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Siemens and Intel Deliver Enhanced Scalability, SecurityStress, load, and scalability testing has verified that Siemens Melior can now scale to over 9,000 concurrent users.Sweden, along with Scandinavian countries in general, is recognized worldwide for its advanced healthcare delivery systems, effective adoption of healthcare IT, widespread deployment of electronic medical systems and care coordination, and its emphasis on improved access to quality healthcare. To achieve these goals, the Nordic region recognizes the need for continuous improvement and reinvestment in supporting technologies.The Nordic region also shares many of the challenges faced by healthcare institutions worldwide:• Data center consolidation and development of shared services across a region, demanding increased scalability of applications• Expansion to include new eHealth services and underlying interoperability requirements across a diverse set of information sources• Implementation of stringent security and data protection regulations.Together, Siemens Melior and Intel deliver a solution that can meet these challenges, preparing Nordic healthcare institutions for the future.Read the full Siemens and Intel Deliver Enhanced Scalability, Security.
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EMC Data Domain* and Avamar Data Store* provide backup efficiency and data center reduction.
Accelrys and Diagnomics executives discuss new medical therapies and science using Intel® technology.
WHO panel discusses the cultural and economic shifts happening as populations age.
See how Intel® Atom™ processor-based intelligent fitness solutions improve gym workouts.
Intel's vision of the future at the Chandler fitness center features intelligent, connected equipment.
Follow a patient through discharge and emergency transport to see how real time data sharing and communications with secure mobile devices can make a "meaningful difference."


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We proudly serve this community with free wi-fi, public computers, a meeting room, classes, activities, and summer reading programs.
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Worland is the county seat and serves over 5.000 people. We have free wifi, public computers, story time, activities, and summer reading programs. Open M-W-F 8-5, T-Th 8am-9pm, Sat. 9-1
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July 4 Parade Marshall Craig Johnson. Wyoming's best selling author Craig Johnson is the Grand Marshall for Ten Sleep's Fourth of July Parade. After the parade, he will be speaking in the city park. Everyone is welcome!
Wyoming Grasslands Exhibit. Ten Sleep Library will be hosting photographs of Wyoming Grasslands, July 1-31. Opening reception is July 2, 6-8 pm. More details on Ten Sleep's page.
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Afghanistan: News Releases
Read news releases related to Afghanistan.
Latest News Releases
The Presidents of the Islamic Development Bank Group and the Asian Development Bank have called for closer cooperation to enhance the impact of their development work in common member countries.
The eight countries of the Central Asian Regional Economic Cooperation (CAREC) Program today agreed on a Trade Policy Strategic Action Plan (TPSAP) to expand trade through regional cooperation.
Eight countries today adopted strategy documents outlining ambitious plans to deepen regional economic cooperation in transport, trade facilitation, trade policy, and energy in Central Asia and neighboring states.
Uzbekistan, Afghanistan, and the Asian Development Bank have signed a Memorandum of Understanding to expand trade and economic opportunities through railway transport.
ADB President to Attend Seventh Ministerial Conference on Central Asia Regional Economic Cooperation (CAREC)Asian Development Bank President Haruhiko Kuroda will attend the Seventh Ministerial Conference on Central Asia Regional Economic Cooperation in Baku, Azerbaijan on 20-21 November.
The Asian Development Bank is providing financial support for the improvement of a key Central Asian transport corridor, which will help boost trade, tourism, and economic cooperation in the region.
The President of the Asian Development Bank, Mr. Haruhiko Kuroda, will visit the nations of Armenia, Georgia and Azerbaijan between 18 - 21 November.
The Asian Development Bank is providing a $3 million grant to expand its clean energy program to involve more developing member countries.
The Asian Development Bank is aiming to enhance regional cooperation on vital Central Asian waterways whose management and protection are crucial to the region's economic growth and social well-being.
The Asian Development Bank is providing $4.5 million to foster closer cooperation among participating countries of the Central Asia Regional Economic Cooperation by establishing a virtual institute that will coordinate research, training, and dialogue within the regional organization.


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Click here to print the obituary below
Rose HAMMERMEISTER
HAMMERMEISTER, Rose Martha
September 5, 1918 - January 13, 2013
Rose Hammermeister, beloved mother, grandmother, great-grandmother and friend, passed away on Sunday, January 13, 2013, at the age of 94. Born and raised in Edmonton, she lived in a number of places in the United States and Canada during her life. She made Calgary her home in 1982, where she spent over 30 of her happiest years. Rose loved spending time with family and friends and her career as mother and homemaker was by far her most rewarding. She loved to cook and bake and her spare time was spent working on arts and crafts. Rose was a friend to all and generously gave of her time and energy to those around her. She will be missed by all who were blessed to have known her. Rose is survived by her daughters, Linda Olson and Joyce Hammermaster, her son Ron Hammermeister (Elena); her grandchildren, Karin Santos (Al) and Michael Olson, and her great-grandchildren, McKenzie Rose Santos, Ashlyn Santos and Molly Olson. She is also survived by numerous extended family. She is predeceased by her parents Adolph and Bertha Fester, husband Adolph Hammermaster, daughter Phyllis Mellquist; sisters, Evelyn Brown and Norma Whyte, and brother Norman Fester. A special thank you to Rose's beloved daughter-in-law, Elena Halmaciu, who lovingly and tirelessly cared for her during her final years. It was Elena's daily visits that put a smile on Rose's face and it was Elena who was blessed with Rose's final smile. Rose will be laid to rest at Mount Pleasant Cemetery in Edmonton. Condolences may be forwarded through
. In lieu of flowers, donations can be made in Rose's memory to the Alzheimer Society of Calgary (
). In living memory of Rose Hammermeister, a tree will be planted at Fish Creek Provincial Park.
Published in The Calgary Herald from Jan. 16 to Jan. 18, 2013


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Press release from Business Wire
Fiserv to Release Fourth Quarter and Year-End Earnings Results for 2012 on February 5, 2013
Wednesday, January 09, 2013
Fiserv to Release Fourth Quarter and Year-End Earnings Results for 2012 on February 5, 201317:00 EST Wednesday, January 09, 2013 BROOKFIELD, Wis. (Business Wire) -- Fiserv, Inc. (NASDAQ: FISV), a leading global provider of financial services technology solutions, will announce its financial results for the fourth quarter and full year of 2012 after the close of regular market trading on Tuesday, February 5, 2013. The company will provide a live webcast of its earnings conference call over the Internet at 4 p.m. CT on February 5. The live webcast and archived replay can be accessed at www.fiserv.com. Those wishing to listen to the discussion should log on ten minutes before the start of the call. An archive of the call will be available approximately one hour after the call concludes. About Fiserv Fiserv, Inc. (NASDAQ:FISV) is a leading global technology provider serving the financial services industry, driving innovation in payments, processing services, risk and compliance, customer and channel management, and business insights and optimization. For more information, visit www.fiserv.com. FISV-G Media Relations:Judy DeRango WicksVice President, CommunicationsFiserv, firstname.lastname@example.com Relations:Eric NelsonVice President, Investor RelationsFiserv, firstname.lastname@example.com


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27 Russian Dating Site Members You Don't Want to Mail Order
25 Most Unbelievable Coincidences
Why You're Lucky You Don't Work For This Company
The Hairiest Girl in the World
12 Real People Who Look Just like Cartoon Characters
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The Largest Ship On the Planet Will Amaze You
Simple Method 'Regrows' Hair. Do This
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Why Every Traveler Has to Visit Japan
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Top 10 "Useless" Organs
25 Things You Probably Google When You're All Alone
Prepare to Have Your Mind Blown
Stolen Car Returned To St. Louis Owner Pimped-Out and Painted
These Mars Rover Photos Will Make You Think Twice About Alien Life
Brutal Female Bodyguards Training in China
The 11 Most Disgusting Fair Foods of All-Time
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You Won't Believe What This Shadow in the Sea Is!
The Terrifying Moment a Spider Jumps Out of a Banana
5 Popular Houseplants That Actually Purify Your Air
10 Household Items That Will Pull A Double Shift For You
46 Perfectly Timed Photos You'll Have to Look Twice to Understand.
21 Outrageous Customized Motorcycles
Well, That Was a Close One!


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- Product Information
Product Description
Make any graduation event shine a little brighter with our Congrats Grad Logo LED Bottom Lit Cup! Whether you use this bottom lit cup as a special glass just for the grad or stock up so all the guests can have one, you can bet the Congrats Grad Logo LED Bottom Lit Cup will be a hit with everyone! This 16 ounce plastic cup features a festive graduation cap design on the bottom that is illuminated by the LEDs installed in the base of the glass. When the sun goes down, this glass will really start to shine! A fun party favor or a festive way to stock your bar, this bottom lit cup is bound to be a crowd pleaser. Our Congrats Grad Logo LED Bottom Lit Cup comes with three AG13 batteries that are included, installed, and replaceable. Our bottom lit cups are hand washable and replaceable. This Congrats Grad Logo LED Bottom Lit Cup is sold by the piece.
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How to Host a Summer Movie Night in Your Backyard
Do something different for your weekly movie night by watching it in your backyard on a big screen, and inviting friends and family to join in on the fun! Follow these party ideas to assist you in putting together a successful summer movie night.
Minnie Mouse Birthday Party Ideas
Her birthday party will be a huge hit with a Minnie Mouse theme! Use these decorating ideas to help your put together the most adorable Minnie Mouse party.
1950s Sock Hop Party Ideas
Invite friends over for a rockin' good time when you throw a fifties theme party! Use these party tips to assist you in putting together the best sock hop anyone's ever seen.


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Date: 1978-02
Description: "Eric Schaumberg has his 'bangs' cut by barber, Jan Reuter."
Date: 1955 ca.
Description: Robert Krueger stands near a driveway next to a Chevy automobile with his hand on the driver's side door knob. A cat is walking on the ground in front of h...
Date: 1952
Description: This map of Beaver Dam shows state trunk highway system, county trunk highway system, town roads, rivers, and Beaver Dam Lake and includes an additional at...
Date: 1983-08
Description: "Threshing on the Batzler farm."
Date: 1952-08-16
Description: Gladys Leichtle helps her sister Carol prepare for Carol and Dave Luedtke's wedding.
Description: Workers at the Malleable Iron Range Company prepare to rivet the top of a stove. The top is fitted to the body of the stove with a powerful leverage system...
Date: 1997-04-05
Description: "Cynthia Neitzel weds Scott Bernhard at St. Peter's Lutheran Church, Theresa."
Date: 1943
Description: Field of shocked hemp with stock pile and J. Leroy Farmer Hemp Mill in the background.
Date: 1979-08
Description: "This old milk wagon will be in Theresa Firemen's parade."
Date: 2004
Description: Richard and Gayle Christian live at W1624 Allen Rd (Section 21). This barn was built in 1914, and they are still milking.
Description: Ken and Sandy Gerlach live at N8684 Bancroft Rd (Section 15).
Date: 1904-04-04
Description: Jennie and Edgar Krueger suspending a tree limb on their shoulders on which hang a dead rabbit and two birds. Each of them holds a rifle over their right s...
Description: This ancient Pomeranian-style building, made of bricks and stone, still stands on the Gebhard property.
Date: 1906
Description: Emil Kuney's sister, Emil Kuney, Sarah Krueger, and Henry Bigalk standing outdoors in front of a fence during winter.
Date: 2003-10-03
Description: "Diane was our waitress when we visited The Wharf in Fox Lake." From left to right; Diane, Shirley Widmer, John Bodden, and Ralph "Buddy" Ruecker.
Date: 1934 ca.
Description: Shirley Krueger standing in the hay barn holding a kitten in a basket. In the background on the left is a wagon full of hay.
Date: 1904-12-04
Description: William Wendorf and Alexander Krueger standing together in an open barn doorway. William is dressed in his white suit from Costa Rica, with a straw hat. He...
Date: date unknown
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Description: Jeff Adams is in the car repair and restoration business, as is evidenced by the many cars on the property. This property was formerly owned by the Ted Eck...
Date: 1996-06
Description: "When Theresa celebrated their sesquicentennial, Village President Chris Giese accepted a proclamation from Assemblyman Bob Goetsch."
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Recent exon sequencing studies have revealed that over 19% of human tumors have mutations in subunits of mSWI/SNF (BAF) complexes. To investigate the underlying mechanism we studied human synovial sarcoma (SS), in which transformation results from the translocation of exactly 78 amino acids of SSX to the SS18 subunit of BAF complexes. We demonstrate that the SS18-SSX fusion protein competes for assembly with wildtype SS18, forming an altered complex lacking the tumor suppressor BAF47 (hSNF5). The altered complex binds the Sox2 locus and reverses polycomb-mediated repression, resulting in Sox2 activation. Sox2 is uniformly expressed in SS tumors and is essential for proliferation. Increasing the concentration of wildtype SS18 leads to reassembly of wildtype complexes retargeted away from the Sox2 locus, polycomb-mediated repression of Sox2 and cessation of proliferation. This mechanism of transformation depends on only two amino acids of SSX providing a potential foundation for therapeutic intervention.
Exon sequencing in human malignancy has provided paradigm-changing insights into pathogenesis (Lander, 2011) but is often limited by the fact that mutation frequencies are correlative, leaving open the possibility that other primary events are responsible for tumor initiation. This correlative aspect has emerged particularly from recent exon sequencing studies of human cancers, which have defined frequent mutations in chromatin regulators(Dawson and Kouzarides, 2012). By contrast, precise chromosomal translocations which define cancer subsets provide strong support for an initiating role. Chromatin regulation has often been thought to play supportive roles and hence a potential instructive or initiating function for chromatin regulators in human cancer is less clear.
Chromatin regulation is essential for appropriate and timely gene expression. This process is achieved by several mechanisms including DNA-methylation, histone modifications and ATP-dependent chromatin remodeling. One of the most well characterized chromatin remodeling complexes studied to date is the SWI/SNF (BAF) complex, which was discovered in yeast (Peterson and Herskowitz, 1992) and plays a general role in gene activation through nucleosome remodeling, thereby allowing accessibility of transcription factors to their recognition sites. In flies, the Swi2/Snf2 ATPase homologue, Brahma was discovered in screens for trithroax genes (Tamkun et al., 1992) and opposes polycomb function. Mammalian complexes have two SWI2-like ATPases (Brg1 and Brm) and a second ATPase, β-actin, and are combinatorially assembled from gene families that encode the 15 subunits. Fewer than half of the subunits are related to yeast SWI/SNF, others are related to RSC and SWR1 subunits (Cairns et al., 1996; Krogan et al., 2003; Mizuguchi et al., 2004) and hence the name BAF (Brg/Brm-associated complexes) is commonly used. The complexes appear to have undergone evolutionary changes in response to the emergence of multicellularity, polycomb mediated repression, DNA methylation, and a larger genome size (Wu et al., 2009). The role of combinatorial assembly is seen most clearly in the mammalian nervous system in which neurons have a family of highly specialized neuron-specific complexes involved in dendritic morphogenesis (Lessard et al., 2007; Wu et al., 2007; Yoo et al., 2009). Recent genetic studies in flies have suggested that the fly homologue of the neural specific BAF (nBAF) subunit, BAF53b has an instructive role in targeting dendritic trees to their correct termini (Tea and Luo, 2011). Instructive roles are also suggested from studies demonstrating that forcing the formation of nBAF complexes leads to the conversion of fibroblasts to neurons (Yoo et al., 2011). Specialized complexes are also found in pluripotent cells (esBAF complexes) (Ho et al., 2009) and recreating the esBAF complex subunit composition in fibroblasts facilitates iPS cell formation (Singhal et al., 2010). These recent studies suggest an instructive role for these ATP-dependent chromatin regulators that was not anticipated from earlier studies.
Recent exome sequencing studies of primary, early human cancers have repeatedly discovered mutations to subunits of polymorphic BAF complexes. Indeed, analysis of the 44 exome sequencing studies published to date indicate that 19.6% of all human cancers have mutations in at least one subunit (Kadoch et al., submitted). For example, BAF250a is mutated in 57% of clear cell ovarian cancers, BAF180 (polybromo) is mutated in 41% of renal cancers (Varela et al., 2011), and medulloblastomas have frequent mutations in Brg, BAF53a or BAF60b (Jones et al., 2012). The significance of perturbation to ATP-dependent chromatin remodeling complexes in tumorigenesis has been most strongly demonstrated in studies focusing on a particular class of tumors, malignant rhabdoid tumors (MRTs), in which the subunit BAF47 (hSnf5) is biallelically inactivated in nearly 100% of cases reported (Versteege et al., 1998). Patients often have inherited a defective SNF5 allele and the remaining wild-type allele is lost in the tumors, implicating them as tumor suppressors. Conditional biallelic inactivation of Snf5 in mouse models results in a fully penetrant phenotype with median onset to tumor development at only 11 weeks (Roberts et al., 2002). The preference for mutation of specific subunits in specific malignancies suggests that different combinatorial assemblies have roles in tissue-specific oncogenic processes, consistent with roles for specialized BAF complexes in neurogenesis and other biologic processes (de la Serna et al., 2001; Lickert et al., 2004; Wu et al., 2009). Because of the possibility that the frequent BAF subunit mutations might be playing a relatively non-specific role in oncogenesis, we initiated studies on a cancer type, human synovial sarcoma (SS) where nearly all tumors have a precise translocation involving a specific subunit, indicating that the translocation is the initiating oncogenic event.
Human synovial sarcoma accounts for 8-10% of all soft tissue malignancies and most commonly arises in the extremities of young adults (Weiss, 2001). A recurrent chromosomal translocation, t(X;18)(p11.2;q11.2) fuses the SS18 gene on chromosome 18 to one of three closely related genes on the X chromosome, SSX1, SSX2 and rarely SSX4, resulting in an in-frame fusion protein in which the eight C-terminal amino acids of SS18 are replaced with 78 amino acids from the SSX C-terminus(Clark et al., 1994; de Leeuw et al., 1995; Skytting et al., 1999). This remarkably precise translocation is present in greater than 95% of cases and has been established as pathognomonic for the disease with clinical diagnosis confirmed by karyotyping and RT-PCR for SS18-SSX transcripts (Hiraga et al., 1998; Sandberg and Bridge, 2002). The presence of this translocation is the defining feature of synovial sarcomas and is often the only cytogenetic abnormality (dos Santos et al., 1997; Limon et al., 1991); hence, this is very likely to be the driving oncogenic event in the development of these tumors. However, the mechanism of SS18-SSX transformation has been unclear.
Both SS18 and SSX proteins lack known DNA binding motifs, yet they appear to be acting through transcriptional regulatory mechanisms. SS18 is a nuclear protein which has been suggested to interact with chromatin remodeling factors such as Brg/Brm containing complexes (Nagai et al., 2001; Thaete et al., 1999), and the transformation potential of the SS18-SSX fusion has been shown to require Brg/Brm(Nagai et al., 2001). Fusion partners SSX1, 2, and 4 are members of a family of nine human SSX genes which encode highly similar proteins with 73-92% homology (cDNA homology 87-96%) (Smith and McNeel, 2010) and conserved intron/exon junctions. SSX3 and SSX5 have not been found as fusion partners in tumors although they are highly similar to the oncogenic fusion partners. mRNA expression of SSX genes are restricted to the testes and have been detected at low levels in the thyroid.
Here we demonstrate that SS18 is a dedicated, highly stable subunit of BAF complexes. We find that the fusion of SS18 with SSX produces a protein that binds to the complex and evicts both the wild-type SS18 and the tumor suppressor BAF47 (hSNF5). This altered complex then binds to Sox2, relieving H3K27me3 repression thereby activating Sox2, which we find is required for proliferation. Importantly, SS18-SSX-driven complex disruption is determined by a 2 amino acid (aa) hydrophilic region of SSX. Assembly of wild type complexes and proliferative quiescence can be produced by increasing the concentration of the wild type SS18, making this region an excellent drug target.
To better understand the composition of BAF complexes, we used a rapid biochemical/affinity purification approach to isolate endogenous complexes from non-transformed cells. Ammonium sulfate fractionation was followed by rapid affinity purification using a highly specific antibody to a genetically non-essential epitope in the Brg/Brm ATPase subunits (Ho et al., 2009). SS18 (synovial sarcoma translocation, chromosome 18) peptides were found in highly pure, endogenous BAF complexes in all tissue types examined, with the exception of post-mitotic adult neurons. Numbers of peptides and percent coverage for the protein SS18 were comparable to those of established BAF complex subunits, suggesting it is a subunit of BAF complexes (Figure 1A). Immunoprecipitation studies using anti-Brg as well as antibodies specific to other established mSWI/SNF complex components including BAF250a, BAF155 and BAF47 confirmed the association of SS18 with native BAF complexes; similarly, reciprocal immunoprecipitation using an antibody to SS18 revealed known components of BAF complexes(Figure 1B, Figure S1A). Two bands are detected for human SS18 due to alternative splicing(Figure S1B). Purification of complexes using anti-Brg and anti-SS18 antibodies revealed similar banding patterns upon silver stain analyses (Figure S1C). In order to determine whether SS18 was dedicated exclusively to BAF complexes, we performed glycerol gradient sedimentation analyses, which demonstrated the presence of SS18 only in fractions containing Brg and other BAF complex subunits (fractions 12-15). SS18 did not associate with polycomb repressor complexes PRC1 or PRC2, as indicated by Bmi1 or Ezh2 immunoblots, respectively, or as a free monomer in earlier fractions of the gradient (Figure 1C). Results were comparable in several cell types assayed including cell lines ES E14, Raji, 293T, and CCRF-CEM as well as primary human fibroblasts. Using urea-based denaturation studies, we determined that SS18 was remarkably stably bound to the complex, to a greater extent than most other subunits including BAF47, BAF155 and BAF 170, requiring denaturing conditions of greater than 5M urea to dissociate (Figure 1D, Figure 1E), similar to ribosomal subunits. The observation that SS18 remains bound when other subunits have dissociated indicates that SS18 binds directly to a stable core complex of Brg, BAF53a, and beta-actin (Zhao et al., 1998). These results demonstrate that SS18 is a dedicated subunit of mSWI/SNF or BAF complexes with binding characteristics similar to those of ribosomal subunits.
The invariant molecular feature of human synovial sarcoma is the SS18-SSX fusion protein in which the C-terminal 78 amino acids of SSX are fused in frame with amino acids 1-379 of the SS18 subunit (Figure 2A). To investigate the oncogenic mechanism we used two biphasic synovial sarcoma (SS) lines, Aska-SS and Yamato-SS, both of which bear the SS18-SSX1 chromosomal translocation (Naka et al., 2010). Anti-Brg immunoprecipitation studies performed on nuclear extracts isolated from synovial sarcoma cell lines, as compared to control 293T cells (and various other cell types), demonstrated that when SS18 was fused to its translocation partner SSX, the SS18-SSX1 fusion protein was indeed bound to BAF complexes, as reflected by an appropriate upshift in molecular weight of SS18 from 55kDa to 66 kDa upon immunoblot analysis (Figure 2B, left). Remarkably, we observed that both synovial sarcoma lines, as compared to several other cell types assayed, exhibited lower to absent total protein levels of the tumor suppressor subunit BAF47 (hSNF5 or INI1) (Figure 2B, right; Figure S2A, left), while transcripts were largely comparable (Figure S2A, right). Immunoprecipitated BAF complexes containing the SS18-SSX1 fusion protein showed nearly absent levels of wild-type SS18 on the complex. Input protein levels of the wild-type sized SS18 protein were also lowered, as were mRNA levels, suggesting reduced transcription (Figure S2B), and consistent with previously reported findings (Brodin et al., 2001). In addition, a prominent Brg peak is located at the promoter and in an intronic region of the SS18 gene as determined by ChIP-seq analysis in murine ES cells (Figure S2C) (Ho et al., 2011), suggesting auto-regulation of this locus. Density sedimentation analyses performed on nuclear extracts isolated from Aska-SS and Yamato-SS lines revealed disruption of BAF complex composition, in that wild-type SS18 protein no longer associated with the BAF complex fractions (fractions 15,16), and rather existed in fractions 3 and 4 suggesting its presence as a monomer (Figure 2C, Figure S2D). Quantitative densitometry of an anti-SS18 immunoblot of the glycerol gradient, revealed that only a small percentage (2-8%) of BAF complexes contains the wild-type SS18 protein in these cells (Figure S2E). Side-by-side molecular weight comparisons indicated that the SS18-SSX fusion protein, in both SS lines, was almost entirely associated with the BAF complex (denoted by Brg peaks in fractions 15,16) and the wild-type SS18 protein was present, albeit at lower protein levels, in the monomeric fractions of the gradient (fractions 3,4) (Figure 2D). This was further confirmed by immunoblotting using an anti-SSX1 antibody, which demonstrated the presence of SSX1 only in fractions containing Brg. As shown above, in SS lines containing the SS18-SSX fusion, BAF47 no longer associated with BAF complexes and was nearly absent from nuclear extracts indicative of degradation. This is particularly interesting given that BAF47 is a known tumor suppressor; loss of this subunit from the complex as a result of the integration of SS18-SSX might produce functional consequences similar to those of SNF5 inactivation. In order to further assess the degree of dedication of SS18 and SS18-SSX to the BAF complex, we performed depletion studies using two rounds of immunoprecipitation with polyclonal antibodies specific to a known complex subunit, BAF155, as well as to SS18's fusion partner, SSX1 (Figure 2E). In 293T cells, BAF155 antibodies depleted SS18 protein from the nuclear extracts; SSX1 antibody did not deplete the lysate, as expected, in the wild-type setting. In the Aska-SS synovial sarcoma cell line, immunodepletion using the SSX1 antibody significantly depleted complex subunits Brg, BAF155, and SS18-SSX proteins from nuclear extracts to comparable levels as with anti-BAF155 antibody. These results collectively demonstrate that both wild-type SS18 and in synovial sarcoma, the SS18-SSX1 fusion protein, are dedicated to BAF complexes, but that the fusion protein alters subunit composition.
To understand how incorporation of SS18-SSX alters the biochemical subunit composition of BAF complexes, we produced N-terminally GFP-tagged constructs of SS18 FL (full length, aa1-387), SS18 aa1-379 (lacking the last C-terminal 8 aa which are lost in the fusion), and SS18-SSX using a pEGFP-based expression system (Figure 3A). Previous studies have established that the N-terminal SNH domain of SS18 is responsible for its BAF complex association (Nagai et al., 2001). Anti-GFP immunoprecipitations were performed to isolate BAF complexes which had incorporated the exogenously introduced SS18 or SS18-SSX variants. Intriguingly, we noted that expressing the SS18-SSX fusion protein resulted in the loss of BAF47 from the complex at 72 hours post-transfection (Figure 3B). Wild-type SS18 FL or SS18 1-379 both incorporated into BAF complexes but did not alter BAF47 binding to the complex. Input levels of BAF47 at this time point (72 hours) following introduction of SS18-SSX (and all variants tested) were comparable to those of untreated cells. Immunoblot analysis performed on total input protein harvested at 96 hours post-transfection with SS18-SSX indicated a marked decrease in BAF47 levels, with mRNA levels held stable, suggesting that BAF47 is first lost from the complex upon integration of SS18-SSX and subsequently degraded (Figure 3C, Figure S3). To understand the means by which BAF47 is degraded under normal conditions, we performed cyclohexamide (CH) chase experiments over 24 hours, plus and minus proteasome inhibitor treatment using MG-132 at the 24 hour time point. The protein half-life of BAF47 was approximately 10 hours after the addition of CH; BAF47 levels could be rescued from CH treatment with MG-132 to >85% of control levels, indicative of proteasome-mediated degradation (Figure 3D). Treatment of Aska-SS cells with MG-132 resulted in a substantial increase in BAF47 total protein levels (Figure 3E). Upon infection of SS18-SSX1 into 293T fibroblasts, wild-type SS18-containing complexes were readily replaced by SS18-SSX-containing complexes (fractions 14,15,16) and BAF47 levels were reduced as determined by glycerol gradient analyses (Figure 3F). Wild-type SS18 was observed in free, monomeric fractions of the glycerol gradient, as well as in transient lower molecular weight, Brg-associated fractions 9-11. These studies indicate that the SS18-SSX fusion incorporates into BAF complexes, replacing wild-type SS18, and ejecting and destabilizing BAF47.
To understand whether low protein levels of BAF47 results specifically from the presence of the SS18-SSX1 fusion in SS cells, we generated shRNA-based knock down (KD) constructs specific for the 3′ UTR of SSX (based on (Takenaka et al., 2010)) to exclusively target SS18-SSX, but not wild-type SS18. Remarkably, we noted a substantial increase in BAF47 total protein levels upon KD of the SS18-SSX oncogenic fusion (Figure 3G). In addition, wild-type SS18 protein levels increased, suggesting relieved repression of SS18 upon KD of the SS18-SSX fusion. We assessed the effect of SS18-SSX KD on proliferation of both synovial sarcoma cell lines. Importantly, KD of the SS18-SSX fusion and of Brg, to which the SS18-SSX fusion was bound, resulted in a profound decrease in proliferation of synovial sarcoma cells (Figure 3H). By contrast, KD of wild-type SS18 and BAF47, subunits not contained in the SS18-SSX-containing BAF complexes, wild-type SS18 and BAF47, had little to no effect on synovial sarcoma cell proliferation (Figure 3H), suggesting that the aberrant residual complex is responsible for driving and maintaining cell proliferation. In human primary fibroblasts with wild-type complexes, KD of Brg, SS18 and BAF47 reduced proliferation; KD of SS18-SSX1 did not alter proliferation as compared to control hairpin (Figure 3I). These studies indicate that the eviction of BAF47 inactivates it and that it is no longer required for proliferation of the SS cell lines. Hence, the free BAF47 protein does not acquire a new function enabling transformation.
Several studies have demonstrated that SS cells harbor stem-cell like gene expression profiles (Garcia et al., 2012) (Naka et al., 2010). Moreover, Roberts and colleagues observed that tumors lacking the BAF47 tumor suppressor subunit also express stem cell-like signatures (Wilson et al., 2010). Naka and colleagues demonstrated that Aska-SS and Yamato-SS lines as well as 15/15 human tumor specimens of synovial sarcoma tested express mRNA transcripts of pluripotency factors Sox2, Oct4 and Nanog (Naka et al., 2010). We focused on Sox2 because of its role in oncogenesis (Bass et al., 2009). Introduction of SS18-SSX dramatically induced Sox2 mRNA in primary, untransformed human neonatal foreskin fibroblasts by 15 days post-infection and selection (Figure 4A). This induction was specific to the full SS18-SSX1 fusion and did not occur when the C-terminal 34 aa of the conserved SSXRD domain was removed from SSX1. To determine if Sox2 mRNA induction was driven by the partially formed complexes, we tested the effect of shRNA-mediated KD of SS18 and BAF47 in fibroblasts on Sox2 mRNA induction. Intriguingly, KD of SS18 and BAF47 both resulted in a statistically significant increase in Sox2 mRNA to levels nearly comparable to those resulting from overexpression of SS18-SSX (Figure 4A). At the protein level, BAF47 and SS18 appear to reciprocally regulate one another's stability in fibroblasts as determined by KD of BAF47 and SS18 and immunoblot analysis for protein levels of each (Figure S4A). KD of Brg alone resulted in >70% reduction in protein levels, but did not induce Sox2. Collectively, these data suggest that the activity of aberrant complexes, which lack BAF47 and wild-type SS18, are responsible for Sox2 mRNA induction. Sox2 mRNA levels increased 23-fold by day 25 post-infection with SS18-SSX1 as compared to control (Figure 4B). Oct4 and Nanog mRNA were not induced significantly.
We sought to determine whether Sox2 was important for synovial sarcoma cell proliferation. To this end, we generated lentivirus containing two different shRNA hairpins to Sox2 which both effectively reduced Sox2 mRNA and protein in Aska SS cells (Figure 4C) and assessed proliferative capacity in vitro. shRNA-mediated KD of Sox2 profoundly reduced proliferation of Aska-SS cells as compared to scrambled shRNA control (Figure 4D). Intriguingly, Sox2 mRNA and protein levels were reduced in Aska-SS cells upon KD of the SS18-SSX1 fusion to levels comparable to those of cells treated with Sox2 shRNA itself (Figure 4E), indicating elevated levels of Sox2 were specifically due to the presence of SS18-SSX fusion.
To understand the mechanism of Sox2 induction by SS18-SSX we assessed BAF complex occupancy at the Sox2 promoter as well as two clusters of transcription factor (TF) binding sites within the Sox2 exonic region using our affinity-purified BAF155 polyclonal antibody. Intergenic regions were selected as normalization controls. SS18-SSX1-infected primary human fibroblasts demonstrated a significant increase in BAF complex occupancy at all three sites within the human Sox2 locus as compared to control fibroblasts (Figure 4F). In MEFs, there is a prominent H3K27me3 peak over the Sox2 locus as shown by MEF ChIP-seq studies (Mikkelsen et al., 2007), consistent with absent Sox2 expression in these cells (Figure S4B). Lentiviral introduction of SS18-SSX1 into primary human fibroblasts resulted in a striking decrease in H3K27me3 enrichment at all three sites tested within the Sox2 locus (Figure 4F).
To determine if the 78 aa tail of SSX was itself responsible for the targeting of BAF complexes to the Sox2 locus (perhaps by binding a transcription factor) we infected human fibroblasts with V5-tagged SSX78aa (as well as SS18FL and SS18-SSX). However, we did not find that the 78aa SSX fragment localized to the Sox2 locus (Figure S4C). These studies indicate that the SS18-SSX fusion functioning within the altered BAF complexes binds to and activates the Sox2 locus in fibroblasts by disrupting H3K27me3-mediated repression, which is likely directed by the actions of PRC2, the only complex known to place this mark(Chamberlain et al., 2008).
Because expression of SS18-SSX1 resulted in the ejection and subsequent degradation of the BAF47 subunit, we aimed to understand the features of the 78 amino acid SSX tail that could be responsible for this. We generated a series of truncation mutants: deleting the conserved SSXRD domain of 34 aa, deleting ½ of the SSXRD domain (17aa, hydrophobic) and adding amino acids in increments of 10 amino acids to the SS18 C-terminus(+10 through +70). We noted that SS18+10 through SS18+70 did not result in significant ejection of BAF47 from the complex as determined by immunoblot analysis and quantitative densitometry performed on immunoprecipitated complexes (Figure 5A). This implies that a region in the last 8 amino acids (SDPEEDDE) is required for BAF47 ejection. Deleting 1/2SSXRD resulted in slightly decreased levels of BAF47. Upon introduction of these variants into human fibroblasts, Sox2 mRNA induction was only observed with SS18-SSX1 (Figure 5B). Because none of these truncation mutants fully recapitulated the SS18-SSX1-induced BAF47 ejection and Sox2 mRNA induction phenotype, we turned to the fact that the only translocations that have been observed in human synovial sarcoma are SS18-SSX1, SS18-SSX2, and SS18-SSX4. SS18-SSX3 has never been observed in a human tumor. This family of 9 genes (SSX1-9) located at ch Xp11.2 is highly similar; protein homology among members ranges from 73-93% (Smith and McNeel, 2010). We used Kyte-Doolittle hydrophobicity analysis to compare the 78 C-terminal amino acids of SSX1,2,4 versus SSX3, which revealed a significant difference in hydrophobicity between amino acids 40-50 (Figure 5C), as highlighted. Upon peptide alignment of the 78 amino acids of SSX1- 4 it became clear that the amino acid composition at position 43, 44 was most discrepant between the SSX members observed in human SS tumors (1,2 and 4) and the non-oncogenic SSX3. SSX1,2, 4, contains lysine (K) and arginine (R), glutamic acid (E) and arginine (R), and lysine (K) and threonine (T), respectively at position 43,44, while SSX3 contains a methionine (M) and isoleucine (I) at these positions (Figure 5D, arrows). Given that SSX1 is a common fusion partner of SS18 in synovial sarcoma and SSX3 is not, we then sought to understand if SSX3 fused to SS18 could result in BAF47 ejection and Sox2 induction. To this end, we generated an SS18-SSX3 fusion protein (379 amino acids of SS18 fused to 78 amino acids of the SSX3 C-terminus). SS18-SSX3 was able to integrate into BAF complexes, as assessed by anti-GFP immunoprecipitation of BAF complexes, but failed to eject BAF47 (Figure 5E) from the complexes. Remarkably, replacement of amino acids 43,44 of SSX1 (KR) with those of SSX3 (MI) in the SS18-SSX1 fusion resulted in substantial loss of the ability to displace BAF47 (Figure 5F). Reciprocal amino acid substitution at position 43,44 in SS18-SSX3 (MI to KR) resulted in the gained ability of SS18-SSX3 to eject BAF47. Comparative densitometry accounting for the BAF47/Brg ratio is shown, representative of n=3 experiments (Figure 5G). Intriguingly, SS18-SSX1 as well as SS18-SSX3 (Δ43,44 MI→KR) significantly induced Sox2 mRNA; no other variant produced this phenotype (Figure 5H), lending further evidence that the loss of BAF47 (hSnf5) from mSWI/SNF complexes is necessary for the induction of Sox2 mRNA expression in synovial sarcoma. All three fusions reported in human synovial sarcomas (SSX1,2,4) produced BAF47 eviction, while SS18-SSX3 and SS18-SSX5 (which bears an amino acid change in the last 8 aa of SSX) fusions did not (Figure S5A,B).
Our observation that SS18 was displaced or failed to assemble into BAF complexes in the presence of somewhat higher concentrations of the SS18-SSX fusion protein (Figures 2C, ,3F)3F) lead us to investigate the possibility that the transforming fusion protein and the wild-type protein might exist in a concentration-dependent equilibrium or could be competing for assembly into newly formed complexes. Urea-based denaturation experiments demonstrated that SS18 and SS18-SSX are both stably bound to BAF complexes and dissociate to comparable degrees from 0 to 8 M urea as shown by immunoblot and quantitative densitometry analyses (Figure 6A, 6B). BAF complex components dissociated at comparable levels across the urea denaturation series from V5-tagged SS18 and SS18-SSX, indicative of equal affinity binding of wild-type SS18 and SS18-SSX (Figure 6C). Moreover, Brg and β-actin remained bound to V5-SS18/SS18-SSX-purified complexes to >5M urea, suggesting that SS18/SS18-SSX is part of a highly stable core complex of Brg, BAF53a and β-actin.
Given these findings and having observed that shRNA-mediated KD of the SS18-SSX1 fusion could restore BAF47 total protein levels (Figure 3G), we sought to determine whether overexpression of wild-type SS18 could also be sufficient to allow normal complexes to reform in synovial sarcoma cell lines and whether this could reverse the misassembly of synovial sarcoma BAF complexes and correct the gene expression phenotypes. Intriguingly, introduction of SS18 FL or SS18 1-379 resulted in a profound increase in BAF47 total protein levels by Day 10 post-infection (Figure 6D, left). Moreover, BAF complexes in Aska-SS cells infected with SS18 regained normal incorporation of wild-type SS18 and BAF47 subunits, suggesting concentration-driven re-integration of SS18 (Figure 6D, right). Introduction of SS18-SSX1 into 293T fibroblasts resulted in reduction of BAF47 total protein to a comparable degree as shRNA-mediated KD of BAF47 (Figure 6E). These studies indicate that the SS18-SSX fusion protein and the wild type SS18 protein compete for assembly into BAF complexes and that the transforming mutant protein can be displaced from BAF complexes to yield wild-type complexes by increasing the concentration of the wild-type protein.
Proliferation of SS cells was inhibited by introduction of wild-type SS18 and SS18 1-379, to a similar degree as in cells treated with shRNA-mediated KD of the SS18-SSX1 fusion (Figure 6F). In contrast, introduction of SS18-SSX into SS18-SSX-bearing synovial sarcoma Aska-SS cells had no appreciable effect on proliferation as compared to control. Sox2 mRNA expression levels in Aska-SS cells were reduced by 3- and 4-fold, upon overexpression of SS18FL and SS18 1-379, respectively (Figure 6G). In contrast, overexpression of SS18-SSX1 in these lines already bearing one translocated allele caused Sox2 mRNA levels to increase 1.7-fold above control levels relative to empty vector control indicating that the levels of Sox2 produced by the SS18-SSX fusion protein were not at maximum. Finally, Aska-SS synovial sarcoma cells infected with SS18FL to reverse the BAF complex phenotype exhibited a dramatically decreased occupancy of BAF complexes at the human Sox2 locus with a concomitant increase in H3K27me3 occupancy (Figure 6H). These studies indicate that normal BAF complexes can be reassembled in malignant cells by over expression of the wild type SS18 protein, leading to BAF complex removal from the Sox2 gene and resumption of normal repression of Sox2 by H3K27 trimethylation.
Finally, we aimed to test the potential for BAF47 overexpression to promote reassembly of wild-type BAF complexes containing BAF47 and SS18 in SS cells and its effect on proliferation. Notably, overexpressed V5-tagged BAF47 was unable to bind SS18-SSX containing complexes in both SS cell lines tested, as evidenced by low protein levels on complexes detected by anti-Brg and anti-V5 immunoprecipitations as well as by total protein immunoblots, suggestive of rapid degradation (Figure 7A). To test whether shifting aberrant complex assembly back to that of wild-type would allow for integration of the exogenous BAF47-V5 into complexes we infected SS cells containing BAF47-V5 with either SS18FL or shSS18-SSX. Indeed, in both lines, overexpression of SS18FL or KD of the SS18-SSX fusion resulted in increased incorporation and stabilization of BAF47-V5 as indicated by anti-Brg immunoprecipitation (Figure 7B). Intriguingly, BAF47 overxpression had no effect on SS cell proliferation in culture; however, proliferation was dramatically attenuated upon co-introduction of overexpressed SS18FL or KD of SS18-SSX, suggesting that BAF47 can only assemble into wild-type SS18-containing complexes and not complexes bearing the SS18-SSX fusion (Figure 7C).
Our studies demonstrate that in the two synovial sarcoma cell lines we have used, the fusion of SS18 with SSX, which is diagnostic of this tumor type, leads to assembly of aberrant BAF complexes that become targeted to the Sox2 locus, with loss of repressive H3K27me3 marks, driving Sox2 expression and proliferation of these cells (Figure 7D). The observation that Sox2 is activated in all SS studied ((Naka et al., 2010)) suggests this is a general mechanism of oncogenesis in these tumors. We find that the SS18-SSX fusion incorporates into BAF complexes and activates Sox2 expression, explaining the uniform activation of this gene in SS. But how do complexes containing the SS18-SSX fusion activate Sox2? BAF complexes containing the SS18-SSX fusion could be targeted by the interaction of SSX with a factor that binds the Sox2 locus. Alternatively, an incorrectly assembled complex could target the Sox 2 locus by changes to bromo-, chromo- and PHD domain presentation. We find that the 78aa of SSX alone is not targeted to the Sox2 locus when expressed in human fibroblasts (Figure S4B), indicating that it is the aberrantly assembled complex that targets the inactive Sox2 locus, reversing H3K27Me3-mediated repression, and leading to Sox2 activation.
Remarkably, the wild type SS18 protein is capable of replacing the SS18-SSX fusion in BAF complexes when expressed at somewhat higher levels than the fusion protein. The incorporation of wild-type and mutant proteins is unlikely to be due to direct binding competition. This conclusion arises from the fact that 8 M urea is required to remove either the wild-type SS18 protein from the wild type complexes or the SS18-SSX fusion from the malignant complexes. Hence, the two proteins most likely compete for assembly into complexes, with the product of the fusion allele winning in SS cells due to increased concentration.
The ability of SS18-SSX to disrupt BAF complexes maps to two regions of the SSX protein: The C-terminal 8 amino acids (SDPEEDDE) and a polar region of two amino acids present in the oncogenic members of the SSX family of proteins. Substitution of KR with MI, found in the non-transforming SSX3, restores normal complex assembly and gene regulation; substitution of MI with KR in SS18-SSX3 results in BAF47 ejection and increased Sox2 mRNA. In this regard, SSX5 is interesting in that it has KT at position 43, 44, combined with an amino acid substitution of P for E in the 8 terminal amino acids; SS18-SSX5 has not been found in translocations and does not eject BAF47, confirming the importance of both regions for oncogenicity. These two regions could interact to facilitate complex dissolution or form dimers in the malignant complexes. Structural studies will be necessary to define the precise mechanism. However, the ability of such a small region to lead to complex dissolution and the observation that the wild type and malignant protein are in a dynamic equilibrium indicates that the fusion containing the two amino acid essential region in the SSX tail (K43, R44) is an excellent target for developing therapeutics for this disease. A decoy molecule that causes SSX1 to resemble SSX3 would be expected to prevent eviction of BAF47, and thereby reverse the effects of the aberrant SS-BAF complex. This notion is consistent with the precision of the oncogenic translocation, in that all translocations discovered to date add exactly 78 amino acids of SSX1,2 or 4 to the SS18 protein at position 379.
In SS cells, the partially assembled complex gains the ability to bind the Sox2 gene, reversing H3K27Me3-mediated repression. Forcing correct assembly by expressing the wild-type SS18 causes the reassembly of wild-type complexes without the fusion thus reestablishing normal repression of Sox2 by polycomb. The fly Brahma protein was discovered from its ability to oppose polycomb and hence is known as a trithorax gene, however the underlying biochemical mechanisms have been controversial. In some studies, polycomb was found to prevent Brahma (BAP) complex binding, while in others it seemed that BAP or SWI/SNF directly recruited PolII, thereby opposing polycomb. Our studies suggest that somehow BAF complexes evict polycomb, however our temporal resolution is limited to the infection times (24-72 hrs) and hence we are unable to determine if the mechanism is direct physical eviction, or dilution of H3K27Me3 by nucleosome exchange with cell division since the measured rates of nucleosome turnover (Deal et al., 2010) are sufficient to remove most H3K27Me3 if methylation were prevented by the SS BAF complex. Evidence for BAF-polycomb opposition in malignancy has also been found with inactivation of BAF47 (hSNF5 or Ini1) in human malignant rhabdoid sarcoma (MRTs). In these tumors and in mouse models, polycomb was found to be removed from the INK4a locus upon introduction of BAF47 (hSNF5) (Kia et al., 2008). Understanding the underlying mechanism of polycomb opposition will require techniques that allow rapid recruitment of BAF complexes with a high degree of temporal and spatial control (Hathaway et al., 2012).
Synovial sarcoma is largely resistant to conventional, chemotherapy-based forms of treatment, underlining the need for an understanding of its pathogenesis. Disease-specific biologic agents which target SS18-SSX or its interactions have to date not been developed. Here we have shown that the SS18-SSX1 oncogenic fusion usurps SWI/SNF-like BAF complexes, resulting in activation of Sox2, which drives proliferation. Remarkably, the oncogenic fusion and wild-type SS18 bind to BAF complexes with comparable affinities, allowing directed assembly of oncogenic or wild-type complexes. Moreover, the composition of SS18-SSX-containing BAF complexes (lacking BAF47 and wild-type SS18) can be reversed by reducing the levels of SS18-SSX or by increasing levels of wild-type SS18. The observation that eviction of BAF47 from the complexes is dependent upon only 2 amino acids in SSX demonstrates an unusual mechanism of oncogenesis and opens a potential therapeutic avenue.
Nuclear extract (NE) preparation and immunoprecipitation (IP) studies were performed as described in Ho et al, 2009 and Extended Experimental Procedures. Antibody specifications are presented in Table S1.
Briefly, cells were plated in 6-well plates to 80% confluence prior to transfection using PEI (Poly(ethylenimine) in a 3:1 PEI:DNA ratio and were harvested at the appropriate time points thereafter.
Cells were assessed for >95% viability prior to being plated at 10^5 cells/well in triplicate/condition in 12-well plates. Cell counts were determined using trypan blue exclusion-based methods.
NE (150 μg) were subjected to partial urea denaturation ranging from 0.25M to 8M urea (in IP buffer) for 15 min at RT prior to anti-Brg IP. The co-precipitated proteins were analyzed by immunoblot. Quantitative densitometry analyses were performed with the LiCor Oddessy Imaging System (Li-COR Biosciences).
800 μg NE were resuspended in 300 μl of 0% glycerol HEMG buffer, and carefully overlaid on to a 10ml 10-30% glycerol (in HEMG buffer) gradient prepared in a 14×89 mm polyallomer centrifuge tube (Beckman # 331327). Tubes were centrifuged in an SW40 rotor at 4°C for 16 hrs at 40K RPM. 0.5 ml fractions were collected and used in analyses. See Extended Experimental Procedures.
MG-132 (Calbiochem, #474790) (10mg/ml in DMSO) was used at 1:1000, cyclohexamide (Sigma, #C4859) (100mg/ml) at 1:100 in cell culture media. Briefly, cells were plated in 6-well plates and treated with the above agents for 0 to 24 hours and harvested with RIPA lysis buffer.
Total RNA was isolated using TRIzol® reagent (Invitrogen) and reverse transcribed into cDNA (SuperScript® III RT kit (Invitrogen)). Real-time PCR was performed using TaqMan Universal Master Mix with Taqman probes and/or SYBR green method with custom designed primers, normalized to GAPDH and/or 18S rRNA expression. All primers are listed in Supplemental Table S2.
shRNAs specific for human Brg1, BAF47, SS18, and Sox2 were purchased from Open Biosystems (Table S3). shRNA KD constructs for SS18-SSX and shScramble control were generated by annealed oligos (Table S3) and subsequent cloning into the pLK0.1 vector. LV was produced as described by Tiscornia et al., 2006. See Extended Experimental Procedures.
Briefly, cells were crosslinked in formaldehyde, washed, and sonicated as described in Extended Experimental Procedures. ChIP antibodies: anti-BAF155 (in house generated), anti-H3K27me3 (Millipore, 07-449), V5 (Invitrogen, 46-0705). Primers used for real-time PCR listed in Table S2.
This work was supported in part by NIH grants HD55391, RO1NS046789, and R01CA163195 (G.R.C.). G.R.C. is an Investigator of the Howard Hughes Medical Institute. C.K. is supported by the National Science Foundation (Graduate Research Fellowship Program). The authors are grateful to Kazuyuki Itoh, Norifume Naka, and Satoshi Takenaka (Osaka University, Japan) for kindly providing the Aska-SS and Yamato-SS synovial sarcoma cell lines and cDNA clone of the SS18-SSX fusion. We are also grateful to Clara Lee for technical assistance and N. Hathaway, O. Bell, A. Shalizi, and J. Ronan for helpful discussions and critical review of the manuscript.
Supplemental Information
Supplemental information includes 6 Supplemental Figures and 3 Supplemental Tables.
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|Federal trial next month won't address NC voter ID mandate
|Tuesday, June 30, 2015
|RALEIGH – A federal trial next month on several provisions of North Carolina's 2013 elections law won't consider challenges to the state's upcoming voter identification requirement in light of recent changes to the mandate, a judge has ruled. U.S. District Court Judge Thomas Schroeder decided that claims against the photo ID provision set to begin in
|String of fires at 6 predominantly black churches scrutinized
|Tuesday, June 30, 2015
|A string of churches with predominantly black congregations – from Florida to Tennessee -- has reported fires in the past week, officials say. The circumstances surrounding the six fires in five states differ in each case, but their occurring in the past eight days has prompted closer scrutiny. So far only two of the six cases are being investigated
|Changes to NC voter ID rankle some
|Monday, June 29, 2015
|RALEIGH – Federal and state lawsuits filed nearly two years ago challenging what's in North Carolina's election overhaul law are finally heading to trial starting next month. But unexpected moves on the way to the courthouse could break down arguments of plaintiffs seeking to overturn the law's most high-profile component: a photo identification
|Is Medicaid expansion next for North Carolina?
|Friday, June 26, 2015
|RALEIGH – Health care groups say it's time for Gov. Pat McCrory to make good on his promise to release a plan to expand Medicaid in North Carolina. The governor delayed a decision pending the Supreme Court's ruling on the constitutionality of state health-care exchanges. With Thursday's favorable ruling, Adam Linker, co-director of the North Carolina
|Pressure builds for feds to ban the box
|Thursday, June 25, 2015
|WASHINGTON – Seventeen states and Washington, D.C., have passed laws making it easier for ex-offenders to find jobs in the labor market. Now civil rights and community groups that advocate for returning citizens are pushing the White House to do the same. Even though politicians on both sides of the aisle have warmed up to criminal justice reform and
|GOP leader calls for 'swift resolution' of rebel flag debate
|Monday, June 22, 2015
|CHARLESTON, S.C. — Calls to remove the Confederate flag from the grounds of South Carolina's Statehouse grew on Monday, days after a young white man who embraced the banner as a symbol of white power was charged with murdering nine people inside their historic black church. Religious and political leaders including Joseph P. Riley Jr., Charleston's
|NC Senate unveils education budget that guts teacher assistants
|Friday, June 19, 2015
|Senate leaders unveiled portions of a 2015-17 budget proposal Monday that gives teachers an average 4percent pay raise and lowers class sizes in the early grades, But much like last year's initial Senate proposal, the budget also would substantially gut funding for teacher assistants by eliminating more than 8,500 TA jobs over the biennium. The
|Obama on Charleston: It�s too easy to get guns in America
|Thursday, June 18, 2015
|President Barack Obama on Thursday expressed profound "sadness and anger" at the Charleston church shooting as well as deeply personal frustration that America's political climate makes it virtually impossible for now to tighten restrictions on who can buy firearms. "We don't have all the facts, but we do know that once again,


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Tag: trout
Lake Yellowstone: Promising News for Native Trout Recovery
Great news on the invasive species front: lake trout in Yellowstone National Park are on the decline, offering a more hopeful future for native fish. A two-year control effort based on the latest fisheries science is paying dividends, researchers say.
Conserving Lake Trout Among the Philosophers
Can well-managed lakes in the Adirondacks provide important refuges for lake trout in the face of climate change? That's the focus of a new intensive research effort being conducted at Follensby Pond, where philosophers once pondered life's mysteries.
Where the River Meets the Road: Reconnecting Adirondack Brook Trout Streams
Road culverts may not be sexy. But in the Adirondacks, simple fixes in culvert design could reconnect miles of habitat for brook trout, one of the most iconic fishes of eastern streams, and prevent flooding during severe storms.
Salmon Cam: A Live Look at Migrating Fish
Watch salmon return after a long absence to California's Shasta Big Springs Ranch, thanks to Nature Conservancy restoration projects! Enjoy our Salmon Cam for a live, underwater look at migrating steelhead trout and Chinook and coho salmon.
Notes from Silver Creek: Computer Modeling for Stream Conservation
What effects will land use changes have on a stream and its wildlife? How do conservation managers know what will happen in a stream when a restoration project takes place? Will it really lower water temperatures? Will fish thrive?
Surely conservationists can't see into the future?
Actually, stream managers now use sophisticated computer modeling to predict the outcomes of their activities. These models allow them to see how planting native shrubs, for instance, will alter stream flows and water temperatures.
In 2010, The Nature Conservancy was contacted by Maria Loinaz, a PhD candidate at the Technical University of Denmark and the University of Idaho. She was interested in developing a hydrologic model of the Silver Creek watershed using software called MIKE SHE/Ecolab.
This software is changing the way stream managers engage in restoration. It incorporates data on both groundwater and surface water, including stream flow, precipitation, vegetation and soils to accurately predict the effects of a new activity on a stream.
Maria proposed using the MIKE SHE program to model the groundwater and surface water systems and use the EcoLab program to build a water temperature model. Together these would allow her to model what happens to stream temperatures when riparian buffers were planted or stream flows increased. Maria also wanted to incorporate fish data to see whether she could model where, based on the hydrology and temperature, fish would thrive in the system.
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What is Cool Green Science?
noun 1. Blog where Nature Conservancy scientists, science writers and external experts discuss and debate how conservation can meet the challenges of a 9 billion + planet.
2. Blog with astonishing photos, videos and dispatches of Nature Conservancy science in the field.
3. Home of Weird Nature, The Cooler, Quick Study, Traveling Naturalist and other amazing features.
Cool Green Science is edited by Matt Miller, the Conservancy's deputy director for science communications, and managed by Lisa Feldkamp, an American Council of Learned Societies fellow with the TNC science communications team. Email us your feedback.
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Call for Inclusive Conservation
Join Heather Tallis in a call to increase the diversity of voices and values in the conservation debate.
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Where will energy development hit hardest? And where can conservationists make a difference?
Not a sci-fi movie. A true story of nanotechnology & clean water.
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The Top 10 In Action Sports
01Brick By Brick: Camping Lego Style
02Booze and Bikes: 14 Tours That Let Combine Two of the Best Things in the World
03Who Needs Hollywood? These Incredible Videos Were All Filmed on the Simplest Cameras
04Sam Pilgrim vs. The World: The Champion Aiming To Reclaim His Crown In A New Era Of Biking
05Eccentric Adventures Episode 2: Britain's Most Brutal Illegal Event is All About Cheese
06A Journey Into Darkness: We Went Mountain Biking in the Abandoned Mines of Slovenia
07After the Apocalypse: Inside The World's Most Breathtaking Ghost Towns
08Debate: Let's Stop Hating On Parents Who Want To Raise Adventurous Kids
09Guns, Roses, And Eating Insects: Adventure Cycling in Vietnam
10From Intense Trails to Intensive Care: Dicing with Death In a Mountain Biking Paradise
Florian Haffner Edit
A BMX video by lenaicmarechal - 5,611 views
Florian twisted his ankle while we were filming for its web edit during winter, we came back at the spot 2 months later when his ankle was healed and spring on. I had a hard time trying to match the grey sky/bad lights clips of the winter and the stunning clips filmed during spring, so i decided to go for a strong B&W;as it was the best way to harmonizes the clips together.
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My favorite hypothetical virus
I took and TA-ed my college's Computer Security course, where we did things like write malware and put it on VMs, or sillier projects like port knocking. Who knows what they're doing now.
Anyways, I always had one idea for a virus I wanted to write to maximize chaos:
Every n hours, the virus finds two files on the filesystem that have identical filetypes. Then, it swaps the files, but also swaps their filenames. Let's see this in action:
# Find two files of similar types, then do something like:
mv $File1 $HOME/secret/gigli_ben_afflect_j_lo.mp4.tmp
mv $File2 $HOME/Videos/wedding_video.mp4
mv $HOME/secret/gigli_ben_afflect_j_lo.mp4.tmp $HOME/secret/gigli_ben_afflect_j_lo.mp4
What this does is: over time, the user's filesystem will appear identical by most human measurement (the 'Movies' directory will contain a bunch of video files, with the same names as it's ever had), but when if and when the user ever tries to open/use it, it contains something else entirely. In the example above, when the owner wants to watch Bennifer's Gigli, they'll see a wedding video instead. When they want to watch their wedding video, they'll get Gigli. Do this slowly, across the entire filesystem and any filetypes.
I never even wrote a prototype, but when I considered doing anything like that, I always envisioned keeping something like a 'transaction table' so it would be easy to roll back.


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Indie Game Watch List2
I love indie games. These are some upcoming indie games that I'm keeping an eye on. I'll put the release date (or lack thereof) in the comments of each game.
Top 10 System Start-Up Sounds4
A few of us were talking about this in the Olympic Olympiad chat. At the time, I said the Dreamcast had the best one, but after going through them all, I think the PlayStation has the best. It's just...sort of magical. That echo of the orange swirl i...
TG16/PCE Collection1
So I'm trying to keep track on the TG16/PC Engine games I own to prevent myself from screwing up and buying a game I already have. OK?HARDWARE(2) TurboGrafx-16 Base Units(1) Turbo CD Attachment(2) TG16 controllers(1) TG16 TurboTapADDITIONAL SOFTWAREC...
Best of 20120
Jeff's Top 10 of 20122
My top 10 for 2012!
Humble Obsession Bundle4
Hello, my name is StarFoxA, and I have a problem.
PlayStation 4 Collection14
Top 25 Games of the Generation37
I've whittled it down to the Top 25, and I think that's the best I can do. List can include games from: Nintendo DS PlayStation Portable Xbox 360 XBLA PlayStation 3 PlayStation Network (PS3/PSP) Wii PC (from 2005 forward) Only one game per franchise...
Wii U Collection12
I bought a Wii U on Black Friday...2013. Why? I don't know. I wanted to play Super Mario 3D World I guess. And turns out the GameStop I went to was sold out of it actually. Perfect. But I figured it was probably my last chance to get one of the Zelda...
Best of 201314
We're contractually obligated to do this, right? And, yeah, The Last of Us is not on my list. I played the first few hours of it and wasn't really enjoying the game part of it. I love Uncharted, so...I don't know. I DON'T HAVE TO EXPLAIN MYSELF TO YO...
Xbox One Collection1
I actually pre-ordered an Xbox One on the first day they started offering it on Amazon (during E3), but I did not get it until Christmas. Despite my age, my mother is still somewhat obsessed with Christmas and always wants to have that one "big" gift...
Name This Genre9
minimalist esports min-eSports?
A Return to Kombat?49
The dawn of a new generation.I've been a fan since the beginning of Mortal Kombat and I probably know way too much about it. Does anyone else remember rat.org from about 20 years ago? Entering info and creating pages for the original actors that port...
Game of The Year 2014 Users Choice0
Best of 20149
It's been a weird year. Despite all the delays, broken-ass games, and utter disappointments, there were still some great games this year. In my preliminary list, I had twenty games, and whittling them down ended up being harder than I thought it woul...
Bundle Backlog3
This is getting out of hand.
Twenty Fifteen4
Pay no mind. Just keeping track of what I play this year. This may or may not possibly might have something to do with Game of the Year stuff eventually maybe.
Ranking of Fighters79
According to the cast and crew of the smash hit web series "Ranking of Fighters," this is a list of the greatest fighting games of all time... in order!
Arcade Games I Currently Possess34


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This job posting is no longer available on Indeed.
Find similar jobs:IT Application Project Support jobs
- NCHS Holdings jobs
Title:IT Application/Project Support (Business Systems Analyst)Responsibilities:Work with partners and in-house staff to gather requirements and determine process improvements and/or system changes needed to accommodate business needs. Will be given project tasks to perform and also asked to proactively seek any departmental areas needing improvement and suggest solutions to the team.Work closely with Business Systems Analyst Manager and other departments acting as support for projects ranging from minor software updates to the planning and rollout of company-wide infrastructure or application changes.Collaborate in or perform the research or design, testing, and implementation of new or upgraded applications needed for identified gaps in technology.Create, modify and/or optimize SQL queries, views, triggers and stored procedures for business process and analysis needs.Create or revise Crystal and/or SQL Reporting Services Reports as requested.Create and update system documentation and user guides for current systems and processes, both for IT and other departments as needed.Under limited supervision, manage daily work activities to ensure projects and tasks have continued and appropriate focus.Nurture and maintain strong relationships with IT vendors and contractors.Present a positive, professional, customer-focused image, communicating effectively to key team members and partners.Provide end user support for various business management applications, either by direct testing and troubleshooting or working with software vendor support.Assist in providing technical direction and guidance to users; assess and recommend training needs.Delegate tasks to Helpdesk team when necessary to ensure support requests are handled in a timely manner.May serve as backup to Helpdesk team on basic support tasks.
Qualifications:Minimum of AA degree or technical certification required.Must have a minimum of 3 years of experience in a business analyst or similar role.Knowledge of concepts and methods of systems analysis, including information needs and requirements analysis.Knowledge of concepts and methods of project management.Self-starter.Familiarity with basic network concepts and structure.Knowledge of relational database and querying; strongly recommend MSSQL.Ability to multi-task and manage multiple concurrent activities.Strong interpersonal and communication skills, both verbal and written.Solid time management and organizational skills.Experience with business intelligence software, preferably Crystal Reports or MSSQL Server Reporting Services.Comfort working on projects both independently and as part of a team.Strong knowledge of Microsoft Office and basic understanding of Microsoft Windows XP and 7.Must demonstrate critical thinking and analytical reasoning skills.Experience in the healthcare industry would be a plus. (Not required.)
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Lack of govt support reason for delay in justice: SC judge Madan B Lokur
Top Stories
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- Louisiana Governor Bobby Jindal announces run for US presidential elections 2016
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- All above the age of 75 in BJP declared brain dead by Modi: Yashwant Sinha
Lack of government support in states is one of the reason for delay in justice delivery, Justice Madan B Lokur of the Supreme Court today said.
He said only 0.4 per cent of the budgetary outlay is allocated to the judiciary.
"Is justice delivery so unimportant that there is only 0.4 per cent of the GDP as budget for the judiciary"? he said and termed it as "quite insufficient".
The apex court said budgetary allocation for the judiciary was insufficient when there was a proposal for improving the infrastructure by increasing the number of courts and modernising the existing ones by using technology.
"The total number of courts in the country is going to be increased from 14,000 as of now to 18,847. Apart from more judges, we require, land, new courts, modernization of existing courts, staff to help judges, resources etc., which will be hard to come by in this situation when there are many other challenging agenda before the government ," he said.
Justice Lokur, who was speaking at a seminar on 'Technology to Enable Accessible and Speedy Justice,' said increasing the number of judges will not help in speedy
disposal of cases as the judiciary is not able to handle the existing 14,000 courts due to lack of infrastructure and state governments does not have money for it.
"Justice delivery system here, often, is not responsive and technology will help in making things better", he said.
"Look at our justice delivery system, I don't think it delivers justice in time. Well, there could be an opinion whether it delivers justice at all, but certainly not in time.
"Very often, it's not responsive and although technology can speed up justice delivery and can make justice available and affordable to people, we would still have to look at justice delivery in a larger context," Justice Lokur, who is also in-charge of the E-Committee of the apex court, said.
... contd.


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Looking for One Star hotels in Jackson? Hotels.com Australia offers a wide selection of One Star Jackson hotels. You can search any of the star ratings to find the perfect hotel for your travels. It has never been easier to find and reserve One Star hotels in .
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El Rancho Motel
Good 3.1 / 5 ( 211 reviews )
14 people looked at this hotel in the last hour
Most recent review
"Rooms are older, but very clean, comfortable, a good size, and had a fridge/microwave. A simple breakfast of fruit, yogurt, granola bars, milk, juice, and coffee was included, and we were given a pass to the community pool/hot tub. The staff was very friendly, the location near town square is gre..."
A Traveller, Mar 2015, US
Anvil Motel
Good 3.5 / 5 ( 142 reviews )
8 people looked at this hotel in the last hour
Most recent review
"Good accommodations within easy walking to the heart of Jackson Hole. Staff was friendly and accommodating."
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One Star hotels within 15 kilometers of Jackson
The Hostel
Good 3.4 / 5 ( 11 reviews )
39 people looked at this hotel in the last hour
Most recent review
"Cheapest place by far in Teton Village. If you're at Jackson Hole to ski and meet a few interesting people with a story or two to tell, this place is perfect. Rustic, simple, clean, ski storage, friendly people, game room and fire place, very convenient location, ski-in, next to the best bar ..."
A Traveller, Jan 2015, US


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Latvia News
News on Latvia continually updated from thousands of sources around the net.
Canada Day festivities in Swift Current draw a large crowd
Thursday | Southwest Booster | Comment?
Grindeks (GRD1R) - Pharmaceuticals & Healthcare - Deals and Alliances Profile - New Market Report
Sunday Jun 28 | PR-inside.com | Comment?
Concert brings bliss, beauties take a rest at Pintakasi 2015
Friday Jun 26 | Manila Bulletin | Comment?
24 Celebrities Who Are Real Jerks In Real Life
How Should the US Government Respond to ISIS?
Are Warning Labels Needed on Soda?
News 66
12:58 EU and U.S. urge Turkmenistan to cooperate with UN and stop human rights violations
Friday Jun 26 | AkiPress | Comment?
NATO Looks to Expand Mission and Membership
Thursday Jun 25 | CFR.org | Comment?
Belgium warns talks on Britain's EU renegotiation will be tough
Thursday Jun 25 | Globe and Mail | Comment?
Award-winning Nordic Cinema Group chooses Barco 4K laser-illuminated...
Wednesday Jun 24 | Boxoffice | Comment?
EuroPride in Riga goes smoothly despite fears of counter-protest
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Conservative Activists Drive Popular Rock Festival Out of Russia
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UN banking on Juncker Cyprus visit
Saturday Jun 20 | The Cyprus Weekly | Comment?
Riga Russian Orthodox Church
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Saturday Jun 20 | Erie Times News | Comment?
Biography a vivid picture of yoga pioneer
Friday Jun 19 | The Recorder | Comment?
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Updated: Fri Jul 03, 2015 04:01 am
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Available in Heather Grey/Black
Love 21 - A timeless piece (the striped sweater), reimagined with an arsenal of unexpected details like dropped long sleeves, asymmetrical seams, tonal topstitching, and a crisp notched hem - this lightly heathered knit is a polished-meets-laid-back update to your sweater drawer (and it's proof that classic pieces don't have to be boring). Lightweight59% acrylic, 41% cotton29" full length, 42" chest, 40" waist, 26" sleeve lengthMeasured from SmallDry cleanImported
Available in Black, Heather Grey
This oversized sweater is the perfect leggings and slip-ons pairing! We love the turtleneck that elevates the conservative vibe of this piece. The dolman sleeves narrow into slim, ribbed knit forearms that complement the narrowed hemline with ribbed trimming. KnitLight-midweight80% cotton, 20% polyester27.5" full length, 39" chest, 40" waist, 29" sleeve lengthMeasured from SmallMachine wash coldImported
Available in Black/Grey, Cream/Black
We are constantly on the hunt for those oversized marled knit sweaters, because lets face it, we can wear them with pretty much anything! This long sleeve, round neck piece will look great paired with leggings and tall boots. Throw it over a patterned skater skirt with ankle booties and a statement necklace for a slightly dressier feel. Medium weight100% cotton25.5" full length, 34" chest, 32" waist, 29" sleeve lengthMeasured from SmallHand wash coldImported
Available in Black/Grey
F21 Contemporary - Love the layered look but hate the bulkiness that usually accompanies it? Then you'll absolutely love this long-sleeved top. Crafted from a marled knit, this piece boasts a fluid crepe hem with a split accent in back to give the look of two tops in one. A truly versatile piece, this sweater will be your saving grace on mornings when you've hit the snooze button one too many times. Lightweight knit and ...
Available in Black, Grey, Heather Blush, ...
Basics are the building blocks to the foundation of any great wardrobe, but we decided to elevate the norm with this sweater. Maintaining all the classic elements we love (like a crew neck, long sleeves, ribbed trim), the straight-line silhouette and a tight-knit construction are the considered details that make it an undeniable favorite. Lightweight60% cotton, 40% acrylic25" full length, 40" chest, 40" waist, 26" sleeve lengthMeasured from SmallHand wash coldImported
Love 21 - Our favorite chilly weather essential, this striped sweater is complete with long sleeves, a V-neck, and ribbed accents. Battle the elements by pairing it with a pea coat, jeggings, and riding boots. Lightweight knit69% acrylic, 23% cotton, 8% polyester24" full length, 34" chest, 34" waist, 20" sleeve lengthMeasured from SmallHand wash coldImported
With the message "I LITERALLY DO NOT CARE" emblazoned on its front, it doesn't come cheekier or (fittingly) more carefree than this short-sleeved sweater. Its unfinished hem lends even more nonchalant appeal, but the ultimate laid-back touches? Its fleecy interior, boxy fit, and classic crew neckline. Rolled cuffsLightweight knit60% cotton, 40% acrylic23.25" full length, 45" chest, 42" waist, 10.5" sleeve lengthMeasured from SmallHand wash coldImported
Available in Black/Grey, Taupe/Black
With a cropped boxy cut and a luxuriously comfy ribbed knit fabrication, this long-sleeved sweater will be topping our must-have list as soon as the cooler weather rolls in. It features a more relaxed turtleneck, unlike the restricting fit of traditional styles. We're taking this super-warm piece and pairing it with all our favorite high-waisted bottoms (think leather mini skirts, woven trousers, and pencil skirts) and piling on longline necklaces for a boho-chic version of ...
Available in Black/Cream, Taupe/Grey
We love knit sweaters for their cuddly comfort, what we don't love is their all too common appearance as soon as temperatures fall - we like to stand out! This piece blends the best of both worlds. With a crew neckline, long sleeves, and ribbed trim, this has all the classic aspects of our favorites, but its longline silhouette and exposed back zipper give it a sleek edge to make it a subtle statement-maker. Toss ...
Available in Grey/Black
Add some updated elegance to your wardrobe with this long-sleeved sweater, featuring colorblocked accents and a scoop neckline. We love this piece with lace shorts and strappy sandals for daytime. LightweightKnit56.6% acrylic, 43.4% cotton24" full length, 36" chest, 38" waist, 25" sleeve lengthMeasured from SmallMachine wash coldImported
Available in Black/Cream, Burgundy/Grey
We love pieces that we can take from lounging around the house to watching a movie with friends, like this chunky cardigan! It features a two-tone knit construction, open-front, and long sleeves. Complete with two front pockets wear this casually with some leggings, tall boots, and a simple knit tee (throw on a some jewelry to glam it up a bit). UnlinedMedium weight60% cotton, 40% acrylic 31.5" full length, 46" chest, 52" waist, 29" sleeve ...
Available in Black, Blush, Brick, Charcoal ...
Fall is quickly approaching, which means we can stop daydreaming about this classic button-front V-neck cardigan and finally start wearing it! Perfect for layering over dresses or tee shirts, this piece features ribbed trim and a slightly slouchy silhouette. KnitLightweight60% cotton, 40% acrylic25.5" full length, 35" chest, 34" waist, 25.5" sleeve lengthMeasured from SmallHand wash coldImported
Adding a little sass to a classic hoodie, this lightweight number features a contrast drawstring, a zippered front, a split kangaroo pocket, and a "Yes Please, No Thank You" graphic on the back. Super comfy and perfectly polite, this piece is perfect to throw on for a casual day out. Lightweight knit54% cotton, 42% rayon, 4% spandex23" full length, 38" chest, 38" waist, 25" sleeve lengthMeasured from SmallHand wash coldImported
When it comes to closet staples, cardigans top our list time and time again. Take this roll-tab 3/4-sleeved one, for example. It's an easy go-to layer with its open front and front patch pockets. Keep it on hand in case a breeze (or the AC in the office) picks up. Lightweight knit53% cotton, 43% viscose, 4% spandex26" full length, 36" chest, 34" waist, 23" sleeve lengthMeasured from SmallHand wash coldImported
From moonlit beach bonfires to strolling down the sunlit street, you want to wear a piece that's comfy, cute, and pairable with all your bottoms. Enter this pullover. Crafted from lightweight heathered knit and outfitted with a drawstring hood, a kangaroo pocket, and a "L&ver" graphic across the chest, this dropped long-sleeved topper will keep you cozy and cool all at once. 53% cotton, 43% viscose, 4% spandex21" full length, 42" chest, 40" waist, 20" ...
Available in Black, Heather Navy, Heather ...
Forever 21+ - This fitted V-neck cardigan features a buttoned front and long sleeves. Thrown on over a cami dress or pair wit denim and a basic tee for chilly days. Ribbed trimLightweight knit62% cotton, 28% acrylic23.5" full length, 41" chest, 41" waist, 28.5" sleeve lengthMeasured from 1XHand wash coldImported
Available in Black, Heather Grey, Light Blue, ...
Your fall wardrobe is not complete without this long-sleeved cardigan! Its V-neckline and button-down front definitely lends an old-school charm. However, it's the popcorn knit construction that adds just the right amount of texture to this classic piece. Finished with two patch pockets, this piece is the perfect layer over collared blouses or basic t-shirts. Lightweight100% cotton26.5" full length, 38" chest, 34.5" waist, 25" sleeve lengthMeasured from SmallHand wash coldImported


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Does this make me a bad cross-dresser? i dont wear bras nor wigs but i do shave my legs and other parts.
No, it just makes you a cross-dresser who doesn't wear bras or wigs. If bras and wigs don't add to your experience, why bother?
every one has their own desires, needs, and interests. Do you enjoy what you are doing? Yes? Great then keep enjoying and F all the people who tell you that you are doing it wrong.
Yeah, you're terrible at it- you seem kinda' half-assed
I think it's a really individual kind of thing. Are you happy and having fun? If you're getting what you need out of it, that's all that counts.

Some girls need to pass and get lucky. If you're doing that without bras and wigs, that makes you a really superior crossdresser. If you've got the right face and hair and build, then maybe you're just a natural.

If you need to pass, but you're not succeeding at it, then you'll have to try harder.

If you're doing it for your partner, and he or she wants you to do more, then that's just a relationship issue.
Do you go out as a male in female clothes? You maybe Androgyne......A male who in part of their make up (Mental, Gender) may have some feminine aspects to your personality. If you pass yourself off as such then really, I (This is my opinion) would not call you a Cross-dresser instead a male with certain female traits, am I cutting the pie slices here really thin? To many the answer would be yes. There is a difference though, type in Androgyne, read the paper then decide. If you truly want to be a full CD'er look into articles on how to do so with out much fuss. Good luck to you.
how the hell should i know... to each their own i guess
Related Questions
or whatever that head to toe covered robe thing is called,,,,,,,,,,,,,,,,,,,,,,,,,
crossdresser31 26-30, M 20 Answers 2 Aug 3, 2013 in Community
Men that wear dresses are Cross dressers...does that mean that all women that wear jeans and T-shirts are too? lmao!!!!LadyLouisiana 56-60, F 17 Answers 5 Oct 17, 2013 in Doing Good
Incideofme 36-40, M 3 Answers 1 Mar 9, 2014 in Dating & Relationships
What types of dresses and skirts will look good on a cross-dresser? Cause im a cross-dresser and i would some help.Renee99 31-35, M 18 Answers 6 Jul 24, 2012


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1998 Honda Civic Q&A
1998 Honda Civic Question: Car will not start
Thanks for your answer. More info. My car has about 65,000 miles on it. It has a short ram air intake, new. I have already fouled 3 sets of spark plugs, so I know that there is a lot of fuel getting to the cylinders. I unplugged the injectors and the car started. When the fuel was burned off, I plugged the injectors back in. The car spins over easily but still will not start. Any thoughts now. - golfman
Answer 1
What model Civic have you is it a VTEC? Any Check Engine Light on? Your car has on board diagnostics (OBD2). There is a 16 pin connector under the dash that communicates with a scan tool. Information available on the scan tool will show Long term and Short term fuel trim, and live Coolant temperature sensor (CTS)data. If say it is 75 degrees out side and the CTS is telling the computer it is 30 degrees outside well the computer is going to dump in a lot of gas because it thinks it is freezing. The scan tool will also read how uch air the computer thinks is going into the engine, if it thinks there is a lot of air going into the engine when in actual fact you are at idle it again will try sending a lot of fuel into the engine. Engine read airflow to the engine by using a Manifold Absolute Pressure Sensor (MAP). - patrick mannion
Related Items:
...would just turn on the lights just to see what would happen without the fuse (stupid). now my car will not start. i believe it is a wiring issue and i was just wondering if you guys knew if i should complete...
Last night I went out and the car started fine. I drove through a bank drive-tru and then to a red light. When I took off from the light the car sputtered and almost stalled. I was able to get the ...
was here a few weeks ago with problem of car not starting but was cranking. raiderron figured out the problem. ecu was bad. thank you raiderron. now we have replaced it and the capacitor blew in ne...
So, car started fine last night. And again this morning when I got off work. Went to start in again and nothing. Its clicking, and everything turns on but no engine start. Got new battery, still no...
in the morning the engine will not start.
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: [Weekly Poll]
Serperior vs Zebstrika (NU Matchup #6)
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December 15th, 2012 (09:55 AM).
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Serperior wins, hands down. Unlike Zebstrika, it eventually got decent coverage via Move Tutors in the sequel, and it also has Sword Dance and Coil to power up its other non-Grass moves like Rock Smash. It stinks to be a quadpedal Electric type, as you're limited to some useful moves for coverage.
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Jason āMayhemā Millerās recent antics have sparked fears for his mental health. First he was arrested apparently naked in a church which he had broken into and vandalised, then he appeared on Ariel Helwaniās āMMA Hourā show and gave a bizarre turn supposedly āin characterā as Lucky Patrick, the bit part he plays in the Kevin James movie Here Comes The Boom.
Helwani claimed he had no prior knowledge of the interview being āin characterā and he seemed lost as Miller spiralled out of control before storming out of the studio. But communications between the two continued and so this week Miller became the second subject of what looks set to be an ongoing series of āwalk and talkā extended interviews.
Its a fascinating piece of film. Even if you arenāt a fan of Millerās public persona - I personally find the zaniness somewhat forced and contrived - this near-hour conversation is completely different to anything youāve seen of him before and there are some genuine insights to be had not only into his mindset but also into life some aspects of being a mid-tier UFC employee.
Video: Jason 'Mayhem' Miller on mental health, Dana, the fight game
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