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replySubmitted by Guest teacher (not verified) on Tue, 01/10/2012 - 12:29am.
Teachers have always had the choice to join and pay dues, or to not join NCAE. The decision that was made is a part of an ongoing effort the Republicans have in their agenda to dismismantle NCAE. I do not understand why they are so intent to destroy the education system in NC. Taking away the rights of teachers to join an organization that will allow us to pay with a payroll deduction, but allowing other professions to do exactly the same thing is just plain unfair targeting of teachers. This needs to stop! When are they going to realize that they are supposed to represent teachers as well as the rest of the people in NC?? »
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Structured Health Improvements through Nutrition and Exercise is a collection of 12 projects designed to make choosing a healthy lifestyle even easier in the Royal Borough.
Walking is just one of the many SHINE programmes operated by the Royal Borough giving access to some of the picturesque surroundings of the Windsor, Maidenhead and Ascot area.
Take your first steps - choose of the the following SHINE walks packages.
Did you know walking

subset: commoncrawl


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Oregon police arrest man for arson after he bragged about the blaze to his hairdresser
SALEM, Ore. - Salem police said one of the two men accused of setting a fire to an old training center bragged about the blaze to his hairdresser. According to court documents, a 20-year-old man told his hairdresser that he went into an old building on the former Fairview Training Center campus to smoke marijuana, then set the Jan. 27 blaze that destroyed the building.
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CLICK HERE for a full detailed PDF resume.
Hulisani Cecilia Ravele is one of the most recognised youth personalities in the country. Having joined the entertainment industry at the tender age of 9, CC (as she is fondly known to fans across the country) has grown to be a favourite face of the South African television industry. Now at the age of 21, she is currently a presenter on SABC 1's Quiz me and a graduate with a B.Comm Marketing Management degree from University of Johannesburg (Auckland Park Kingsway campus). Those that know and love Hulisani can attest to her fun-loving, outgoing and bubbly personality that goes everywhere with her.
Born and raised in Chiawela, Soweto, Hulisani grew up loving the stage and a natural-born entertainer. After joining the Professional Kids Casting Agency in 1997, she went for her first audition just 2 weeks after she had completed her training and clinched it. It was here where she appeared as 'Sabrina' on the Yotv kiddies' programme 'Pula and Friends'. This continued until the show came to an end in 1999. During this period from 1997-1999, Hulisani also appeared in adverts for Jet and Sales House.
Hulisani's career took off when she joined the Yotv family in June of 2000. In the 8 years that she has been there she has excelled in presenting a wide variety of shows which include a talk show, environmental show, quiz show and game shows. The shows have both been live and prerecorded and Hulisani executes both genres with style and panache.
Some of the countries biggest youth shows have had enjoyed Hulisani's talent as a contributing factor to their success. These shows include the widely popular Yotv's Wildroom (a live interactive show) and Yotv's Big Breakfast (a live weekend sports show).
For bookings and more information on Hulisani please get in contact with an agent from Owen S Management on 011 021 9882.


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Sean P. Colgan1, Richard S. Pitman2, Takashi Nagaishi2, Atsushi Mizoguchi3, Emiko Mizoguchi3, Lloyd F. Mayer4, Ling Shao4, R. Balfour Sartor5, John R. Subjeck6 and Richard S. Blumberg2
1Center for Experimental Therapeutics, and2 Division of Gastroenterology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA3 Immunopathology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA4 Immunobiology Center, Mount Sinai School of Medicine, New York, New York, USA5 University of North Carolina, Chapel Hill, North Carolina, USA6 Department of Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, New York, USA
Address correspondence to: Richard S. Blumberg, Division of Gastroenterology, Brigham and Women's Hospital, Thorn Building, Room 1410, 75 Francis Street, Boston, Massachusetts 02115, USA. Phone: (617) 732-6912; Fax: (617) 264-5185; E-mail: firstname.lastname@example.com.
Published September 1, 2003Submitted: October 24, 2002; Accepted: June 3, 2003.
CD1d is expressed on the surface of professional and nonprofessional APCs, including intestinal epithelial cells (IECs), for a role in the presentation of glycolipid-based antigens to subsets of T cells. The mechanisms that regulate CD1d expression in any cell type are unknown. To investigate the possibility that expression of CD1d is influenced by exogenous factors present within the intestinal lumen, CD1d expression was analyzed in several IEC lines after culturing in the presence of lumenal contents (LC) of the normal human intestine. Exposure of the colon-derived cell lines T84, HT-29, and Caco-2 to soluble LC resulted in a marked induction of CD1d expression as determined by RT-PCR, confocal microscopy, cell surface ELISA, and Western blot analysis. Similarly, exposure of human IECs to LC isolated from mice bred in both specific pathogen–free and germfree conditions also resulted in the induction of CD1d expression, with the maximum CD1d-inducing activity observed in the small intestine. Biochemical and biophysical characterization of the human CD1d-inducing activity identified heat shock protein 110 (Hsp110) as a major functional component of the LC that contributes to CD1d surface regulation, and immunolocalization studies revealed Hsp110 expression in subsets of human IECs in vivo. These data support the presence of a novel autocrine pathway of CD1d regulation by Hsp110.
See the related Commentary beginning on page 646.
CD1d belongs to the CD1 family of proteins that share structural and functional homology to the MHC class I and class II molecules (1, 2). CD1d is expressed by a majority of thymocytes, resting B cells, DCs, and monocytes (1, 3). In addition, CD1d expression is observed in a variety of epithelial cell types, including hepatocytes and intestinal epithelial cells (IECs) (4, 5). Although its role has not been clearly defined in epithelia, CD1d is known to associate with glycolipid antigens and present them to subsets of CD1d-restricted T cells bearing NK cell markers (NK-T cells) (2). Activation of CD1d-restricted cells in vivo by association of CD1d with a model lipid antigen, α-galactosylceramide, regulates colitis in a chemically induced disease model (6, 7). In these studies on colitis, α-galactosylceramide was observed to localize to the surface of the intestinal epithelium, raising the possibility that CD1d was functional in this site in vivo. Moreover, intestinal epithelial CD1d can signal the production of IL-10, which, through autocrine pathways, can protect epithelia from the deleterious influence of cytokines such as IFN-γ (8). Such data have implicated CD1d as a molecule capable of facilitating mechanisms that regulate mucosal inflammation and highlight the CD1d-bearing IEC as a candidate cell type in this immune regulation.
Immunostaining and biochemical analysis of tissues from the human small and large intestine have revealed the presence of CD1d on both the apical and the basolateral membranes of IECs (9). In addition, CD1d is observed intracellularly within subapical regions in these cells. Relative to the levels of CD1d observed in freshly isolated tissues, epithelium-derived cell lines, such as T84 cells, express low levels of CD1d. The reasons for such differences remain unclear. In these studies, we hypothesized that extracellular components derived from the intestinal milieu exist that are capable of influencing the levels of CD1d expressed by IECs. The lack of such components under in vitro culture conditions could explain, therefore, the relatively low level of CD1d expression observed in model IEC lines. However, other than the cytokine IFN-γ, essentially nothing is known about what factors regulate CD1d expression on any cell type (10). We now report that incubation of intestinal epithelium–derived cell lines with soluble lumenal components of the human and mouse gastrointestinal tract results in upregulation of CD1d expression. Biochemical and functional characterization of this CD1d-inducing activity from mouse and human samples identified it to be soluble heat shock protein 110 (Hsp110). The data presented from these studies predict a novel function for Hsp110, indicate the ability of lumenal components to influence cell surface molecule expression on IECs, and provide important insight into the mechanisms of CD1d regulation in vivo and in vitro.
Antibodies. Two antibodies specific for human CD1d were used in these studies. D5 is a mouse mAb of the IgG2a isotype. This antibody was raised against a glutathione-S-transferase-CD1d fusion protein containing the α1–α3 domains of CD1d (kindly provided by Steven Balk, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA; ref. 11). The mAb 51.1.3 (mouse IgG2b) was raised against an Fc-fusion protein of CD1d (kindly provided by Steven Porcelli, Albert Einstein Medical School, New York, New York, USA; ref. 12). P2A4 is a mouse anti–human ICAM-1 mAb and was obtained from the Developmental Studies Hybridoma Bank (Iowa City, Iowa, USA). L243 is a mouse mAb specific to human MHC class II (American Type Culture Collection, Rockville, Maryland, USA). Polyclonal anti-Hsp110 antibodies were previously characterized (13).
In vitro cell culture. The colon-derived human epithelial cell lines T84, HT-29, and Caco-2 were grown in a 1:1 mixture of Dulbecco/Vogt modified Eagle's medium and Ham's F-12 medium, supplemented with 15 mM HEPES buffer (pH 7.5), 14 mM NaHCO3, 40 mg/ml penicillin, 8 mg/ml ampicillin, 90 mg/ml streptomycin, and 6% FCS. Cultures were maintained at 37°C in a humidified incubator containing 5% CO2. Cells were passaged every 7–14 days with 0.1% trypsin and 0.9 mM EDTA in calcium- and magnesium-free PBS. Where indicated, human microvascular endothelial cells were used as described previously (14).
Exposure of epithelial cells to lumen-derived factors. Confluent monolayers were incubated for varying periods of time with preparations of lumen-derived samples or indicated concentrations of purified recombinant Hsp110 (15) on the apical and/or basolateral surface, as indicated. Prior to their addition to cell cultures, all samples were sterilized by passing of their contents through a 0.22-μm sterile filter. The protein concentration of each sample was also assessed by use of the Bio-Rad protein assay reagent in accordance with the manufacturer's instructions (Bio-Rad Laboratories Inc., Hercules, California, USA).
Preparative methods for purification and characterization of lumenal contents. Six volunteers donated approximately 40 g of human feces each. Individual samples were resuspended in 50 ml of PBS. Insoluble particulate matter was sedimented by centrifugation at 13,000 g for 10 minutes at room temperature and subsequently discarded. Soluble components represented within the supernatant were further clarified by passage through a 0.22-μm sterile filter. The filtrate was defined as the crude lumenal contents (LC) and was used as a starting point from which to proceed with purification. In order to remove LC factors of less than 10 kDa in molecular weight, crude samples were dialyzed against 100 times their volume of PBS for a period of 4 hours at 4°C. Molecules over 10 kDa were retained within the confinement of the dialysis tube and were used in subsequent stages of the fractionation procedure. Aqueous and hydrophobic factors from LC samples were resolved by chloroform-induced phase separation. Briefly, 1 volume of chloroform was added to an equal volume of LC material. The mixture was vortexed for 10 minutes, and phases were resolved by centrifugation at 20,000 g for 20 minutes at room temperature. The upper phase comprised those molecules that partitioned to the aqueous solution and could be incubated with epithelial cells without the need for buffer exchange. The lower phase, containing hydrophobic factors, was dried and resuspended in PBS prior to subsequent use. To fractionate LC material on the basis of size, samples were passed under pressure through filters of varying molecular weights, ranging from 100 kDa to 10 kDa. Both filtrate and retentate from filtration procedures were applied to epithelial monolayers to assess elevations in CD1d expression.
Peptide microsequencing. In subsets of experiments, partially purified fractions of LC were resolved by SDS-PAGE and stained by the Coomassie method, and the resulting approximately 100-kDa band (∼100 pmol) was extracted and submitted to the Dana-Farber Cancer Institute Peptide Core Facility (Boston, Massachusetts, USA) for trypsin digestion and microsequence analysis, using previously described protocols (16).
Preparation of intestine-derived material from mice. Groups of 6- to 12-week-old female mice of the BALB/c strain, bred under either specific pathogen–free (SPF) or germfree conditions, were used as previously described (17). All mice were sacrificed by asphyxiation in a carbon dioxide–saturated chamber followed by cervical dislocation. Lumenal components were then isolated from the stomach and the small and large intestine. Mucosal scrapings, representing the mucin-rich epithelial mucosa, were also isolated from the small and large intestine. Both mucous material and lumenal material were suspended in PBS (pH 7.4) and tested for their ability to upregulate CD1d on epithelial monolayers.
ELISA. Expression of epithelial cell surface proteins was evaluated by use of a cell surface ELISA, as previously described on 96-well plates (18) or on permeable supports (19). The anti-CD1d antibody 51.1.3 was used at a final concentration of 30 μg/ml in HBSS containing 0.5% BSA. Antibodies specific to ICAM-1 (P2A4) and MHC class II (L243) were used as undiluted hybridoma supernatants. All incubations were performed at 4°C for a period of 2 hours. Control samples were stained with isotype-matched antibodies and the secondary peroxidase conjugate. Data are presented as mean ODs at 405 nm blanked against values obtained from isotype-matched control samples. Variations between samples treated under one condition are represented by SEM.
Analysis of mRNA levels by RT-PCR. Epithelial cells were lysed in Trizol reagent (GIBCO BRL; Life Technologies Inc., Rockville, Maryland, USA), and total cell RNA was purified according to the manufacturer's instructions. RNA samples were then treated with DNase I (GenHunter Corp., Nashville, Tennessee, USA) to remove any contaminating DNA. Reverse transcription was performed using a cDNA synthesis kit from BD Biosciences Clontech (Palo Alto, California, USA). Briefly, 1 μg of total RNA was added to a reaction mixture of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 μM of each dNTP, 20 pmol oligo-(dT)18 primer, 20 IU RNase inhibitor, and 200 IU Moloney murine leukemia virus reverse transcriptase in a final volume of 20 μl. Single-stranded cDNA was synthesized using a Thermocycler (model PTC-200; MJ Research Inc.) set to perform one cycle at 42°C for 60 minutes followed by a second cycle at 94°C for 5 minutes. Amplification of CD1d-encoding transcripts by PCR was performed as described previously (10) using the Platinum Taq High Fidelity enzyme mix containing 1 μM each of the CD1d-specific sense primer (5′-TTATCGAAGCAGCTTCACCAGG-3′) and the antisense primer (5′-GATACCATGTCTCGTCAGCATT-3′). Amplification reactions consisted of a 2-minute denaturation step at 94°C and a 1-minute annealing step at 60°C, followed by repeated cycles of 68°C for 1 minute, 94°C for 45 seconds, and 60°C for 1 minute. At the end of the reaction, a final extension step was performed at 72°C for 10 minutes. Two rounds of amplification were required to detect CD1d-encoding transcripts, as previously described (10). The first round consisted of 25 cycles, and the second round consisted of 15 cycles. Products of PCR were resolved on a 1% agarose gel containing ethidium bromide. A 518-bp fragment corresponding to CD1d transcript expression was visualized under ultraviolet light. To ensure that equal quantities of template had been loaded between samples, the housekeeping gene GAPDH was amplified using 1 μM of the gene-specific primer pair purchased from CLONTECH Laboratories Inc. GAPDH fragments appeared as a 983-bp band when resolved by electrophoresis on a 1% agarose gel.
Immunoprecipitation and Western blot analysis. Following experimental conditions, epithelial monolayers were washed with ice-cold HBSS and lysed in 500 ml of RIPA buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM phenylmethanesulfonyl fluoride, and 1 mg/ml each of pepstatin, aprotinin, and leupeptin. Insoluble material was removed by centrifugation at 13,000 g for 10 minutes at 4°C. Supernatants, normalized for protein concentration, were precleared by rotation with normal mouse serum and protein A–conjugated Sepharose beads. CD1d from cell lysates was precipitated with 20 μg of either D5 or 51.1.3 bound to protein A-Sepharose. Incubations with CD1d-specific antibodies were performed overnight at 4°C. Immunoprecipitates were washed with RIPA buffer, boiled in 1× reducing Laemmli buffer, and resolved by 12% SDS-PAGE. Proteins were transferred to nitrocellulose and blocked overnight in PBS containing 5% nonfat dry milk and 0.05% Tween-20. Nitrocellulose blots were then incubated for 1 hour with D5 antibody (4 μg/ml) followed by species-matched peroxidase-conjugated IgG (1 μg/ml) for 45 minutes. In subsets of experiments, Hsp110 in LC was determined by Western blot using rabbit polyclonal antisera, as previously described (13). HRP detection on nitrocellulose blots was achieved using ECL.
Localization of Hsp110 in human tissue. Cryopreserved human large and small intestinal tissue samples were obtained from the tissue bank of the Brigham and Women's Hospital and Massachusetts General Hospital. Samples were frozen in OCT compound (Bayer, Elkhart, Indiana, USA) and stored at –80°C until use. Four-micrometer-thick frozen sections were air-dried for 20 minutes and stained by the avidin-biotin complex method as previously described (20). Briefly, the sections were fixed in acetone for 10 minutes, air-dried, and incubated with purified rabbit anti–human Hsp110 polyclonal antibody at 1:100 dilution for 1 hour. As a negative control, parallel sections were incubated with 2% rabbit serum. After incubation, specimens were incubated for 30 minutes with 0.3% hydrogen peroxide in PBS to block endogenous peroxidase activity. Endogenous biotin was blocked by sequential incubations with avidin (Vector Laboratories Inc., Burlingame, California, USA) and biotin (Sigma-Aldrich, St. Louis, Missouri, USA). Specimens were then incubated with biotinylated goat anti-rabbit Ig (Vector Laboratories Inc.) at 1:200 dilution followed by avidin-biotin HRP complex (DAKO Corp., Carpinteria, California, USA). After washing, the specimens were developed in a solution of 3-amino-9-ethylcarbazole (Aldrich Chemical Co., Milwaukee, Wisconsin, USA). After developing, specimens were counterstained with hematoxylin and mounted with Glycergel (DAKO Corp.).
In subsets of experiments, Hsp110 was localized to the surface of primary culture enterocytes. Briefly, enterocytes were isolated by a method described previously (21). Surgical specimens were obtained from the operating room. Specimens were washed extensively with PBS containing 1% penicillin/streptomycin and 1% Fungizone (Flow Laboratories, McLean, Virginia, USA). The mucosa was stripped from the submucosa, minced, and placed in 1 mM dithiothreitol (Sigma-Aldrich) for 5 minutes at room temperature to remove the mucus. The pieces were then washed in PBS and incubated in dispase (3 mg/ml in RPMI 1640 medium; Roche Applied Science, Indianapolis, Indiana, USA) for 30 minutes in a 37°C shaking incubator. This was repeated four times. The tissue pieces were removed and the cell suspension collected, pooled, and centrifuged on a Percoll density gradient (Amersham Biosciences Corp., Piscataway, New Jersey, USA). Enterocytes located at the 0–30% interface were washed three times with PBS and resuspended in RPMI medium with 0.1% BSA. Preparations of purified enterocytes were greater than 90% viable, free of macrophages and B cell contamination as determined by staining with anti-CD14 and anti-CD20 mAb's (Coulter Corp., Hialeah, Florida, USA), and contaminated with only 2–4% intraepithelial lymphocytes (CD3+ cells). Enterocytes were stained with anti-Hsp110 (1:100 dilution of rabbit polyclonal) or an equivalent dilution of normal rabbit serum, followed by FITC-labeled goat anti-rabbit secondary antibody. Stained enterocytes were analyzed on an EPICS Profile III flow cytometer (Beckman Coulter Inc., Fullerton, California, USA), as described previously (21).
Soluble lumenal components induce expression of CD1d on IECs. Monolayers of epithelial cells form a selective barrier between the lumen of the intestine and the underlying intestinal mucosa. As a consequence, IECs are constantly exposed to the lumenal environment and its contents (22). To assess whether such exposure could result in alterations in epithelial CD1d expression, T84 cells were cultured for varying periods of time with soluble LC prepared as described and diluted in media to final protein concentrations of 3 μg/ml and 6 μg/ml. Figure 1a depicts cell surface ELISA using the CD1d-specific antibody 51.1.3. OD values at 405 nm reflect the degree of HRP-bound secondary antibody association with 51.1.3 and therefore represent the relative levels of cell surface CD1d expression. Of note, since basolateral access is limited with cells grown on plastic (19), these assays likely reflect a predominant apical CD1d signal. Exposure of T84 cells to LC on the apical surface resulted in a time-dependent increase in the level of cell surface CD1d expression, peaking by 48 hours of exposure. The degree of surface CD1d upregulation was also dependent on the concentration of LC material present in cultures, being significantly higher when cells were exposed to 6 μg/ml (OD = 0.45 ± 0.029) rather than 3 μg/ml (OD = 0.27 ± 0.032), compared with T84 cells incubated alone without exposure to LCmaterial (OD = 0.05 ± 0.015, n = 6 at 48 hours, P < 0.01 by ANOVA in comparison to 3 μg/ml/LC and 6 μg/ml samples). The upregulation of CD1d by lumenal material was observed with all LC samples analyzed, including those from subjects with inflammatory bowel disease (data not shown). As a control for the induction of CD1d expression, cells were cultured in the presence of IFN-γ for 48 hours at a concentration of 1,000 IU/ml. As in previous studies (10), IFN-γ elevated levels of CD1d on the surface of T84 cells (OD = 0.32 ± 0.042, P < 0.01 compared with no IFN-γ). To determine whether the effects of LC components on epithelial cell phenotype were specific to CD1d, levels of cell surface MHC class II and ICAM-1 were also assessed by ELISA (Figure 1b). Upon exposure to IFN-γ at 1,000 U/ml for 48 hours, T84 cells displayed elevated levels of both MHC class II and ICAM-1 expression. In contrast, however, no significant alterations in the surface expression levels of these two markers were detected in cultures treated with 6 μg/ml LC material over the same time period. These results demonstrate that upregulation of CD1d by LC is relatively specific.
Soluble components present within human LC selectively enhance CD1d expression on the surface of IECs. (a) Cell surface ELISA of T84 monolayers following incubation with either 6 μg/ml (black bars) or 3 μg/ml (gray bars) of LC sample for varying periods of time. (b) Relative levels of CD1d, MHC class II, and ICAM-1 expression were compared by ELISA on T84 cells incubated alone (CNT) or in the presence of 6 μg/ml LC sample (LC) for a period of 48 hours. As a positive control for the detection of all inducible epithelial markers, monolayers were exposed to IFN-γ for 48 hours at a final concentration of 1,000 IU/ml. ODs at 405 nm ± SEM, generated by ELISA, were blanked against isotype-matched control antibodies.
To determine whether the LC response might be polarized, we subjected T84 cells to LC exposure on the apical or basolateral surface and assessed CD1d expression by ELISA. To do this, T84 cells were grown to electrical confluence (transepithelial resistance >1,000 ohms × cm2) on permeable supports, and ELISA was performed as described previously (19). Interestingly, some degree of polarity was apparent. While apical exposure to LC resulted in a greater than fourfold induction of CD1d (OD = 0.37 ± 0.011 with LC compared with OD = 0.08 ± 0.01 without LC, n = 5 at 48 hours, P < 0.01 by ANOVA), a less than twofold change was observed when LC was delivered to the basolateral surface (OD = 0.15 ± 0.005 with LC compared with OD = 0.08 ± 0.01 without LC, n = 5 at 48 hours, P < 0.05 by ANOVA). Such findings suggest that a degree of polarity may exist in the LC response.
ELISA data shown in Figure 1 revealed the increased levels of cell surface CD1d expression following exposure of IECs to LC. To determine whether elevations in surface CD1d levels were accompanied by an overall increase in CD1d transcription, CD1d mRNA levels were analyzed by RT-PCR. As shown in Figure 2a, exposure of T84 cells to 6 μg/ml LC resulted in an increase in the level of mRNA encoded by the CD1d gene. Increased mRNA levels were detectable by 4 hours of cell culture and reached maximal levels by 12 hours. Equal loading of total cell RNA was confirmed by parallel examination of the housekeeping gene GAPDH.
LC-induced upregulation of cell surface CD1d expression is accompanied by an elevation in the levels of CD1d-encoded mRNA transcripts and protein. (a) RT-PCR of total RNA isolated from T84 cells treated for varying lengths of time with media containing 6 μg/ml human LC sample. Primers specific to CD1d and the housekeeping gene GAPDH were used as described. (b) Immunoprecipitation (IP) of whole-cell lysates following treatment with LC material, using the CD1d-specific antibodies D5 and 51.1.3. Immunoprecipitates were resolved by 12% SDS-PAGE under nonreducing conditions. Proteins were transferred to nitrocellulose, and CD1d was detected by immunoblotting with the D5 antibody. Locations of 48-kDa (filled arrowhead) and 37-kDa (open arrowhead) forms of CD1d are indicated. The molecular weight is indicated on the left. (c) Confocal microscopy of T84 cells following incubation for 48 hours alone or in the presence of LC sample (6 μg/ml) or IFN-γ (1,000 IU/ml). CD1d (green) and the tight-junction marker ZO-1 (red) were stained with FITC- and Texas red–conjugated antibodies, respectively.
Detection of CD1d at the protein level was then assessed by immunoprecipitation of CD1d from whole-cell lysates using the antibodies D5 and 51.1.3. Contents of immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose, and CD1d was detected by probing of membranes with the D5 antibody. The results of immunoprecipitation using D5 are shown in the left panel of Figure 2b. T84 cells, which had been stably transfected with the cDNA-encoding CD1d (T84d), have been previously characterized and are known to express significantly higher levels of CD1d than untransfected cells (8). Extracts from T84d cells were used as a positive control. Blots of the proteins immunoprecipitated from T84d cells with the D5 antibody revealed two major bands. The 37-kDa (open arrowhead) and 48-kDa (filled arrowhead) bands have been previously characterized and have been shown to correlate with nonglycosylated and glycosylated forms, respectively (9, 11, 23). After immunoblotting with the D5 antibody, immunoprecipitated extracts of T84 cells cultured in media alone revealed low levels of these two CD1d protein bands. Incubation of cells with LC for 48 hours resulted in the detection of increasing amounts of the 37- and 48-kDa forms of CD1d as defined by D5 immunoprecipitation and Western blotting. By 48–72 hours of culture with soluble LC, the levels of CD1d protein detected were similar to that detected in the T84d samples. Immunoprecipitation with 51.1.3, which primarily recognizes mature, fully glycosylated CD1d (11), also revealed an increase in the level of the 48-kDa form of CD1d (Figure 2b, right panel, filled arrowhead).
Characterization of CD1d protein expression was also assessed, using confocal microscopy (Figure 2c). T84 cells grown to confluence and cultivated under varying conditions were permeabilized and stained with a combination of the 51.1.3 antibody and an antibody specific for the tight-junction protein ZO-1. Incubation of cells with isotype-matched antibody controls resulted in no specific staining (data not shown). Under all conditions, cell outlines were distinguished by ZO-1 border staining. Cells cultivated in the presence of media alone displayed a low degree of CD1d staining (Figure 2c, left panel), while culture of cells with IFN-γ (1,000 U/ml) for 48 hours resulted in increased levels of CD1d observed throughout cell cross sections (Figure 2c, right panel). CD1d-specific staining of IFN-γ–treated samples appeared granular and diffuse throughout the monolayer. Similarly, exposure of T84 cells to LC material for 48 hours resulted in a significant increase in the degree of CD1d-specific staining above that observed in control monolayers. Taken together, the results illustrated in Figures 1 and 2 demonstrate the ability of soluble components present within LC to upregulate CD1d protein and mRNA expression. Elevated levels of CD1d by LC are comparable to the levels observed in cells treated with IFN-γ or transfected with a construct containing the CD1d gene and controlled with an exogenous promoter (8).
LC-induced CD1d is not limited to T84 cells. To establish that the effects of LC on CD1d expression were not specific to the T84 cell line, levels of CD1d were examined on other epithelium-derived lines, namely HT-29 and Caco-2, following exposure to LC material. As shown by ELISA in Figure 3a, culture of HT-29 cells with LC material at a concentration of 6 μg/ml resulted in a time-dependent increase in cell surface CD1d expression. As with T84 cells, CD1d expression reached maximal levels by 48 hours of culture and was quantitatively comparable to the levels observed in samples treated with 1,000 U/ml IFN-γ.
Soluble LC material elevates CD1d expression in the colonic epithelium-derived cell lines HT-29 and Caco-2. (a) CD1d-specific cell surface ELISA of confluent monolayers of HT-29 cells following incubation with LC samples. As a positive control, cells were exposed to IFN-γ at a final concentration of 1,000 IU/ml for a period of 48 hours. (b) Western blot of whole-cell lysates isolated from HT-29 and Caco-2 cells following treatment with soluble LC material at a concentration of 6 μg/ml. Proteins contained within the lysates were resolved by 12% SDS-PAGE and transferred to nitrocellulose. The presence of CD1d was subsequently assessed by probing with the anti-human CD1d-specific antibody D5. CD1d was observed as dominant bands of 48-kDa (filled arrowhead) and 37-kDa (open arrowhead) forms. No bands were detected when nitrocellulose membranes were probed with normal mouse serum (data not shown). The blots were reprobed for the presence of β-actin (bottom panels).
In subsets of experiments, we also examined whether LC induced CD1d on cell types other than epithelia. Specifically, we addressed whether microvascular endothelial CD1d might be regulated by LC. Such experiments revealed that while IFN-γ (300 U/ml, 48 hours) increased CD1d on the surface of microvascular endothelia (2.5 ± 0.6–fold increase in OD by ELISA, P < 0.01 compared with no IFN-γ), LC did not influence endothelial CD1d (0.3 ± 0.2–fold increase in OD by ELISA, P = not significant compared with no IFN-γ).
Cell surface ELISA of Caco-2 cells was not performed, since adherence of epithelial monolayers proved insufficient to withstand extensive washing necessary for the procedure (data not shown). Therefore, whole-cell CD1d levels were next examined for both HT-29 and Caco-2 cultures by probing of lysates resolved by Western blot. Immunoblots of extracts obtained from HT-29 cells cultivated in media alone revealed the presence of two bands of approximately 46–48 kDa when probed with the D5 antibody. Following exposure of HT-29 cells to LC material for 24–48 hours, a band of 37 kDa and increased intensity of the 46- to 48-kDa bands were detected in the cell extracts. Of note, and as we have previously reported (11), whereas the 51.1.3 antibody recognizes mature CD1d, the D5 mAb preferentially recognizes immature CD1d that has not yet acquired maturation of the carbohydrate side-chain modifications. Therefore, it is likely that the middle bands observed within the D5 Western blot represent immature CD1d with partially processed carbohydrate side-chain modifications. The migration pattern of these inducible bands matched the patterns observed in the immunoblots of T84d extracts (data not shown) and presumably represents forms of CD1d that vary in their degree of glycosylation. Immunoblots of lysates obtained from Caco-2 cells also revealed the induction of CD1d-specific bands following exposure to LC material. As with the T84 and HT-29 cells, a band of 37 kDa and bands between 46 and 48 kDa became readily visible after 48 hours of cultivation in the presence of soluble LC. In both cases, β-actin Western blotting was used to demonstrate equal loading (Figure 3b). Thus, such LC-induced upregulation of CD1d expression was not specific for T84 cells but may be rather specific for intestinal epithelia.
Non–microbe-derived, heat-labile components isolated from the lumen of the murine intestinal tract stimulate CD1d upregulation on the surface of T84 cells. To better define the origin of CD1d-inducing activity in LC, soluble LC were isolated from the intestinal tract of mice and tested for their influence on CD1d expression by T84 cell ELISA. Incubation of T84 cells with 6 μg/ml lumenal extracts obtained from mice housed under SPF conditions resulted in the induction of CD1d cell surface expression by 48 hours of culture (Figure 4a). To determine whether the murine CD1d-inducing factor was derived from components of the microbial flora, lumenal samples isolated from mice bred under germfree conditions were incubated with T84 cells. Exposure of T84 cells to germfree lumenal extracts led to elevations in surface CD1d levels that did not differ significantly from the levels induced by SPF-derived lumenal samples. These data demonstrate that lumenal extracts devoid of microbial content are capable of upregulating CD1d expression on epithelial cells, implicating a non–microbe-derived component as the active CD1d-inducing factor.
Non–microbe-derived factors are present within the lumen of the murine small and large intestine that upregulate CD1d expression on human T84 cells. Cell surface ELISA was performed on confluent T84 monolayers using the CD1d-specific antibody 51.1.3. (a) BALB/c mice housed in either germfree (GF) or SPF environments were sacrificed and the LC of the colon isolated. T84 cells were cultured alone (–) or exposed to the soluble lumenal preparations for 48 hours at a final concentration of 6 μg/ml. (b) Lumenal material from the stomach (St), small intestine (SI), or large intestine (LI) of SPF-housed mice was tested for its ability to enhance epithelial cell surface CD1d expression. The CD1d-inducing activity of both soluble lumenal components (black bars) and mucosal scrapings (gray bars) was assessed relative to untreated controls (–).
It is also possible that LPS contributes to this CD1d-inducing activity, even in germfree animals (e.g., LPS in autoclaved food, water, etc.). Therefore, we examined whether LPS might induce CD1d. T84 cells exposed to LPS (derived from Salmonella minnesota; range 0.01–100 ng/ml in the presence of 5% normal human serum) resulted in no change in CD1d expression compared with controls (maximal increase 0.2 ± 0.1–fold, P = not significant). As controls, monolayers treated in a similar fashion and assessed for ICAM-1 induction resulted in a rapid and concentration-dependent elevation in ICAM-1 surface expression (maximal increase 18 ± 0.8–fold, P < 0.01). Such findings demonstrate that, while epithelia can respond to LPS, it is unlikely that CD1d-inducing activity in LC represents LPS.
To establish the region of the gastrointestinal tract displaying the highest degree of CD1d-inducing activity, lumenal samples and mucosal scrapings were isolated from the small intestine, the large intestine, and the stomach. Figure 4b reveals that induction of CD1d expression on T84 cells was greatest when cells were incubated with mucosal and lumenal extracts from the small intestine. Upregulation of CD1d was clearly detectable in cultures containing large bowel–derived samples, but not across monolayers cultured in the presence of stomach-derived material. These data demonstrate that the active CD1d-inducing component is present both along the mucous layer lining the epithelium and in the lumen of the gut itself. Maximum activity is present within regions of the proximal small intestine. In contrast, no activity could be detected in contents obtained from the stomach.
Identification of Hsp110 as the CD1d-inducing activity. To begin to characterize the active component within LC responsible for induction of CD1d expression, we initially examined heat stability, hydrophobicity, and nominal molecular size of the active LC component(s). To determine whether the activity was heat sensitive, soluble LC components were incubated at 80°C for 10 minutes and then exposed to cell monolayers. In contrast to unmodified LC material, heat-treated samples did not elicit a detectable elevation in CD1d expression (Figure 5a). Hydrophobicity of the active LC component was determined by chloroform extraction or by C18 column chromatography. For both separation techniques, the CD1d-inducing activity partitioned into the aqueous phase (Figure 5b). Molecular weight fractionation was performed using the upper aqueous phase of chloroform extracts as starting material. This fraction was passed through a series of consecutively lower–molecular weight cut-off filters. Retentates and filtrates from each filtration procedure were subsequently tested on T84 cell monolayers for their ability to stimulate CD1d expression. As indicated in Figure 5c, the retentate generated from filtration using the 100-kDa filter possessed the vast majority of activity, as reflected by the ability of this fraction to upregulate CD1d expression to a level comparable to that for whole untreated LC material. The 100-kDa filtrate and the fractions generated by its filtration through lower–molecular weight filters displayed very little CD1d-inducing activity. Identical results were obtained with murine LC fractions from both SPF and germfree mice (data not shown).
Preliminary biochemical characterization of the active CD1d-inducing factor present within soluble LC material. ELISA was performed on T84 cells treated for a period of 48 hours with various preparations of LC isolates. (a) Monolayers were cultured alone (CNT), in the presence of unmodified whole LC samples (–), or with LC samples that had been preincubated at 80°C for 10 minutes. (b) LC material was separated into aqueous (AQ) or hydrophobic (HP) phase by chloroform extraction and C18 column chromatography, respectively, as indicated. Individual fractions were subsequently assessed for their ability to induce CD1d expression. (c) LC samples were passed through a Centricon filter (Millipore Corp., Bedford, Massachusetts, USA)of 100-kDa molecular weight cut-off. The 100-kDa filtrate was then passed consecutively through 30-kDa and 10-kDa Centricon filters. The filtrates (white bars) and retentates (gray bars) from each filtration step were subsequently resuspended in PBS, tested for their CD1d-inducing activity (black bars), and compared with those from the no-treatment (CNT) and crude-fraction (LC) groups. (d) Coomassie-stained SDS-PAGE gel of partially purified material, indicating the major bands selected for microsequencing (arrows), and the migration position of the band with sequence homology to Hsp110. A control (CNT) derived from a100-kDa filtrate fraction is also shown.
Using partially purified extract (aqueous phase following chloroform extraction eluted from C18 chromatography with molecular weight >100 kDa), SDS-PAGE analysis revealed three distinct bands of Mr 85, 110, and 125 kDa by Coomassie staining (Figure 5d). No specific sequence was obtained from the 85- or 125-kDa bands. Microsequence analysis of tryptic peptides from the approximately 110-kDa protein revealed three peptides (sequences IEVPLHSLMEQTHLK, ELNNVCEPVVNQPKPK, and KPVTDCVISVPSFFTDAER) with direct sequence homology to Hsp110 (15). As shown in Figure 6a, Western blot analysis of unfractionated human LC material revealed the presence of an antigen consistent with purified Hsp110, with slightly slower mobility by SDS-PAGE, likely related to an as-yet undetermined posttranscriptional and/or posttranslational modification. Similar analysis with primary antibody omitted revealed no detectable signal. Taken together, these studies suggest the presence of relatively high concentrations of Hsp110 in our LC fractions.
Role of Hsp110 in induction of epithelial CD1d. (a) Western blot analysis of LC for Hsp110. LC or purified recombinant Hsp110 (0.3 μg) was resolved by SDS-PAGE under nonreducing conditions, and blots were probed with polyclonal rabbit anti-Hsp110. Also shown is the control with primary antibody omitted from the reaction mixture. A nonspecific band (NS) of approximately 70 kDa was observed in both LC and control samples. (b) Cell surface ELISA of T84 monolayers following incubation with 6 μg/ml LC in the presence of indicated concentrations of anti-Hsp110 or control anti-Hsp30. (c) Cell surface ELISA of T84 monolayers following incubation with indicated concentrations of purified recombinant Hsp110 in the presence of anti-Hsp110 or control anti-Hsp30. OD values generated by ELISA were blanked against isotype-matched control antibodies. (d) Flow cytometric analysis of Hsp110 on native small intestinal enterocytes (black line) compared with equivalent concentrations of an isotype-matched control (gray line). (e and f) Immunohistochemical localization of Hsp110 in normal small intestinal tissue (e) and in normal colon (f). Positive staining with anti-Hsp110 but not with control Ig (Control) is seen in epithelial cells and diffusely in the lamina propria. Magnification, ×400.
As proof of principle for Hsp110-mediated induction of CD1d, two antibody-antigen approaches were used. First, as shown in Figure 6b, the addition of increasing concentrations of anti-Hsp110, but not control anti-Hsp30, to LC extracts resulted in decreased CD1d-inducing activity in a concentration-dependent fashion (P < 0.025 by ANOVA). Second, as shown in Figure 6c, the addition of purified mammalian Hsp110 to epithelial monolayers resulted in a concentration-dependent induction of CD1d by surface ELISA (P < 0.025 by ANOVA). The addition of anti-Hsp110, but not anti-Hsp30, significantly diminished CD1d-inducing activity of purified Hsp110 protein (P < 0.05). Taken together, these data identify Hsp110 as a component of the LC extract, presumably derived from the IEC itself, in the autocrine regulation of epithelial CD1d cell surface expression.
Finally, given the nonmicrobial origin of Hsp110 (Figure 4a) consistent with the absence of known prokaryotic Hsp110 isoforms (15), and the relative abundance of CD1d-inducing activity in the small intestine (Figure 4b), we questioned the origin of Hsp110 within LC. First, we addressed whether Hsp110 was detectable on native enterocytes. As shown in Figure 6d, flow cytometric analysis of native small intestine enterocytes revealed dominant Hsp110 surface expression. Interestingly, exposure of native epithelia to recombinant Hsp110 (10 μg/ml, 24 hours) did not induce CD1d expression above base line (data not shown), suggesting that in the native state, this signaling pathway may already be saturated. To morphologically confirm these findings, immunohistochemistry was performed on normal small intestine (n = 3) and large intestine (n = 2) tissue samples (Figures 6, e and f, respectively). These studies revealed abundant expression of Hsp110 in small intestinal samples, with prominent localization to the epithelium and patchy staining within the lamina propria. Hsp110 expression within large intestinal epithelia was also evident, although less prominent than in the small intestine (Figure 6f). Taken together, these findings indicate that the epithelium is a rich source of Hsp110 in the intestine and, as such, likely reflects the CD1d-inducing activity evident in LC.
The ability of CD1d to stimulate immune responses that regulate intestinal inflammation implicates this molecule as an important component of the mucosal immune system (8). Characterization of the events that regulate CD1d expression in general, and on the surface of IECs in particular, promises to reveal further information as to the functional role of CD1d. Similar to other MHC class I–related molecules, CD1d expression and function are regulated by IFN-γ (10). However, little is known about other endogenous pathways for such regulation. We demonstrate here that lumenal, IEC-derived Hsp110 represents a novel regulatory component for CD1d expression on epithelia.
A number of previous studies have suggested discordant in vivo and in vitro expression of CD1d; namely, that CD1d expression is higher in vivo than in in vitro culture systems. Such studies suggest the possibility that additional endogenous components may be necessary to induce and/or maintain surface levels of epithelial CD1d. As such, we screened human and murine LC for the existence of CD1d-inducing bioactivity. Such experiments revealed a prominent factor(s) that partitions into high–molecular weight, aqueous-phase components. A possibility was that LPS may represent the CD1d-inducing activity. However, four observations ruled out LPS. First, we directly demonstrated that LPS does not regulate CD1d expression on T84 epithelium. Second, LPS has been shown to induce ICAM-1 on IECs (24), and our results indicated that LC fail to regulate the expression of epithelial ICAM-1. Third, LC derived from both conventionally housed mice and germfree mice readily induced CD1d on human epithelia, likely indicating that this activity is not LPS but is of eukaryotic origin. Finally, the sensitivity of CD1d-inducing activity to heat denaturation argued against LPS activity, since LPS is heat stable. Using biochemical and biophysical properties of this bioactivity, we identified Hsp110 as an LC-derived agonist for CD1d expression. Subsequent studies revealed that epithelia from the normal human intestine prominently express Hsp110, and that purified Hsp110 can recapitulate the activity observed in crude LC, suggesting an autocrine pathway for Hsp110 function.
The mechanism(s) of CD1d induction by Hsp110 is not known. It is possible that Hsp110 binds directly to epithelial surface receptors and couples to intracellular signaling. While much attention has been paid to understanding the role of extracellular Hsp signaling in a variety of disease states (25), mechanisms or surface targets for Hsp's are only recently appreciated. Of distinct interest, several studies suggest that Toll-like receptors (TLRs) may represent one class of receptors for Hsp family members (26, 27) and that Hsp70 can function as a "cytokine-like" molecule in association with CD14 (28). We do not know whether Hsp110 binds to TLR-2 or TLR-4 or whether CD14 is necessary for CD1d-inducing activity. IEC lines are known to express functional TLRs (29, 30) and have been demonstrated to signal in a CD14-dependent manner (31). Similarly, since it was recently shown that CD40 is a receptor for the Hsp70 family (32), it is possible that CD40 provides a receptor for Hsp110. However, studies directed at this issue have revealed no detectable CD40 on intestinal epithelia (L. Mayer, unpublished observations). Another candidate molecule is CD91 (also called α2-macroglobulin receptor or the LDL-related protein), as it has been demonstrated to bind directly to a number of Hsp's (33, 34). However, CD91 has not been shown to directly bind Hsp110, and little is known about CD91 expression on intestinal epithelia. Alternatively, it is possible that Hsp110 does not bind directly to an epithelial surface receptor. Thus it is possible that functional Hsp110 is in complex with an as-yet unidentified peptide(s) that activates CD1d via chaperone function and/or costimulation of other surface molecules. All Hsp's described to date have attributable chaperone function (25), raising the distinct possibility that such chaperone function provides an accessory signal for induction of CD1d.
At present, it is not known whether Hsp110-mediated regulation of CD1d represents a physiologic regulatory pathway or a process induced by immune activation. Given the constitutive presence of Hsp110 in the lumen of all individuals examined, it might be argued that Hsp110-mediated regulation of CD1d is a physiologic process. In the context of gut-associated lymphoid tissue, it could similarly be argued that Hsp110 regulation of CD1d represents the "physiologic inflammation" associated with a number of mucosal tissue sites (35). Some evidence exists that similar pathways may be associated with pathological processes. It was recently shown, for example, that increased expression of Hsp family members (especially Hsp70) in atherosclerotic lesions coincides with specific expression of CD1d, but not other MHC molecules (36). Whether such observations can be extended to other Hsp molecules is not known, but they suggest a relatively specific activation pathway for CD1d that involves Hsp-related molecules. Our studies also shed some light on this subject. Indeed, while IFN-γ regulates both MHC class II and ICAM-1 on IECs (18), LC failed to modulate either of these molecules. Thus, additional work will be necessary to define the details of Hsp110-induced CD1d expression.
In summary, these studies identify a novel function elicited by LC of the intestinal milieu. Specifically, results from these studies suggest that epithelia may liberate Hsp110 and thus provide an autocrine signal for CD1d regulation on this cell type. These studies also predict a major role for Hsp110 in CD1d expression in other cells and provide evidence for yet another link between innate and adaptive immunity in the immunobiology of CD1d.
This work was supported by NIH grants DK-50189, DK-44319, DK-51362, and DK-53056 and the Harvard Digestive Diseases Center. R.S. Blumberg, L.F. Mayer, and R.B. Sartor were supported by a grant from the Broad Medical Research Program.
Sean P. Colgan and Richard S. Pitman contributed equally to this work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Nonstandard abbreviations used: intestinal epithelial cell (IEC); heat shock protein 110 (Hsp110); lumenal contents (LC); specific pathogen–free (SPF); Toll-like receptor (TLR).


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Romney visits Western Wall in Jerusalem
(CNN) -- Presumptive GOP nominee Mitt Romney, joined by his wife Ann, made a previously unannounced stop at the Western Wall on Sunday while in Jerusalem meeting with Israeli leaders.
Sunday also marks the Jewish holiday of Tisha B'Av, a holy day of mourning and fasting in remembrance of the destruction of the first and second temples.
According to pool reports, the Romneys were joined by Rabbi Shmuel Rabinowitz of the Western Wall, who also accompanied President Barack Obama's visit to the site during his 2008 trip to Israel as a presidential candidate.
Wearing a yarmulke and a dark suit, Romney put a piece of paper believed to be a prayer into the wall after being read a passage by the rabbi.
Meanwhile, Ann Romney was on the other side of a partition reserved only for women. She stood before the wall for several moments and also placed a piece of paper in the wall.
The Romneys were flanked by hundreds of Jewish worshipers praying at the site. The Western Wall, a highly important religious site for Jews, sits below the location where the second temple stood.
As Romney walked out, he greeted a crush of people and media.


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Join Fr. Rocky Hoffman Executive Director of Relevant Radio for a Marian Day of Reflection and May Crowning at the Shrine of Our Lady of Guadalupe on Saturday, May 19. The Day of Reflection commences with prayer, procession and the May Crowning of Our Blessed Mother. The morning session will include meditations, Holy Mass, Rosary and time for Confession and reflection. Following a Relevant Radio lunch will fellow pilgrims, the Day of Reflection continues with Exposition of the Blessed Sacrament, the final meditation and the Chaplet of Divine Mercy. The full schedule for the Day of Reflection is below.
Printable Flyer – Help us Promote!
The Day of Reflection is free and open to the public.
1. Sit-down Lunch in Shrine Crypt - $15. Limit 200. Registration is full. Thank you!
2. Restaurant – Call Culina Mariani Café for reservations (608) 788-8400.
3. Shrine will have brat booth on site.
4. Bring Your Own. There is space to eat outside.
8:30-9:30 am Registration (for those who ordered the $15 crypt lunch)
9:30 am Procession and Opening Prayer
10:15 am First Meditation
10:45 am Break
11:00 am Second Meditation / Confessions begin
11:45 am Rosary
12:15 pm Holy Mass
1:15 pm Lunch
2:00 pm Confessions resume
2:30 pm Exposition/Third Meditation
3:00 pm Divine Mercy Chaplet/Benediction
3:30 pm Rosary/Vespers/Benediction/Reposition


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Kitty sends a kind of original email messages to her friend Garf. To write a message, shechooses a word W and a number n and replicates W n times horizontally. Then she repeats thisstring in the next line, but rotating the characters once to the left. And she repeats this 'rotate-and-output' process until the word W appears displayed as the rst column of the rectangularpattern that she produces.As an example, when she chooses the word Hello and the number 3, she gets the pattern:
Kitty has been sending such emails during the last three years. Recently, Garf told her thatperhaps her work may be automatized with a software to produce Kitty's patterns. Could youhelp her?
The input le contains several test cases, each one of them in a separate line. Each test casehas a word and a positive integer that should generate the corresponding rectangular pattern.The word is a string of alphabetic characters (a..z). The number is less than 10.A line whose contents is a single period character means the end of the input (this last lineis not to be processed).
Output texts for each input case are presented in the same order that input is read. For eachtest case the answer must be a left aligned Kitty pattern corresponding to the input.
Input:Love 1Kitty 2.Output:LoveoveLveLoeLovKittyKittyittyKittyKttyKittyKityKittyKityKittyKitt


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Great can't-pick-a-colour shade. It's a strange colour; at first it just looks like gold pearl, but in some light it's more silvery, and the undertone is a pinky mauve taupe. Not so pale that it looks strange on my pinky skin; not so warm or cool that it clashes with anything I might wear; two coats are opaque on me; wears well; nice pearlized sheen.
3 out of 3 people found this review helpful. Did you? Yes No
I'm another one who didn't particularly like this at first. My skin is quite tanned and I thought the taupey-ness of this polish made it look a bit like I had no nails. Somewhere along the line it became my favourite polish - there's a sweet pinkness mixed in with the taupe, and soft pearlised finish. It's also beautiful to apply. After 2 coats the whites of my nails are still easily discernible which isn't a dealbreaker but I wouldn't complain if it were more opaque.
I have to admit this color wasn't my fave at first, but it grew on me LOADS. The opaque beige base has lots of teensy-tiny gold-beige mica in it that shimmers like crazy. I thought it was too much at first, but I love it on my little toes! Shiny and sweet with one layer or multiple layers.
Revlon nail polishes are my go-to polishes. The color selection is great, the color payoff is great, the finish is great and the durability is AMAZING! Best for durability I've ever tried. Where I normally get chippage on the ends of my nails from every other brand I've tried so far, Revlon wears down just around the end edges after a week and a half. I've worn these without top coat and had the same result, just sooner. The longest wear ever!
2 out of 2 people found this review helpful. Did you? Yes No
Great polish. Looks like a champagne color to me, shimmer/frosh finish. Opaque in 2 coats, and dries pretty fast.
Will definitely repurchase if I ever use up the bottle.
A really nice nail polish to wear during the summer, looks really nice in the sun.
It applies really nicely like all the other Revlon nail polishes and dries really quickly. It lasts a nice amount of time with or without a top coat.
The glossy finish is really nice and is also available in Matte/Suede which is just as good but is also more suited to work and everyday rather than going out with friends.
Great nail polish, recommended.
2 out of 2 people found this review helpful. Did you? Yes No
I love this nail polish on my toes during summer. It matches any open toe shoe and looks gorgeous with a tan. Champagne color with gold iridescent shimmer is the best way for me to describe the color.
This nail polish was one of my first purchases of the Revlon Nail Polish line. I fell in love with it! It is a nice opaque colour with two coats. The drying time I great, you don't have to wait hours. It doesn't chip that easily either.
This is a lovely nail polish. I've never bought any polishes by Revlon before, but I'm definitely planning on picking up more in the future. The quality is amazng: it's a fairly thin formula that goes on in a very opaque coat and doesn't take forever to dry. I usually do two coats, but one still looks pretty darn good. As a piano-player I tend to chip nail polishes quite quickly, but this (with a top-coat) lasts me close on a week before any chipping happens.
In terms of this particular colour: it's a lovely subtle metallic/shimmery nude gold, that can also look slightly silver or bronze depending on the light/colours around it. I like to use it as an accent nail as well, as it goes with so many colours.
And last but not least - price! The Revlon polishes only cost around NZ$20, which is amazing when you consider that OPI polishes retail for $32 here in NZ. Love! :)
Have to disagree... my skintone is neutral (neither cool nor warm) and this colour is too warm for me - just looks too brown. Makes me think of nicotine-stained nails. As always, the quality of Revlon n/p is great, so 2 lippies for that. EDIT JULY 2012: I just added 2 lippies. Here's what happened: my favourite Revlon polish from the wintertime, Gray Suede, was all of a sudden looking all wrong on my nails and I realized it was because it's summer and I have a tan. So I thought, I wonder if this colour would look different now too? And yes it does! It doesn't look too brown or warm anymore, just like a shimmery nude neutral nail. It's a keeper - for summertime only!


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Daddy & Son
This Italian chain moved from DC's Dupont Circle to Bethesda earlier this year; it's the only Daddy & Son store in the United States. The men's shop stocks house-brand classic and European-fit dress shirts, including extra-slim cuts. Ties—all standard width—come in a wide array of colors and patterns; they're displayed on wall-mounted racks for easy browsing. And if you need help tying a European knot, owners Roberto and Francesca Inferrera happily give tutorials. 4836 Bethesda Ave.; 301-652-0595.
Daisy Too
The stylish clothes at this women's boutique can go easily from work to date night. Look for basic dresses, blouses, and slacks plus all the accessories you could need to dress them up. If your wallet's feeling light, check out the consignment corner—a recent visit turned up a Tory Burch jacket for $150 and a Nanette Lepore strapless dress for $120. 4940 St. Elmo Ave.; 301-656-2280.
Denim Bar
With more than 40 designer brands for men and women, this boutique caters to serious denim lovers. The prices aren't cheap—you could pay $300 for a pair of jeans—but the helpful staff will work with you to find the right brand, size, wash, and style. Denim Bar offers alteration services, but for an even more custom look, try a pair made from raw denim ($150 to $300), an unwashed fabric that molds to your shape after about six months of unlaundered wear. 4939 Cordell Ave.; 301-986-5260.
Cute cocktail dresses and stylish office-ready ensembles are the draws at this Bethesda Row women's boutique. Eco-friendly touches show up in everything from bamboo hangers to clothes made with organic fabrics. Owner Gretchen Hitchner carries pieces from established and up-and-coming designers. Look for jackets, tops, shoes, and accessories from Wendy Mink, Calypso St. Barth, White & Warren, and more. 7114 Bethesda La.; 301-664-9242.
J. McLaughlin
Prepsters like the madras and gingham prints, ribbon belts, and pastel accents at this cheery storefront. But you can also find more updated looks: We like the pencil skirts and slim-cut trench coats for women and the hipster-style button-downs for men. A sale rack downstairs has basics from this season at a discount. 4851 Bethesda Ave.; 301-951-5272.
Trade those shapeless sweats and tees for more stylish workout clothes from Lucy. This activewear retailer has clothes and accessories for all kinds of movers, from yogis to runners to outdoor types. Fans love the store's "compression" pants, which pull the legs in and smooth problem areas. 7112 Bethesda La.; 301-656-3106.
Lululemon Athletica
Yoga addicts flock to this chain for well-made, durable sportswear. The secret is Lulu's preshrunk fabric, called luon, which stretches without becoming misshapen. Pants, which start around $75, come in lots of cuts and colors. Lululemon also has goods for men, including T-shirts, hoodies, running shorts, and bags. The Bethesda Row location hosts a free yoga class on Sunday, group runs on Wednesday, and Pilates on Saturday. 4856 Bethesda Ave.; 301-652-0574.
BCBG, Trina Turk, French Connection, and Citizens of Humanity are just some of the nearly 40 labels you'll find at this small, well-stocked shop. Designer denim generally runs between $150 and $250, and a wide selection of pretty jewelry starts around $20. Sign up for the e-mail list and you'll receive alerts about Luna's quarterly customer-appreciation sales, when everything is 20 percent off. 7232 Woodmont Ave.; 301-656-1111.
Well-dressed women swear by Pirjo Jaffe's collection of classic separates. She stocks lots of European labels, including designers from her native Finland. Clothes and accessories are unique and feel art-inspired. Stylish women of any age will like Jaffe's leather handbags and glitzy costume jewelry. 4821 Bethesda Ave.; 301-986-1870.
Urban Chic
A local chain that started in Georgetown, Urban Chic carries high-end designers such as Alice & Olivia, Juicy Couture, and Diane von Furstenberg. Displays show shoppers creative ways to pair designer separates, and racks are well maintained by helpful staff. Looking for wallet-friendly options? Head to the sale rack. A recent visit turned up in-season Nanette Lepore and Milly dresses for 30 to 50 percent off. 7126 Bethesda La.; 301-657-0290.
Wear It Well
This tiny women's boutique is brimming with sophisticated separates. The style appeals to a fashionable 35-and-over set, with jackets, slacks, and casual weekend wear by Gerard Darel, Babette, and more. Check out the jewelry, handbags, and gloves for an easy way to glam up an outfit. 4816 Bethesda Ave.; 301-652-3713.
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Why is Fashion so Anti-social?
LONDON, United Kingdom — Facebook, Twitter, MySpace. Many large fashion brands have pages or accounts on at least one of the big three in social media. As a whole however, the fashion industry has been slow to embrace these tools — certainly a lot slower than their customers. Of the 38 tweeting fashion brands listed by WWD, only nine are based in Europe. Of those nine, Henry Holland is the only one not associated with a large


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United Kingdom & Ireland > Products & Services > Manufacturing and Industry > Corrosion Protection > Chemical Coatings > Bunds
3M™ Scotchkote™ liquid coatings provide chemical resistant protection designed to help spills and leaks. Concrete repair screeds can be used to resurface bund floors, walls and covings, prior to the application of a chemical resistant coating. The prevention of leaks enables compliance with legislation.
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Pacific Regional Office: Field Reports
Who We Are | Parks | Regional Publications | Meet Our Staff | Field Reports
Field Report Summer 2014
In This Issue:
- Preserving Los Angeles' Last Great Wilderness
- Thirteen Compelling Reasons for Congress to Work Together
- King of the Forest…or Not
- More Camping, Less Traffic at Yosemite?
- Clearing the Air at Joshua Tree
- Hiker's Shuttle at Joshua Tree National Park?
- Eco-Divers & Bubble-Spotters: NPCA Sponsors a Colorado River Clean-Up
- California School Kids Share What They Love about National Parks
- Preserve Manzanar's History
- Mountain Lions at Risk
- Right Project, Wrong Place
- Making the Case in D.C.
- See America Without Leaving Home
Previous Issues
Winter 2014 (PDF, 1.1 MB)
- Tower Trouble
- National Parks and Local Economies
- A Birthday Present for the Parks
- One Year Later: Recommendations for Expanding César Chávez Monument Into a National Historic Park
- Mammoths & the Military
- NPCA Welcomes New Program Manager for Los Angeles
- West Coast's First Marine Wilderness One Step Closer to Protection
Summer 2013 (PDF, 761 KB)
- Victory Achieved: Eagle Mountain Landfill Finally Trashed
- Victory on the Way
- Two Steps Forward and a Big Cloud
- Yosemite's Merced River Plan
- Death Valley Night Skies
- YOU Did It!
- Pacific Regional Staff Visits Rim of the Valley Corridor
Autumn/Winter 2012 (PDF, 761 KB)
- NPCA and Allies Challenge Cadiz Water Project in San Bernardino County Superior Court
- A Japanese-American WWII Incarceration Site: NPCA's Role
- Honoring Cesar Chavez
- Restoring Hetch Hetchy — Opportunity for a Step in the Right Direction
- Congress Sequester Damaging to "America's Best Idea"
- An Opportunity for Cleaner Air
- Investing in Solar Energy While Protecting Our Desert National Parks
- The Wild and Scenic San Joaquin


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I know what you're thinking. Is it April Fool's Day again? Why does this headline feature the seemingly completely unrelated topics of Doctor Who, Jane Austen and crime thrillers? Well, today's BBC story is so interesting that I couldn't have made it up on my own if I tried. Doctor Who star Jenna-Louise Coleman has signed on to appear in the three-part mini-series, Death Comes to Pemberley, which is based on a P.D. James novel that includes classic Jane Austen characters. No foolin'!
Death Comes to Pemberley is a drama, which was commissioned by BBC's controller Ben Stephenson along with former BBC One Danny Cohen. Cohen was recently promoted to director of television.
In front of the camera, the BBC One min-series has added Jenna-Louise Coleman to play Lydia Wickham, who is the sister of Pride and Prejudice protagonist Elizabeth Bennet. That famous role has been taken by Anna Maxwell Martin. Matthew Rhys will play Elizabeth's husband, Fitzwilliam Darcy and Matthew Goode has been cast as Lydia's husband, George Wickham.
So, the Jane Austen characters we know and love are married and enjoying happily ever after. The story kicks of six years into the wedding bliss of Elizabeth and Darcy when a muder takes place at the couple's annual ball. How is that for intriguing?
THR had the news of Coleman's involvement and released words that other cast members now include Trevor Eve of Waking the Dead, James Fleet from Sense and Sensibility, Rebecca Front, Penelope Keith, James Norton of Rush, Joanna Scanlan, Eleanor Tomlinson from The White Queen and Tom Ward of Silent Witness.
I'm tickled because I never heard of the P.D. James novel before. I may need to add Death Comes to Pemberley to my reading list.
Coleman is known for her work as Jasmine Thomas on the British soap, Emmerdale, and has become the Eleventh Doctor's companion Clara Oswald for the indomitable Doctor Who.


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My Army: Reloaded Review
+ Universal App - Designed for iPhone and iPadMy Army: Reloaded makes it easier for everyone to check out this quirky little shooter.
Read The Full Review »
Posts Tagged ReloadedMy Army: Reloaded Review+ Universal App - Designed for iPhone and iPadMy Army: Reloaded makes it easier for everyone to check out this quirky little shooter. Read The Full Review » Rock Band Reloaded ReviewiPhone App - Designed for the iPhone, compatible with the iPadRock Band Reloaded is like so many so-so musicians, great technical skills but where's the soul? Read The Full Review » Rhythm gamers who hate leaving their plastic instruments behind when they leave the house will be pleased to hear that Rock Band Reloaded is now available on the App Store. The game features 29 new songs from the likes of Billy Idol, Nirvana and the Steve Miller band, as well as several new gameplay features and enhancements. It would seem the time has come to once again take the rock on the road. Of the new features the one with the most potential (and the most potential embarrassment) is vocal recognition which will let you sing along with the songs. Much like its console brethren, Reloaded will score your performance based on how closely your pitch matches that of the song. Don't worry if you don't know the words, the game only cares that you're hitting the right notes, not saying the right thing. Other new features include a landscape mode so you can play the game horizontally and a new Expert difficulty setting. While we're happy to see a new Rock Band on iOS we can't help but feel a little underwhelmed by the set list. Even though Reloaded packs more songs than last year's edition and a bunch more than Guitar Hero, most of them are retreads and rehashes from the console versions. Yes, we still love Hungry Like the Wolf, but after playing it six dozen times in Rock Band 2 we're not particularly thrilled to see it yet again. Also, while the inclusion of vocals is cool, how functional is it going to be on a mobile version of the game? The only place where you can really let lose and sing along is at home, and if you're in your living room why not fire up one of the console versions of Rock Band instead? But hey, if rhythm games are your thing then don't let us talk you out of it. Rock Band is still a ton of fun, so by all means stick it on your iOS device and enjoy. $4.99 iPhone App - Designed for the iPhone, compatible with the iPad Released: 2010-12-02 :: Category: Games $9.99 iPad Only App - Designed for the iPad Released: 2010-12-02 :: Category: Games [via SlidetoPlay] |


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With over 750 channels of entertainment, from movies and television to music and games, you'll never run out of things to watch and do with ice. Here's a preview of what's playing this month.
Sorry, entertainment information for your date of travel is currently unavailable. Please see the following entertainment listing for what's on this month.
Recounting the inspiring and entertaining life of world-renowned movie critic and social commentator Roger Ebert. Watch preview
Directed by: Steve JamesStarring: Roger Ebert, Chaz Ebert, Gene Siskel


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This Means War, one of the upcoming movies Abigail is in, has been moved back to Feb 17th!
"This Means War" moved to Feb 17th
Comments are closed. Latest ImagesAdvertisementFamily SitesAffiliatesKatee Sackhoff and Michael Trucco Fansite Kathryn Morris Kyra Sedgwick Powered by Affiliationally Abigail on TwitterCurrent Projects
The Haunting in Georgia (2012)
Role: Lisa Wyrick Status: Released Official Site / IMDb / Photos Chasing Mavricks (2012) Role: Of Men and Mavericks Status:On DVD Official Site / IMDb / Photos Oz: The Great and Powerful (2013) Role: May Status: March 8th 2013 Official Site / IMDb / Photos Rectify (2013) Role: unknown Status: April 22nd on Sundance Channel Official Site / IMDb / Photos One Heart (2013) Role: Amy Hogan Status: finished Official Site / IMDb / Photos CausesSite Stats
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Online: Dec 2010 Host: Fan Sites Network - Privacy Policy - DMCA Abigail Spencer Fan is no way affiliated with Abigail. This is merely a fansite and I claim no rights to any celebrity endorsed material. No copyright infringement intended. All images are copyrighted to there original owners. If you find anything that is yours and you want it taken down, feel free to send me an email and I will take it down immediately. Designed by: modified designs |


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Antarctic rescue: US ice-breaker to help stuck ships
A US ice-breaker is to help two ships that are stuck in thick ice in Antarctica following a rescue mission.
The Polar Star has been asked to cut a path through the ice in order to free the Xue Long and the Akademik Shokalskiy, the US Coast Guard said.
The Chinese boat, Xue Long, became ice-bound after helping to rescue 52 passengers stranded on Akademik Shokalskiy on Thursday.
The Russian research vessel has been trapped since 24 December.
Safety of lifeThe US ice-breaker is responding to requests for assistance from the Australian authorities as well as from the Russian and Chinese governments, the US Coast Guard said in a statement.
"Our highest priority is safety of life at sea, which is why we are assisting in breaking a navigational path for both of these vessels," said Vice Adm Paul Zukunft, commander of Coast Guard Pacific Area.
The Polar Star, which is specifically designed for open-water icebreaking, was due to leave Sydney, Australia, on Sunday and is expected to arrive at the stranded ships in a week's time, according to the Australian Maritime Safety Authority (Amsa).
It comes after the Xue Long confirmed it was trapped following a rescue mission to remove passengers on board the Akademik Shokalskiy.
On Thursday, a helicopter from the Chinese ship transferred 52 people from the Akademik Shokalskiy to the Australian vessel, the Aurora Australis.
Most of the Russian crew-members stayed on board the Akademik Shokalskiy.
The Russian research vessel became trapped by thick floes of ice driven by strong winds, about 1,500 nautical miles south of Hobart - the capital of the Australian state of Tasmania.
It was being used by the Australasian Antarctic Expedition (AAE) 2013 to follow the route explorer Douglas Mawson travelled a century ago.
The Return to Mawson's Antarctica
- The 2013 Australasian Antarctic Expedition repeats scientific investigations made by Douglas Mawson and his team between 1911 and 1914
- Andrew Luck-Baker and Alok Jha are reporting on the expedition for Discovery on the BBC World Service
- Listen to Part One, Part Two and Part Three of their story, or get the Discovery podcast
Several attempts to break through to the ship by sea - by the Xue Long, Aurora Australis and French-flagged L'Astrolabe - failed because of the thickness of the ice.
The team on board said the ship was stocked with food and was in no danger.
There are 111 individuals on the Xue Long and 22 remaining crew on the Shokalskiy.
The Aurora Australis will now transport the rescued passengers to Hobart, once it has completed a re-supply mission at the Casey research base on the Windmill Islands, just outside the Antarctic Circle.
Meanwhile Prof Chris Turney, co-leader of the AAE 2013, has defended the scientific value of the expedition and rejected claims it was a "tourist trip" hampered by poor preparation.
Writing in the UK's Observer newspaper, he said the trip had been struck by bad luck as opposed to human error. He said it was an important scientific expedition and its success would ultimately be measured by peer-reviewed studies.
The Polar Star is 399ft (120m) long with a maximum speed of 18 knots, the US Coast Guard said. It can continuously break 6ft (1.8m) of ice at three knots, and can break 21ft (6.4m) of ice by backing and ramming.
The vessel has been diverted from its mission to break a channel through the ice to resupply the US Antarctic research station on Ross Island - but will return to that task once it has freed the trapped ships.


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Seven wonder of World.
Ganga Aarti.
Kashi Vishwanath temple.
The Capital of Uttar Pradesh.
Allahabad Maha Kumbh Mela.
Map of the Uttar Pradesh.
Uttar Pradesh Association & Suburbs of North America (Or well known as 'UPASNA') is the biggest UP'iate organization in North America. UPASNA was founded in Dallas, TX in 2013 with UP'iate's from across North America and was incorporated as a non-political, non-religious and non-profit organization. UPASNA's mission is to unite and organize the people belonging to the Indian State of Uttar Pradesh, people who have connections with the Indian State of Uttar Pradesh across North America under single umbrella with the goal of preserving their cultural heritage, to identify and address social, cultural and educational needs of North American UP'iates. Read more..


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Democracy Now! (9 am), for April 26, 2012 - 9:00am
Download this clip (mp3, 10.27 megabytes)
Play this clip in your Computer's media player
CISPA Critics Warn Cybersecurity Bill Will Increase Domestic Surveillance and Violate Privacy Rights; Targeted Hacker Jacob Appelbaum on CISPA, Surveillance and the "Militarization of Cyberspace"; Obama Admin Challenges Arizona Anti-Immigrant Law While Overseeing Unprecedented Mass Deportations.
April 26, 2012
U.S. Approves Expansive Drone Strikes in Yemen
Military Judge Rejects Dismissal of Manning Charges, Sets Trial Date
Supreme Court Weighs Arizona Anti-Immigrant Law
Connecticut Abolishes Death Penalty for Future Cases
Ex-Liberian President Taylor Convicted of War Crimes
Up to 69 Killed in Syria Blast
Israeli General Doubts Iran Seeking Nukes
Murdoch Appears Before U.K. Phone Hacking Panel
Mexico Probes Wal-Mart Bribery Claims
Occupy Activists Protest GE Shareholders' Meeting
Senate Backs Postal Service Debt Bill
Pentagon Orders Review for Anti-Islam Material
Transgender African-American Woman Faces Controversial Murder Trial
ACT UP Activists Detained at 25th Anniversary Protest
Protests Held Nationwide as Student Debt Hits $1 Trillion
More from this show: About the Show | Archives of this Show | Playlists from this Show


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2.10.14 (via Race Chaser Online) - If you want an example of how to make a difference in the world both on and off the race track, look no further than Andretti Autosport driver Zach Veach.
In just four years, Veach has gone from go-karts to the cockpit of an open-wheel Indy Lights machine, handpicked by Michael Andretti for a coveted spot on one of the most successful American open-wheel teams, Andretti Autosport, beginning in 2010.
The talented 19-year-old from Stockdale, Ohio, has proven in a short period of time that he is one of the top young prospects in open wheel racing today, working his way through the U.S. F2000 championship and Star (now Pro) Mazda Series on the Mazda Road to Indy. Veach has recorded top ten points finishes in three of the four years he has competed on the Road to Indy, finishing fifth and fourth in U.S. F2000 in 2010 and 2011 and tenth in Star Mazda in 2012 before making the leap to Indy Lights competition in 2013. Veach also scored the U.S. F2000 Winterfest championship in 2011.
"We've really just moved right up the ladder, and it's been a lot of fun," Veach said in a recent interview with Race Chaser Online. "Working with Andretti Autosport the last several years, I've really found a place where just being a part of the team is as fun as what I actually get to do on the race track."
Veach came into Indy Lights with high aspirations in 2013, his fourth season with the Andretti operation, but mechanical failures in the first three races derailed the team's title hopes. Veach rebounded in the second half of the season to score a total of six top five and twelve top ten finishes, including a pole effort at the season finale in Fontana and a season best third-place finish at the Milwaukee Mile.


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Abramson Cancer Center of the University of Pennsylvania
Ultima Vez Modificado: 21 de marzo del 2007
Conference Dates: May 11, 2007
Conference Location: Hilton Hotel, City Line Avenue, Philadelphia, PA
Sponsoring Group: Abramson Cancer Center of the University of Pennsylvania
Who Should Attend: Patients and families whose lives have been affected by melanoma.
Conference Agenda: Join us for this informative patient and family conference focusing on diagnosis, treatment, survivorship issues, research advances and complementary therapies for melanoma.
Conference Information: Please view the Conference Brochure [PDF].
Jun 23, 2011 - Screening for early melanoma detection should be focused on the at-risk population, according to a study published in the June issue of the Archives of Dermatology.


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5 A casino in your pocket!Zomb1e | 23/07/2008 | See all Zomb1e's reviews (67)make yourself a (virtual) millionaire.lots of different games, hours of fun.
2 A casino no, a childs archade maybe.Kaci007 | 19/01/2011 | See all Kaci007's reviews (8)Ok so maybe I expected a bit much from this game. I love casino games, I play poker and go have the occasional gamble, so my expectations are pretty high.This game only has a handful of games and can get very repetative very quickly. Ok so you strive to make yourself a millionaire and probably enter a competition to do so, I didn't get that far even. It bored me to death too quickly.Considering the rating is 18 I wouldn't bother suggesting this to adults and would imagine my 11 year old nephew would find it interesting for a while. (I know the rating is to protect kids from suggestive behaviour)I wouldn't bother with this and would seek an alternative, use your money wisely on another game because this really isn't worth the money.


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Protecting credit unions and the consumers who own them through effective regulation
The mission of the NCUA is to facilitate the availability of credit union services to all eligible consumers, especially those of modest means, through a safe and sound credit union system.
In this section, you can find credit unions and research general and financial information. Other statistics, data, and reports include the archived Quarterly Call Reports, Financial Performance Reports, and Share Insurance Queries.
Find and research specific credit unions; locate educational resources on financial literacy, privacy, and identity protection; and read about the regulations that govern credit unions and corporate credit unions.
This section details the laws and regulations that govern NCUA-insured credit unions. Here you can access current and proposed regulations, regulatory alerts and policy statements, as well as guides, manuals and forms.
View the latest news, media and events at NCUA, including newsletters, speeches, and press releases.
Home > Regulatory, Publications, and Reports > PEFCO Securities
You have asked if secured notes issued by the Private Export Funding Corporation (PEFCO) are permissible investments for federal credit unions (FCUs). As discussed below, the answer is yes.
FCUs are authorized to invest in, among other things, "obligations of the United States . . . or securities fully guaranteed as to principal and interest thereby" and "obligations, participations, securities, or other instruments of, or issued by, or fully guaranteed as to principal and interest by any . . . agency of the United States." We understand that interest payments on the PEFCO notes are explicitly guaranteed by the Export-Import Bank of the United States, which is an agency of the United States. We further understand that principal payments on the PEFCO notes are backed by the principal of a collateral pool of assets that are either issued or fully guaranteed by the United States or an agency of the United States.
Accordingly, as principal and interest payments on PEFCO secured notes are issued by or fully guaranteed by the United States or an agency of the United States, such notes are permissible investments for FCUs.
Please contact Staff Attorney Lisa Henderson or me with any questions.


text: Jakpak does Colorado View full-size image < previous next > Is this photo a winner? "My husband and I took our family for a spring break trip this year. We haven't been able to travel for several years, so this was extra special for everyone. I surprised the family with the hoodies and everyone was so excited! The kids wore them everyday. This was my first order with CustomInk and it will not be my last. Such wonderful customer service and exceptional product. Thank you for making our memories even better! " - Jakpak Spring Break (Mar 23, 2013) Share this photo on: Twitter Facebook Pinterest Similar Photos: colorado | event vacation | snow | spring break | hoodies | sweatshirts Feeling inspired? Visit the CustomInk Design Lab to start your own group order. Design Your Own


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Hi there. I've got a 2001 Mk3 Mondeo, a Ghia X 2.5l V6.
First of all is the brake light supposed to come on when I've got the clutch depressed? Seems to come on only when the clutch is very lightly touched then go off again or it comes on and stay on if I've got the clutch pressed right down.
Secondly, the central locking seems to have developed a mind of it's own, but only in damp or cold weather (it does occasionally do it on hot sunny days but seeing as we haven't had many of them lately...). Basically it has this habit of locking itself again after I unlock it. Sometimes it'll do it up to three times after I unlock it, other times it'll just stay unlocked for about a minute or so before locking itself again (usually by that point I've got in the car). On other occasions if I'm in the car and press the lock in on the door handle it will lock [i]that[/i] door but the central locking doesn't lock the other doors, as if it wasn't work. Usually when this happens after a while only that door unlocks itself. It's most peculiar and I presume it's some sort of fault due to the damp or perhaps there's water somewhere it shouldn't be.
Anyway thanks for any insights you can provide.


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Since I am retired for the season I thought I would share my defensive thoughts on my personnel set up and the formation I run out of.
I use the Atlanta Falcons defensive playbook.
It has 4-3, 46, and Nickel 3-3-5.
I mostly run out of the 46.
Here is how I set up my defensive personnel.
LE I want this to be my best pass rusher
DT run stuffer
DT run stuffer
RE Probably look for a combo run stuffer and pass rusher
MLB I want this to be my best OVR LB. He will be on the field for me at all times. Looking for hit power, speed and zone, and AWR.
LOLB I tend to send this guy after the QB a bit, so I look for some Speed and pass rushing stats. Also looking for zone
ROLB look for hit power and zone coverage
When it comes to my secondary I am quite a bit different than most people. I typically try and take my 2 best secondary players and play them at FS and SS because the middle of the field is abused on this game and also people tend to throw to their slot WRs and TEs more than the outside.
FS I am wanting my best AWR and Zone guy here.
SS I am looking for man coverage, AWR and typically I make this my best cover guy.
CB 1 I am looking for press coverage and speed
CB2 press coverage and speed.
Honestly when it comes to the outside CBs I feel like SPD and press are the two most important things because they are either coverage an curl route, out route or a deep route. Most of the time you cant stop the curl or the out, so I just want a guy who will not get toasted deep.
CB3 and CB4 I typically use my original FS and SS in these spots, because when I got 46 speed package they are my underneath coverage and since they are safeties they arent terrible against the run.
An example of a team depth chart I have used. Dallas Cowboys.
LE - D. Ware
DT - Ratliff
DT - Hatcher
RE - Spencer
MLB - Sean Lee
LOLB - Bruce Carter
ROLB - Ernie Simms
CB1 - Mike Jenkins
CB2 - O. Scandrick
CB3 - Sterling Moore
CB4 - Barry Church
FS - Brandon Carr
SS - Mo Claibourne
Plays I run out of the 46 defense.
Normal package
Cover 3 zone with man align. I use this as a base coverage, sometimes I will hot route the light blues to purples to protect against the corner or curl route.
Cover 2 zone with man align and backed off coverage. I like to back off the coverage because if you move the FS back to normal depth it looks like cover 4 and since I run a lot of cover 4, I like to use it as a change of pace to try and get a quick throw. Always user your MLB on this play to cover the middle deep of the field.
Cover 4 Typically I will change to the speed package and hot route 2 purples on this play. There are 3 underneath yellows and I leave the MLB in his yellow and change the other two to purples. This is a good long down and distance coverage.
Cover 2 man with base align. Here is your basic man coverage. Sometimes I will hot route the SS to a purple and then user the FS and it turns into a modified cover 1.
I also will drop a DL on certain cover 2 man plays and rush 3 guys.
SS Blitz typically I just bring down the SS to the LOS outside of the LE and rebltiz him
Inside blitz all I usually do is man align and press coveage. Key is to take away their first option and the blitz should hit home.
Cover 3 with man align and then re-blitz the MLB and the LOLB. This is the zone version of the inside blitz. I like to user the FS and bring him closer to the LOS to try and take away the quick hitter in the vacting zone. Remember not to get to sucked in though, if your blitz doesnt hit him.
Fire zone 3 I typically base align, then I show blitz, then I spread and crash out the DL and then I reblitz the LE. The LE should come free. Typically I like to hot route the SS to a purple zone on this play and then I user the FS and just kind of play ball hawk.
So these are the plays I like to use and I typically will mix them up and set them up as much as possible. I try to stay in the 46 and 46 speed as much as I can because the formation all looks the same and it becomes harder to know what I am doing.
Thought I would share.


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God's wisdom (Proverbs 8:25): "Before the mountains were settled, Before the hills I was brought forth."
Originally from the eastern coast of Sumatra, the Kerinci fled from local Muslim Sultanates in an ancient war and moved into their existing homeland high in the Bukit Barisan Mountains. They speak Kerinci and Bahasa Indonesian.
Most of the 260,000 Kerinci are farmers harvesting rice, potatoes, coffee, cinnamon, and even resin and rattan from the jungle areas. Those living near Lake Kerinci and other small lakes are fishermen. The Kerinci have been able to resist assimilation with the stronger lowland peoples.
Mass Relocations
Today their isolation is being changed by government-sponsored mass relocations of Jawa, Sunda, and Bali people for plantation projects on their rich soil. In addition, a world-class national park is being developed by the World Wildlife Fund to preserve the rainforest, flora, and fauna. Islam is the majority religion of the Kerinci, but they still hold to animism, especially as it is exhibited by their use of traditional healers and magic to bless their crops.
A young woman from a conservative Muslim background is now a follower of Christ. Her brother heard of this, but was sure she would soon return to Islam. When he saw that she hadn't, he demanded that she renounce her faith and say the Islamic creed. She refused, and he grabbed a metal rod and threatened to kill her. The family stopped him, but her mother sobbed and pleaded with her to say the creed. She finally did, but deeply regretted it afterward. When she shared her sorrow with another believer, her friend explained that the Apostle Peter also denied Jesus, but was forgiven and restored. She has now experienced this same grace and says, "I never want to deny the Lord ever again."
Dreams and Visions
A recently baptized believer just out of prison was greatly troubled by questions which another person brought up about the reliability of the New Testament. He said he was full of doubts and fell asleep with a Bible next to his bed. In the middle of the night he was awakened by a man who pointed to the Bible and said, "Don't be afraid. Read my book. I'm always with you." Later, he realized it was Jesus who had spoken to him, and he was greatly encouraged.
Prayer Starters:
- Ask the Lord to give wisdom and favour to believers going among the Kerinci of Indonesia.
- Pray for God's wisdom and authority in dealing with the spiritual forces that have kept the people bound for generations (1 Cor. 1:30, 2 Cor. 10:4).
Listen to the Podcast – DAY 18 – The Kerinci People of Indonesia
Video: Church Planting Among Muslims (1:20)
Indonesia Trailer from Techinc Productions on Vimeo.


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Instagram Users Are Perfectly Parodied Using Nickelback's 'Photograph'!
We totally don't take all of these pictures ourselves…
Boy With Asperger's Is Ecstatic After Train Set Surprise!
Everybody loves Nickelback, right? Right?! Well, the people over at CollegeHumor have taken Nickelback's infamous song "Photograph" and put a hilarious Instagram twist on it.
Thanksgiving Claims Title As Instagram's Biggest Day Ever
Hitting almost every Instagram stereotype including food, cute kittens, airplane wings, and all those wonderful filters, it has taken the Internet by storm. We love it, but still think our pictures are art. Check it out below!