 or $700 a month, much less than renting an apartment," Curt Hartog, Habitat for Humanity of Bay County board member, said.
The non-profit is finishing up six houses in Callaway, one of them being their 100th home. Just down a few blocks, their first fundraiser house is underway.
"We wanted to be able to generate some more funds, things are tight for everybody. So the idea was we're going to build this as a spec house under speculation, hoping that we can sell it for the true value of the home," Hartog said.
That means if the home is worth about $350,000 and they build it for under $200,000, they can put that extra $150,000 towards building another home. It helps make the American dream a reality for one more family.
"So every time we build a house, we sell it to the homeowner, they're making payments, all that money goes back into building more houses and more houses," Hartog said.
Habitat for Humanity is asking for the public's help to make the dream of homeownership possible for more people in our community.
There are levels of support through tax-deductible donations. Groundbreakers are $1-$250, Wall Framers are $250-$499, Roof Raisers are $500-$999, House Blessers are $1,000-$2,500, and Developers are $2,500 and up.
For information on how to get involved or donate, you can head to https://habitatbay.org/.
Two arrested after attempting to flee during arrests
Man charged for stabbing, firearm possession
Hundreds race to raise money for breast cancer patients
Get ready for the 36th Annual Bay County Job Fair


Yep, one of those threads where we say what we've been up to the past few months sense it's apparent we've all found better things to do than spend our time here!
Since school got out I've been doing nothing but working, hanging out at a friend's house, and doing manual labor at my great grandma's house for extra cash. Exciting, yes?
Oh, I've also been on 4chan alot, thus explaining (kind of) why I'm never here.
I've been involved in an endless recursion of time.
An endless recursion of time.
To avoid looking too bad for brining up something that probably only my sister would know of, I'm still at University.
Really busy in Japan, traveling, studying, and hanging out with friends, as well as wasting some time on the internetz. Once I get back home, I go almost straight back to college, where I'll have a horse dung! load of classes for my final year, band, and I'm an RA, oh and I have to launch a new pokemon competitive battling site and a forum before september.
So yeah, my plate's full.
I actually will be helping out at band camp for my high school (even though I graduated, my younger brother goes there now).
Com'ere son, I'm going to tell you of the times where we used to spend our time on this littl' forum with our littl' bunch of merry friends.
Why, there was a lot of people. Most of them you wouldn't know anyway, so it's not that important.
When not creating fictional sons or characters in general, Raijuu has had exams and spending the days doing nothing in particular. You can contact Raijuu through the little PM icon at the side top of the post.
Lol late reply is late.
If anyone's still alive in this darnit ghost forum, Miror is almost 16.
I've been having the urge to get on and see if anyone has been posting for like 2 weeks now, and just forced myself not to due to believing it would be a waste of effort.
Miror, you're a goddamn liar. You're still 12 years old just like you were 4 years ago.
I actually think I know what you were talking about in your first post now, Raijuu. Melancholy of Suzumiya Haruhi? I got to the third episode of that endless recursion of time and rage quit.
Now, what the hell have I been doing? I was in a welding class and getting certified for Arc Welding, but the class ended like 2 weeks ago and I passed it, leaving me with no obligations besides working. Since then I've been bullshitting: playing WoW again, sitting on /b/, keeping up with the manga I follow once a week, and trying to find either a second job or a new job so that I can support myself and move out on my own.


text:
A List of the Ladies Niall Horan May Have Put a Little Love On
Who Has Niall Horan Dated?
15 January, 2020 by Toria Clarke
Niall Horan has been in the spotlight since the formation of One Direction back in 2011. Now, trailblazing down his own path as a solo artist, he continues to set the bar high for himself and his music. When you're a celebrity in the limelight, one of the most frequent questions you're asked is, "Are you dating anyone?" followed by "Who are you dating?" When you're Niall, answering those questions might not be as easy as they would be for you or me. Niall has been particularly low-key about his personal life, especially when it comes to romance. But, we know that there have been some lovers in his life here and there (looking at you, Ellie Goulding). Scroll through the gallery to find a list of everyone Niall may have put a little love on over the years.
Hailee Steinfeld's New Song Is Called "Wrong Direction" — and It Might Be About Niall Horan
by Danielle Jackson 2 weeks ago
Save the Date: These Are the Artists Coming to a City Near You in 2020
by Maggie Boyle 19/12/19
Late Night Highlights
Niall Horan Reading Twas the Night Before Christmas in a Southern Accent Is Surprisingly Spot On, Y'all
by Angela Law 18/12/19
Jennifer Lopez and Scarlett Johansson Are Both Returning to Host SNL in December
by Danielle Jackson 21/11/19
Niall Horan's Stripped-Down Version of "Nice to Meet Ya" Might Be Better Than the Original
by Quinn Keaney 19/11/19
All I Want to Listen to Today Is Niall Horan's Beautiful Cover of Post Malone's "Circles"
OK, Diplo and Niall Horan's Deepfake in the "Nice to Meet Ya" Remix Video Is Too Trippy
No One Loves Niall Horan and Lewis Capaldi as Much as Lewis Capaldi and Niall Horan
by Ashley Gale 04/11/19
Niall Horan Is Smooth, Suave, and Incredibly Thirsty in the "Nice to Meet Ya" Music Video
If the Whole World Was Watching, We'd Still Pick These Performances as Niall Horan's Best
by Hedy Phillips 03/10/19
Celebrate 9 Years of One Direction With 99 of Their Best Photos Ever
by Hedy Phillips 23/7/19
Latest Celebrity

redpajama_set_name: RedPajamaCommonCrawl


Here are seven morning prayers I use in my daily devotions.
The first prayer is a brief prayer I have said daily when I awake for the past twenty years or so: Psalm 119:133 (NIV) along with a three-part petition for personal expression of the Lord Jesus in my life which I adapted from a sermon by Charles Stanley, the American Baptist pastor of the First Baptist of Atlanta and founder of In Touch ministries, his television teaching and book ministry. On July 1, 2017, I added an introductory prayer to my first prayer, which is from A Diary of Private Prayer, by John Baillie, originally published in 1949, recently updated by Susanna Wright chosen by Baillie's son to do this. This is a fine daily morning and evening devotional prayer book, with 31 daily morning and evening prayers, and a Sunday morning and evening prayer.
The second prayer I added recently and posted here April 2, 2017, is a prayer for concentration during prayer which I adapted from a prayer for lack of concentration included in a collection of Prayers for Focus which I found on the website Knowing Jesus, and its Prayer Room.
The third prayer, "To Reflect the Master" is from William Barclay, the British theologian and Bible scholar who wrote insightful and useful commentaries on the New Testament books.
The fourth prayer, "A Morning Resolve," was is an Episcopal prayer given to me by my friend, Rev. Phil Rountree, a retired Episcopal priest in Novato, California, now a member of the Novato rock band, The Role Models.
The fifth prayer, "Face the Day with Peace," is a wonderful comprehensive and concise prayer for a day of peace from the Metropolitan Philaret of Moscow (19th Century) from "A Pocket Book of Prayers for Busy People".
The sixth prayer is a special prayer for sensitivity in our daily relationships. It was written by Lucy H.M. Soulsby who was a headmistress of a private girl's school, Oxford High School, from 1879 to 1889, and was a women's leader in the suffragette movement, from "A Pocket Book of Prayers for Busy People".
The seventh prayer, "A Daily Confession" I composed from Scripture and the Book of Common Prayer. The Daily Confession is discussed here in a blog post from April 7, 2013.
Eternal Father of my soul, let my first thought today be of you, let my first impulse be to worship you, let my first word be your Name, let my first action be to kneel before you in prayer.
And Father God, thank you for another day. "Direct my footsteps according to your word; let no sin rule over me." and make me an extension of the life of Jesus, an expression of His love, and an exhibition of His power, in all that I do. In Jesus name, Amen.
Dear Lord, I find that my mind too often starts to wander when I am needing to concentrate on my prayer. Keep me, I pray, from becoming distracted when I am at prayer. Help me to develop the ability to concentrate during my prayer sessions. Thank you, Lord, for listening and answering my petition, in Jesus name. Amen.
enthusiasm or to increase anyone's doubts.
whose I am and whom I seek to serve.
Teach me to treat all that comes to me throughout the day with peace of soul, and with the firm conviction that Thy will governs all.
Direct my will. Teach me to pray. Pray Thou Thyself in me.
O Lord, grant that each one who has to do with me today may be happier for it. Let it be given me each hour what I shall say, and grant me the wisdom of a loving heart that I may say the right thing rightly.
Help me to enter into the mind of everyone who talks with me, and keep me alive to the feelings of each one present. Give me a quick eye for little kindnesses, that I may be ready in doing them and gracious in receiving them. Give me quick perception of the feelings and needs of others, and make me eager-hearted in helping them.
Lucy H. M. Soulsby, a headmistress of Oxford High School, a private girl's school in England 1879-1889, and suffragette.
In addition to my daily Bible study, I also include with my morning and evening prayers, readings from various sources from which I read a little each day, and which I change from time to time including Sarah Young's Jesus Calling; Martin Lloyd-Jones, Walking with God Day by Day: 365 Daily Devotions; Christoper Webber's Give Us Grace: An Anthology of Anglican Prayers; Day by Day with the English Puritans: Selected Readings for Daily Reflections; Tim Keller and Kathy Keller, God's Wisdom for Navigating Life: A Year of Daily Devotions in the Book of Proverbs and The Songs of Jesus: A Year of Daily Devotions in the Psalms; Oswald Chambers, My Utmost for His Highest (Updated Version); Andrew Murray's Humility; John Calvin's Institutes of the Christian Religion; Phillip Schaff's History of the Christian Church, Louis Berkhof's or Wayne Grudem's Systematic Theology.
I encourage all who read this to develop their own daily devotional life with regular disciplined daily Bible Study, prayer and devotional Christian readings I am thankful to God that in my senior years I have been blessed to be able and willing to devote time daily to Bible Study, Prayer and devotional reading for the past 15 years that I failed miserably to do in the in the prior 31 years since I was saved in 1971 reading the Bible throughout for the first time. See my 2008 Spiritual Memoir.
Thanks for sharing your daily prayers. I keep close A Morning Resolve and A Prayer for Today from ForwardMovement. I checked out the link to St Francis of Assisi and liked their vision. The prayer attributed to St. Francis is my other favorite, so a nice tie in this morning, my 56th birthday. Hope all is well with you!
Happy Birthday, James! Here is what I wrote on my 60th Birthday.
What beautiful and poignant prayers these are! I just stumbled on your blog today as it was suggested by WordPress and I noticed the beautiful painting by Beirstadt. I look forward to reading more here!


NO HOA, large home sites! Beautiful enlarged Pecan B plan. Open living areas, split bedrooms, front porch and 6'x23' rear screened porch. Living area has vaulted ceiling, ceiling fan with light, spacious dining area, large kitchen with lots of solid wood custom cabinets with crown molding and knobs, stainless steel appliances, including gas stove, wrap around breakfast counter and pantry closet. Large and private master bedroom has tray ceiling, fan with light, 2 walk-in closets, garden tub plus separate walk-in shower and two raised height vanities each with its own sink. Split bedroom floor plan. Spacious guest bedrooms are 12'6 x 12'5. Rinnai tankless gas water heater, gas heat. Upgraded interior trim includes 3 1/4" casings and 5 1/4" baseboards. Low E glass windows, energy efficient homes. Boat and RV parking allowed. Spacious finished/painted 2-car garage, with automatic door opener, pull down stairs to attic storage above. Irrigation system, gutters, sodded and landscaped yard. Natural gas community. Close to International Drive, quick/easy access to Myrtle Beach. Basic Restrictive Covenants. Photos are for illustrative purposes only and may be of similar home built elsewhere. Square footage is approximate and not guaranteed. Buyer is responsible for verification.


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Oregon Senate President Peter Courtney, 76, hospitalized with hip pain
Oregon State Senate President Peter Courtney.Dave Killen
SALEM — Oregon Senate President Peter Courtney, the longest-serving legislator in Oregon history, is in a Portland hospital after experiencing severe hip pain.
Courtney's office in the Oregon State Capitol said Tuesday that the 76-year-old Democrat hurt his hip while spinning last week while attending a conference in Arizona.
"He was in a considerable amount of pain, but was able to get back to Oregon, and is currently being checked out at OHSU," his office said in a statement, referring to OHSU Hospital. He is expected to be released in several days.
Courtney is missing events this week as so-called Legislative Days in the capitol but expects to be back in time for the 2020 legislative session, which starts Feb. 3, the statement said.
Courtney was first selected Senate president in 2003 and has been re-elected as the chamber's presiding officer numerous times. He is in his fifth term as a State Senator, representing portions of Salem, Gervais and Woodburn. He previously served seven terms in the Oregon House of Representatives, including four terms as House Democratic leader.
-- Associated Press


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Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells
Birdal Bilir1,2,
Omer Kucuk2,3 &
Carlos S Moreno1,3
Triple-negative breast cancer (TNBC) is an aggressive clinical subtype of breast cancer that is characterized by the lack of estrogen receptor (ER) and progesterone receptor (PR) expression as well as human epidermal growth factor receptor 2 (HER2) overexpression. The TNBC subtype constitutes approximately 10%–20% of all breast cancers, but has no effective molecular targeted therapies. Previous meta-analysis of gene expression profiles of 587 TNBC cases from 21 studies demonstrated high expression of Wnt signaling pathway-associated genes in basal-like 2 and mesenchymal subtypes of TNBC. In this study, we investigated the potential of Wnt pathway inhibitors in effective treatment of TNBC.
Activation of Wnt pathway was assessed in four TNBC cell lines (BT-549, MDA-MB-231, HCC-1143 and HCC-1937), and the ER+ cell line MCF-7 using confocal microscopy and Western blot analysis of pathway components. Effectiveness of five different Wnt pathway inhibitors (iCRT-3, iCRT-5, iCRT-14, IWP-4 and XAV-939) on cell proliferation and apoptosis were tested in vitro. The inhibitory effects of iCRT-3 on canonical Wnt signaling in TNBC was evaluated by quantitative real-time RT-PCR analysis of Axin2 and dual-luciferase reporter assays. The effects of shRNA knockdown of SOX4 in combination with iCRT-3 and/or genistein treatments on cell proliferation, migration and invasion on BT-549 cells were also evaluated.
Immunofluorescence staining of β-catenin in TNBC cell lines showed both nuclear and cytoplasmic localization, indicating activation of Wnt pathway in TNBC cells. iCRT-3 was the most effective compound for inhibiting proliferation and antagonizing Wnt signaling in TNBC cells. In addition, treatment with iCRT-3 resulted in increased apoptosis in vitro. Knockdown of the Wnt pathway transcription factor, SOX4 in triple negative BT-549 cells resulted in decreased cell proliferation and migration, and combination treatment of iCRT-3 with SOX4 knockdown had a synergistic effect on inhibition of cell proliferation and induction of apoptosis.
These data suggest that targeting SOX4 and/or the Wnt pathway could have therapeutic benefit for TNBC patients.
Breast cancer is the most common malignancy and the second leading cause of cancer death among women in the United States. About 226,870 new cases of breast cancer and 39,510 breast cancer deaths are estimated to occur among US women in 2012 [1]. It is a heterogeneous disease comprised of distinct subtypes based on clinical, pathological, and genetic findings [2]. One of these subtypes is triple-negative breast cancer (TNBC), which is characterized by lack of expression of estrogen receptor (ER) and progesterone receptor (PR) as well as absence of human epidermal growth factor receptor 2 (HER2) overexpression [3]. TNBC accounts for approximately 10%-20% of all breast cancer cases [3–5]. TNBC patients exhibit a more aggressive clinical course, and have a higher rate of distant recurrence and a poorer prognosis than women with other breast cancer subtypes [3, 6, 7]. Population-based studies identified several risk factors for TNBC including young age at diagnosis, African-American race, high body mass index, young age at menarche, high parity, young age at time of first birth, and lack of breast feeding [3–5]. TNBC tumors are typically larger in size and of higher grade [8]. Due to the lack of well-defined molecular targets in TNBC, chemotherapy remains the mainstay of systemic treatment for this disease [9].
Cluster analysis of gene expression profiles from 587 TNBC cases identified six distinct subtypes, including two basal-like, an immunomodulatory, a mesenchymal, a mesenchymal stem-like, and a luminal androgen receptor subtype [10]. Signaling pathway abnormalities implicated in the pathogenesis of TNBC include DNA damage response, apoptosis, proliferation, epithelial-mesenchymal transition, immune response, and angiogenesis [10, 11].
Several components of the Wnt pathway as well as the Myc pathway are upregulated in the basal-like 2 and mesenchymal subtypes of TNBC such as WNT5A, SOX11, SOX4, LRP6, FZD4, and FZD7 [10, 12, 13]. Wnt signaling regulates cell proliferation, survival, and differentiation, and plays key roles in embryonic development and tumorigenesis [14–17]. In the absence of Wnt ligands, cytoplasmic β-catenin is recruited into a destruction complex that consists of adenomatous polyposis coli (APC), glycogen synthase kinase-3β (GSK3β), axin, and casein kinase 1 (CK1). This complex formation induces the phosphorylation of β-catenin at the amino terminus by GSK3β and CK1, resulting in the ubiquitination and the subsequent degradation of β-catenin [16–19]. When Wnt ligands are secreted, they bind to their receptors, low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled (FZD), to activate the Wnt signaling pathway. This binding leads to the recruitment of the scaffolding protein Dishevelled (Dsh) and axin to the cell membrane, and inactivation of the destruction complex. Inhibition of the degradation of β-catenin allows the cytoplasmic stabilization and translocation of the protein to the nucleus where it binds to members of the T-cell factor/lymphoid enhancing factor (TCF/LEF) family of transcription factors, and induces the expression of Wnt target genes. Sex-determining region Y-box 4 (SOX4), which is a highly conserved developmental transcription factor, has been implicated in playing an important role in Wnt signaling pathway in cancers [20, 21]. SOX4 contains a high-mobility group DNA-binding domain that is structurally related to TCF/LEFs [22]. Sinner et al. demonstrated that SOX4 stabilizes β-catenin, and enhances Wnt signaling pathway in colon carcinoma [23], while Scharer et al. demonstrated cooperativity between SOX4 and β-catenin in prostate cancer cells [21]. SOX4 was also shown to have a role in Wnt signaling in malignant melanoma by regulating β-catenin [24, 25].
Genistein, the major isoflavone in soybean, has been shown to have anticancer effects [26–28]. Genistein treatment of prostate cancer cells upregulates the expression of GSK3β, enhances GSK3β binding to β-catenin, and increases β-catenin phosphorylation, resulting in inactivation of Wnt/β-catenin and inhibiting cancer growth [29, 30]. Genistein was also shown to diminish Wnt1-induced proliferation and decrease the expression of Wnt targets, namely c-myc and cyclin D1 [31, 32]. Genistein also suppresses β-catenin transcriptional activity in colorectal carcinoma cells, and reduces cell proliferation through theWnt pathway in mesenchymal stromal cells isolated from human umbilical cord [33, 34].
Other recently identified inhibitors of the Wnt pathway include the inhibitors of catenin-responsive transcription (iCRT) compounds, iCRT-3, iCRT-5, and iCRT-14, which were identified in an RNAi modifier screen [35]. The Novartis compound XAV-939 stimulates β-catenin degradation by inhibiting Tankyrase and thereby stabilizing axin [36]. The IWP-4 compound was identified in a small molecule screen for Wnt antagonists and inhibits Porcupine, the membrane-bound acyltransferase that is essential to the pamitoylation of Wnt ligands [37]. In this study, we investigated the potential of these Wnt pathway inhibitors in effective treatment of TNBC cells. In addition, we tested the effects of reduced SOX4 levels in combination with Wnt inhibitor and/or the soy isoflavone genistein treatment on cell proliferation, migration and invasion on TNBC cells. We found that iCRT-3 and SOX4 knockdown have potential for therapy of TNBC.
Cell culture and reagents
MCF-7 and TNBC cell lines (BT-549, MDA-MB-231, HCC-1143 and HCC-1937) were obtained from American Type Culture Collection (Manassas, VA). MCF-7 and MDA-MB-231 cells were maintained in DMEM (Cellgro, Manassas, VA) while BT-549, HCC-1143 and HCC-1937 cells were grown in RPMI 1640 medium (Gibco, Grand Island, NY). Both media were supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 2 mM L-Glutamine (Gibco, Grand Island, NY) and 50 U/ml Penicillin-50 μg/ml Streptomycin antibiotics (Gibco, Grand Island, NY). Medium for BT-549 was also supplemented with 0.023 IU/ml bovine insulin (Sigma-Aldrich, St. Louis, MO). Cell lines were cultured in a 37°C incubator with humidified atmosphere of 5% CO2.
XAV-939 and genistein were purchased from Sigma-Aldrich (St. Louis, MO). iCRT-3, iCRT-5 and iCRT-14 were obtained from ChemDiv (San Diego, CA). IWP-4 was purchased from Stemgent (San Diego, CA). Each compound was reconstituted in dimethyl sulfoxide (DMSO) (EMD, Germany). Recombinant human Wnt-3a (5036-WNP) was purchased from R&D Systems (Minneapolis, MN), and reconstituted in PBS containing 0.1% BSA. Puromycin was obtained from Enzo Life Sciences (Farmingdale, NY). Matrigel was purchased from BD Biosciences (San Jose, CA). Trypan blue solution was obtained from Thermo Scientific (Waltham, MA). Rabbit anti-SOX4 (LS-B3520) antibody, mouse monoclonal antibody against active β-catenin (clone 8E7, #05-665), and mouse monoclonal antibody against β-actin (8H10D10, #3700) were purchased from LifeSpan BioSciences (Seattle, WA), Millipore (Billerica, MA) and Cell Signaling Technology (Danvers, MA), respectively. Mouse monoclonal antibody against β-catenin (E-5, sc-7963) and rabbit polyclonal antibody against Dvl-2 (H-75, sc-13974) were procured from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). IRDye 680RD goat anti-rabbit and IRDye 800CW goat anti-mouse secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE). Alexa Fluor® 488 conjugated goat anti-mouse secondary antibody (A11017) and Hoechst 33342 (H3570) were procured from Molecular Probes (Eugene, OR). Fluoromount-G medium was purchased from SouthernBiotech (Birmingham, AL).
Immunofluorescence staining and confocal microscopy
Cells were grown on sterile coverslips placed in 6-well plate, and serum-starved for 24 hours prior to the treatment with 200 ng/ml Wnt-3a for 4 hours. Cells were then fixed with 4% paraformaldehyde for 15 minutes at room temperature, and permeabilized with 0.5% Triton X-100 for 10 minutes. After blocking with 3% BSA for 30 minutes, cells were incubated with the primary antibody (mouse monoclonal antibody against β-catenin, 1:100 dilution) for overnight at 4°C. Cells were then incubated with Alexa Fluor® 488 conjugated goat anti-mouse secondary antibody at 1:1,000 dilution for 1 hr at room temperature in the dark. To ensure specificity of our results, negative controls with no primary antibody or no secondary antibody were included. For nuclear counterstaining, cells were incubated with Hoechst 33342 (1:10,000 dilution) for 15 minutes. Coverslips were then mounted with Fluoromount-G. Cells were visualized using Zeiss LSM510 Meta confocal microscope (Carl Zeiss Microscopy GmbH, Germany). Images were acquired at 200× total magnification using Zeiss Zen 2009 software.
Generation of stable SOX4 knockdown cell line
Knockdown of SOX4 expression was performed using MISSION short hairpin RNA (shRNA) lentiviral transduction particles (Sigma-Aldrich, St. Louis, MO) according to manufacturer's protocol. BT-549 cells were transduced with scrambled control shRNA or SOX4 shRNA lentiviral construct at 60-70% confluency. Puromycin (1 μg/ml) was administered for two weeks for selection of transduced cells. Knockdown of SOX4 was verified by Western blotting and quantitative real-time RT-PCR.
Western blot analysis
Whole cell lysates were prepared from cells on 100 mm-culture dish in lysis buffer containing 137 mM NaCl, 20 mM Tris–HCl (pH 8.0), 10% glycerol, 1% NP-40, and protease inhibitor cocktail (Promega, San Luis Obispo, CA). Protein concentrations in the supernatants were determined using Micro BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). 50 μg total protein was separated on 7.5 or 10% SDS-polyacrylamide gel, and transferred to nitrocellulose membrane. Membrane was blocked in 1× PBS buffer containing 0.1% Tween-20 and 5% non-fat dry milk for 1 hour at room temperature, and then incubated with primary antibody (mouse anti-active β-catenin, 1:1,000; rabbit anti-Dvl-2, 1:400; rabbit anti-SOX4, 1:1,000) overnight at 4°C. Mouse monoclonal antibody against β-actin (1:5,000) was used as normalization control. Membrane was then incubated with fluorescence-conjugated secondary antibodies (IRDye 680RD goat anti-rabbit and IRDye 800CW goat anti-mouse) at 1:5,000 dilution for 1 hour at room temperature, and signals were visualized and quantitated using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Immunoblots were repeated three times with new lysates from independent experiments.
Quantitative real-time RT-PCR analysis
Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen, Valencia, CA), quantitated using NanoDrop 1000 (NanoDrop, Wilmington, DE), and reverse transcribed into cDNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Quantitative real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories) on a Bio-Rad iCycler. Sequences of the primers for Axin2 were 5′-CAGGACACTGCTCTCTCAGATTCA-3′ (forward) and 5′-TCACAACAGCCTTTGCAGGG-3′ (reverse). Sequences of the primers for SOX4 were 5′-CCGAGCTGGTGCAAGACC-3′ (forward) and 5′-CCACACCATGAAGGCGTTC-3′ (reverse). Sequences of the primers for β-actin were 5′-CTGGAACGGTGAAGGTGACA-3′ (forward) and 5′-AAGGGACTTCCTGTAACAATGCA-3′ (reverse). The relative changes in gene expression data were analyzed by the 2-ΔΔCT method. β-actin was used as an internal control. Triplicates were run for each sample in three independent experiments.
Apoptosis assay
Analysis of apoptosis was performed using Caspase-Glo 3/7 assay (Promega) according to manufacturer's protocol. BT-549 cells that were transduced with scrambled or SOX4 shRNA lentiviral particles were seeded in 96-well plate, and incubated overnight. Cells were then treated with DMSO or 25 μM iCRT-3 for 12 hours. Caspase 3/7 activity was measured using FLUOstar OPTIMA (BMG Labtech, Cary, NC) microplate reader. Each sample was assayed in triplicate in three independent experiments.
Dual-luciferase reporter assay
BT-549 cells were seeded into 12-well plates. After overnight incubation, cells were transiently transfected with 0.5 μg of TOP-FLASH firefly luciferase reporter vector (Promega) and 0.04 μg of Renilla luciferase vector (Promega) as an internal control for transfection efficiency using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 24 hour-transfection, cells were treated with DMSO or 25 μM iCRT-3 for 48 hours. Cells were then lysed, and luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega) and TD-20/20 luminometer (Turner Design). The relative luciferase activity was calculated by firefly luciferase activity/Renilla luciferase activity. Data were presented as mean ± SEM from three independent experiments.
Cell proliferation, migration, and invasion assays using xCELLigence system
xCELLigence experiments were performed using the RTCA (Real-Time Cell Analyzer) DP (Dual Plate) instrument according to manufacturers' instructions (Roche Applied Science, Mannheim, Germany and ACEA Biosciences, San Diego, CA). The RTCA DP Instrument includes three main components: (i) RTCA DP Analyzer, which is placed inside a humidified incubator maintained at 37°C and 5% CO2, (ii) RTCA Control Unit with RTCA Software preinstalled, and (iii) E-Plate 16 for proliferation or CIM-plate 16 for migration and invasion assays. First, the optimal seeding number for each cell line (B-T549, MDA-MB-231, HCC-1143 and HCC-1937) was determined by cell titration and growth experiments. After seeding the respective number of cells/well (BT-549: 10,000 cells/well, MDA-MB-231: 20,000 cells/well, HCC-1143: 5,000 cells/well, and HCC-1937: 12,500 cells/well), the cells were automatically monitored every 15 minutes. Cells were treated with the compounds about four hours after seeding, when the cells were in the log growth phase. For cell proliferation assay in each cell line, cells were treated with DMSO as the vehicle or different concentrations of each Wnt inhibitor: iCRT-3 (25, 50, 75 μM), iCRT-5 (50, 100, 200 μM), iCRT-14 (10, 25, 50 μM), IWP-4 (1, 2.5, 5 μM), and XAV-939 (5, 10 μM). For cell proliferation, migration and invasion assays in BT549 cells with SOX4 knockdown, cells were treated with DMSO or 25 μM iCRT-3. The upper chamber of CIM-plate 16 was coated with Matrigel (1:40 dilution) for cell invasion assay. In addition, cell proliferation was measured in BT-549 cells with SOX4 knockdown that were treated with 50 μM genistein for six days, and 25 μM iCRT-3 at the time of the experiment. Each sample was assayed in triplicate, and three independent experiments were performed. Cell proliferation assays were run for 48 hours, and cell migration and invasion experiments for 24 hours. Cell index value, which is used to measure the relative change in electrical impedance to represent cell morphology, adhesion or viability, was calculated for each sample by the RTCA Software Package 1.2.
Cell viability assay
Cells were seeded at 20,000 cells/well into 96-well plates. After overnight incubation, cells were treated with DMSO or each Wnt inhibitor (iCRT-3, 75 μM; iCRT-5, 200 μM; iCRT-14, 50 μM; IWP-4, 5 μM and XAV-939, 10 μM) for 48 hours. Cell viability was determined using the Cell Titer-Glo luminescent cell viability assay kit (Promega) according to the manufacturer's instructions. Luminescence was measured using FLUOstar microplate reader. All treatments were performed in triplicate, and each experiment was repeated three times.
Data obtained from three independent experiments performed in triplicate were presented as mean ± SEM. Student's t-test (two-tailed, equal variance) was used to determine significant differences between two groups of data. p values of <0.05 and <0.01 were considered as statistically significant, and are indicated by asterisks (* and **, respectively).
Bioinformatics meta-analysis
Gene expression data was downloaded from the Gene Expression Omnibus (GEO) repository using series accession GSE12790 derived from two studies of breast cancer cell lines [38, 39]. Data was also obtained from the Cancer Cell Line Encyclopedia (CCLE) [40]. For the GSE12790 dataset, 43 luminal breast cancer cell lines were compared to 12 TNBC cell lines of mesenchymal, mesenchymal stem-like, or basal-like 2 subtypes of TNBC. For the CCLE dataset 22 luminal cell lines were compared to 21 TNBC cell lines. Differentially expressed genes were identified by Significance Analysis of Microarrays [41] with a false discovery rate of 5%, and pathway enrichment was determined by Ingenuity Pathway Analysis.
Wnt signaling pathway is activated in TNBC cells
Previous studies have shown that Wnt pathway genes are upregulated in TNBC tumors [10]. To verify these earlier studies, we performed pathway enrichment analyses on two independent datasets. The first dataset (GSE12790) derived from two studies of breast cancer cell lines [38, 39] included microarray data from 43 luminal breast cancer cell lines and 12 TNBC cell lines of mesenchymal, mesenchymal stem-like, or basal-like 2 subtypes. The second dataset included microarray data from the Cancer Cell Line Encyclopedia (CCLE) [40] with microarray data from 22 luminal breast cancer cell lines and 21 TNBC cell lines. For both the GSE12790 dataset and the CCLE dataset, Wnt-pathway genes were strongly enriched (p = 8.5e-6 and p = 3.0e-4, respectively). Genes differentially regulated in TNBC cell lines in both analyses included CD44, CDH1, DKK3, FZD7, SFRP1, SOX9, TGFB2, TLE4, WNT10A, and WNT5B (see Additional file 1: Table S1 and Additional file 2: Figure S1).
This prompted us to first assess whether the Wnt pathway was active in human TNBC cell lines by confocal microscopy. We selected two mesenchymal subtype TNBC cell lines (BT-549 and MDA-MB-231), and two basal-like 2 subtype TNBC cell lines (HCC-1143 and HCC-1937), as well as the non-TNBC ER+ control cell line MCF-7. Immunofluorescence staining of β-catenin showed both nuclear and cytoplasmic localization in TNBC cell lines whereas β-catenin was observed only in the cytoplasm of MCF-7 cells (Figure 1; see Additional file 3: Figure S2). We further explored the subcellular localization of β-catenin by treating the cells with Wnt-3a ligand (100 ng/ml) for four hours. We observed increased nuclear localization of β-catenin in MDA-MB-231 and BT-549 cells treated with Wnt-3a but not in MCF-7 cells, suggesting responsiveness of the canonical Wnt pathway in TNBC cells. To evaluate Wnt pathway activation, the expression levels of active β-catenin and phosphorylated Dvl-2 were examined in TNBC and non-TNBC cell lines by Western blot analysis (Figure 2). Active β-catenin was detected in both mesenchymal and basal-like subtypes, although the levels were much higher in basal-like cells than in mesenchymal cells. Activated β-catenin was lower in non-TNBC MCF-7 cells than in basal-like TNBC cells, but surprisingly was higher than in mesenchymal TNBC cells. Wnt-induced phosphorylation of Dvl-2 associated with mobility shift was differentially observed in all cell lines, with the highest ratio in HCC-1937 and the lowest in MDA-MB-231 cells.
Wnt signaling pathway is activated in TNBC cells. Subcellular localization of β-catenin in TNBC cells treated with or without human recombinant Wnt-3a (200 ng/ml) for 4 hours was examined using confocal microscopy. Immunofluorescence staining of β-catenin (green) showed cytoplasmic localization in MCF-7 cell line, and was both nuclear and cytoplasmic in TNBC cell lines, MDA-MB-231 and BT-549. Treatment with Wnt-3a resulted in increased nuclear localization of β-catenin in MDA-MB-231 and BT-549 cells. Nuclei were counterstained with Hoechst 33342 (blue). Total magnification was 200×, and the images were zoomed in 500%.
Relative expression levels of active β-catenin and phosphorylated Dvl-2 in non-TNBC (MCF-7) and TNBC (MDA-MB-231, BT-549, HCC-1143 and HCC-1937) cell lines. Whole cell lysates were prepared, and analyzed for protein expression using Western blotting. β-actin was used as normalization control. Data represent mean ± SEM of three independent experiments.
iCRT-3 effectively inhibits cell proliferation in TNBC cells
To investigate the effectiveness of five different compounds targeting the Wnt pathway in breast cancer cells, we first tested the inhibitory effects iCRT-3, iCRT-5, iCRT-14, IWP-4, and XAV-939 on cell proliferation in BT-549, MDA-MB-231, HCC-1143 and HCC-1937 cell lines using the xCELLigence system that allows continuous and quantitative monitoring of cell status in real-time. Cells were treated with increasing concentrations of each compound and assayed for 48 hours. The concentration range for treatment with each inhibitor was determined based on previous studies (34–36). This analysis showed that each compound induced differential effects on proliferation of these TNBC cells in a dose- and time-dependent manner (Figure 3; see Additional file 4: Figure S3, Additional file 5: Figure S4 and Additional file 6: Figure S5). These findings were confirmed using an alternative cell viability assay, the Cell Titer-Glo luminescent cell viability assay (see Additional file 7: Figure S6). Taken together, these data indicated that iCRT-3 was the most effective compound that we tested for inhibiting proliferation in all of these TNBC cells.
iCRT-3 effectively inhibits cell proliferation in BT-549 cells in a dose- and time-dependent manner. Cells were treated with vehicle (DMSO) or each of five Wnt inhibitors (iCRT-3, iCRT-5, iCRT-14, IWP-4, and XAV-939) at the indicated concentrations. Cell index values were continuously measured for 48 hours at intervals of 15 minutes using an xCELLigence instrument. Data represent mean ± SEM of three independent experiments (**p < 0.01).
Wnt pathway is antagonized by iCRT-3 in BT-549 cells
To evaluate whether the inhibitory effects of iCRT-3 are mediated through canonical Wnt signaling in TNBC, BT-549 cells were serum-starved for 24 hours, and then treated with Wnt-3a (200 ng/ml) and/or iCRT-3 (25 μM) for four hours. Quantitative real-time RT-PCR analysis of Axin2 in these cells showed that Wnt pathway is significantly activated and iCRT-3 efficiently blocked the expression of Axin2, which is a Wnt-induced target gene (Figure 4A). However, none of the other Wnt inhibitors had inhibitory effect on Axin2 expression (see Additional file 8: Figure S7). Previous studies have reported that iCRT-3 efficiently blocks the transcriptional function of β-catenin [22, 34]. To assess the effect of iCRT-3 treatment on transcriptional activity of β-catenin in BT-549 cells, dual luciferase assay was performed using the TOP-FLASH reporter vector. Cells were transfected with TOP-FLASH reporter and Renilla control vectors. After 24 hour-transfection, cells were treated with DMSO or 25 μM iCRT-3, and luciferase activity was measured at 48 hours post-treatment. iCRT-3 treatment of BT-549 cells resulted in significant decrease in transcriptional activity of β-catenin, suggesting that iCRT-3 inhibits the canonical Wnt pathway (Figure 4B). These data demonstrate that iCRT-3 antagonizes Wnt pathway signaling.
iCRT-3 inhibits Wnt signaling in TNBC cells. (A) Wnt pathway is antagonized by iCRT-3. BT-549 cells were serum-starved for 24 hours, and treated with Wnt-3a (200 ng/ml) and/or iCRT-3 (25 μM) for 4 hours. Total RNA was prepared, and assessed for Axin2 expression using quantitative real-time RT-PCR. β-actin was used as normalization control. Data represent mean ± SEM of three independent experiments (*p < 0.05). (B) iCRT-3 significantly inhibits transcriptional activity of β-catenin in BT-549 cells. BT-549 cells were transiently transfected with TOP-FLASH firefly luciferase reporter vector and Renilla luciferase vector. After 24 hour-transfection, cells were treated with DMSO or 25 μM iCRT-3 for 48 hours. Luciferase activities were measured using Dual-Luciferase Reporter Assay System. The relative luciferase activity was calculated by firefly luciferase activity/Renilla luciferase activity. Data represent mean ± SEM of three independent experiments (**p < 0.01).
SOX4 knockdown synergizes with iCRT-3 to induce apoptosis in BT-549 cells
Previous studies have shown that the oncogenic SOX4 transcription factor plays an important role in Wnt signaling pathways in many cancers including TNBC [10, 18, 19]. Therefore, we hypothesized that knockdown of SOX4 could inhibit cell viability and induce apoptosis in TNBC cells. To test our hypothesis, we first transduced the BT-549, MDA-MB-231, HCC-1143 and HCC-1937 cells with scrambled or SOX4 shRNA lentiviral particles. However, generation of stable SOX4 knockdown was successful only in BT-549 cells, possibly because SOX4 knockdown may be lethal to the other lines tested. Western blotting and quantitative real-time RT-PCR analyses demonstrated that the expression of SOX4 protein in BT-549 cells transduced with SOX4 shRNA was significantly decreased in comparison with that of cells transduced with scrambled shRNA, verifying that the expression of SOX4 was successfully knocked down in BT-549 cells (Figure 5A and B). Moreover, Caspase 3/7 activities showed that while knockdown of SOX4 alone did not enhance apoptosis of these cells (Figure 5C), combined treatment of iCRT-3 with SOX4 knockdown has a synergistic effect in inducing apoptosis.
SOX4 knockdown enhances the effects of iCRT-3 on apoptosis in BT-549 cells. BT-549 cells were transduced with scrambled shRNA or SOX4 shRNA lentiviral particles. For stable knockdown, cells were kept under puromycin selection for two weeks. Whole cell lysates and total RNA were prepared, and assessed for SOX4 expression using (A) Western blotting and (B) quantitative real-time RT-PCR. β-actin was used as normalization control. Data represent mean ± SEM of three independent experiments (**p < 0.01). (C) Apoptosis levels were quantitated by Caspase-Glo 3/7 assay. Data represent mean ± SEM of three independent experiments (**p < 0.01, *p < 0.05).
SOX4 knockdown inhibits cell proliferation and migration of BT-549 cells, and cooperates with iCRT-3 to inhibit proliferation
To characterize the mechanism underlying the synergism between SOX4 knockdown and iCRT-3 treatment, we next assessed the effect of SOX4 knockdown in combination with iCRT-3 treatment on cell proliferation, migration and invasion in BT-549 cells using the xCELLigence system. Cells that were transduced with scrambled or SOX4 shRNAs were treated with DMSO or 25 μM iCRT-3, and monitored continuously for 48 hours in a proliferation assay, and for 24 hours in migration and invasion assays. As shown in Figure 6, SOX4 knockdown inhibited cell proliferation and migration. Effects on invasion were not statistically significant, although the trend was towards lower invasion with SOX4 knockdown. Combination treatment of iCRT-3 with SOX4 knockdown induced further decrease in cell proliferation, but had no increased effect over SOX4 knockdown in cell migration or invasion.
SOX4 knockdown inhibits proliferation, migration and invasion of BT-549 cells, and cooperates with iCRT-3 to inhibit proliferation. Cells that were transduced with scrambled shRNA or SOX4 shRNA lentiviral particles were treated with vehicle (DMSO) or iCRT-3 (25 μM), and cell index measurements were continuously taken for 48 hours in (A) proliferation assay, and 24 hours in (B) migration and (C) invasion assays using an xCELLigence instrument. Data represent mean ± SEM of three independent experiments (**p < 0.01).
Genistein has been shown to have anticancer effects through targeting multiple signaling pathways, including Wnt signaling [31, 32]. Therefore, we tested the effect of genistein in combination with SOX4 knockdown and/or iCRT-3 treatment on proliferation in BT-549 cells. Cells that were transduced with scrambled or SOX4 shRNAs were treated with DMSO or 50 μM genistein for six days. Cells were then treated with 25 μM iCRT-3, and monitored for 48 hours (Figure 7). Genistein treatment did not enhance the inhibitory effects of either SOX4 knockdown or iCRT-3 treatment on cell proliferation in BT-549 cells.
Genistein has no inhibitory effect on cell proliferation in BT-549 cells. (A) Cells that were transduced with scrambled shRNA or SOX4 shRNA lentiviral particles were treated with vehicle (DMSO) or genistein (50 μM) for six days. (B) These cells were further treated with iCRT-3 (25 μM) at the time of the proliferation assay, and cell index measurements were taken for 48 hours using an xCELLigence instrument. Data represent mean ± SEM of three independent experiments (**p < 0.01).
Due to the lack of hormone receptors and HER2 overexpression in patients with TNBC, hormonal therapies and HER2-targeted agents are ineffective, and chemotherapy is the main current systemic treatment in this subtype of breast cancer [9]. Although they are sometimes responsive to neoadjuvant chemotherapy, TNBC patients have a higher rate of relapse with distant metastases and a poorer prognosis than women with other breast cancer subtypes [3, 4, 6–8]. These factors highlight the urgent need to develop more effective and targeted therapy options for patients with TNBC. Aberrant activation of Wnt signaling, which plays a key role in the regulation of cell growth, development and differentiation, has been associated with many cancer types including colorectal, prostate, liver, and breast cancers [16–19]. Thus, inhibition of Wnt signaling could potentially be an effective approach to the treatment of many types of human cancers.
In the present study, we investigated, for the first time, the effects of five different small-molecule inhibitors that target different components of the Wnt signaling pathway on cell proliferation in four TNBC cell lines. These representative cells belong to mesenchymal or basal-like 2 subtypes of TNBC in which Wnt pathway-associated genes are specifically overexpressed. While mesenchymal TNBC cells (MDA-MB-231 and BT-549) responded to Wnt-3a treatment (Figure 1), basal-like 2 TNBC cells (HCC-1143 and HCC-1937) did not even though they did respond to iCRT-3 treatment. One hypothesis to explain this observation is that basal-like 2 TNBC cells may respond to different Wnt family ligands such as Wnt-5a, Wnt-5b, or Wnt-10a. The small compound Wnt inhibitors that we utilized in this study included (i) iCRT-3, iCRT-5 and iCRT-14 which inhibit catenin responsive transcription (CRT) [35], (ii) IWP-4, an inhibitor of Wnt production (IWP) that targets the acetyltransferase porcupine [37], and (iii) XAV-939, which induces β-catenin degradation by stabilizing axin through inhibition of poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2 [36]. Our findings indicated that each inhibitor had differential effects on proliferation of each cell line. The mesenchymal MDA-MB-231 and BT-549 cells were more sensitive than the basal-like HCC-1143 and HCC-1937 cells. We speculate that these differentials are due to the different levels of basal Wnt activation and different genetic backgrounds of the cell lines that could provide for different mechanisms of constitutive Wnt activation. Surprisingly, the cell lines with the lower constitutive Wnt activity are more sensitive to the inhibitors. It is not clear why this is the case, but we can speculate that the inhibitors may be less effective when Wnt activation levels are above a certain threshold. Alternatively, the less sensitive cell lines may have activation of additional anti-apoptotic, pro-survival pathways that confer resistance to Wnt pathway inhibition.
Our data also demonstrated that of these five compounds, iCRT-3 was the most effective and consistent one in inhibiting cell proliferation in all TNBC cell lines tested. Because iCRTs, IWP-4 and XAV-939 inhibit Wnt signals through different mechanisms, the effectiveness of each inhibitor would be expected to vary in these TNBC cell lines depending on the genetic changes they have. Previous reports have shown that IWP-4 and XAV-939 are effective in cells which have loss of APC tumor suppressor function, and this effectiveness could be explained by the rate-limiting role that Axin proteins play in canonical Wnt pathway. Our finding that there was no significant inhibitory effect of IWP-4 and XAV-939 on proliferation of the TNBC cell lines examined in this study may correlate with the fact that none of these TNBC cell lines has a mutation in APC gene. In addition, iCRT-3 resulted in increased apoptosis in BT-549 cells, whereas knockdown of SOX4 expression did not have a significant effect on apoptosis. Combined treatment of SOX4 knockdown with iCRT-3 synergistically induced apoptosis in BT-549 cells. It is noteworthy that in the combination experiments, we treated the cells with a suboptimal concentration of 25 μM iCRT-3 in order to enable detection of synergistic effects of combination treatments. Luciferase reporter assays showed that iCRT-3 significantly antagonized canonical Wnt pathway in BT-549 cells, consistent with our finding that expression of Axin2 was suppressed by iCRT-3 in these cells. Another important finding in this study is that knockdown of SOX4 in BT-549 cells had inhibitory effects on cell proliferation and migration. Moreover, iCRT-3 treatment enhanced SOX4 knockdown-induced inhibition of cell proliferation, but did not have an additive effect over SOX4 knockdown on migration and invasion of BT-549 cells.
We show in this study that iCRT-3 treatment inhibits proliferation and induces apoptosis, whereas SOX4 knockdown effectively inhibits cell proliferation and migration, suggesting potential therapeutic roles for iCRT-3 and SOX4 in targeting TNBC. These findings highlight the importance of the Wnt signaling cascade in TNBC progression, and provide a strong rationale for future in vivo studies of these agents in TNBC. Further investigation of the molecular mechanisms of TNBC will provide a better understanding of the pathogenesis, and thus additional insight into the development of new, targeted therapeutics for TNBC.
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This work was supported by grant R01CA106826 from the National Cancer Institute (NCI) of the National Institutes of Health (NIH) to CSM and funding from the Georgia Cancer Coalition. Roche xCelligence analysis was performed at the Integrated Cellular Imaging (ICI) Core of the Emory Winship Cancer Institute supported by NCI Cancer Center Support Grant P30CA138292. OK is a Georgia Research Alliance Distinguished Scientist.
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA
Birdal Bilir
& Carlos S Moreno
Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, Georgia, USA
& Omer Kucuk
Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
Omer Kucuk
Search for Birdal Bilir in:
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Correspondence to Carlos S Moreno.
Authors have no competing interests.
BB contributed experimental design, acquisition and analysis of data, interpretation of findings, and writing of the manuscript. OK contributed experimental design and editing of the manuscript. CSM contributed experimental design, analysis of data, interpretation of findings, and editing of the manuscript. All authors read and approved the final manuscript.
Additional file 1: Table S1: List of genes differentially expressed in TNBC cell lines in microarray analyses for Wnt pathway genes. (XLSX 52 KB)
Additional file 2: Figure S1: Wnt pathway analysis of TNBC. (JPEG 435 KB)
Additional file 3: Figure S2: Subcellular localization of β-catenin in HCC-1143 and HCC-1937 cells treated with or without human recombinant Wnt-3a (200 ng/ml) for 4 hours was examined using confocal microscopy. Immunofluorescence staining of β-catenin (green) showed cytoplasmic localization in both cell lines. Treatment with Wnt-3a did not have an effect on subcellular localization of β-catenin in HCC-1143 and HCC-1937 cells. Nuclei were counterstained with Hoechst 33342 (blue). Total magnification was 200×, and the images were zoomed in 500%. (JPEG 245 KB)
Additional file 4: Figure S3: iCRT-3 effectively inhibits cell proliferation in HCC-1143 cells in a dose- and time-dependent manner. Cells were treated with vehicle (DMSO) or each of five Wnt inhibitors (iCRT-3, iCRT-5, iCRT-14, IWP-4, and XAV-939) at the indicated concentrations. Cell index values were continuously measured for 48 hours at intervals of 15 minutes using an xCELLigence instrument. Data represent mean ± SEM of three independent experiments (**p < 0.01). (JPEG 424 KB)
Additional file 5: Figure S4: iCRT-3 effectively inhibits cell proliferation in MDA-MB-231 cells in a dose- and time-dependent manner. Cells were treated with vehicle (DMSO) or each of five Wnt inhibitors (iCRT-3, iCRT-5, iCRT-14, IWP-4, and XAV-939) at the indicated concentrations. Cell index values were continuously measured for 48 hours at intervals of 15 minutes using an xCELLigence instrument. Data represent mean ± SEM of three independent experiments (**p < 0.01). (JPEG 322 KB)
Additional file 7: Figure S6: iCRT-3 effectively inhibits cell proliferation in MDA-MB-231, BT-549, HCC-1143 and HCC1937 cells. Cells were treated with vehicle (DMSO) or each of five Wnt inhibitors (iCRT-3, iCRT-5, iCRT-14, IWP-4, and XAV-939) at the indicated concentrations. Cell viability was measured using Cell Titer-Glo luminescent cell viability assay. Data represent mean ± SEM of three independent experiments (**p < 0.01). (JPEG 268 KB)
Additional file 8: Figure S7: Wnt pathway is not antagonized by iCRT-5, iCRT-14, IWP-4 or XAV-939. BT-549 cells were serum-starved for 24 hours, and treated with Wnt-3a (200 ng/ml) and/or iCRT-5 (50 μM), iCRT-14 (10 μM), IWP-4 (1 μM) or XAV-939 (5 μM) for 4 hours. Total RNA was prepared, and assessed for Axin2 expression using quantitative real-time RT-PCR. β-actin was used as normalization control. (JPEG 310 KB)
Bilir, B., Kucuk, O. & Moreno, C.S. Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells. J Transl Med 11, 280 (2013) doi:10.1186/1479-5876-11-280
DOI: https://doi.org/10.1186/1479-5876-11-280
iCRT-3
SOX4
Patient-targeted molecular therapies


May 15, 2014, 2:29 a.m.
The main battleground between Samsung Electronics Co. and Apple Inc. in the global smartphone market is moving from courtrooms to the laboratory, amid a race for patents on at ..
May 15, 2014, 2:21 a.m.
Citigroup Inc.'s Mexican unit showered loans on a troubled oil-services company as other banks were pulling back, according to people familiar with the matter, setting the sta ..
May 15, 2014, 12:51 a.m.
It may soon be easier for the average homeowner to buy a house. But, is it too little, too late for home builders? ..
May 14, 2014, 11:23 p.m.
The New York Times Co (NYT) on Wednesday abruptly ousted the newspaper's top editor, Jill Abramson, after less than three years in the job and named managing editor Dean ..
May 14, 2014, 10:11 p.m.
Malaysian Airlines will report first-quarter earnings later on Thursday that are set to provide confirmation of just how badly the already loss-making carrier's finances ..
May 14, 2014, 9:43 p.m.
U.S. stocks fell on Wednesday, with the Dow and S&P 500 retreating from recent record highs, as small caps resumed their sell-off and consumer discretionary shares lagged. ..
May 14, 2014, 9:18 p.m.
Network equipment maker Cisco Systems Inc posted a lower-than-expected 5.5 percent decline in quarterly revenue, as the company faces sluggish demand. ..
May 14, 2014, 8:59 p.m.
The New York Times on Wednesday announced that executive editor Jill Abramson is being replaced by managing editor Dean Baquet after two and a half years on the job. The compa ..
Fannie and Freddie to the rescue!
May 14, 2014, 8:40 p.m.
It was once unthinkable, but the two wayward agencies may now save the housing market. ..
May 14, 2014, 8:24 p.m.
Stocks edged lower in early afternoon trading Wednesday, a day after a record high close. Investors favored safer stocks over riskier ones. ..


Comprehensive style mixed with all around versatility makes it an outstanding choice! Kia prioritized fit and finish as evidenced by: delay-off headlights, automatic temperature control, and remote keyless entry. Smooth gearshifts are achieved thanks to the 3.3 liter 6 cylinder engine, and for added security, dynamic Stability Control supplements the drivetrain.


The regular fare at $166 allows for normal size carry-on and personal item in addition to advanced seat assignment.
The $116 fare is a Basic Economy fare. United's Basic Economy fares do not allow for any advanced seat assignment. Rather, your seat assignment will be randomly assigned during check-in. In addition, normal size carry-on that does not fit under the seat is charged a $25 fee each way with applicable gate check fees (but free if you have an United co-branded credit card or if you have United elite status).


Many different parties share responsibility for maintaining the miles of pavement and sidewalks in New York City. When damage to those sidewalks leads to injuries, the hardest part of recovering rightful compensation may be determining just who is legally responsible for the patch of sidewalk that caused the injury. Fortunately, the experienced personal injury attorneys at the New York City law firm of Segan, Nemerov & Singer, PC can guide you through the injury claims process from start to finish.
Just who is responsible for maintaining this sidewalk?
But the question of responsibility for a safe sidewalk has more to do with the nature of the adjacent property than the reason for the damage.
The city is responsible for a sidewalk when the adjoining property is a one-, two- or three-family residential dwelling occupied by its owner and used only for residential purposes.
For all other properties, the adjacent property owner shoulders sidewalk maintenance responsibility. This includes any commercial building or residential structure that houses more than three families.
New York City courts have also ruled that the owner of a building cannot delegate or defer responsibility for their sidewalks to any other party, including the city.
What types of compensation can an attorney help recover in an uneven sidewalk injury case?
The responsible party knew about the hazardous sidewalk and failed to repair it within a reasonable timeframe.
The hazardous sidewalk directly caused your injury.
You suffered financially due to your injury.
If you or someone you love has been injured by a fall on broken or uneven pavement, time is not on your side. This is especially true when the liable party in your case is the city of New York. Contact the experienced injury lawyers at Segan, Nemerov & Singer, PC today to arrange a free consultation. You can reach us online or at (212) 696-9100.


The next BSTH conference will take place on 28 – 29 November 2019. Please save the date en keep up to date via our newsletter or website. We will inform you when about the program and registration as soon as possible!


text:
Cambodia paper's closure illustrates abuse of tax laws
IPI concerned government effort based on disputed debts part of growing global trend
IPI Contributor Renan Akyavas and IPI staff
A man reads newspapers on display at the Cambodia Daily Newspaper office in Phnom Penh, Cambodia on Sept. 4, 2017. EPA-EFE/MAK REMISSA
Observers worry that this month's closure of an independent Cambodian newspaper in the face of an astronomical tax demand is the latest incident illustrating a global trend in which governments abuse tax laws to harass independent news media outlets and silence critical reporting.
The English-language Cambodia Daily published its last print edition on Sept. 3 after Cambodia's government ordered it closed unless it failed to pay €5.3 million in disputed tax debts.
The Daily, which reported for more than 20 years, denies the government's claim and it has decried the fact that it was not allowed to appeal the tax bill.
The paper printed only a few thousand copies per day, but it had a reputation for breaking news about sensitive topics such as corruption, waste, environmental issues and land rights.
The government claims that the Daily owes a tax bill of €5.3 million dating back a decade, but the paper denies that, claiming that it has operated at a loss since 2008. The paper accuses the government of inflating the amount owed and failing to look at the paper's accounts or audit its books before levying the bill.
The Daily was just one of a number of media organisations that Cambodia's government has targeted amid a broader crackdown on independent media.
Nineteen radio stations were shut down in the last month, including the U.S.-funded Radio Free Asia and Voice of America. The government has been targeting not only independent media, but also opposition politicians and rights activists as part of a widening crackdown seen as a move to bolster Prime Minister Hun Sen ahead of an election next year.
In August, the government expelled the U.S.-funded, pro-democracy National Democratic Institute (NDI), also for alleged failure to pay taxes,
Cambodia Daily Editor-in-Chief Jodie DeJonge said the efforts were part of a broad crackdown.
"The closure of the Daily combined with the closure of the 19 radio stations and the NDI, the expulsion of foreign workers and the threat against the civil society was an attempt to silence independent voices," DeJonge told the International Press Institute (IPI).
She added that harassment, intimidation and surveillance of human rights defenders in Cambodia have intensified. Viewed along with the arrest of the main opposition leader, who is accused of treason, the developments point towards an attempt by the government to maintain power.
Cambodia's government's use of tax bills to pressure critics appears to be in line with a pattern around the globe. Over the last year, two similar tax cases against media outlets have played out in the neighbouring southern African countries of Zambia and Malawi.
In June 2016, Zambia's government seized the offices and printing press of The Post newspaper, demanding approximately €4.8 million in allegedly unpaid taxes. However, the Post disputed the amount owed and accused authorities of trying to silence it ahead of an August 2016 general election.
IPI and the African Media Initiative (AMI) sharply criticised the Zambian government's move in statements and a report following a joint press freedom mission to the country in July 2016. However, the paper folded in November 2016 after a court appointed a trustee to liquidate it amid former employees' complaints for back pay – complaints The Post argues were brought with government encouragement.
Three months later, Malawi's government took a similar tack when its revenue authority seized the Blantyre offices of the opposition Times Group – which publishes the Daily Times and Malawi News, and owns Times Radio and Times TV – and demanded approximately €783,000 in allegedly unpaid taxes.
The seizure of the Times Group's office came on Jan. 13, less than a week after Malawi's Agricultural Development and Marketing Corporation (ADMARC) sought an injunction preventing the media outlet from publishing stories on so-called "maize gate" scandal, over allegations of corruption in Malawi's maize imports from Zambia.
The Malawi Chapter of the Media Institute of Southern Africa condemned the seizure, arguing that it was intended to deny Malawians access to relevant information and was likely to tarnish the image of the administration of President Peter Mutharika.
IPI recently spoke to Joan Chirwa, the former managing editor of The Post in Zambia, about similarities between the closure of her former paper and the Cambodia Daily. Chirwa said that she was not surprised by the Daily's closure and expressed a belief that both cases were the manifestations of a troubling trend.
"The [Cambodian] government is afraid that the people in the Cambodia Daily are going to expose a lot of wrong things for the upcoming elections," she commented.
Expressing concern that government aggressiveness towards media often precedes elections, Chirwa emphasised that journalists and rights groups should unite to protect independent journalism around the world.
"What we will do is to protect what we believe in, to protect the independence of the media, not only for our own sake but also for the people," she explained.
IPI Director of Advocacy and Communications Steven M. Ellis, noting that a number of other governments have misused tax laws similarly in recent years, called on governments around the world to implement safeguards ensuring that tax powers are not politicised or used to target and harass independent media.
"This type of abuse severely impacts media freedom in a country and, if allowed to stand, invites further violations," he said. "Allowing journalists to report freely and without fear that they will be targeted is not only required under international human rights standards, it's absolutely vital to provide an environment where independent voices can be heard and citizens can hold government accountable."
Press freedom is under threat. But you can help.
To Our Readers: We need independent news and information more than ever, but journalists around the globe are increasingly under attack for doing their jobs. IPI works every day to support independent journalism and the free flow of news. But we need your help. By becoming an IPI member or supporter, you strengthen our mission to protect everyone's right to be informed.
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Two years of legal limbo for Cambodia journalists charged with espionage
Two years after their arrest in Cambodia on groundless esiponage-related charges, former R…
IPI joins Washington Post Press Freedom Partnership
The International Press Institute (IPI), a global network of editors, publishers and leadi…


DEVIN THE DUDE AMPED UP SMOKE FEST FEATURING SALUTE Club Forty7 July 17th 2015 - Duration: 58 seconds.
ALL NIGHT Salute feat Devin the Dude - Duration: 4 minutes, 14 seconds.
Check out the new video from Salute featuring Devin the Dude...Brace yourself, you may end up high all night!
Eyes Closed - Duration: 4 minutes, 33 seconds.
This is the next smash hit that's going to tear up the streets. Watch And Learn Something!
Salute ft. Jessica Rappit-Me and My Baby - Duration: 4 minutes, 42 seconds.
Salute - Come Up Gang - Das Aight Official Video - Duration: 4 minutes, 49 seconds.
Check out the latest hit from Salute of Come Up Gang, Das Aight!
SALUTE "TRYING TO COME UP" (COME UP ANTHEM) - Duration: 4 minutes, 47 seconds.
Hot new music from Salute from his mixtape Contraband War and Peace Volume 1.
SALUTE - PLANTATION MENTALITY [Official Music Video] - Duration: 5 minutes, 51 seconds.
Salute - 25 NIGGAZ - Duration: 4 minutes, 25 seconds.
Salute - Hell Nawl - Duration: 3 minutes, 53 seconds.
Salute - Aint No Way - Duration: 3 minutes, 39 seconds.
Salute - Barz - Duration: 3 minutes, 29 seconds.
Salute - Thang 4 You - Duration: 5 minutes, 5 seconds.


Ms. Zerrougui brings more than 30 years of experience in rule of law and protection of civilians, as well as a demonstrated record of management and leadership. She served as Special Representative of the Secretary-General for Children in Armed Conflict (2012 - 2016) and as Deputy Special Representative of the Secretary-General in MONUSCO (2008 - 2012), where she spearheaded the Mission's efforts in strengthening the rule of law and protection of civilians.
Born in 1956, Ms. Zerrougui graduated from the Ecole Nationale d'Administration (Algiers) in 1980. Since 1993, she has held various academic positions at law schools in Algeria, and was associate professor of the Ecole Supérieure de la Magistrature (Algiers).
United Nations Secretary-General António Guterres today announced the appointment of Kim Bolduc of Canada as his Deputy Special Representative for the United Nations Stabilization Mission in the Democratic Republic of the Congo (MONUSCO), where she will also serve as United Nations Resident Coordinator, Humanitarian Coordinator and Resident Representative of the United Nations Development Programme (UNDP).
Ms. Bolduc succeeds Mamadou Diallo of Guinea, who completed his assignment in October and to whom the Secretary-General is grateful for his leadership and dedicated service during his tenure.
Ms. Bolduc brings to the appointment more than 30 years of experience in international development and humanitarian work, as well as a demonstrated record of management and leadership. She served since 2014 as Special Representative of the Secretary-General and Head of United Nations Mission for Referendum in Western Sahara (MINURSO). She previously served as the United Nations Resident Coordinator and UNDP Resident Representative in Panama from 2010 to 2014, having previously been Deputy Special Representative of the Secretary-General, Resident Coordinator and Humanitarian Coordinator for the United Nations Stabilization Mission in Haiti (MINUSTAH), and Resident Coordinator and UNDP Resident Representative in Brazil. She held various field appointments within the United Nations system since she joined the Organization in 1987, including positions in Honduras, Iraq and Mozambique.
Ms. Bolduc has a master's degree in international development and cooperation from Ottawa University in Canada.
Born in 1952, she is married and has a son.
United Nations Secretary-General António Guterres today announced the appointment of Lieutenant General Elias Rodrigues Martins Filho of Brazil as Force Commander of the United Nations Organization Stabilization Mission in the Democratic Republic of the Congo (MONUSCO). He succeeds Lieutenant General Derrick Mbuyiselo Mgwebi of South Africa, who completed his assignment on 31 January, to whom the Secretary-General is grateful for his dedicated leadership as head of the MONUSCO military component.
Lieutenant General Martins Filho has had a distinguished military career with the Brazilian Armed Forces spanning more than 35 years. He is currently serving as the Chief of Office of the International Organizations Office at the Ministry of Defence of Brazil since 2017. Previously, he served as Chief of Defence Intelligence in the Intelligence Office of the Ministry of Defence, and as Director of the Brazilian Command, War and Staff College, from 2015 to 2016. Between 2013 and 2015, he was Chief of Operations at the Brazilian Western Border, having been Commanding Officer of the Brazilian Presidential Guard battalion between 2009 and 2011.
Lieutenant General Martins Filho also has extensive peacekeeping experience, having served as Planning Officer in the Force Generation Service in the Department of Peacekeeping Operations from 2005 to 2008. He also served as Deputy Military Adviser to the Permanent Mission of Brazil to the United Nations, between 2001 and 2003, and as Staff Officer at the United Nations Angola Verification Mission III from 1995 to 1996.
Lieutenant General Martins Filho holds a post-graduate degree in International Relations from the University of Brasilia. He graduated from the Defense War College of the Escola Superior de Guerra.
Born in Fortaleza in 1960, he is married and has three children.
Awale Abdounasir, a national of Djibouti with effect from 22 February 2016 became the current MONUSCO Police Commissioner. He just left MINUSMA as the Head of the Police component after 29 months of service.
Before Mr. Awale's appointment as the MINUSMA Police Commissioner, he served in UNOCI for five (5) continuous years in various capacities starting as an IPO (Patrol officer), a Team Leader at a Station, Special Assistant to the Police Commissioner, Head of Reform and Restructuring Section responsible for building the capacities of national Security Forces, the Deputy Police Commissioner and finally Acting Police Commissioner.
He holds a master's degree in law and security policy from the University of Lyon III and a master's degree in biochemistry from the University of Rennes.
He was a Professor before joining the police where he has more than 18 years' experience. He is a graduate of National Police Academy of Saint Cyr au Mont d'Or (FRANCE). He has also attended several training programs on antiterrorism and disaster management in USA.
Mr. Awale, during his career at the national level, held various managerial positions including Director of Forensic Police Laboratory, Director of Police Academy, Advisor and Chief of Staff to the Minister of Interior.


text:
<!DOCTYPE html>

    <meta charset="utf8">
    <title>test</title>

it works!

redpajama_set_name: RedPajamaGithub


Bobbi Kristina Brown Sued Following Car Crash, Deathbed Photos Being Shopped By Family?
A few days prior Bobbi Kristina Brown entered the hospital after being found in her bathtub, the daughter of the Late Whitney Houston was involved in a car accident with a man who claims he suffered serious injuries from the crash.
Bobbi Kristina then allegedly lost control of the 2005 Jeep Liberty she was driving, crossed the center line and collided with Eckerman's Ford Taurus.
According to PEOPLE, Eckerman claims he sustained "significant injuries" from the crash, including facial, spinal and skull fractures, a laceration on his scalp and a left wrist sprain that landed him in the hospital where he was placed in a medically induced coma and on a ventilator.
According to the papers, he underwent several procedures and his medical bills exceeded $732,610.22.
Bobbi Kristina Brown may be in her final days of life, but apparently that does not stop extended family members from taking advantage of her. Pictures of Bobbi Kristina in a hospice facility outside of Atlanta are now surfacing. Reports state that these pictures, supposedly taken by an extended family member, are extremely disturbing. They show her laying down, very obviously deteriorating, and feature a woman hovering over Bobbi Kristina. The photos were reportedly offered to editors at TMZ, and shopped to many other magazines and websites.
Leolah Brown, paternal aunt of the 22-year old, was so upset over the leaked photos that she suggested everyone who had been anywhere near Bobbi Kristina, including herself, take a lie detector test.
Bobbi Kristina spent months in the hospital and in a rehab center, after being found unconscious in a bathtub at the end of January this year. She was moved to a hospice facility recently after a brain specialist told the family that there was no hope of her condition improving or that she would wake up.
Although Bobby Brown historically does not get along with the Houston side of the family, all family members are coming together to work for Bobbi Kristina's good during her final days.
Now he's suing? Never cared for the Brown family. I thought Whitney was an amazing singer who's biggest flaw was hooking up with Bobby. But, after everything that's happened he thought best to sue because she definitely can't discuss anything. Sounds like he's a scumbag.
Wow be picked a perfect time to sue. Its all about the money with these people.
What an inconsiderate jerk!! Her family is going through enough right now!! Unreal!! They r watching her die before their eyes and this piece of garbage is worrying about a payday!! He could at least wait awhile he has waited this long to file suit what's a couple more months so the family grieves and have a funeral.


Cooking a delicious ribeye roast for dinner? In this video, learn how to slow cook a ribeye roast. Advice: "500 degrees for 15 minutes and then reduce temp. to 250 degrees. Takes around 3 hours for the internal temp. reaches 130 degrees. Then let it stand for 20 minutes."
In this video, learn how to combine citrus with savory in a perfect way. You will learn to cook English specialty - roast quail - with cured lemons for a unique and interesting taste. Enjoy this dinner at your next party and you'll have everyone asking for seconds and thirds!
There are few recipes as American as the basic pot roast. This video shows you how to take a basic cut of beef and turn it into a delicious dinner. All you need besides the beef is some flour and olive oil, as well as your marinating spices.
Accompanied with an adzuki bean puree, bok choy and a soy-mustard-miso aioli, this dish is guaranteed to impress on your next dinner date.


In an ongoing effort to keep up with their closest competition, the Big Four -- T-Mobile, Sprint, AT&T, and Verizon -- have continually innovated and directed wireless industry trends over the years.
As such, the challenge of an ever-evolving carrier landscape has made it increasingly difficult for regional carriers to keep pace. But, with a little creativity and perseverance, regional carriers still have opportunities to stand out.
Broadly, one of the primary challenges for all wireless carriers is that consumers are now keeping devices longer, at an average of nearly three years. Who can blame them with phone prices on the rise? The latest iPhone, for example, rings in at a whopping $1,000. And since customers are now visiting stores to upgrade less often, it reduces the opportunity for regular interaction consumers might have with carriers every few years. With customer touchpoints limited, wireless carriers should tap in to tools like warranty protection plans. Not only can warranty products provide added value for customers looking to protect devices long term, but they can also generate additional carrier revenue.
While it's easy for smaller, regional carriers to get caught up in the news of major industry changes -- like Sprint and T-Mobile merging, or the hype around 5G rollouts -- it's essential that they not get distracted and focus on highlighting the advantages of their business. Regional carriers have a unique opportunity to connect with their community in ways the Big Four can't. By embracing that niche position, they can provide innovative offerings and programs for their local customer base. A great example is a regional carrier who equips remote school buses with Wi-Fi capabilities so students can complete assignments during their long rides home. Large carriers don't have the resources to offer these services to rural communities, and aren't nimble enough to devote time to build these kinds of close customer relationships.
With so many options on the market, it's important to ensure customers feel valued -- it's an investment of effort that builds long-term loyalty… and revenue. In fact, when customer retention increases by just five percent, companies can see up to a 95 percent increase in profit. It's essential for regional carriers to prioritize their customer relationship strategy. With a specific customer base to focus on, regional carriers can offer a more personalized service experience, utilizing approaches like custom social media messaging or encouraging call center employees to go off-script. These kinds of locally-focused efforts can keep customers coming through the door long-term.
At the end of the day, there's no reason for regional carriers to be intimidated by the Big Four. By focusing on customer relations and how they can uniquely serve their community, regional carriers can enjoy unique advantages versus their bigger competitors.
Jon Mikow is Vice President of Wireless at Fortegra Financial Corporation (a Tiptree Inc. company). Fortegra and its subsidiaries comprise a single-source insurance services provider that offers a range of consumer protection options including warranty solutions, credit insurance, and specialty underwriting programs. Delivering multifaceted coverage with an unmatched service experience for domestic and international partners and their customers, Fortegra solves immediate, everyday needs, empowering consumers to worry less and Experience More.


zend-mvc integrates with zend-console; the integration also works with modules loaded with the Module Manager.
arguments could not be matched by any routes.
The strategy currently supports two types of information: application banners and usage information.
As you can see, the application banner should be a single line string that returns the module's name and (if available) its current version.
If several modules define their own banner, they are displayed one after the other in the order in which the modules are loaded. This allows you to see at a glance which modules expose console commands.
Application banners provided by modules are displayed as-is — no trimming or other adjustments will be performed on the text. As you can see, banners are also automatically colorized as blue.
Similar to application banners, multiple modules can provide usage information, which will be joined together and displayed to the user. The order in which usage information is displayed is the order in which modules are loaded.
As you can see, the zend-mvc integration also prepends each module's usage with the module's name. This helps to visually separate commands by module — which is particularly useful when you have multiple modules providing commands. By default, the component colorizes the module names in red.
Usage info provided in modules is not connected with console routing. You can describe console usage in any form you prefer, and it does not affect how MVC handles console commands. In order to handle real console requests, you still need to define console routes.
The text provided is displayed as-is; no trimming or other adjustments will be performed. If you'd like to fit your usage information inside the console window, you could check its width with $console-getWidth().
If getConsoleUsage() returns an associative array, it will be automatically aligned in 2 columns. The first column will be prepended with script name (the entry point for the application). This is useful to display the various commands exposed.
Commands and their descriptions will be aligned in two columns that fit inside the terminal window. If the window is resized, text may wrap, but all content will be aligned. If you don't like the behavior, you can always return free-form text instead, as free-form text is never transformed or aligned.
All information will be aligned in one or more columns that fit inside the terminal window. If the window is resized, some text may wrap, but all content will remain aligned. In case the number of columns changes (e.g. the array contains different number of elements), a new table will be created, with its own alignment and column widths.
If you don't like this behavior, you can always return free-form text.
You can use mix together all of the above styles to provide comprehensive usage information.
Your module's getConsoleBanner() method should return a one-line string containing the module's name and its version (if available); do not provide any other information.
Your getConsoleUsage() method should not return the module's name, as it is prepended automatically for you by the zend-mvc integration.


You'll never know all you give me.
And it gives me butterflies.
No one knows of my dependence on you.
Let alone share in my wonder at the world.
You see me and act surprised at what I reveal.
Like you haven't poked your finger in every crack I've exposed in myself.
But I know you read it – every single word.
It doesn't matter that you don't tell me so.
There is excitement in the not knowing.
When I see you, I pretend too.
Like the mere truth of your being doesn't terrify and ignite me.
And so, in ignorance we are safe.
When perhaps that is not true.


text:
Q: How to mix Java Swing and JavaFX in Swing application? I am developing a Java Swing application but I want to also use JavaFX with Swing. Is there any resource for that told how to do that?

A: See here. The short story is that embedding JavaFX in Swing is now possible and supported via JFXPanel, but the other direction is not supported.

A: The "JavaFX in Swing" link given above only works with JavaFX 1.1. Here is how to do it in JavaFX 1.2 JavaFX in Swing - 1.2. However, as Rastislav mentioned above, this is also using private APIs and will most likely break in the next major version. JFXtras has added some classes to support this, and hopefully, this will insulate the developer from the underlying release changes.

redpajama_set_name: RedPajamaStackExchange


OAKLAND, Calif. (AP) - Nearly a year after a deadly warehouse fire, Oakland has announced a permanent fire chief.
The Northern California city announced Thursday that interim Chief Darin White has been appointed to the post.
He succeeds Teresa Deloach-Reed, who retired in May after coming under pressure from a fire last December at the Ghost Ship warehouse that killed 36 people.
The Fire Department was criticized for failing to conduct annual inspections of the warehouse, which had been illegally converted to living space.
White has 30 years in fire service, including 20 in the local department.
Those included a shooting that killed an off-duty firefighter and wounded another and a massive fire at an unfinished housing project.