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You've worked hard to keep your restaurant clean, yet when the health inspectors visited, they found signs of pest infestations. Of course, you realize that this is a huge concern. Pests like cockroaches and rodents can spread diseases and contaminate your food. Although you've set traps in your food storage areas, you're still having a pest problem. So what could it be? The answer may lie with something that is outside your restaurant — your dumpsters. Check Your Dumpsters Out A dumpster, in good condition and used properly, can effectively keep pests at bay. But if your current dumpster has holes and dents or is missing one or more of its covers, you may have inadvertently created a perfect breeding and feeding ground for all sorts of pests. Unfortunately, these pests aren't going to remain outside, especially if the weather turns colder or wet. They're going to seek shelter in a warm, dry place such as your restaurant. And in some areas, such as Chicago, you could face fines if rats from your dumpster become a nuisance to the neighborhood. So to prevent pests from turning your dumpster area into their own personal playground, you need to: Always shut the lids to your dumpsters tightly. Check your dumpster to make sure that the lids are still fitting correctly and are intact. If you see gaps that could allow pests in, call your service to request a new dumpster. You should also request a new dumpster if yours is showing any signs of rust or has holes. If your service is slow in responding, you may want to check out other dumpster businesses to see if you might be able to find one that can offer better service and, possibly, lower prices. Bag all of your food waste before putting it in the dumpster. Rotting food attracts pests, so try to mask the scent by placing all of your garbage in plastic bags. Have your trash picked up on a regular basis. Is your trash overflowing or are their bags of waste piled up near your dumpster? Then you may need to request another trash pickup and/or a larger dumpster. Speak with your employees. They need to understand the importance of keeping the lids down on your dumpster and to never leave trash bags lying on the ground, either near your business or by the dumpsters. Secure your dumpster. If you are having problems with neighbors using your dumpsters and causing them to overflow, you may want to consider securing them with a lock. Clean out your dumpster on a regular basis. A smelly dumpster is going to attract more pests, so take the time to wash out your dumpster and to also clean debris that may have fallen under your unit, as well. In addition, you should consider hiring a pest management service that specializes in working with restaurants to place traps in and around your restaurant and the dumpster. Placement of Your Dumpsters The placement of your dumpsters is also important for pest management. For example, your dumpster should: Be placed on a concrete pad and/or have wheels so that rodents cannot burrow underneath your dumpster. Not be placed near tall grass or shrubbery. Rats like to make their burrows under bushes and plants. Because a pest infestation...read more
A septic tank allows you to turn the most remote plot of land into a homestead the whole family can enjoy. When picking out your brand new septic system, you'll need to choose between a simple one chamber tank or a unit with two or more separate spaces. Figure out which design best fits your budgets and plans so you don't need to get a new tank within the first few years of moving onto your property. Solid Settling The number of chambers in a septic tank has the biggest effect on how well the solid waste in the tank settles at the bottom instead of flushing out into the drain line and clogging it. Without proper settling, you'll end up a clogged system that sends sewage backing up into the house. Two or more chambers cause solid waste to end up in the first chamber, while the water waiting in the second and third spaces gives smaller particles a chance to settle out too. If the chambers are separated properly, the sludge at the bottom won't wash into the other chambers as easily either. A dividing wall along the bottom forces only the water at the top to spill over into the next part of the tank. Cleaning Schedule The biggest maintenance task for owning a septic tank is the pumping process. No matter what type of tank you choose, you must get it pumped at least once every few years or it could end up cracked and in need of replacement. Since multiple compartments help you gather solid waste at a higher rate, you need to get that chamber cleaned out more regularly. The small divided chambers also fill up faster. However, even the biggest single chambered tank eventually needs a complete pump out to keep working properly. Soil Accommodation What kind of restrictions is the local health department putting on your septic tank due to the soil conditions? Slow drainage and high water tables make it unsafe to use the usual standard tanks with open drain fields. Two, three, and four compartment designs allow for the extra space needed to accommodate alternative septic systems like, Solid waste grinding pumps that save space in the bottom of the holding tank Aerobic digestion systems that rely on oxygen to break down waste faster Baffles and filters that keep solid and thick liquid waste from draining out into the field Dosage chambers for systems based around bacteria additives that do the hard work of neutralizing your waste. Water Velocity Each flush or sudden drainage of the washing machine sends a jet of water shooting into the water holding tank. Sudden movement stirs up all the particles and solid waste trying to settle safely at the bottom of the system. Two-chambered designs can slow down the velocity of water entering the septic system by putting baffles and walls around the inlet area. Most single chamber designs feature the same basic water slowing baffles too now, so the two types of tanks don't differ too much in this respect. Chamber Proportions The only proportion important in single chamber designs is the overall size. If there's a second chamber added on, the first holding space should account for two-thirds of the total volume. This allows you to accumulate plenty of waste before...read more
If your home has become damaged in a flood, you will need to quickly get to work and clean up the mess. Once you have prevented any further water from flooding your home and removed the excess water, clean up any water-damaged items and deal with possible mold growth. Here are two tips to help make your flood clean-up an easier process. Rent a Dumpster When you are cleaning up the inside and outside of your home after a flood, there are going to be a lot of damaged items and debris that you will need to dispose of. It can be helpful to rent an appropriately-sized dumpster from a local waste company to accommodate all the debris you need to throw away. A representative with the waste company can help you estimate the size of dumpster you will need for the clean-up. Don't try to overfill your dumpster to save money. If you go over the waste company dumpster's weight limit, you can end up paying extra fees to the dumpster rental company. The reason for this is because the waste company pays the landfill a tipping charge according to the weight of the refuse they are dumping. As you decide which furniture items need to be thrown out and which ones can be saved, determine the value of any household items that have become damaged and wet from the flooding. Then, decide if it will cost more to clean, refinish, and reupholster an item or to replace the item with a new one. Upholstered furniture can absorb contaminants from the flood water and may need to be discarded. Also, some wood furniture can become warped from sitting in water and be damaged beyond repair. Items such as mattresses cannot be salvaged after they have become wet in a flood and will need to be discarded. Mold can begin to grow in wet conditions within 24 hours. Because the interior of a mattress can not completely be dried out within 24 hours, it is best to toss it in the dumpster. Carpet padding cannot be dried within this time frame and should be discarded as well. Drywall that has become soaked needs also to be pulled out and discarded. Drywall contains gypsum and, when it gets wet, will pull in a great deal of moisture and hold it in. Then, when drywall does dry out, it will become crumbly to the touch, losing its strength. After cutting out any wet drywall, also pull out and throw away wet insulation batting. Once the air pockets inside insulation batting become full of moisture, the insulation loses its ability to effectively insulate your home. Hire Mold Remediation Specialists After a flood occurs in your home, it is important to remove the water and dry all wet surfaces as soon as possible to prevent mold growth. Some types of mold, such as black mold, can be dangerous to your health and cause many health issues from breathing in its spores. When the mold covers more than ten square feet of area in your home, it is best to hire a mold remediation specialist to professionally and safely remove the mold from your home. If you try to clean up a large mold growth on your own, you can risk spreading the spores throughout your home and having further mold growth and health problems. Mold remediation specialists first contain the area in...read more

subset: commoncrawl


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ERβ1 represses basal-like breast cancer epithelial to mesenchymal transition by destabilizing EGFR
- Christoforos Thomas1Email author,
- Gayani Rajapaksa†1,
- Fotis Nikolos†1,
- Ruixin Hao1,
- Anne Katchy1,
- Catherine W McCollum1,
- Maria Bondesson1,
- Phil Quinlan2,
- Alastair Thompson2,
- Savitri Krishnamurthy3,
- Francisco J Esteva4 and
- Jan-Åke Gustafsson1
DOI: 10.1186/bcr3358
© Thomas et al.; licensee BioMed Central Ltd. 2012
Received: 1 July 2012
Accepted: 12 November 2012
Published: 16 November 2012
Epithelial to mesenchymal transition (EMT) is associated with the basal-like breast cancer phenotypes. Sixty percent of basal-like cancers have been shown to express wild-type estrogen receptor beta (ERβ1). However, it is still unclear whether the ERβ expression is related to EMT, invasion and metastasis in breast cancer. In the present study, we examined whether ERβ1 through regulating EMT can influence invasion and metastasis in basal-like cancers.
Basal-like breast cancer cells (MDA-MB-231 and Hs578T), in which ERβ1 was either overexpressed or down-regulated were analyzed for their ability to migrate and invade (wound-healing assay, matrigel-coated Transwell assay) as well as for the expression of EMT markers and components of the EGFR pathway (immunoblotting, RT-PCR). Co-immunoprecipitation and ubiquitylation assays were employed to examine whether ERβ1 alters epidermal growth factor receptor (EGFR) protein degradation and the interaction between EGFR and the ubiquitin ligase c-Cbl. The metastatic potential of the ERβ1-expressing MDA-MB-231 cells was evaluated in vivo in a zebrafish xenotransplantation model and the correlation between ERβ1 and E-cadherin expression was examined in 208 clinical breast cancer specimens by immunohistochemistry.
Here we show that ERβ1 inhibits EMT and invasion in basal-like breast cancer cells when they grow either in vitro or in vivo in zebrafish. The inhibition of EMT correlates with an ERβ1-mediated up-regulation of miR-200a/b/429 and the subsequent repression of ZEB1 and SIP1, which results in increased expression of E-cadherin. The positive correlation of ERβ1 and E-cadherin expression was additionally observed in breast tumor samples. Down-regulation of the basal marker EGFR through stabilization of the ubiquitin ligase c-Cbl complexes and subsequent ubiquitylation and degradation of the activated receptor is involved in the ERβ1-mediated repression of EMT and induction of EGFR signaling abolished the ability of ERβ1 to sustain the epithelial phenotype.
Taken together, the results of our study strengthen the association of ERβ1 with the regulation of EMT and propose the receptor as a potential crucial marker in predicting metastasis in breast cancer.
In the last decade, genomic studies have identified five breast cancer intrinsic subtypes (Luminal A, Luminal B, HER2 (overexpressing the ERBB2), basal-like and claudin-low) [1, 2]. In a recent study, an integrated analysis of copy number and gene expression split the intrinsic subtypes revealing novel subgroups with distinct clinical outcome, including a high-risk ERα-positive subgroup and a subset of ERα-positive and ERα-negative cases with a favorable outcome. According to this analysis, the majority of the basal-like tumors formed a high-genomic instability subgroup with relatively good long-term outcomes (after five years) [3]. Basal-like phenotypes represent tumors that express markers that are characteristic of the myoepithelium of the normal mammary gland, such as epidermal growth factor receptor (EGFR), p63 and the basal cytokeratins CK14, CK5/6 and CK17 [1, 4]. They show partial overlap with the triple-negative breast cancers that are characterized by a lack of HER2 gene amplification and estrogen and progesterone receptor expression. Approximately 75% of triple-negative breast cancers are classified as basal-like tumors on the basis of their overall gene-expression profile. The basal-like phenotype represents a more homogeneous group of cancers than the group of cancers defined by triple negativity [5]. Basal-like tumors are often resistant to chemotherapy and develop distant metastases in characteristic tissues, such as lung and brain [6]. Recent studies have suggested a correlation between the basal phenotypes and epithelial to mesenchymal transition (EMT) [7].
EMT has been reported to promote invasion during the progression of breast carcinomas and it is considered as an essential early step in tumor metastasis [8, 9]. EMT is characterized by loss of cellular adhesion, which is mediated by down-regulation of adhesion molecules, such as CD44 and E-cadherin [10, 11]. The expression of E-cadherin is regulated by a number of transcriptional repressors, which include SNAIL, SLUG, SIP-1 (ZEB-2), δEF1 (ZEB-1) and TWIST [12–15]. The family of microRNAs 200 (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) and the miR-205A regulate the expression of the transcriptional repressors of E-cadherin ZEB-1 and ZEB-2 and, consequently, the levels of E-cadherin in breast cancer cells and tissues. A decrease in the expression of these microRNAs has been observed in cells that have undergone EMT and in mesenchymal regions of metaplastic breast cancer lacking E-cadherin expression [16]. Up-regulation of components of the EGFR signaling pathway, such as ERK2, has also been reported to influence the levels of E-cadherin by regulating the transcriptional repressors ZEB-1 and ZEB-2 [17, 18].
The potential role of estrogen receptors in regulating EMT and aggressive behavior in breast cancer has recently been under investigation [19]. Although a decline of ERα levels is detected in invasive breast cancers, a few studies have shown regulation of cell migration and invasion by ERα [20, 21]. Recent studies have also associated the ERβ isoforms ERβ1, ERβ2 and ERβ5 with the regulation of cell migration and invasion in prostate cancer [22, 23]. Down-regulation of the fully functional ERβ isoform ERβ1 (also known as wild-type ERβ) promoted EMT in prostate cancer cells and this correlated with the loss of ERβ1 in high Gleason grade invasive prostate carcinoma [22]. Interestingly, patients with triple-negative breast cancer that were treated with adjuvant tamoxifen have been shown to have significantly better survival when the tumors were positive for ERβ1 [24]. In addition, clinical findings showed an inverse correlation between ERβ1 positivity and expression of EGFR, a crucial component in basal-like cancers that drives proliferation and EMT [25]. Given that down-regulation of ERβ1 has been observed in invasive breast cancers, in this study we hypothesized that ERβ1 functions to maintain an epithelial phenotype in breast cancer and examined whether ERβ1 reduces the invasiveness of basal cancer cells by repressing EMT [26].
Materials and methods
Cells, reagents and transfections
The breast cancer cell lines (MDA-MB-231, Hs578T and MCF-7) and the lung cancer cell line (H1299) were obtained from the ATCC. In 17β-estradiol (E2) experiments, cells were maintained in phenol red-free media containing two or five percent dextran-coated charcoal (DCC)-treated fetal calf serum (FCS). Transforming growth factor β (TGF-β) and EGF experiments were performed in serum-free or 0.5% FCS media with recombinant human TGF-β1 (5 ng/ml; R & D Systems, Minneapolis, MN USA) for one to three days or EGF (10 ng/ml; Sigma) for 24 h. MDA-MB-231 and Hs578T cells were infected with lentiviruses containing the plenti6/V5 empty vector or the recombinant pLenti6/V5-D-FLAG-ERβ1 and pLenti6/V5-D-FLAG-ERα plasmids as described previously [27]. Cells were transfected twice with ERβ-specific siRNAs (Invitrogen, Carlsbad, CA USA), target sequences 1# 5'-TTAGCGACGTCTGTCGCGTCTTCAC-3'; 2# 5'-TTACGACATTAAGTAGTGTCGTCCC-3'; 3# 5'-TATTGACCGCTACCTGGTGATTTCC-3'. siRNA targeting luciferase was used as a control (Cat. No. 12935-146, Invitrogen). For the expression of wild-type EGFR, cells were stably transfected with the pBABE-EGFR construct (Addgene, plasmid # 11011, Cambridge, MA USA), using the empty pBABE vector (Addgene, plasmid # 1764) as a control. Cells were transfected with microRNA inhibitors at a final concentration of 300 nM (100 nM of each of miR-200a, miR-200b and miR-429 2'-O-Methylmodified oligonucleotides, Dharmacon, Waltham, MA USA) or a negative control inhibitor (300 nM). The complementary sequences for miR-200a, miR-200b and miR-429 were cloned in the 3' end of the luciferace gene into the PGL3-promoter vector (Promega, San Luis Obispo, CA USA). A total of 2 × 105 cells were seeded at 24-well plates and transfected with 800 ng DNA/well (PGL3, β-gal) as well as microRNA inhibitors. Twenty-four hours after transfection, cells were lysed and analyzed using a Luciferase Assay (Promega). Luciferase units were normalized to β-galactosidase units. For ERE-luciferase reporter assays, cells were incubated in DCC-FCS media for 48 h and transfected with 800 ng DNA/well (3-ERE-TATA-LUC reporter plasmid, β-gal plasmid) using LipofectamineTM 2000 (Invitrogen). Cells were mock treated (EtOH) or treated with E2 for 24 h in 2% DCC-FCS media. Reporter gene activity was normalized to β-galactosidase enzyme activity.
Migration and invasion assays
In the wound-healing assay, cells were allowed to form monolayers at 24-well plates. The monolayer was scratched with a pipette tip to form the wound. Twelve hours later, images of the wound were taken using a 10× objective in an OLYMPUS IX51 microscope equipped with an OLYMPUS camera (OLYMPUS, Center Valley, PA USA) and cells in the wound area in five independent fields were counted.
In the invasion assay, cells were seeded in matrigel-coated 6.5 mm Transwell champers (8 μm pore size; BD Biosciences, San Jose, CA USA). Six hours later, the cells that had been translocated to lower compartments of the wells and attached to the lower surface of the filter were fixed in methanol and stained with crystal-violet. The stained cells were counted in five independent fields in each Transwell.
Immunofluorescence and microscopy
Cells were plated onto 18 mm2 coverslips, fixed in 3% paraformaldehyde (PFA) and 2% sucrose for 15 minutes at room temperature (RT), permeabilized in 20 mM Tris HCI pH 7.5, 75 mM NaCl, 300 mM sucrose, 3 mM MgCl2 and 0.5% Triton-X-100 for 15 minutes at RT and blocked with 5% goat serum in phosphate-buffered saline (PBS) for 1 h at RT. Slides were stained with an E-cadherin antibody (BD Biosciences) at 4°C overnight, washed, incubated with secondary antibody and images were collected on an OLYMPUS BX51 microscope equipped with an OLYMPUS XM10 camera (OLYMPUS, Center Valley, PA USA).
RNA extraction and real-time PCR
Total RNA was isolated using TriZol reagent (Invitrogen) and reverse-transcribed to cDNA using a SuperScript™ II reverse transcriptase kit (Invitrogen). Real-Time PCR was performed using the SyBr green PCR kit (Applied Biosystems, Grand Island, NY USA). EGFR mRNA levels were additionally analyzed using TaqMan mRNA assay according to the manufacturer's instructions (Applied Biosystems). All quantitative data were normalized to GAPDH and actin-β. For microRNAs, real-time PCR was performed as above using TaqMan microRNA assays (Applied Biosystems). All microRNA data are expressed relative to a U6 small nuclear (sn) RNA TaqMan PCR performed on the same sample. The sequences of the primers used for qPCR are listed in the Additional file 1, Table S1.
Immunoblotting and immunoprecipitation
Cells were lysed in RIPA buffer, including protease and phosphatase inhibitors as previously described [28]. For separation of cytoplasmic and nuclear fractions, cells were suspended in a cold buffer containing 10 mM Hepes pH 7.0, 10 mM KCI, 0.1 mM EDTA, 1 mM DTT and 0.5 mM PMSF. After 15 minutes' incubation on ice, the homogenate was mixed with 10% NP-40 and centrifuged for 30 sec. The nuclear pellet was resuspended in a cold buffer containing 10 mM Hepes-KOH pH 7.9, 400 mM NaCI, 0.1 mM EDTA, 5% glycerol, 1 mM DTT and 0.5 mM PMSF and the nuclear extract was isolated by centrifugation. The blots were performed as previously described [28]. Primary antibodies used in immunoblotting include: ERα, E-cadherin, N-cadherin, cadherin-11, vimentin, ZEB-1, SIP1, Lamin A/C and Tubulin (Santa Cruz Biotechnology, Santa Cruz, CA USA), actin-β (Sigma St. Louis, MO USA), EGFR (Santa Cruz Biotechnology, Cell Signaling, Danvers, MA USA), ERβ1 (14C8; GeneTex, Irvine, CA USA), which detects an N-terminal epitope and recognizes the ERβ isoforms derived from alternative splicing of the last exon, including ERβ1 and an in-house antibody that detects an epitope in ligand binding domain of ERβ1 (amino acids 320 to 527)) [29], SNAIL (Abcam, Cambridge, MA USA), p-ERK1/2 and total ERK1/2 (Cell Signaling), c-Cbl (BD Biosciences). Recombinant ERβ1 (Invitrogen) was loaded in SDS-PAGE gels and used as a positive control. For ubiquitylation analysis, cells were lysed in RIPA buffer containing protease inhibitor cocktail (Roche, Branchburg, NJ USA). The lysates were briefly sonicated and cleared by centrifugation at 4°C. Supernatants were incubated with anti-EGFR antibody overnight at 4°C and A/G agarose beads for 2 h at 4°C. The immunocomplexes were washed three times, boiled in 2× sample buffer and immunoblotted with anti-ubiquitin antibody (Santa Cruz Biotechnology). For the EGFR-c-Cbl co-immunoprecipitations, cells were lysed in a buffer containing 50 mM Hepes pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 1% glycerol including protease and phosphatase inhibitors. Lysates were incubated on ice for 30 minutes without sonication, cleared by centrifugation and the cleared lysates were subjected to immunoprecipitation as described.
Zebrafish tumor model and generation of fluorescent cells
Animal work was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Houston. Control (Lenti) and ERβ1-expressing (ERβ1) MDA-MB-231 cells were stably transfected with either the pAmCyan vector or the pCMCV-DsRed vector (Clontech, Mountain View, CA USA). A tumor cell suspension (5 nL) of approximately 300 to 500 cells containing a mixture of equal numbers of either DsRed-Lenti:AmCyan-ERβ1 cells or AmCyan-Lenti:DsRed-ERβ1 cells were injected into the perivitelline cavity of each 48 h post-fertilization casper Tg(Flk-1;EGFP) anesthetized embryo using a pressure injector (Harvard Apparatus, Holliston, MA USA) and Manipulator (MM33-Right, Märzhäuser Wetzlar, Wetzlar, Germany). Glass needles (1.00 mm in diameter, Sutter Instrument Company, Novato, CA USA), were used for the microinjection. Injected embryos were kept at 32°C and were examined every day for tumor invasion using a fluorescent microscope (OLYMPUS IX51) equipped with an OLYMPUS XM10 camera. Information for the zebrafish lines is included in the Additional file 2, Supplementary materials and methods.
Patient information
A tissue microarray consisting of 240 breast cancer samples was constructed by the Tayside Tissue Bank. Access to tumor samples was approved by the Tayside Regional Ethics Committee with written informed consent from contributing patients. Clinical history and tumor characteristics were available for 238 cases. The clinicopathological characteristics of these patients are summarized in the Additional file 3, Table S2. The majority of the patients received adjuvant endocrine therapy or combined endocrine therapy and chemotherapy, with or without radiotherapy. Among these patients, 74.7% were ERα-positive, 53.7% were PR-positive and 14.5% were HER2-positive. Histologically, 192 invasive ductal carcinomas (80.6%), 14 invasive lobular carcinomas (5.8%), 5 tubular carcinomas (2.1%), 5 mucinous carcinomas (2.1%) and 22 other histological or mixed types (9.2%) were included.
Antibody validation and immunohistochemistry
The anti-ERβ1 antibody (clone PPG5/10, Dako, Carpinteria, CA USA), which is specific for the C-terminal amino acid sequences of ERβ1, was used for immunohistochemistry (IHC). This antibody was validated by immunocytochemistry. Briefly, H1299 human lung cancer cells were stably transfected with the pIRES empty vector (Clontech) or the recombinant pIRES-ERβ1 or pIRES-ERβ2 plasmids. Control, ERβ1 and ERβ2-expressing cells were fixed with 10% formalin. The cell suspension was centrifuged and the cell pellet was folded in sharkskin filter paper using four overlapping edges and placed within the base of a tissue cassette. The cassette was placed in a specimen bucket with 10% formalin. The formalin-fixed cell material was embedded in paraffin, cut at 5 μm intervals and used for H&E staining and IHC.
For immunohistochemistry, formalin-fixed, paraffin-embedded sections were de-paraffinized with xylene and rehydrated through a graded alcohol series. For antigen retrieval, the slides were immersed in 10 mM sodium citrate buffer (pH 6.0) and maintained at a sub-boiling temperature for six minutes. The endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide solution for 20 minutes. The slides were first incubated with 1% bovine serum albumin (BSA) to block non-specific staining and then with the primary antibody overnight at 4°C in a humidified chamber. The sections were then processed according to the Dako DAB detection kit.
The results of the immunohistochemistry were assessed by a pathologist (SK) in a blinded fashion. Each specimen was assigned a score according to the intensity of the nuclear staining (for ERβ1) and cytoplasmic and membrane staining (for E-cadherin) (no staining = 0, weak staining = 1, moderate staining = 2, strong staining = 3) and the extent of stained cells (0% = 0, 1 to 24% = 1, 25 to 49% = 2, 50 to 74% = 3, 75 to 100% = 4). The final immunoreactive score was determined by multiplying the intensity score with the extent of the score of stained cells, ranging from 0 (the minimum score) to 12 (the maximum score). We defined ERβ1 expression as low (score 0 to 4), medium (score 5 to 8) and high (score 9 to 12). For E-cadherin, we defined a 0 score as negative and a 1 to 12 as positive.
Statistical analysis
The correlation between expression of ERβ1 and E-cadherin, respectively, was determined using Pearson's correlation test. All statistical tests were two-sided and P-values less than or equal to 0.05 were considered as statistically significant. The statistical analyses were performed using SPSS 20.0 software (SPSS, IBM, Armonk, NY USA).
ERβ1 is required for the epithelial breast cancer phenotype
miR-200 and ZEB1/2 are involved in ERβ1-mediated regulation of E-cadherin
A number of transcription factors (SNAIL, SLUG, TWIST, SIP-1 and ZEB-1) have been shown to promote EMT in vitro by acting as transcriptional repressors of E-cadherin [34, 35]. Nuclear translocation of SNAIL has been shown to repress E-cadherin expression in ERβ1 knockdown prostate cancer cells [22]. Based on these data, we examined whether SNAIL inhibition is involved in the ERβ1-mediated induction of E-cadherin expression that we observed in breast cancer cells. Surprisingly, induction of ERβ1 expression in MDA-MB-231 cells neither altered the expression nor the intracellular localization of SNAIL as assessed by immunoblotting using cytoplasmic and nuclear extracts from control and ERβ1-expressing cells as well as immunofluorescence microscopy using a SNAIL Ab (Additional file 7, Figure S3A, B). Instead, up-regulation of ERβ1 in MDA-MB-231 and Hs578T cells repressed the expression of the transcriptional repressors of E-cadherin ZEB-1 and SIP-1 (Figure 2C). Given that recent studies have reported that the microRNA-200 family and miR-205 regulate EMT by targeting ZEB-1 and SIP-1, we examined whether the expression of members of the microRNA-200 family and miR-205 were up-regulated prior to repression of ZEB-1 and SIP-1 expression in ERβ1-expressing cells [16]. Using quantitative real-time PCR we found that the cluster of miR-200b-200a-429 was up-regulated by more than 7-fold in the ERβ1-expressing MDA-MB-231 and Hs578T cells (3.5-fold increase only for miR-429 in Hs578T cells) (Figure 2D; Additional file 8, Figure S4A). In addition, reduction of endogenous ERβ1 expression in MDA-MB-231 and Hs578T cells by ERβ siRNA led to a decrease in the expression of miR-200a, miR-200b and miR-429 (Figure 2E; Additional file 8, Figure S4B). In contrast to the cluster of miR-200b-200a-429, the cluster miR-200c-141 and the miR-205 were unchanged in ERβ1-expressing MDA-MB-231 cells (Additional file 9, Figure S5). We also examined how important is the up-regulation of miR-200a-b-429 for the ERβ1-mediated repression of EMT. We transfected the ERβ1-expressing MDA-MB-231 cells with inhibitors of miR-200a, miR-200b and miR-429 and assessed the level of functional knockdown of miR200a-b-429 by a reporter assay, in which the complementary sequence of miR200a-b-429 was introduced in the 3' UTR of a luciferase reporter gene. Transfection of the cells with miR200a-b-429 inhibitors resulted in a more than two-fold increase in luciferase activity compared with the negative control inhibitor suggesting that a greater than 50% inhibition of the miR200a-b-429 function had been achieved by the miR200a-b-429 inhibitors (Figure 2F). Inhibition of miR200a-b-429 partially reversed the ERβ1-mediated epithelial phenotype and caused a 50% reduction in the expression of E-cadherin (Figure 2F). These data strengthen the role of ERβ1 in regulating EMT and suggest a mechanism through which the receptor may regulate E-cadherin expression.
ERβ1 inhibits EMT by repressing EGFR signaling
ERβ1 induces degradation of EGFR by enhancing the EGFR-c-Cbl interaction
ERβ1 inhibits invasion of breast cancer cells in vivo
ERβ1 and E-cadherin levels are positively correlated in breast cancers
To better understand the correlation between ERβ1 and E-cadherin, we divided the breast cancer samples into three groups based on ERβ1 levels defined by their expression scores and studied the difference of E-cadherin expression among the three groups. The expression of E-cadherin in each group was represented by its median score (Figure 6C) or positive percentage (Figure 6D). As shown in Figure 6C, the median scores of E-cadherin in tumors expressing low levels of ERβ1 were significantly smaller than in those with higher ERβ1 levels. Similarly, the positive percentage analysis for E-cadherin showed a positive correlation with ERβ1 levels (Figure 6D). These results are consistent with our findings that ERβ1 up-regulates E-cadherin in breast cancer cell lines.
Although basal-like breast cancers in general are associated with relatively poor prognosis, they are heterogeneous, including diverse subgroups in terms of chemotherapy response and risk of developing distant metastases [2, 6, 7]. Interestingly, ERβ1 positivity has recently been associated with better survival in triple-negative cancers that were treated with tamoxifen and inversely correlated with the expression of EGFR, an important marker in the immunohistochemical identification of basal-like cancers [24, 25, 36].
One process that has been attributed to primary tumor metastasis is EMT. Here we examined whether ERβ1 through regulating EMT can influence invasion and metastasis in basal-like cancers. ERβ1 repressed the mesenchymal spindle-shaped morphology of the MDA-MB-231 and Hs578T cells and enhanced cell-cell contact. ERβ1 altered the morphology of these cells in the absence of ligand. This is in agreement with our previous data showing increased transcriptional activity following expression of ERβ1 in MDA-MB-231 cells in the absence of ERβ agonists. The increased transcriptional activity in the absence of ligand was correlated with the phosphorylation of ERβ1 at ser-87 [28]. As a result of the changes in the morphology, ERβ1 inhibited migration and reduced the invasiveness of MDA-MB-231 cells. When control and ERβ1-expressing cells were injected into zebrafish embryos, only the control cells disseminated to distant sites suggesting that ERβ1 functions as a crucial anti-invasive factor. Given that expression of EMT markers and cadherin switching have been reported to correlate with the basal-like phenotypes in in vitro model systems and in specimens from patients [7], we examined whether ERβ1 inhibits invasion and migration by regulating EMT in cells with basal characteristics. ERβ1 was found to induce the expression of E-cadherin by inhibiting its transcriptional repressors ZEB1/2 and up-regulating the miR-200a, miR-200b and miR-429, which correlate with the epithelial breast cancer phenotype (Figure 6E).
ERK2 has recently been shown to affect the ZEB1/2 regulatory pathway of E-cadherin expression in human mammary cells [17, 18]. ERK1/2 are activated by diverse pathways including that initiated by EGFR [38]. Overexpression of EGFR promotes migration and invasion of basal cells and its expression correlates with poor survival in basal-like cancers [36, 37]. Since ERβ1 was found to inhibit EMT by down-regulating the ZEB1/2 pathway in basal-like cells, we tested whether repression of EGFR and ERK1/2 signaling are involved in ERβ1-mediated up-regulation of E-cadherin and the subsequent inhibition of cell migration and invasion. Indeed, ERβ1 induced a decrease in EGFR protein levels without altering the transcription of the EGFR gene followed by down-regulation of the phosphorylated ERK1/2 forms. Induction of EGFR signaling in ERβ1-expressing cells through up-regulation of EGFR or treatment of the cells with EGF reversed the ERβ1-dependent epithelial phenotype, suggesting that EGFR is a critical factor in the ERβ1-mediated regulation of EMT.
Given that inhibition of transcription was not involved in ERβ1-mediated down-regulation of EGFR, we examined whether ERβ1 promotes degradation of the tyrosine kinase receptor. EGFR degradation is a complex process that involves ubiquitylation of the activated receptor by the E3 enzyme Cbl and subsequent proteolysis by proteosomal and lysosomal hydrolases [39]. ERβ1 was found to induce ubiquitylation and degradation of EGFR by enhancing the EGFR-c-Cbl association. Ubiquitylation is an important process of a negative regulatory circuit that terminates EGFR signaling by targeting the receptor for degradation [42]. Our data show for first time that ERβ1, by inducing these negative feedback pathways, is likely to exert a role of EGFR inhibitor and tumor suppressor function.
Interestingly, it has recently been shown that ERβ decreases the expression of insulin-like growth factor II-mRNA binding protein 3 (IMP-3) by repressing EGFR transcription in MDA-MB-231 cells [43]. In our study, the transcription of EGFR was not altered when ERβ1 was expressed or knocked down in MDA-MB-231 and Hs578T basal-like cells. Instead, as mentioned above, ERβ1 promotes degradation of EGFR by inducing its ubiquitylation in both MDA-MB-231 and Hs578T cells.
By examining 208 clinical breast cancer specimens, we found that the expression of ERβ1 was significantly associated with the expression of E-cadherin. This correlation has not previously been reported. However, since the discovery of ERβ, it has been shown that the association of ERβ to other clinicopathological indicators is likely to be divergent in different breast cancer cohorts analyzed by IHC using different ERβ antibodies. The tumor cohort examined in our study included a different number of HER2-positive (14.5%) and probably triple-negative breast cancers compared with the cohorts utilized in some of the recent studies that examined large number of samples with well-validated antibodies (HER2-positive, Honma et al. 2008: 5.25% and Novelli et al. 2008: 31.9%) [24, 25, 44, 45]. Such differences in the characteristics of the clinical cancer samples as well as differences in the specificity of the ERβ antibodies used in these studies, may explain why the correlation between ERβ1 and E-cadherin expression has not been previously observed. This positive ERβ1-E-cadherin association is consistent with the ERβ1-mediated up-regulation of E-cadherin observed in breast cancer cells. It is possible that there are some limitations in the relevance of these results since the level of ERβ1-expression achieved in our cells may not reflect the levels of expression seen in clinical samples. Despite these limitations, taken together, our results propose a role for ERβ1 in up-regulating E-cadherin in breast cancer cells. This suggests that the low ERβ1 levels may be the primary cause of low E-cadherin expression and induction of EMT in some breast cancers. Since EMT correlates with a group of basal-like breast cancers that often develop metastases in distant sites [7], ERβ1 may play a crucial role in repressing invasive behavior and inhibiting metastasis in this subset of breast cancers. Our data show that ERβ1 impedes EMT and influences invasion by down-regulating EGFR, which is expressed in basal-like cancers. These results strengthen the possibility that ERβ1 can help to identify patients with basal-like cancer with lower risk to develop metastasis.
Basal-like breast cancers that show unfavorable prognosis and often develop distant metastases are associated with EMT. Our findings indicate that ERβ1 inhibits EMT and reduces the invasiveness of basal-like breast cancer cells by up-regulating the epithelial marker E-cadherin. ERβ1 induces the expression of E-cadherin by down-regulating EGFR, an oncogenic factor that is expressed in basal-like cancers. ERβ1 was found to terminate EGFR signaling by targeting the receptor for degradation. Our data support the notion that ERβ1 can serve as a clinical marker to identify patients with basal-like cancer that have lower risk to develop metastasis.
bovine serum albumin
dextran-coated charcoal
ductal carcinoma in situ
- dpi:
days post-injection
epidermal growth factor receptor
epithelial to mesenchymal transition
- ERα:
estrogen receptor α
- ERβ:
estrogen receptor β
- ERKs:
extracellular-signal-regulated kinases
fetal calf serum
green fluorescent protein
- hpf:
hours post-fertilization
Institutional Animal Care and Use Committee
insulin-like growth factor II mRNA binding protein 3
phosphate-buffered saline
room temperature
reverse transcription polymerase chain reaction
- TGF-β:
transforming growth factor β
We thank Igor Bado, Anders Strom, Cecilia Williams, Ivan Nalvarte, Luisa A. Helguero and Ryan Butler for technical help, and Sang-Hyuk Chung and Chiara Gabbi for help with statistical analysis. This paper was supported by grants from The Emerging Technology Fund of Texas, CPRIT, The Welch Foundation (E-0004) and The Swedish Cancer Society.
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text:
For a while back then, there was a chance that Kevin Pietersen would become a great cricketer. The time has gone. As he prepared to leave England's World Cup campaign yesterday, 48 hours after it was announced that he would struggle through the tournament, the overwhelming feeling was of relief.
His team-mates did not exactly club together for the plane fare home but nor did they accompany him to the airport singing We'll Meet Again. The decision was made to replace Pietersen after he informed the team management that increasing pain caused by a hernia made it impossible for him to continue.
Although the medical advisers were firm in their view that the condition could be managed during the World Cup, they could hardly ignore the player's own assessment. Eoin Morgan, the team's most proficient one-day batsman (a mantle once worn by Pietersen), will rejoin the team in Chittagong today.
Morgan was omitted from the original squad after breaking a finger during the one-day series in Australia in January. Initially, it was expected he would require surgery but Morgan has recovered much quicker than expected and has recently been batting in the nets at Middlesex.
There was a feeling as the team travelled to Bangladesh yesterday that Pietersen might have given the injury a few days to settle down given what is at stake. England have only two matches in the next 10 days.
The player exchange was probably welcomed in the dressing room. Pietersen is simply not the player he was in the one-day game and has gone 27 innings without a hundred. Morgan was one of the most complete finishers of a one-day innings and England have missed him here.
Despite his lack of recent cricket he will almost certainly be drafted into the team for the match against Bangladesh on Friday. Victory will confirm England's quarter-final place after their gripping six-run triumph against South Africa in Chennai on Sunday.
Pietersen's career has dealt him some poor hands since he was deposed from the captaincy in January 2009. He missed most of that year's Ashes with a severe Achilles tendon injury which took its time to heal after complications set in. At no time since has he been quite the swashbuckler of his early days and last summer he was dropped from the one-day squad for the series against Pakistan.
It was typical of him that he should court controversy by revealing his displeasure to the world via Twitter. The feeling is that he does not set out to cause offence or disturb the carefully constructed equilibrium but simply does not think what the possible repercussions might be.
Pietersen is never less than courteous. He was propelled into the stratosphere after his imperishable, blazing century against Australia at The Oval in 2005 when the Ashes were won after an 18-year gap. It was a place he liked being.
The loss of the captaincy undoubtedly hurt him deeply and it was to his credit that he returned to the ranks and continued to wear his England heart on his South African sleeve. A glittering double century in the second Test at Adelaide on England's recent Ashes tour did not have much else to accompany it.
He has remained England's highest profile cricketer but has never been able to shed the perception that he is also high-maintenance. This is easier to accommodate for all concerned when you are scoring most of the side's runs and in a fashion that empties bars but when you have come back to the pack it is more difficult to tolerate. Alone among most of the squad, he has no close pals in it.
Pietersen's departure will only enhance the theory that he will soon retire from one-day cricket to concentrate on Test cricket and Twenty20 to ensure he can take part in the lucrative Indian Premier League. It is not a combination that will sit well in the committee rooms of the ECB where there are already rumblings that his central contract should not be renewed later this year.
Andy Flower, the team coach, fell some way short yesterday of giving Pietersen a ringing endorsement but he said of Pietersen's place in England's future one-day plans: "It might fuel the speculation, but Kevin's told me he wants to carry on playing one-day international cricket and I'll take him at his word."
The pity is that Pietersen looked determined to play his part as he had in the World Twenty20 last year when he was player of the tournament. Briefly, the sunny days were back. England revealed Pietersen's hernia injury only on Saturday night, stipulating that it could be managed until surgery after the World Cup.
The injury was diagnosed after he returned from Australia. Having kept it within house for so long, it seemed odd that they should put it in the public domain. Pietersen was relatively upbeat although the recuperation period would mean his missing the IPL for which the Deccan Chargers were paying him $650,000 (£418,000).
"Representing England in a World Cup is something I've looked forward to for the last four years and I'm excited by the prospect of helping the team achieve something special here against the best teams in the world," he said on Saturday night. "I'm very disappointed to have picked up this hernia injury but I've been able to manage it so far throughout the World Cup and will continue to do so under the guidance of the England team's medical staff."
After the tense six-run win against South Africa on Sunday in Chennai when he was out for two but bowled eight crucial overs of serviceable off-spin, Pietersen told management he felt it was unmanageable after all. Flower said: "The medical advice was that it wouldn't get significantly worse and he was at no risk of damaging himself further, therefore it was a pain-management situation. Kevin says it has got worse, pain-wise, and he can't carry on playing like this. So it was a pretty simple decision to replace him.
"I empathise with anyone with a serious injury. You never know exactly what they're feeling, so you've got to listen to what they're saying. Of course we're disappointed to lose him. He's a world-class player."
England have to move on quickly. While it will be fascinating to see if Pietersen dare fulfil his IPL contract now after the six-week recovery period (surely not?) they have a World Cup to get on with. Pietersen's departure means they must find yet another opening partner for Strauss.
Pietersen was drafted into the role only after Matt Prior, who had replaced the original candidate, Steven Davies, failed to make any sort of impression. The options are that England return again to Prior, or move Ian Bell or Ravi Bopara up the order. Both would be missed in the middle order. Flower made it clear that he is prepared to continue changing the order.
"We've got a number of potential openers," said Flower. "We're not going to let you or the opposition know who it's going to be, but we've got a clear idea of who we want for the game against Bangladesh. I don't think it does have to be the same for the rest of the tournament because opening against Bangladesh requires a different set of skills from opening against Australia. I think in this tournament we're going to have to be flexible, and if that means shuffling the order again we'll do that."
Pietersen might just be yesterday's man in many senses and he will have a long retirement to ponder his withdrawal from the fray yesterday.
England's exchange: how the two batsmen compare
Kevin Pietersen
Eoin Morgan
Strike rate
116 v South Africa
High score
115 v Canada
South Africa
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Relationship Manager, Bank at Work - Front Range (North Denver, Boulder, Cheyenne)
United States-Colorado-Boulder
What sets Bank of the West apart from other banks is our team members–they embody the optimistic spirit of the West. There is a spirit here that drives us to do more. Our team of more than 10,000 employees is vital to the success of our Bank. They reflect our modern western values—straightforward, entrepreneurial and optimistic. We seek to create a corporate culture that fosters and rewards excellence, encourages creative thinking and respects diversity – an environment where team members are engaged, supportive of one another and enthusiastic about serving our customers. Bank of the West offers the stability of a company that has a 135 year history and is part of BNP Paribas, a European leader in global banking and financial services and one of the 6 strongest banks in the world. We offer opportunities across our diverse business lines – Retail Banking, Commercial Banking, National Finance, and Wealth Management.
Position Summary
Responsible for focusing on new consumer relationship acquisition. The Bank at Work Relationship Manager is responsible for Bank at Work program implementation, effectiveness and sales results within an assigned territory. Sources Bank at Work companies through prospecting/cold calling, branch and business partner referrals, centers of influence and personal network. Conducts sales presentations, follows up to enroll companies, and schedules onsite account opening sessions and financial education events at enrolled companies. Works closely with branches to achieve consumer acquisition/account opening, campaign, cross-sell and other related goals. Provides training and sales coaching. As the BAW subject matter expert, conduct program training and provide coaching for assigned branches.
Position Accountabilities
1. Prospect and sell BAW program to sourced companies within assigned territory.
2. Coordinate with enrolled companies and local branches to schedule consumer account opening events (a/k/a "onsites") and employers place of business.
3. Conduct financial education seminars and activites with enrolled participants and prospects.
4. Develop and manage a pipeline of BAW company prospects through business development efforts (prospecting, cold calling, referrals, business partners, centers of influence, personal network).
5. Provide training and coaching to assigned branches on BAW program, sales effectiveness/techniques, and presentation skills.
6. Provide ongoing relationship management to maximize sales of bank products and services to employees with enrolled companies.
7. Develop and implement sales plans; utilize BAW and Bank marketing and collateral materials effectively; monitor BAW results reporting and tracking.
8. Provide input and feedback to improve BAW program; may conduct competitive shops, research and gather intelligence about similar programs at other financial institutions.
9. Manage expenses and travel costs effectively.
10. Complete all assigned training in a timely manner (by required completion/due date)
11. Participate in Division, Region, Area or Branch Meetings as required.
12. Performs other duties as assigned.
Required Education or Equivalent Experience
· Bachelor's Degree in Business, Marketing, Finance or similar
Required Experience
· 3 Years of functional/professional experience
· 2 Years of supervisory or managerial experience
· 5 years combination of college education, financial services experience, management or selling to individuals and businesses.
Field of Experience
Bank of the West is an Equal Opportunity employer and proud to provide equal employment opportunity to all job seekers without regard to any status protected by applicable law. Bank of the West is also an Affirmative Action employer - Minority / Female / Disabled / Veteran.
Bank of the West will consider for employment qualified applicants with criminal histories pursuant to the San Francisco Fair Chance Ordinance subject to the requirements of all state and federal laws and regulations.


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Lot 74: (3) Larger Goebel Germany Hummel Figurines
Palm Beach Modern & Antique Estate Clearance Auction
by MG Neely Auction
November 13, 2016
Lantana, FL, USALive Auction
Looking for the realized and estimated price?
Description: (3) Larger Goebel W. Germany porcelain Hummel figurines. Excellent condition. Measure 7" - 7.5" tall, each.
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Nicholas Gonzalez
He showed up on The OC looking like this and wearing that (nothing!) and suddenly we had a very big interest in gardening. What was Marisa (Mischa Barton) thinking when she chose Ryan (Ben McKenzie) over him?
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Learn how to make your own liqueur from scratch with Luscious Liqueurs by A. J. Rathbun, including recipes like Persephonia; Limoncello; Basil Grappa; and Simple Syrup.
Makes about 1-3/4 pints
Most won't jump right into the idea of infusing grappa because most, or at least many, are a tad scared of grappa. I can understand this, having been nervous the first time I tried it (and I like bitter-and biting-drinks on occasion). I was even more nervous the second time, because my first grappa had a bit of the taste of gasoline. But the second time it was more what I've come to expect with grappa: a serious nature and rewarding flavor. If you haven't come around yet to enjoying grappa straight, this softer version may be right up your after-dinner-drink alley.
1. Put the basil and lemon juice in a glass container with a tight-fitting lid. Using a muddler or wooden spoon, muddle the basil leaves and lemon juice.
2. Add the grappa, stir, and seal. Place the container in a cool, dry spot away from sunlight. Let sit for 1 week, swirling occasionally.
3. Add the simple syrup, stir, and reseal. Return to its spot. Let sit for 2 more weeks, swirling occasionally.
4. Strain the liqueur through a double layer of cheesecloth into a pitcher or other easypouring vessel. Strain again through 2 new layers of cheesecloth into 1 large bottle or a number of small bottles or jars.
A Serving Suggestion: Serve Basil Grappa chilled or at room temperature in small glasses; it's especially good for alleviating that over-full feeling when you've had a couple bites too many.
Grappa is a fragrant grape-based pomace brandy of between 50% and 80% alcohol by volume (100 to 160 proof), of Italian origin. See the full article on grappa on Wikipedia.
Luscious Liqueurs
50 Recipes for Sublime and Spirited Infusions to Sip and Savor
by A. J. Rathbun
Harvard Common Press, 2008
Hardcover, $12.95
Information provided by the publisher.
This page created 2008
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text: Posted December 20, 2012 · Report post Source has it that the New York Jets are looking to trade Sanchez, and release Tebow. Okay. After watching the last few games of the Jets -- my question is -- is what team is honestly going to want to trade for Sanchez? For Tebow it seems more like he is just unhappy with his role in New York, and wants out. Once again, I honestly question what other team will possibly want him. And if this all holds true, what made them wait so long? Is this more of a strategy for Rex to help save his job as he may feel on the "[i]hot seat[/i]"? [url="http://espn.go.com/new-york/nfl/story/_/id/8763411/new-york-jets-shop-mark-sanchez-part-ways-tim-tebow-according-sources"]http://espn.go.com/n...cording-sources[/url] 0 Share this post Link to post Share on other sites


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Posts: 244
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StRiKeR said:
NOTE: Would anybody be interested in composing a MIDI song for my DOOM level?
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ShadowRunner said:
I would suggest ralphis,But hes an asshole.
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ShadowRunner said:
ralphis is the biggest asshole ive seen around here bad I mean Blackfish.
StRiKeR said:
Wow, off-topic guys. I been to /idgames. Nothing stands out. Oh well, guess I'll go back to playing HR.
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When I decided to go natural, I was not 100% sure of my decision. I wasn't sure what my natural hair looked like — I'd been getting relaxers since elementary school — and I wasn't sure if I had the skills to maintain my it. However, I decided to go natural was because my hair obviously thinning.
It needed all the help it could get.
That said, when I finally made the decision to go natural, the one thing I knew for sure was that I was not getting the BIG CHOP. (This is when a person cuts off their hair to return to its natural state, cutting out perms, relaxers, and hair dye.) I have seen the BIG CHOP look absolutely gorgeous on some people. And then again, I have not liked it on others. And for me personally, I could not imagine myself with a little Afro. Plus, I did not know what my hair texture was really like…there were too many unknowns.
So I decided to just grow my hair out, and while I was doing it, I avoided the hairdresser for a few months. I did this because I found a few hairdressers to be very discouraging. I had contemplated going natural several times before only to have my hairdressers discourage me from doing it. I went in to the beauty shop to talk to them about going natural and left with a relaxer in my hair. So, while I was in this delicate and not-so-confident transitional state, I decided to avoid the hairdressers.
Unfortunately, I later found out this was not such a great idea.
You see, when I finally went to the hairdresser to get my hair trimmed, I was informed that my hair was badly damaged. The permed hair was breaking off from the significant amount of new growth that I had, and the hairdresser told me "I can't do anything with this." Then she tried to discourage me from going natural.
She told me that going natural was not for everyone, and she was frowning up — giving me the stank face — as she was talking. But I remained strong in my decision to continue on this natural path that I started.
So she washed, deep-conditioned, cut, flat-ironed and styled my hair into a really cute and really short spiked style. I walked out of there feeling cute and confident that I was now on the right path with my hair.
About 3 days later — when that humidity from the summer's heat and the steam from my showers got over to my head — I realized that I had just gotten THE BIG CHOP!
So here are a few lessons that I learned while trying to avoid THE BIG CHOP:
1. Avoiding the hairdresser may not be such a good idea.
This is especially when you are transitioning as you need to make sure you are still trimming your ends and conditioning your hair properly. If you know how to do that yourself, cool; but if you don't, get help from the professionals.
2. Find a hairdresser that works with natural hair.
If your hairdresser does not think highly of natural hair, then you need to find someone that does. because you need someone that will give you great advice and will help to keep your hair healthy.
3. Don't be afraid of the "Big Chop."
Or at least don't be afraid of a trim every now and then. I have heard people say that clipping your ends is not necessary, but for me, it keeps my hair healthy. And it grew super fast after my big chop, a lot faster than it was growing when I was just trying to grow it out without cutting it. But that is my experience.


text:
In a medium sized mixing bowl place the minced beef and season with sliced green onions, chopped parsley, salt, ground pepper and egg and mix well. Form the mixture in a ball shape and set aside.
Heat a non-stick frying pan, melt the butter and saute the onion rings, add the meatballs and stir very gently, add mixed mushrooms and cover for 2min, dissolve mushroom powder in water and add to the meatballs. Bring to a boil over a low heat and simmer until sauce thickens and mushrooms are cooked.
For more information please visit: http://www.cookingwithpeachy.com/
This video is a creation of Peachy24. You can visit cookingwithpeachy for complete recipes, and more videos.
Serving size: Complete recipe
Calories 1881 Calories from Fat 1214
% Daily Value*
Total Fat 136 g209.2%
Saturated Fat 64.8 g324.1%
Trans Fat 5.3 g
Cholesterol 753.2 mg251.1%
Sodium 3355.7 mg139.8%
Total Carbohydrates 18 g6.1%
Dietary Fiber 5.6 g22.6%
Sugars 7.8 g
Protein 154 g308.6%
Vitamin A 47.3%
Vitamin C 38.6%
Calcium 18.4%
Iron 99.5%
*Based on a 2000 Calorie diet


text:
Basic differentiation challenge
Review your understanding of basic differentiation rules with some challenge problems.
Review: Basic differentiation
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1789 in Canada
From Wikipedia, the free encyclopedia
|This article does not cite any sources. (December 2009) (Learn how and when to remove this template message)
|Years in Canada:
|1786 1787 1788 1789 1790 1791 1792
|17th century · 18th century · 19th century
|1750s 1760s 1770s 1780s 1790s 1800s 1810s
|1786 1787 1788 1789 1790 1791 1792
|Part of a series on the
|History of Canada
|Year list / Timeline
Events from the year 1789 in Canada.
- 1789-93 - Alexander Mackenzie of Canada, seeking northern river route to the Pacific, travels to the Arctic Ocean; on second journey he crosses continent by land, making contact with many tribes.
- Alexander Mackenzie journeys to the Beaufort Sea, following what would later be named the Mackenzie River.
- French Revolution begins
- David Thompson learns surveying from Philip Turnor.
- Lord Grenville proposes that lands in Upper Canada be held in free and common soccage, and that the tenure of Lower Canadian lands be optional with the inhabitants.
- November 13 - Denis-Benjamin Papineau, joint premier of the Province of Canada (died 1854)
|This section is empty. You can help by adding to it. (December 2010)


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Ginger Girl Makeover
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Browse the categories by clicking the hearts next to the category names. Apply make-up by clicking the color you want and then clicking the right spot to apply it. Try on the clothes by clicking them.
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Please visit our Hours of Operation page for detailed Spring hours.
All visitors must have a parking permit when parking on campus. Permits must be displayed in order to avoid a citation. Campus Recreation is not responsible for any citations given by University Police. Guest parking permits can be obtained by contacting UPD.
Attention: Please check out the reservations page for updated information on MRC reservation policies. We will no longer accept old versions of our reservation forms. All requests must be turned in a minimum of 21 days in advance. Contact firstname.lastname@example.com for more information.
The Campus Recreation Advisory Board is a new opportunity for students, faculty/staff, other university leaders, and community members to serve in
an advisory capacity to the Director and staff of Campus Recreation. Its purpose is to involve and provide an opportunity for members to provide feedback in the planning, organization, and administration of recreation programs, services, and facility operations. Please visit the Our Mission page for more details.
Due to University policy, a fire permit must be obtained by the Department of Safety prior to igniting one of the grills at the MRC or Cain. Please contact the Department of Safety at 903-468-3091 or visit them during regular business hours at Ferguson Social Science #137 for more details.
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Field trimmed to 15 at Miss America Pageant
LAS VEGAS (AP) -- Judges, the public and contestants themselves have trimmed the field of contestants at the Miss America Pageant to its 15 finalists.
The public voted in Miss Oklahoma Taylor Treat, Miss Arkansas Sarah Slocum and Miss Kentucky Mallory Ervin.
The judges picked Miss Tennessee Stefanie Wittler, Miss Colorado Katie Layman, Miss California Kristy Cavinder, Miss Indiana Nicole Pollard, Miss Hawaii Raeceen Woolford, Miss Louisiana Katherine Putnam, Miss District of Columbia Jennifer Corey, Miss New Mexico Nicole Miner, Miss Virginia Caressa Cameron, Miss Texas Kristen Blair and Miss Nebraska Brittany Jeffers.
The contestants themselves then voted in Miss Oregon CC Barber.
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- Feb. 20, 2017 Kris Jenner opens up about her future with Corey Gamble


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Guardian Media Group today reported major losses of almost £90m as it complained that its national newspapers and websites were operating in the "toughest trading conditions seen for many years". The group's regional newspapers suffered plunging profits while the radio business made a significant loss.
In its annual report for 2008-2009, GMG, which publishes The Guardian and The Observer newspapers, reported a pre-tax loss of £89.8m, compared with a profit last year of £306.4m, on turnover which was down to £405.4m compared with £502.1m.
Guardian News & Media, the group's national newspaper division, reported an increase in operating losses to £36.8m, compared to £26.4m in 2008, on a reduced turnover of £253.6m compared with £261.9m.
Despite the losses, the GNM editor-in-chief Alan Rusbridger received an 11 per cent rise in salary to £445,000 for the year to 29 March. Rusbridger said in April that he would take a 10 per cent pay cut this year.
The GMG chief executive, Carolyn McCall, took a 39.7 per cent pay cut last year, receiving a total package of £498,000. Her salary was £472,000 and her benefits totalled £26,000.The previous year she received a total of £827,000, including a salary of £424,000 and bonus of £385,000.
Yesterday McCall said: "The sharp decline in the advertising market had an impact on each of the wholly owned businesses. All are reducing costs to a more sustainable level in this harsh new environment."
GMG's 2008 profit figures were inflated by the one-off proceeds from selling a 49.9 per cent of Trader Media Group, publishers of Auto Trader. That joint venture, in which GMG retains a 50.1 per cent share, is the group's most profitable arm with an operating profit of £110.8m (compared with £119.9m in 2008) on turnover of £296m (£309.9m).
The local newspaper division, GMG Regional Media, which includes the Manchester Evening News, saw operating profit down to £0.5m (from £14.3m in 2008) on turnover down to £94.5m (from £120.5m). GMG described the current market as "the most challenging in the history of the local and regional press". GMG Radio, which includes stations such as Smooth and Real Radio, posted an operating loss of £6.6m (compared with £0.1m profit in 2008) on turnover of £46.6m (£48.8m).
- More about:
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Walgreen (WAG) and Carnival Corporation (NYSE:CCL) were the two major large-cap losers on Tuesday. Here are some of the specifics about these stocks and what to expect from them going forward.
Walgreen fell 6.6% on Tuesday. Walgreen reported better than expected Q2 results; however, gross margins fell short of what many analysts had estimated. I believe the gross margin miss was more due to a mismatch in analyst expectations than a fundamental business issue. After two consecutive quarters with 80 to 90 basis points of Y-o-Y FIFO gross margin improvement, some analysts and investors were expecting stronger results. However, Walgreen had significantly tougher comps in the February quarter, which led to the miss. Investors were also concerned about how inflation is affecting Walgreen's front-end margins. However, one should note that drugstores arguably have the most pricing power in retail and are usually least affected by inflation.
The long-term trend is still headed in the right direction, as the company continues to deliver on its $1B cost-cutting program, which along with fewer store openings and the record 2012 generic wave should help gross profit dollar growth exceed SG&A growth, driving margin expansion over the next few years. After yesterday's correction, the stock is trading at ~13x consensus FY12 EPS of $3.01 as compared to its historical average of 15x-17x. Investors should initiate a long position on this dip.
Carnival Corporation fell 4.5% on Tuesday. CCL reported 1Q EPS of $0.19, in line with previously announced guidance. CCL modestly raised 2011 EPS guidance to $2.55-2.65 (vs. previously pre-announced guidance of $2.50-2.60 and the consensus estimate of $2.77) due to changes in spot fuel prices and currency.
While CCL's bookings and prices appear to be going in the right direction, there had been some weakness in the southern European-sourced brands Costa and Iberocruises. This weakness can mainly be attributed to the recent unrest in the MENA region. Although CCL management has taken the proper steps to mitigate the financial impact from the MENA region, I remain concerned about the MENA turmoil and higher energy prices. I will wait for some more clarity on these issues before becoming positive on the stock.
Disclosure: I have no positions in any stocks mentioned, and no plans to initiate any positions within the next 72 hours.


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Editors' Note: Clicking on the Download Now (Visit Site) button above will open a connection to a third-party site. Download.com cannot completely ensure the security of the software hosted on third-party sites.
Publisher's Description+
What's new in this version:
Version 184.108.40.20614: Fixed Toolbar position problem on HiDPI; Improved M4A support; Improved Gapless.