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Host Response and Inflammation
Calprotectin Expression by Gingival Epithelial Cells
Karen F. Ross, Mark C. Herzberg
Karen F. Ross
Department of Preventive Science, School of Dentistry, University of Minnesota, Minneapolis, Minnesota 55455
Mark C. Herzberg
DOI: 10.1128/IAI.69.5.3248-3254.2001
Calprotectin, a heterodimer of MRP8 and MRP14 with antimicrobial properties, is found in the cytosol of neutrophils, monocytes, and human gingival keratinocytes. During inflammation of the oral mucosa, the expression of immunoreactive calprotectin appears upregulated. Given the possible cell sources, we sought to learn if epithelial cells upregulate calprotectin in response to proinflammmatory agents. First, human gingival keratinocytes were maintained in primary culture until senescence. At each passage, cells were harvested and analyzed for quantitative expression of MRP8 and MRP14 subunit mRNA by RNase protection assays and calprotectin complex by enzyme-linked immunosorbent assay. Calprotectin expression was constitutive in the primary gingival keratinocytes, but calprotectin-specific mRNA and protein tended to increase as the cells neared senescence. To test whether calprotectin expression was inducible, immortalized gingival keratinocyte cultures were treated for 2 to 4 h with lipopolysaccharide (LPS) or interleukin-1β (IL-1β). As a positive control for inducible expression, immortalized keratinocytes were incubated with phorbol myristate acetate (PMA) (50 ng/ml) for 24 h. Incubation with PMA stimulated increased expression of MRP8 and MRP14 mRNA within 2 h, peaking within 5 h. MRP8- and MRP14-specific mRNA expression by immortalized keratinocytes appeared to be unaffected by LPS or IL-1β. In contrast, LPS, IL-1β, and PMA each upregulated IL-8. These data show that calprotectin mRNA is expressed constitutively in cultured keratinocytes, while expression by immortalized cells appears to be independent of the exogenous proinflammatory agents LPS and IL-1β.
Calprotectin appears to be constitutively expressed by squamous mucosal epithelia but is expressed by normal epidermis only in certain inflammatory skin diseases. During inflammation of the skin and mucosa, keratinocytes can actively contribute to immunological events that occur by expressing class II major histocompatibility complex (MHC) molecules (2), cell adhesion molecules (4, 14), and various cytokines (22, 40). Concomitantly, calprotectin is induced in skin and may be upregulated in mucosal epithelium as detected immunohistochemically. A major constituent in the cytoplasm of granulocytes and monocytes, calprotectin constitutes up to 45 to 60% of the total cytosolic protein in neutrophils (9, 13) but is absent in resident tissue macrophages. The biological functions of the calprotectin complex are poorly defined.
Calprotectin is a heterodimer of two noncovalently associated polypeptides, MRP8 and MRP14 (10.8 and 14 kDa, respectively). The calprotectin heterodimer is anionic and calcium binding. MRP8-MRP14 (31) synonyms include cystic fibrosis antigen (8), L1 antigen (1, 7), calgranulin A and B (42), and S100A8 and S100A9 (36, 43). MRP8 and MRP14 belong to the S100 protein family, whose members are involved in cell cycle progression, cell differentiation, and cytoskeletal membrane interactions with the plasma membrane (20). MRP14 phosphorylates in both monocytes and neutrophils (9). The phosphorylated amino acid (Thr-113) is within a domain with a sequence identical to neutrophil immobilizing factor (41; P. Freemont, N. Hogg, and J. Edgeworth, Letter, Nature 339:516, 1989), suggesting that calprotectin may be important in localization and adherence of myeloid cells during inflammation. Calprotectin complex, but not its subunits, has been shown to inhibit the activity of casein kinases I and II and protein kinase-mediated stimulation of RNA polymerase (25, 27), suggesting a role for the complex in gene regulation and cell differentiation.
Calprotectin also shows antimicrobial activity in vitro against the periodontopathic bacterium Capnocytophaga sputigena, as well as Candida strains, Escherichia coli, Staphylococcus aureus, and S. epidermidis (24, 28, 38,39). Suprabasal oral gingiva shows constant expression of calprotectin detected by 27E10, a monoclonal antibody that detects the complex form of calprotectin (5, 37). In periodontitis, gingivitis, and other oral mucosal inflammatory diseases, the expression of immunoreactive calprotectin increases in the gingival epithelium (11, 37). With periodontitis, calprotectin in the gingival transudate (crevicular fluid) increases in concentration directly with the severity of the lesions (18). Yet the cellular source(s) of immunoreactive calprotectin in the gingiva during periodontitis and other mucosal inflammatory states is unclear. Increased tissue calprotectin may be produced by neutrophils during infiltration and transmigration through the mucosal epithelium. Similarly, gingival keratinocytes may increase expression in response to proinflammatory molecules. In this study, we tested the influence of the proinflammatory agents lipopolysaccharide (LPS) and interleukin-1β (IL-1β) on the expression of MRP8 and MRP14 mRNA in human gingival keratinocytes.
Cell culture and RNA isolation.Using an informed consent protocol that was reviewed and approved by the Human Subjects Institutional Review Board of the University of Minnesota, healthy human gingiva was excised from sites overlying impacted third molar teeth. Gingival keratinocytes were isolated and cultured by the method of Oda and Watson (30) and passaged in KGM (Clonetics; BioWhittaker, Inc., Walkersville, Md.). The cells were propagated through successive passages to senescence. Cells were plated at 5,000 cells/cm2 in T75 flasks and grown until the epithelial layer reached approximately 70% confluency. Cells were then harvested by trypsinization and seeded equally into two new T75 flasks. One flask was used for analysis of calprotectin mRNA and protein antigen expression, while the other flask was used for further propagation of the cell line.
Immortalized human gingival keratinocytes (a gift from R. Lamont and D. Oda, University of Washington) were passaged in Keratinocyte SFM (Gibco-BRL, Grand Island, N.Y.) containing 50 U of penicillin and 50 μg of streptomycin per ml. Immortalized keratinocytes were stimulated by incubation with lipopolysaccharide from E. coli O55:B5 (Sigma, St. Louis, Mo.), phorbol 12-myristate 13-acetate (PMA; Sigma) or recombinant human IL-1β (R&D Systems, Inc., St. Paul, Minn.). Total RNA was extracted from keratinocytes using TRIzol (Gibco-BRL).
Immunohistochemistry.Human gingival keratinocytes and immortalized human gingival keratinocytes were grown overnight on coverslips, and then cells were washed once with Dulbecco phosphate-buffered saline and fixed with 4% paraformaldehyde for 10 min. The cells were then washed, permeabilized with 0.5% Triton X-100 for 2 min, washed again, and subsequently incubated with 27E10 monoclonal antibody or biotinylated 27E10 (Bachem Bioscience, King of Prussia, Pa.) which was diluted 1:50 in 3% bovine serum albumin in PBS. Cells were incubated overnight with antibody at 4°C, washed, and then incubated for 2 h with either goat anti-mouse Cy3 (Jackson Laboratories, Inc., West Grove, Pa.) or Alexa350-conjugated streptavidin (diluted 1:200; Molecular Probes, Eugene, Oreg.). Cells were mounted with Fluoromount G (Southern Biotechnology, Birmingham, Ala.), examined with a Nikon Eclipse epifluorescence microscope, and photographed using a Spot Camera and software (Diagnostic Instruments, Inc., Sterling Heights, Mich.).
Cytosol extraction and ELISA.Cytosol was released from keratinocytes using digitonin according to the method of Bakouche et al. (3). In brief, keratinocytes were suspended in 500 μl of sucrose-KCl medium (125 mM sucrose, 60 mM KCl, 3 mM K-HEPES; pH 7.1) containing 0.02% digitonin (Sigma), incubated on ice for 5 min, and pelleted by centrifugation at 200 × g for 5 min. The cytosol-containing supernatant was assayed for protein concentration by the BCA Assay (Pierce, Rockford, Ill.). MRP8, MRP14, and MRP8-MRP14 were analyzed in triplicate using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Bachem Bioscience) which detects MRP antigens by a sandwich ELISA.
Labeled RNA probe preparation.Templates for RNA probe synthesis were generated by PCR from cDNA fragments of MRP8, MRP14, IL-8, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cloned into pCR-Script Vector (Stratagene, La Jolla, Calif.). The radiolabeled probes were synthesized under the following reaction conditions: 500 μM concentrations each of rCTP, rGTP, and rATP, and 1 μM rUTP; 3 μM [α-32P]UTP (800 Ci/mmol, 10 mCi/ml) (DuPont NEN Research Products, Boston, Mass.); 0.5 μl of PCR template; 1 U of T7 or T3 RNA polymerase (Stratagene); 2 μl of transcription buffer (Stratagene); 40 U of RNase inhibitor (Promega, Madison, Wis.); and distilled H2O to a total volume of 10 μl. Nonradioactive UTP (1 μM; see above) was added to reduce premature termination of RNA transcripts. To lower the specific activity of the internal control GAPDH, 150 μM rUTP was added. The reaction mixture was incubated for 1 h at 40°C and then inactivated at 95°C for 2 min. After the mixture cooled to 37°C, 0.5 μl of RNase-free DNase (Promega) was added for an additional 15 min to digest the DNA template. The reaction mixture was diluted with an equal volume of gel loading buffer (Ambion, Austin, Tex.). Single-stranded RNA was then purified by gel electrophoresis in a 5% acrylamide–8 M urea gel. After exposure to autoradiographic film the full-length transcript was excised from the gel and eluted overnight in elution buffer (Ambion). Before gel electrophoresis and after elution of the labeled probe from the gel, 1-μl aliquots were sampled for scintillation counting, and the specific activity of the probe and yield of the reaction were determined.
RNase protection assay.RNase protection assays were performed using the RPA II Kit (Ambion) according to the manufacturer's protocol. Briefly, each reaction contained 8 μg of total RNA, 8 × 104 cpm of the 32P-labeled probe (gene of interest), and 2.5 × 104 cpm of the housekeeping gene probe (GAPDH). Control reactions were performed simultaneously using 8 μg of yeast tRNA. After hybridization for 18 h at 42°C, single-stranded RNA was digested for 30 min at 37°C by using a mixture of RNase A and T1. Reaction products were separated on a 5% acrylamide–8 M urea gel and exposed to a storage phosphor screen (Molecular Dynamics, Sunnyvale, Calif.). The screen was scanned using a Storm 840 imager (Molecular Dynamics) in storage phosphor mode. Signal intensity was quantified using ImageQuant software.
Immunohistochemistry.Human gingival keratinocytes and immortalized human gingival keratinocytes were grown overnight on coverslips. In both types of keratinocytes, calprotectin was detected with monoclonal antibody to 27E10, which specifically recognizes the complex of MRP8-MRP14 (5). Reactivity to the 27E10 epitope was seen in the cytoplasm with the strongest immunofluoresence detected in a perinuclear distribution (Fig. 1). A similar distribution of the 27E10 epitope was detected in both the immortalized and the human gingival keratinocytes.
27E10 reactivity in cultured immortalized and human gingival keratinocytes. Gingival keratinocytes were fixed and permeabilized before reaction with the antibody 27E10 or biotinylated 27E10 against the MRP8-MRP14 complex. (A) Human gingival keratinocytes (27E10 secondary antibody Cy3). (B) Immortalized gingival keratinocytes (Alexa 350-conjugated streptavidin). Nonspecific immunoglobulin G controls showed no detectable fluoresence. Magnification, ×400.
Calprotectin protein antigen and mRNA expression by cultured gingival keratinocytes.Healthy gingival keratinocytes were isolated from four individuals. Cells were cultured for successive passages through senescence. Cells survived an average of 9 passages (range, 6 to 16) before death. Keratinocytes harvested at each passage were analyzed for cytoplasmic MRP8 and MRP14 subunit antigens and MRP8-MRP14 complex by sandwich ELISA. The calprotectin complex was expressed at each passage and through cell death by each keratinocyte cell line (Fig. 2A). At later passages, expression of calprotectin tended to increase. At each passage, MRP8 and MRP14 were expressed below the detection limits of the ELISA. At each passage, mRNAs specific for MRP8 and MRP14 was detected by the RNase protection assay. Relative expression of MRP8- and MRP14-specific mRNA appeared to increase in successive passages to about threefold, after which expression appeared to plateau (Fig. 2B and C).
Expression of calprotectin in gingival keratinocytes. (A) Calprotectin complex (MRP8-MRP14) was detected in keratinocyte cytosol by sandwich ELISA. Results are representative of four experiments. (B and C) MRP8 (B) and MRP14 (C) mRNA in keratinocytes detected by RNase protection assay.
Expression of MRP8-, MRP14-, and IL-8-specific mRNAs by immortalized keratinocytes.Immortalized keratinocytes expressed MRP8- and MRP14-specific mRNAs under normal culture conditions; IL-8 mRNA was expressed at relatively low levels (Fig.3). Cells were incubated with 50 ng of PMA per ml for 24 h. Within 1 h, IL-8 mRNA expression was elevated, peaking with a 15-fold increase at 5 h (Fig.4). After 5 h of incubation with PMA, IL-8 mRNA levels declined but remained significantly elevated from the baseline level through 12 to 24 h.
Constitutive expression of IL-8, MRP8, and MRP14 mRNA in immortalized keratinocytes detected by RNase protection assay. IL-8-, MRP8-, and MRP14-specific mRNA are represented by single protected fragments of 374, 282, and 333 bp, respectively. The internal control GAPDH included in each assay tube is represented by a band at 220 bp.
Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative RNase protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total RNA from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation (n = 3) .
IL-8 mRNA expression was then measured in immortalized keratinocytes as a positive control for stimulation of cultured keratinocytes by PMA, LPS, and IL-1β. The same mRNA sample was then analyzed by quantitative RNase protection assay for the levels of MRP8- and MRP14-specific mRNAs. The expression of MRP8- and MRP14-specific mRNAs increased 3- and 1.5-fold above constitutive levels, respectively, upon treatment with 50 ng of PMA per ml (Fig.5). Expression of both mRNAs appeared to maximize at about 8 h. Subsequently, MRP8 mRNA levels declined, while MRP14 mRNA remained elevated through 24 h.
Regulation of MRP8 and MRP14 mRNA expression in immortalized keratinocytes. Quantitation of MRP8-specific (A) and MRP14-specific (B) mRNA in immortalized keratinocytes following induction with 50 ng of PMA per ml for 24 h. The signal intensity determined for the MRP8 and MRP14 protected fragments was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation (n = 3) .
Treatment of the immortalized keratinocytes for 2 h with LPS (10 μg/ml) induced a 2.6-fold increase in the expression of IL-8 mRNA above that of the control (Fig. 6A). Incubation with IL-1β (10 ng/ml) for 2 h resulted in a 1.8-fold increase in IL-8 mRNA expression (Fig.7A). After treatment with LPS (0.1, 1, and 10 μg/ml) or IL-1β (0.1, 1, and 10 ng/ml) for either 2 or 4 h, the expression levels of MRP8- and MRP14-specific mRNAs were similar to the constitutive levels obtained from untreated immortalized cultures (Fig. 6 and 7).
Treatment of immortalized keratinocytes with LPS (0.1, 1, and 10 μg/ml) for 2 or 4 h. Immortalized keratinocytes were incubated with various concentrations of LPS (0.1, 1, and 10 μg/ml) for 2 h (black bars) or 4 h (crosshatched bars). Expression of mRNA for IL-8-, MRP8-, and MRP14-specific mRNA was determined by RNase protection assay. The signal intensity for the protected fragments was normalized to the abundance of the internal control GAPDH. The data shown are the mean ± the standard deviation (n = 3) .
Treatment of immortalized keratinocytes with IL-1β (0.1, 1, and 10 ng/ml) for 2 or 4 h. Immortalized keratinocytes were incubated with various concentrations of IL-1β (0.1, 1, and 10 ng/ml) for 2 h (black bars) or 4 h (crosshatched bars). Expression of mRNA for IL-8-, MRP8-, and MRP14-specific mRNA was determined by RNase protection assay. The signal intensity for the protected fragments was normalized to the abundance of the internal control GAPDH. The data shown are the mean ± the standard deviation (n = 3) .
In cultured keratinocytes derived from oral epithelial mucosa, the data show that calprotectin is expressed constitutively. Normal human gingival keratinocytes in culture express mRNAs specific for the calprotectin subunits MRP8 (S100A8) and MRP14 (S100A9). When expressed constitutively, immunoreactive calprotectin localizes primarily in the cytoplasm, with a higher density in the perinuclear region. The calprotectin was recovered from keratinocyte cytoplasm exclusively as heterodimeric complexes of MRP8 and MRP14. Free or unassociated MRP8 and MRP14 proteins were virtually undetectable by ELISA.
During successive passages, MRP8- and MRP14-specific mRNAs appear to increase in association with a cytoplasmic elevation in immunoreactive calprotectin. As the passage number increases, keratinocytes from patients with cystic fibrosis show a higher turnover rate than the normal controls (17). Detection of immunoreactive MRP14 (cystic fibrosis antigen) increases as keratinocytes from both sources age in culture. Based on pulse-chase experiments, the increase in calprotectin reflects the rate of synthesis, and the rate of degradation and secretion does not change (17). Consistent with these observations, the abundance of calprotectin-specific mRNAs plateaus, and immunoreactive calprotectin levels continue to increase as gingival keratinocytes approach senescence.
In the gingiva and other squamous epithelia, calprotectin localizes to the spinous and subcorneal layers of the stratum spinosum, but it is not seen in the basal or cornified cell layers (10). Immature basal cells and mature cornified cells do not express calprotectin. Indeed, the cornified cells may actually release calprotectin without replenishment by the cell. Since monolayer keratinocyte cultures do not cornify, senescence may modulate expression of calprotectin-specific messages. When grown in serum-free media, gingival keratinocytes express keratins that are typically associated with basal cells (29), yet insufficient information is available to stage the cells in culture to model the maturation of certain epithelial layers. Exposure of epidermal keratinocytes to PMA promotes keratinocyte differentiation (15). Indeed, terminal differentiation of various epithelial cell lines was linked to the expression of calprotectin (35). Therefore, calprotectin expression by epidermal and mucosal keratinocytes may be controlled in association with cellular maturation.
During mucosal inflammation, the epithelial turnover rate increases (32). Higher levels of immunoreactive calprotectin are seen in affected epithelia and in transudative fluids that bathe these tissues (11, 18, 37). In addition to keratinocytes, inflamed mucosal epithelia contain several other cell sources of calprotectin. Calprotectin is expressed by transmigrating neutrophils and monocytes, which increase in number with inflammation. Hence, we tested the influence of the proinflammatory agents LPS and IL-1β on the expression of MRP8 and MRP14 mRNA in human gingival keratinocytes.
To test for the regulation of calprotectin expression by LPS and IL-1β, immortalized gingival epithelial cells were used as surrogates for normal cells in culture. Culture of normal gingival keratinocytes is labor-intensive, with low yields and donor-dependent heterogeneity. The immortalized gingival epithelial cells used in this study were developed from human gingival keratinocytes that had been transformed with human papillomavirus type 16 E6/E7 open reading frames (16). These cells express E- and P-cadherins and show contact-dependent growth inhibition (16). Like normal cells, immortalized keratinocytes were shown to express MRP8 and MRP14 mRNA and immunoreactive calprotectin complex, albeit at lower levels (data not shown). Similarly, normal and immortalized cells constitutively expressed IL-8. Collectively, these data showed that the immortalized keratinocytes were suitable surrogates for normal human gingival keratinocytes.
In immortalized gingival keratinocytes, the expression of MRP8-, MRP14-, and IL-8-specific mRNAs increased in the presence of PMA, a result consistent with the activation of protein kinase C. Expression appears to be tightly regulated since the level of calprotectin upregulation by PMA was significant but modest compared to IL-8. In contrast, incubation with LPS and IL-1β only increased the expression of IL-8. Cells were also incubated with 1 μg of LPS per ml over a 24-h period to learn if an increase might require longer exposure. No change in the expression of MRP8 or MRP14 was observed (data not shown). These data suggest that LPS and IL-1β do not exclusively regulate calprotectin expression. The presence of LPS and IL-1β in inflamed mucosal epithelia may be insufficient to explain the apparent increase in the expression of calprotectin in keratinocytes and the interstitial tissues.
Release of calprotectin into the interstitial tissues from keratinocytes is likely to involve a Golgi-independent process. Calprotectin lacks a signal peptide, membrane anchor, or N-linked glycosylation sequence, suggesting that the protein complex is neither targeted or packaged for export nor routed to the exterior as an integral membrane protein or glycoprotein to be proteolytically cleaved and released into the external environment. In monocytes, calprotectin is released after activation of protein kinase C by a tubulin-dependent mechanism that is Golgi independent (33). In neutrophils, calprotectin is released in complex with arachidonic acid (17,19). Similar mechanisms could stimulate release from epithelial cells, since PMA, which activates protein kinase C, upregulated the expression of calprotectin in immortalized gingival keratinocytes.
Healthy human gingiva expresses calprotectin strictly in the suprabasal cells, as suggested by histopathological studies (10, 11,37). If released by keratinocytes, immunoreactive calprotectin in the tissues would be expected to increase in proximity to the suprabasal layer of cells in the stratum spinosum. As inflammation increases, calprotectin does appear to be expressed by cells throughout the stratum spinosum, within and proximal to the keratinocytes. While IL-1β and LPS would explain the upregulation of IL-8 by epithelial cells in inflamed tissues, these signals and mediators appear insufficient to explain the upregulation of calprotectin.
Calprotectin expression in squamous mucosal epithelium differs from that in skin and intestinal epithelia. Normal skin and intestinal epithelia do not express calprotectin. During inflammation, calprotectin expression is upregulated in the epidermis and intestinal epithelia (21, 23). In inflammatory dermatoses of the skin, calprotectin expression requires de novo mRNA transcription. For example, MRP8 and MRP14 are expressed and protein kinase C is activated in psoriatic epithelial cells (34). In the inflammatory skin diseases lichen planus, lupus erthematosus, and psoriasis vulgaris, 27E10 epitope expression increases, but independently of the MHC class II molecules and the adhesion molecule, ICAM-1 (22). The MHC class II molecules and ICAM-1 are upregulated during epidermal inflammation but apparently in association with keratinocyte turnover. In contrast to gingival keratinocytes, cultured epidermis upregulates calprotectin in the absence of proinflammatory agents and without concomitant upregulation of MHC class II (6). Calprotectin expression is apparently tissue specific and appears to be regulated by a different mechanism than MHC class II molecules and ICAM-1.
Keratinocyte production of calprotectin may contribute to the antimicrobial barrier function and the innate immunity of mucosal epithelia. For example, calprotectin protein and mRNA are found in the upper and middle spinous cell layers in oral pseudomembranous candidiasis (12). Fungal hyphae penetrate the parakeratin layer but appear not to extend beyond the boundary of calprotectin expressed in the spinous layer. Epithelial cells transfected to express calprotectin become significantly resistant to invasion by key mucosal pathogens, including Listeria monocytogenes and salmonellae (K. Nisapakultorn, K. F. Ross, and M. C. Herzberg, unpublished results). In contrast, calprotectin-free superficial mucosal epithelial cells appear to harbor potentially pathogenic oral bacteria (34a). Therefore, constitutive expression of calprotectin may explain in part the relative resistance of the oral mucosa to infection and invasion by commensals, oral pathogens, and enteric pathogens in transit.
We thank Kanakwan Nisapakultorn for the immunohistochemical staining of the gingival keratinocytes.
This work was supported by NIH grants P30DE09737 and RO1DE11831.
Editor: R. N. Moore
Received 26 June 2000.
Returned for modification 7 September 2000.
Accepted 19 January 2001.
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Infection and Immunity May 2001, 69 (5) 3248-3254; DOI: 10.1128/IAI.69.5.3248-3254.2001
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Okay, even in the midst of my rejection, good things are happening. Missy Foy, the ultramarathoner with type 1 diabetes whose essay was featured in this week's Parade, has agreed to contribute an essay to the anthology! I'm very excited about this, she is truly and inspiration with a great story, thank you Missy. Also, Dr. Sheri Colberg-Ochs, Professor of exercise science at Old Dominion University has agreed to contribute. Sheri has written several books about diabetes such as, The Diabetic Athlete and The 7 Steps to a Diabetes Fitness Plan. Check out her website at www.shericolberg.com.
So the anthology moves on, even without skirt! books.
FYI, I see stories about women with diabetes cropping up EVERYWHERE! I mean every time I pick up a publication, which is often!
So, here's a question. Have you been down to your local library to conduct a search on which publications are reporting on the topic?
I bet you'd find more contributors, as well.


The list cab quite literally go on.
And if there is any form of correlation with these great stars, it's that they all had to, at some point, leave the north east to aspire and achieve their dreams. I myself was the exactly same and can completely relate. When I think back to my pop idol days and watching my parents travel week in week out to London and the costs that must have incurred to them just so they can keep supporting me and allow me to follow what I believe. Overall it can get costly and very expensive. And if some people can't make those journeys due to income and costs etc why should they be restricted on their dreams.
So what I intend to do is bring all of that talent that you are expected to chase and travel for, to your door step, Dancers who have worked with some of the world's biggest stars, actors that have worked on some of the world's biggest shows, dancers that have performed and choreographed on some of the world's biggest TV shows and stages. Get these people who have the training, experience and understanding of showbusiness and affectively pass that knowledge on to the aspiring stars of the north east, ignite that spark for them so they can for that one weekend know what it's like, and what they need to do as their next steps to go on achieve as much of their dreams as they desire.


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Zoe Kravitz Recreated Her Mom's Nude 'Rolling Stone' Cover From 1988 & She Looks SO MUCH Like Lisa Bonet
Growing up Zoë Kravitz couldn't have been a simple life. Not only are you under the watchful eye of a rock star dad and then you have a Huxtable for a mom. As it turns out, Kravitz was literally a talent in the making, as a pregnant Lisa Bonet covered Rolling Stones' Hot Issue. 30 years later, Zoë Kravitz now recreates the flawless nude cover as an ode to her famous mama.
Zoë Kravitz has never been one to stick to ordinary tradition, and this steamy cover was no exception. On the original cover Bonet graced, she served readers a whole lot of "bawdy" back in 1988 save for a white shirt. Instead of sticking to the same photoshoot concept, the actress told Rolling Stone she wanted to reach into the pages of the spread to derive inspiration from a fiercer photo.
"I think she was a little bummed when they used the shirt picture as the cover," Kravitz said. "I think she just thought, 'I'm doing it, let's do it!'" Naturally, she gifted fans with a recreation of her mom's desired cover. "It's less about the picture and more about doing the thing my mom intended to do. That feels cool."
The actress decided on shooting the exact same pose and styled with the same wet hair look that her mom sported in her 20s. These shoots look eerily similar. Like, mother-like-daughter kind of similar.
In this upcoming November issue, Kravitz not only shines light on her mother's influence, but her dad Lenny as well. She also admitted that she while she loves being a part of her famous family, she wanted to make a name for herself by herself.
"I've kind of stayed away on purpose from referencing my parents, because I've been just trying to create my own identity," she told Rolling Stone. "But I feel like it's a good time. I feel comfortable in my own skin. And it's nice to do that homage to them. Because I love them! They're my parents!" Awww!
The actress, though, has seriously come into her own in the entertainment industry. Fans first got a glimpse of 21-year-old Zoë in X-Men: First Class with Hunger Games starlet Jennifer Lawrence. After her moment with the Marvel characters, she followed up with an action-packed appearance in Mad Max: Fury Road and then again in the Divergent films. And regardless of her action-adventure resume, she's even made a cameo on IFC's Portlandia to show her more comedic side.
Her last order of business in the interview was to describe her version of what "hot" means. "To me, 'hot' means someone who's vibrant and confident and comfortable with who they are," she said. "When somebody allows themselves to be who they are unapologetically — and they're a good person — that's really hot."
And needless to say, it's very similar to the answer her mom gave the issue back in the 80s. It seems that when it comes to having progressive ideals, they like to keep it all in the family.

redpajama_set_name: RedPajamaCommonCrawl


As many of you living in the Sacramento Valley know, we have had a long run of frosty mornings around here. Our outdoor thermometer has been getting a workout with overnight temperatures dipping below freezing for a few weeks. Yes, it's cold and we may not be used to it here in California, but it makes for some outdoor fun we can't get around here the rest of the year.I've been heading outside early on these cold mornings to practice with my camera and I'm pretty happy with the results. Cold. But happy.
I don't know about you all, but I feel best when I've allowed myself a moment to take in everything around me and really let it sink in. I find that joy, peace and hopefulness are most present when I'm appreciating nature: the white frosty blanket on front yards and rooftops, the way the light hits the frozen bits of dew and the crunch underfoot as I walk across a freshly frozen lawn.
So, I hope to continue capturing these moments of nature at work, paying attention to small details and appreciating things for what they are. Sounds like a life lesson, no?
The challenge is making sure I apply these tiny, cold, beautiful lessons to all things in life.
I think I'm up for the challenge. How about you?


There are only a few brands that achieve ubiquity in popular culture like Nike. In the song "Air Force Ones", rap artist Nelly dedicated his song to buying a pair of Nikes. Sneakerheads went crazy when the company launched their limited edition line called MAG. The shoes, which sold for up to $9,000, were a replica of the shoe worn by Michael J. Fox in the film "Back to The Future II".
No matter where we turn, it feels like there's a swoosh just around the corner. So how does Nike build such a great brand?
#1 Marketing is the product. The product is the marketing.
Nike has a unique skill for sponsoring the right people. From Jordan, LeBron, and Ronaldo in the sports arena to filmmaker Spike Lee or cutting-edge fashion brands such as Commes des Garçons.
Nike has led the way in connecting their product to role models and tastemakers. Their pioneering partnership with Michael Jordan redefines how sports build a brand and sell products. As of today, years after Jordan's retirement, the Jordan brand has retail sales more than $3 billion per year.
Nike has been "Just Doing It" for over 30 years now. Their brand campaign has come to be the benchmark by which all other brands compare themselves.
Just Do it started in 1988 with 80-year-old Walt Stack. Nike chose to create an inspiring and universal message that we are all athletes. And customers were mobilised. Nike's profits almost tripled in the year, and the Just Do It snowball had begun.
Fast forward 30 years and Nike still has its magic touch. This year they featured controversial NFL player Colin Kaepernick. In the three days after launching the campaign, Nike sales increased by 31% and the brand was once again on the front page.
If that's not enough for you, the biggest company on the planet sourced their inspiration from Nike too.


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Animal / birds
Take a look at this adorable baby barn owl as he runs through the forest.
The Internet has been in love with this picture in the last few days. Now, a picture of a cute baby barn owl has gone viral. It looks like it's running late for the most important meeting of its life. Hannie Heere, a Dutch photographer, took a picture of a very special moment. People can't get enough of it now.
The 63-year-old photographer from Dordrecht, Netherlands, has been a big fan of photography for most of her life. However, in the last few years, she has been taking it more seriously. But it looks like it was enough to get a picture of a once-in-a-lifetime event.
A few months ago, Hannie was outside the city looking for a good picture. She saw a barn owl chick. The cute little thing still had white fluffy chick feathers, which meant that it wasn't yet able to fly. They can flap their wings when they're about 7-8 weeks old. However, the chicks' legs are still strong enough to move around their nest.
Then, this is what happened in this case. The little owl is said to have gone for a run, and the photographer captured the exact moment. The answer is that every person on Facebook fell in love with the cute baby owl in the picture, which was very pretty.
Hannie told the PetaPixel that she was lying on the ground. This young bird hasn't learned to fly yet, but it was not afraid. About 16 feet away, I took the picture.
The photographer didn't think her picture would be so popular on social media. Instead, the picture made a lot of noise on the Internet. "I was shocked," the woman said.
If you want to see more of Hannie's work, you can go to her Facebook page.
HEDGEHOG GETS STRIPPED BY A CAR, AND THEN AN X-RAY SHOWS WHY SHE DIDN'T GIVE UP.
Golden Retrievers are kind to firefighters who are fighting wildfires in California.
Next story A cute video shows a bald eagle, a wild fox, and cats on a porch in Alaska.
Previous story Because of his valiant efforts, the adorable baby flamingo has become an overnight sensation on the internet.


From Elon Musk's lively Twitter presence to worries about self-driving cars, there's no shortage of headlines that tie back to Tesla (TSLA). Yet plenty of bulls argue that investors should tune out the noise: Once the electric-car maker starts turning in regular quarterly profits, that's all that will matter. Faith in Musk and the product will be vindicated, the company will be less beholden to debt markets, and the stock will rise.
Datatrek's Nicolas Colas thinks not. If Tesla starts delivering consistent earnings, investors will start to value it like a car maker–a high-growth auto firm, for sure, but a car maker nonetheless. And that doesn't do great things for a stock's valuation. "And after looking at that sector for more than 25 years, we know where that leads: multiple erosion as markets deliberate, 'Is Tesla really 20 times better than General Motors (GM) on a multiple basis, or just 10 times better?'" Colas writes in a report published today.
Then there's the fact that Tesla, like other big companies, will ultimately find itself hampered by its size. Stock "multiples tend to contract as already large companies grow their market share. You can only get to 100%, after all," he writes.
What's fueled Tesla's rise so far, Colas posits, is that markets have been valuing the stock the way a venture capital firm eyes up a new prospect, looking not for steady earnings, but disruption potential and swift user adoption. In this world, moving toward a stage where a company starts reporting regular earnings signals a shift in its evolution that may not be as appealing. "Profits that approximate the company's cost of capital only occur when the business has ended its explosive growth phase and can't scale into new opportunities. Then, rather than seeing the stock rise further in celebration of strong earnings, the vultures start to circle."
Tesla is scheduled to report earnings on Aug. 1, and analysts expect it to lose $2.73 a share. Hooray?
Tesla shares are up 0.6% to $324.50 this afternoon.
From Elon Musk's lively Twitter presence to worries about self-driving cars, there's no shortage of headlines that tie back to Tesla (TSLA).


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Hungry for more
Lifestyles Recent News Reviews
March 27, 2012 Blair Stalder34
Like most books created into movies, the books are usually better. The movie version of Suzanne Collins' "The Hunger Games" is in the same category. While people who never read the book enjoyed this action-sci-fi-drama, I was left wanting more.
Set in the future of North America, the United States is gone. There is only a Capitol and 12 Districts. Because of the Districts' failing attempts to overthrow the Capitol, each District must select a boy and girl (Shirley Jackson's "The Lottery" style) to compete in the Hunger Games — a televised fight to the death.
Only one can win.
The nominees' ages range from 12-to-18; it's Primrose Everdeen's (Willow Shields) first year. When her name is surprisingly called, her sister Katniss (Jennifer Lawrence, "X-Men: First Class") steps up — sprints up, rather — to volunteer.
Having read the book, it was easy to spot the changes in the movie. It's 2 hours and 22 minutes, so it's hard to fit in every little piece of information, I get it; however, there were too many slow parts and long shots of facial expressions that could've been cut down in order to make up for the loss of the important details that were either substituted or dismissed that essentially make the story.
For instance, the character Madge was not in the movie at all, and the relationships between the characters were not very strong; they felt rushed throughout the movie.
There were some other smaller parts that were changed that could have easily been avoided, but the movie's story line was overall along the same lines as the book and portrayed the scenes accurately and in a unique fashion.
As for the acting, job well done. It's not easy showing an excruciating in-pain emotion when not in pain, and Lawrence and Josh Hutcherson ("RV," "Journey to the Center of the Earth") as Peeta Mellark, Katniss' fellow District partner, did a great job. Lawrence also displayed great versatility as she played the role of a terrified and loving, yet sassy and confident older sister. She was very believable.
As for the other well-known names, like Woody Harrelson ("Zombieland") as Katniss' and Peeta's drunken mentor Haymitch Abernathy and singer Lenny Kravitz as Katniss' clothes designer Cinna, I had my doubts.
Haymitch is supposed to be the fat sarcastic slob that you love to hate, and Harrelson nailed it. Cinna, on the other hand, is the colorful and feminine role that I wasn't sure Kravitz was going to be able to play, considering his usual afro, tatted and pierced body and tough attitude. Kravitz was the adorable, caring teddy bear you just want to squeeze.
Oh, and don't forget the unforgettable Stanley Tucci ("Easy A," "Burlesque") as the over-the-top white-toothed grin of the host of the Hunger Games, Caesar Flickman. Tucci's small parts are perhaps some of the funniest, a close tie with the bickering between Haymitch and other mentor Effie Trinkett (Elizabeth Banks, "The 40-Year-Old Virgin").
For fair warning, the camera work is very similar to "Cloverfield." The jittery movements are there to make the audience feel like they are in the movie, but it just leaves an uneasy feeling.
Once again, if you read the book, you'll probably find the book better; but thankfully for the actors, the movie is still entertaining. If you didn't read the book, you will more than likely enjoy the movie version, but I encourage you to take the time to read the book because it's gold.
Western baseball swept in double header
Athlete of the Week — Natalie Bird
'Vagina Monolgue' performance proceeds go to V-Day
April 7, 2012 April 9, 2012 Christian Mengel
Western's car charging stations are go
September 19, 2014 October 1, 2014 Daniel Cobb
Let's get down to business
August 29, 2014 Daniel Cobb


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Art Institute Portland Oregon
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The Art Institute of Portland is one of The Art Institutes, a system of nonprofit schools throughout the united states. programs, credential levels, technology, and scheduling options vary by school and are subject to change.
The Art Institute of Portland is one of The Art Institutes, a system of non-profit schools throughout the United States. Programs, credential levels, technology, and scheduling options vary by school and are subject to change.
Oregon. Some institutions in The Art Institutes system are campuses of Argosy University. The Art Institute of Portland, 1122 N.W. Davis Street, Portland, OR 97209-2911 © 2018.
Eugene Oregon Art Museum All Eugene Restaurants. Restaurants near Jordan Schnitzer Museum of Art. It's unbelievable what you can find at this museum on the University of Oregon campus. It's like a set of samples from top-notch museums all over the world. University of Oregon Museum of Natural and Cultural History. Lists of public art in Oregon. Outdoor sculptures
The Art Institute of Portland, a nonprofit institution owned and operated by Dream Center Education Holdings, LLC, is not currently enrolling students. Located in Portland's Pearl District, the school has twelve computer labs, multiple animation and video labs, a recording studio, library…
oregon culinary institute is Portland's most well-known culinary school, offering Culinary Arts and Baking & Pastry Diplomas & more. Our home of Portland, Oregon, is a restaurant mecca, home to some of the world's best chefs. We recently teamed up with a couple of our top chef partners in…
The following is a partial list of institutions and individuals who are notable and active in the art scene of Portland, Oregon. Museum of Contemporary Craft. Portland Art Museum. Art Institute of Portland.
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When you arrive by ferry to Vashon Island, about 20 minutes from downtown Seattle (because there are no bridges), "modern" may not be what immediately comes to mind. That's because while this 37-square-mile island is rife with artists, organic micro-farms and even some of the best Thai food we've had anywhere (truly), the approximately 10,000 residents have embraced the rustic seaside aesthetic that's so prevalent in the Pacific Northwest islands.
It was into this relaxing setting that Jena Thornton and Scott Shapiro conceived The Lodges on Vashon – a 4.5 acre micro-community designed to welcome visitors to a more modern vision of Vashon Island, but a vision that integrates the established Vashon lifestyle into an achingly cool set of modular, modern retreats for the forward-thinking Vashon visitor.
Take the short walk to your lodge and the units, designed by Seattle firm S+H Works, immediately welcome you into a minimalist but warm embrace. From the easy-on one-button fireplace to the spa-like soaking tub, the Lodges manage to highlight and enhance the restorative sense of the Island with a clean, open design that manages to surprise considering the compact exterior. With a living/dining area anchoring one end and the bedroom opposite, all in soothing whites and neutrals, the Lodges manage to convey a Scandinavian rustic elegance that remains in sync with the rural island location while wholly embracing modern – no small task. Round out each lodge with art and furnishings largely sourced locally, and the task of integrating a modern retreat while maintaining harmony with the established island community has to be considered a success.


Syndrome of the Present is a research based project deriving from the fields of visual art. It aspires to establish a collaborative, cross disciplinary platform, shared between artists and experts from diverse geographies and disciplines. A platform, which enables collective thinking in order to analyse the Present's syndrome and the present as syndrome.
The project's starting moment is the 17th century central Europe, in which the cornerstones of present conflicts were laid. The 17th century was the moment in which the religious reformation, sovereign state, democracy, citizenship and human rights were constituted in response to decades of religious wars. This century was accompanied by outbursts of messianism and scientific development, economic and cultural progress, as signs of the expectation that the world would change for the better. Today it seems that we are heading backwards and progressing towards the state of an internal war; the sovereign state is under attack, democracy is at risk, citizenship is replaced by consumerism and human rights are fully disrespected.
Two 17th century protagonists, the 'Messiah' Sabbatai Zevi and the Philosopher Baruch Spinoza, constitute a 'time tunnel' in the project that establishes links between past and present, eschatology and politic, and create a perspective for insights into contemporary events. Both protagonists offer a revolutionary worldview and both were ahead of their time in their attempts to redefine the relations between God, man and society (state).
The 17th century has been formative for our contemporary reality in many ways: Europe's religious wars ended with the Peace Treaty of Westphalia, which exiled religion from the supreme power. Secular sovereignty inverted the monarchical paradigm: the people appropriated the king's power, turning the sources of political authority upside-down. Tolerance "as a government-sanctioned practice" in Europe's Christian countries was institutionalized at this time. The sovereign ruler and the (national) territorial state became the "administrators of tolerance" in the early modern era, and promised to secure peace within their states.
Westphalian secularization meant the beginning of Europe's institutionalization of religious tolerance and freedom. However, it also sowed the seeds for the modern grand myth of nationalism and its authoritarian suppression of heterodox movements and accounts. International law proved to be a means for the protection of sovereignty as much as a neutralized tool for the systemic oppression of the designated Other. Hence, the myth of Westphalian peace is now a challenge to overcome.
The present crisis also presents a moment of possibilities: the destruction that leads us to face the crises also opens doors to re-imagine a future where equality, dignity, wealth, justice, mobility, and education are equally shared among each one of us. In order to accomplish this, we have to know how to look back, to see what was imagined for us.


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Q&A: 'Enemies of the People' director Rob Lemkin
Filmmaker/investigative journalist Rob Lemkin teamed up with Thet Sambath to co-direct an unsettling look at Sambath's quest for truth concerning Cambodia's Killing Fields. Realscreen spoke with Lemkin about the making of the film and a new follow-up project to be released this year on DVD.
By Kevin Ritchie
Many filmmakers have tackled the unsettling and inexplicable subject of Cambodia's Killing Fields, but few have dared to look as deeply into the madness of the genocidal Khmer Rouge regime as directors Rob Lemkin and Thet Sambath's Enemies of the People.
Lemkin, a filmmaker and investigative journalist, met Thet Sambath when he traveled to Cambodian capital Phnom Penh in 2006 to make a documentary about former Khmer Rouge leaders on trial in a UN-backed court for genocide and crimes against humanity. He quickly realized that Sambath, his interpreter and a reporter for The Phnom Penh Post, had been gathering information on the subject for several years and had a long-standing relationship with former Khmer Rouge leader Nuon Chea, known as Brother Number Two during Pol Pot's genocidal regime. (His trial is set to begin in mid-2011.)
The two decided to co-direct a documentary about Sambath as he doggedly pursues confessions from Chea and a handful of ex-Khmer Rouge soldiers. Sambath, whose own family was killed during the 1975-1979 period, proves an unflappable character by refusing to give into vindictive impulses in his quest for the truth.
Produced by Oxford-based Old Street Films, Enemies of the People was difficult to make for a few reasons. Initially, the directors struggled to get financing from TV broadcasters because several other documentaries based around the UN tribunals were already in production. Moreover, the sensitive and harrowing nature of the interviews meant that the crew only consisted of the two directors.
After premiering at the International Documentary Film Festival Amsterdam in 2009, Enemies of the People won the World Cinema Special Jury Prize at the 2010 Sundance Film Festival. It has screened in theaters across the United States, is shortlisted for an Academy Award nomination in the feature documentary category, and has just received a best documentary screenplay nomination for the upcoming Writers Guild Awards. Realscreen recently spoke with Lemkin about his experience making the film and his plans for a follow-up film to be included on a DVD release in 2011.
How were you able to tell that Sambath and Nuon Chea had such a close relationship?
They felt very familiar with each other because, at that point, they'd already been working together for six years. Sambath had sort of broken the back of getting the information from him and they just seemed to have a certain understanding with each other. They seemed to be quite happy to go from very serious to almost light-hearted exchanges… I sensed that there were many layers to their relationship. The first two times that I went there, Sambath took him into the back room and would lay him down to go to sleep because he was at that point having slight problems with his blood pressure. He would take him in almost like he was his nurse as well as his confessor.
How did you feel about that?
I felt a lot of things. To be honest, I felt that it was amazing because we had a film on our hands. It was an inherent tension in that. You wouldn't expect him to behave like that. Right up until we finished filming the main part in May 2009 I always was expecting him to break out at some point and he never did.
It's a bit like he set in [with] a very ethical idea that he was not going to go and start making judgments or making accusations and start trying to indulge in personal ideas of revenge, or making himself personally feel better about his own situation. He was always going to be doing it in this much more structured way.
You use archival footage from the Khmer Rouge period sparingly. The action is mostly rooted in the present. Why that decision?
And not only is it about the present, it's [also about the] future because [for] any society that's undergone such intense and traumatic violence, the only way it can really go forward is to come to terms with its past in a profound and full-on way. That hasn't happened in Cambodia for 30 years for a number of reasons.
In a way it's more about the present and a road to the future than it is about the past, hence we tended to use the archive in order to explain and give the audience some kind of experience of the past that was being talked about… it's certainly not a history lesson.
Watching the film you get a sense that in the end, Sambath felt satisfied on some level, but I never grasped why these people would blindly follow orders – not that you can ever grasp that. Why is the 'why' so important?
In a way we have to get to the end of our process… We've made a film about Sambath's process and the film is its own process. Sambath's process did result in him feeling that he understands why all of the violence happened. When he says that, it means that he understood the rational decisions, the rational reasons for unleashing the violence in the way that it was unleashed.
In making the film, that has not been something we have so far been able to bring out. That's why we're making a second film which will go into the political complexity. It'll have the same characters in it, but will also have other people that we've also filmed and it will feel a little bit more like a political conspiracy thriller-type thing. You'll see people from both sides of the Khmer Rouge fighting it out and you will begin to see how decisions made in committee meetings over very understandable – scary, but understandable – political considerations about how to maintain power and how to maintain control of this very secret political party, which had taken over the country, [can be made]. You'll begin to see what the impulse [was] and how that impulse towards violence played out.
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We wanted to share some recent news stories that highlight our field, NWSA members and leaders, and call attention to important trends and issues.
Drew Nelson, a business major at Indiana University, never expected he would pick up a minor in the subject.
"I wasn't very interested in the subject, to be honest, but once I started doing a lot of the class readings and having discussions with people in the class, I got kind of interested," Nelson said.
Student activism sporadically reoccurred in the 2000s, when, according to University of Illinois Professor Barbara Ransby, students shape "the conscience of the university" by raising awareness about racial inequality, sexual assault on campus, immigrant rights, homophobia and unequal rights for the LGBTQ community, as well as global issues such as the Palestinian crisis.
A historian, writer and political activist, Ransby's work focuses on the civil rights movement and feminism.
See all of the women selected here.
In the introductory class on women's and gender studies at Lafayette College, students examine how gender shapes their interactions with the world.
Read more about the debate to approve a gender studies elective for high school seniors here.
We look forward to hearing your thoughts about these stories.


Can I install on a machine with no internet connection?
A single electronic license can only work on one computer but it is easy to move it around if you need to. Please contact us for pricing and more information.
Yes you can. It is called a manual installation and if there is no internet connection on registration then you just follow the instructions to get a license working. If you plan to use the machine offline then make sure it is unplugged on registration.
Our software is available directly from us. It is also available with a number of gel documentation systems. Please contact us at info@totallab.com to get more details.
I have further questions. Can I have a demonstration?
Yes we can organise an online demonstration with a member of our team if required. If you have your own images already then they can be used in any analysis we do. We are always interested in our customers questions.
Is it possible to have a trial of CLIQS 1D Pro?
Click here to download a free trial of CLIQS 1D Pro.
I haven't had a chance to use the trial can I have an extension?
If you contact our sales team at info@totallab.com then we may be able to extend your trial if you have not used it yet.
All of the latest TotalLab products will work under Windows Vista, Windows 7 and Windows 8 and 10, 32 and 64 bit versions.
Why should I pay money for software?
TotalLab software is a lot easier to use than free software. It has more automation, more functionality and better reporting. Also you get high quality support from our experienced team. Please get in touch with our sales team to check out any current offers.
Why does Automatic Lane Creation fail on my gel?
The resolution of the image is very low and there is not enough band information, using an AOI for lane detection can help (just draw a box when in automatic detection mode) or simply try to create the lanes manually.
How does Molecular Sizing work?
Each lane has its own calibration curve calculated from where the iso-molecular weight lines(yellow) cross the centre of a lane. With only one standard lane these curves are usually the same as the lines just run horizontally across the gel. However with more than one standard lane it joins up the bands of the same value and so the lines can be slightly off horizontal depending on the gel.
Why use different curve types?
The default curve fit is cubic spline. This curve type creates a smooth curve passing through all points. It has no defining equation. This option can be somewhat over sensitive to inaccurate mappings but will guarantee that the results are as expected from where the iso-molecular weight lines(yellow) cross the lanes. First order Lagrange is similar but uses straight lines.
The other main option used historically is the Linear Log equation which is a straight line calculated from the log of the Molecular Size. This is theoretically correct but can lead to misleading results due to any inaccuracy of the curve fit. Even curves with high R2 results can still have problems.
What is the name of the curve fitting algorithm?
We use an iterative algorithm called Levenberg-Marquardt to get the results for the curve fitting equations. Cubic Spline and First order Lagrange are also well-defined algorithms.
My image does not look like the image I captured. Why?
A histogram of all pixel values is created. Then the default auto contrast is created from the value of the histogram after 0.5% of all pixels and at 99.5% of all pixels. This stops outliers or scanner noise affecting the result, but it can look a little different.
Note: Contrast does not affect the values used in any calculations.
My profile is upside down. What can I do to fix it?
Go to the File menu and select Invert Measurements to reverse the calculation used to generate the Profile. This option is remembered so you should not need to change it again.
What file formats can the software support?
The software can load in .tif, .tiff, .jpg,.jpeg, .png, .gif, .gel, .img (Fuji), .bmp files.
This occasionally happens as part of the loading function. Just press the invert button on the toolbar to see if reversed again.
I cannot load my tiff image?
One type of tiff image we cannot load is a multi-page tiff. These are very rare in life science and are only used for items such as multi page faxes. There are many free tools which can split the tiff into single image files including ImageMagick.
This is defined as the total value of all the pixels in a band with background subtracted. The pixels are shown on the image as between the 2 red lines (the band edges). Another way to look at it is as the area under the profile for the band (with background subtracted) multiplied by the lane width.
Where can you get the total volume of all the bands in a Lane?
This information can be found in the Analysis Report (previously called Gel Report). On the first page total band volume (and total lane volume) are displayed in a summary table of all the lanes in the gel.
This option allows the user to model each band as a gaussian and use these to recalculate the band volume. This is sometimes used to split the volume of overlapping bands in what is believed to be a more accurate manner. Advanced fitting is best but there are problems. They are firstly, that is can be very slow and secondly, without good background subtraction they can be very wrong.
Why do 1D Pro tables show all bands matched even when it is not true?
This happens when Image file names have one or more extra "." in them (e.g. fred.flintstone.tif). This is fixed in latest version when adding lanes to the database.
How do I export images/dendrograms in CLIQS 1D Pro?
To do any exporting to the clipboard or to file just right click on the image to bring up a context menu then choose the function you require.
What is the normal procedure for using Rf lines to aid matching?
I cannot move my Rf lines?
This may be because they are too close to each other. There is a limit to how close they can be and on low resolution images you may not be able to move them close to each other.
In 1D can I select which lanes to do a Dendrogram on?
Yes. Standard lanes are not matched by default and they would not be in a dendrogram (they can also be matched if required). Also, you can use Lane Selection in the Options dialog to switch off some lanes if you wish.
My profile is flat at the baseline, but I can see some background on the image?
This is probably due to Intensity Calibration errors (from Powerscan or Phoretix 1D). If your calibration curve intersects the y intersect at too high a value, then all raw values under the y intersect value will be calibrated to zero. This can lead to the background and some lane material showing as zero. Recalibrate the curve to improve your results or force it to go through the origin.
How many lanes can be stored in a database/library?
The limit is only based on RAM and disk space. We have loaded 500 in simple tests and they should cope with >1000.
Can more than one person access the database at the same time?
As many people as they like can access the database at the same time though it has not been designed for this type of use. However, if one user adds lanes to a database/library or changes bands then the other users may not get to see the changes straight away unless they load the database back in. So if you are careful it should be fine.
Can I create a Principal Component Analysis (PCA) on the database matching results?
You cannot currently do this within the product, but you can use the data generated to send to another package. By going to the Matrix table, you can right click to Copy the table to the clipboard and this can be used to generate a PCA plot.
BSI is short for Band Sharing Index. It is calculated as (2*number of bands matched)/(total number of bands in both lanes) So for example two lanes have 5 bands and 7 bands. 3 bands are matched in each lane so the BSI = (2*3)/(5+7) = 0.5. This is then displayed as a percentage so it is 50%. It is a measure of how similar two lanes are and is best known as the "Dice" similarity.


The Future Of Wearable Sensors.
In today's premium podcast episode, I interview Pierre-Jean, the co-founder of Echo Labs. Echo Labs has developed a wearable sensor that can measure what's in your blood using spectrometry (which means that this wearable can measure actual molecules in your bloodstream), along with heart rate, blood pressure, hydration, heart rate variability, sleep and more.
Do you have questions, comments or feedback about the future of wearable sensors? Leave your thoughts below, and click here to go Premium and listen in!


text:
Top Climate Change "Expert" Admits He Is A Pathological Liar
December 16, 2013 December 16, 2013 by Carlos Eire
John C. Beale: EPA pathological liar
The sky is falling, the sky is falling!
No, you can't make up stuff like this. Life is far stranger than fiction.
John C. Beale, the nation's top expert on climate change– who also happened to be the highest paid official at the Environmental Protection Agency– has admitted in court that he has trouble telling the truth. Well, it's actually worse than that. He actually admitted that he is driven to lie by a fierce compulsion, big time. His "illness" was confirmed by his attorney and his therapist.
Mr. Beale is no Walter Mitty. This man in charge of promoting climate change hysteria told lies so big that they defy comprehension. In the process, he also bilked U.S. taxpayers of over a million dollars.
Question of the day: If this kind of uncontrolled malfeasance can be expected of top government officials, should more and more power be entrusted to them?
Additional question of the day: will this buffoon's pathetic story discredit the anthropogenic global warming panic represented by him?
No mention of Mr. Beale's party affiliation is made in any news report. This omission naturally leads one to suppose that he is not a Republican.
Julia Sweig, Phil Peters, Arturo Lopez-Levy, meet your comrade and potential mentor….
Tommy Flanagan, meet your long-lost fraternal twin…
Tommy Flanagan, SNL Pathological Liar
From NBC News:
Climate change expert's fraud was 'crime of massive proportion,' say feds
The EPA's highest-paid employee and a leading expert on climate change deserves to go to prison for at least 30 months for lying to his bosses and saying he was a CIA spy working in Pakistan so he could avoid doing his real job, say federal prosecutors.
ohn C. Beale, who pled guilty in September to bilking the government out of nearly $1 million in salary and other benefits over a decade, will be sentenced in a Washington, D.C., federal court on Wednesday. In a newly filed sentencing memo, prosecutors said that his lies were a "crime of massive proportion" that were "offensive" to those who actually do dangerous work for the CIA.
Beale's lawyer, while acknowledging his guilt, has asked for leniency and offered a psychological explanation for the climate expert's bizarre tales.
"With the help of his therapist," wrote attorney John Kern, "Mr. Beale has come to recognize that, beyond the motive of greed, his theft and deception were animated by a highly self-destructive and dysfunctional need to engage in excessively reckless, risky behavior." Kern also said Beale was driven "to manipulate those around him through the fabrication of grandiose narratives … that are fueled by his insecurities."
The two sentencing memos, along with documents obtained by NBC News, offer new details about what some officials describe as one of the most audacious, and creative, federal frauds they have ever encountered.
When he first began looking into Beale's deceptions last February, "I thought, 'Oh my God, How could this possibly have happened in this agency?" said EPA Assistant Inspector General Patrick Sullivan, who spearheaded the Beale probe, in an interview with NBC News. "I've worked for the government for 35 years. I've never seen a situation like this."
Keep reading HERE. Much, much more: A three-ring circus of incredible details.
Categories From the editors
Changing the debate on the handshake
Reagan was right on Mandela and South Africa
3 thoughts on "Top Climate Change "Expert" Admits He Is A Pathological Liar"
There's good money in being an eco-red.
When are people going to wise up and realize the so-called 'environmentalist movement' was created by the communists to subvert and destroy America from within.
Funny. Beale looks like a cross between Harry Reid and Joe Biden. I wonder what that means.


Realestate.com.au is Australia's #1 home for property, listing real estate for sale in Burra, and other property listings by real estate agents in Burra. Find buy a property in Burra packages, Southern Tablelands new house and land or Southern Tablelands new apartments in our new homes section where you can also view the latest new land estates in Southern Tablelands. If you're looking to sell or new home designs from Southern Tablelands, Realestate.com.au can provide you with Burra auction times, and auction results for Burra. Property data and house prices in Burra is available as well as recent sold property in Burra. People looking for Burra rentals and houses to share in Burra are catered to as well and everyone visiting Realestate.com.au can take advantage of our free and helpful real estate resource section.


text:
Q: Android TextViews not appearing I need help with this layout file. The problem is that on Android Studio preview this displays as it should, the image has a title at the bottom of the image, and there is text inside the ScrollView under the FAB.
I'm sorry for the lengthy xml.
<?xml version="1.0" encoding="utf-8"?>
<LinearLayout xmlns:android="http://schemas.android.com/apk/res/android"
    xmlns:app="http://schemas.android.com/apk/res-auto"
    android:orientation="vertical" android:layout_width="match_parent"
    android:layout_height="match_parent">

    <RelativeLayout
        android:layout_width="fill_parent"
        android:layout_height="wrap_content">

        <ImageView
            android:layout_width="fill_parent"
            android:layout_height="300dp"
            android:id="@+id/detailed_image_view"
            android:scaleType="centerCrop"
            android:elevation="@dimen/card_height">

        </ImageView>

        <TextView
            android:layout_width="fill_parent"
            android:layout_height="wrap_content"
            android:id="@+id/detailed_title"
            android:background="@drawable/white_gradient"
            style="@style/large_white_text"
            android:layout_alignBottom="@+id/detailed_image_view"
            android:layout_alignParentLeft="true"
            android:layout_alignParentStart="true" />
    </RelativeLayout>

    <LinearLayout
        android:layout_width="fill_parent"
        android:layout_height="wrap_content"
        android:elevation="10dp"
        android:orientation="horizontal"
        android:layout_margin="10dp"
        android:paddingLeft="5dp"
        android:paddingRight="5dp"
        android:background="@color/accent">

        <ImageView
            android:layout_width="0dp"
            android:layout_height="wrap_content"
            android:src="@drawable/location_icon"
            android:layout_weight="1"
            android:layout_marginTop="2dp"
            android:layout_marginBottom="2dp"/>
        <TextView
            android:layout_width="0dp"
            android:layout_height="wrap_content"
            android:id="@+id/detailed_location"
            style="@style/normal_text"
            android:layout_weight="5"
            android:layout_marginTop="8dp"/>

        <ImageView
            android:layout_width="0dp"
            android:layout_height="wrap_content"
            android:src="@drawable/time_date_icon"
            android:layout_weight="1"
            android:layout_marginTop="2dp"
            android:layout_marginBottom="2dp"/>

        <TextView
            android:layout_width="0dp"
            android:layout_height="wrap_content"
            android:id="@+id/detailed_date"
            style="@style/normal_text"
            android:layout_weight="5"
            android:layout_marginTop="8dp"/>

        <ImageView
            android:layout_width="0dp"
            android:layout_height="wrap_content"
            android:src="@drawable/price_icon"
            android:layout_weight="1"
            android:layout_marginTop="2dp"
            android:layout_marginBottom="2dp"/>

        <TextView
            android:layout_width="0dp"
            android:layout_height="wrap_content"
            android:id="@+id/detailed_price"
            style="@style/normal_text"
            android:layout_weight="5"
            android:layout_marginTop="8dp"/>

    </LinearLayout>

    <RelativeLayout
        android:layout_width="fill_parent"
        android:layout_height="fill_parent">

        <ScrollView
            android:layout_width="fill_parent"
            android:layout_height="fill_parent">

            <TextView
                android:layout_width="fill_parent"
                android:layout_height="wrap_content"
                android:text="@string/lorem"
                android:id="@+id/detailed_text"
                android:elevation="10dp" />
        </ScrollView>

        <android.support.design.widget.FloatingActionButton
            android:id="@+id/fab"
            android:layout_width="wrap_content"
            android:layout_height="wrap_content"
            android:layout_gravity="end|bottom"
            app:backgroundTint="@color/accent"
            android:src="@drawable/ic_stat_name"
            android:layout_margin="30dp"
            android:layout_alignParentBottom="true"
            android:layout_alignParentRight="true"
            android:layout_alignParentEnd="true" />

    </RelativeLayout>

</LinearLayout>

Here is the initialization code
ImageView headerImage = (ImageView) linearLayout.findViewById(R.id.detailed_image_view);
        TextView title = (TextView) linearLayout.findViewById(R.id.detailed_title);
        TextView date = (TextView) linearLayout.findViewById(R.id.detailed_date);
        TextView location = (TextView) linearLayout.findViewById(R.id.detailed_location);
        TextView price = (TextView) linearLayout.findViewById(R.id.detailed_price);
        TextView text = (TextView) linearLayout.findViewById(R.id.detailed_text);
        FloatingActionButton fab = (FloatingActionButton) linearLayout.findViewById(R.id.fab);

        EventModel eventModel = bundle.getParcelable("event");

        Glide.with(context)
                .load(eventModel.getImage())
                .diskCacheStrategy(DiskCacheStrategy.ALL)
                .into(headerImage);

        title.setText(eventModel.getTitle());
        date.setText(eventModel.getDateString());
        price.setText("Free");
        location.setText(eventModel.getHost());
        text.setText(eventModel.getText());

This is how it is supposed to look, but what I get on my phone is no "[PLACEHOLDER]" and lorem ipsum text

redpajama_set_name: RedPajamaStackExchange


EvoCreo v1.2.2 Apk Become the Top evoker by capturing and battling over 130 kinds of creo. Test yourself in the arenas and work the way up to the Coliseum. On the way you will be fighting the sinister plot of nefarious organization, Shadow hive.


text:
Goldman Sachs Partners Put Own (Liquidity) Needs Before Those Of The Firm
Every fall, previously in the basement of 85 Broad and more recently at 200 West, Goldman Sachs names a class of new partners. Blindfolded and naked, they pledge their devotion to the firm. To commemorate the event, and for the practical purpose of tagging them so their status at the firm can be quickly verified with one quick drop of trou, these newly-made partners have their nether regions dipped in a vat of gold, which harden while Lloyd Blankfein gives a speech about how to carry oneself differently once they reach the upper echelons of GS (literally, as those things will drag if you're not careful). At the stroke of midnight, as a baby seal barks in the corner, they are inducted into the Brotherhood of the Sach. And while one is more than welcome to benefit, monetarily, from this new position, being a member of the Brotherhood is less about sharing in its huge ass profits than it is making sure the partnership stays long and strong. Some people, apparently, did not read that portion of the fine print.
Goldman Sachs partners have sold $108 million in shares in recent months, driving their total ownership stake in the Wall Street firm down to about 10 percent from 11.2 percent. The sales are a small yet significant dilution of the influence the partners have over the firm. Part of the power of the Goldman partnership is the stake they own collectively, and the agreement they have made to act in unison on shareholder votes. The next annual shareholder meeting has not been set, but it is typically held in May.
And while no one is keeping a list of names....a list is in fact being kept.
The top two sellers in the most recent period were Gregg Felton, a senior executive in the firm's asset management division, who sold shares amounting to almost $8 million, and Philippe Altuzarra, a banker, who sold shares amounting to about $6 million.
No one should be worried that this going to reflect poorly on them. You are free to sell your shares as you please. And hey, everyone's allowed (one) transgression! Keep it up though and don't be surprised when you're pulled from your desk one unseasonably chilly day next September and placed on a bus headed upstate, to a warehouse in Buffalo. Don't be afraid of the the head of HR will come out of the shadows, wearing an executioner's mask and swinging massive clippers. It'll all be over soon enough.
Goldman Sachs Partners Sell Shares, Diluting Stake [Dealbook]
Goldman Sachspartnersliquidity
Would-Be Goldman Partners Will Have To Work Extra Hard This Year To Gain Brotherhood Of The Sach Status
Confidential To The Partners Of Goldman Sachs: Gird Those Loins. Literally.
Goldman Sachs To Offer More Would-Be Partners Opportunity To Go David Tepper On An Executive's Ass This Year
Each year, after a long and very comprehensive background check, a lucky group of Goldman employees are abducted from their desks, blindfolded, gagged, and led by candlelight through a dark hallway and into a subterranean conference room. Standing on the table before them are Lloyd Blankfein, Gary Cohn and the rest of the management committee, who ask if they are prepared to pledge their devotion to the firm above all else. Those who agree have their nether regions dipped in a vat of gold, genuflect before Cohn's groin, and, at the stroke of midnight, are inducted into the Brotherhood of the Sach. While there are many ways that becoming a member of the club will change one's life, the most important one involves the partaking of astronomical profits on payday. As a result, when people are not invited to join the group, they tend to get very upset. For instance, hedge fund manager David Tepper, who became a billionaire many times over after leaving the firm, was still so upset about the snub twenty years later that he bought and bulldozed the house of the guy who passed him over. Others probably wouldn't have even gone to the trouble of buying the place first, and operated the wrecking ball themselves. Which is why we say in full seriousness that the Partnership Committee might want to watch its back. Goldman Sachs has begun vetting potential new partners and is expected to appoint a smaller number of bankers to its upper echelons this year, according to senior executives involved in the process... The nomination process for new partners ended during the summer. The internal vetting process began earlier this month and is expected to last until mid-November when the new class of partners will be announced. The vetting process is known within the bank as "cross-ruffing", in reference to a manoeuvre from the card game bridge and typically sees a team of partners deployed to every division to talk to employees who know the candidates. [FT, related]
Goldman Sachs Mints 84 New Partners In Anticipation Of Needing Human Sacrifices To The Gods Of American Apocalypse
What To Expect When You're Expecting To Make Partner At Goldman Sachs
Stephen Colbert Threatens To Give Out Goldman Sachs Partner's Credit Card Number Unless He Comes On Show
Goldman Sachs Changes 110 People's Lives
Goldman Sachs Changes 78 People's Lives


text:
Bahrain Shia groups quit reconciliation talks
Participation suspended after arrest of senior member of opposition bloc for allegedly instigating violence.
More than 65 people have died in protests since early 2011, Bahrain's government says [AP]
Bahrain's main Shia Muslim groups have suspended participation in reconciliation talks with the Sunni-led government after the detention of an important opposition figure.
Bahrain's public prosecution said Khalil al-Marzooq, a former deputy parliament speaker, was detained on Tuesday accused of instigating violence and having links to bombings and other attacks.
Marzooq, a senior member of Al Wefaq, the main Shia political bloc, was ordered to be held for 30 days during the investigation.
His supporters claim he was targeted in attempts to punish the opposition after recent criticism from European officials about government crackdowns on dissent.
The decision by the Shia groups closes one of the main channels for dialogue.
Repeated rounds of political talks have failed to significantly close the rifts between the Sunni establishment and Shia factions, who began an Arab Spring-inspired uprising in early 2011 to seek greater political rights.
According to the government, more than 65 people have died in the unrest, but rights groups and others put the death toll higher.


Plant Protein Pasta Recipe – With Loads Of Fiber & Protein!
This plant protein pasta dish with arugula, artichoke hearts, and hemp seeds is a delicious way to get your fiber and protein. Unlike traditional wheat pasta, choosing alternative pastas made from beans and legumes significantly increase the protein and fiber.
Chickpea pasta is a favorite as it looks and tastes most like traditional pasta. When paired with other fiber and protein rich ingredients, this makes for one healthy and filling plant-powered meal.
One serving provides approximately 26 grams of protein, from the chickpea pasta (14g) peas (4g), hemp seeds (3g), grilled artichoke hearts (3g), and yes, even the arugula (1.8 g per 3 cups)! The same serving also provides approximately 17 grams of fiber.
Cook the pasta 7 minutes or according to package directions.
Three minutes before the pasta time is up, add in the frozen peas to the same pot. When done, drain the peas and pasta.
Meanwhile, put the baby arugula in a large salad bowl. Chop the grilled artichoke heart and add them to the bowl, along with the drained peas and pasta. Top with hemp seeds, pink salt, and extra virgin olive oil. Toss well before serving.


text:
Betty Hutton (26. februar 1921 – 11. marts 2007) var en amerikansk filmskuespiller og sanger.

Biografi
Betty Hutton blev født som Elizabeth June Thornburg og voksede op med sin mor og søster, da faderen havde forladt familien. Allerede som 3-årig begyndte Hutton at optræde i familiens smugkro. Som teenager sang hun med forskellige orkestre, og som 18-årig blev hun opdaget af en talentspejder, der skaffede hende ind hos et filmselskab, hvor hun begyndte med at lave små musikalske filmklip. Snart efter fik hun roller på Broadway, men i 1942 fik hun sin første egentlige filmrolle.

De følgende år indspillede hun i alt tyve film, heriblandt Annie Get Your Gun, hvor hun erstattede Judy Garland, der var blevet fyret på grund af sygdom. I samme periode indspillede hun plader og optrådte på natklubber, blandt andet i Las Vegas. Hun blev kendt for at være en besværlig stjerne, der ofte afbrød kontrakter i utide, og derfor blev hendes Hollywood-karriere ikke så lang. Hun fortsatte dog med radioshows og pladeudgivelser, og hun fik også en vis succes med sit eget tv-show. I løbet af 1960'erne løb hendes karriere imidlertid ud i sandet, blandt andet på grund af alkohol- og narkomisbrug.

Privatliv
Betty Hutton var gift i alt fire gange, men ingen af ægteskaberne varede længere end syv år. Hun fik tre døtre med to af sine mænd, henholdsvis i 1946, 1948 og 1962.

I 1980'erne konverterede hun til katolicismen.

Filmografi
Betty Hutton indspillede følgende spillefilm:
 The Fleet's In (1942)
 Star Spangled Rhythm (1942)
 Let's Face It (1943)
 Gutter på landlov (1943)
 Miraklet i Morgan's Creek (1944)
 And the Angels Sing (1944)
 En pige med charme (1945)
 Duffy's Tavern (1945)
 Stork Club (1945)
 Cross My Heart (1946)
 På et hængende hår (1947)
 Dream Girl (1948)
 En pige slår sig løs (1949)
 Annie Get Your Gun (1950)
 Så danser vi (1950)
 Verdens største show (1952)
 Sailor Beware (1952)
 Somebody Loves Me (1952)
 Spring Reunion (1957)

Eksterne henvisninger

Skuespillere fra USA
Hollywood Walk of Fame

redpajama_set_name: RedPajamaWikipedia


President Trump endorsed Mitt Romney in the Senate race to replace retiring Sen. Orrin Hatch (R-Utah) on Monday night, despite reportedly urging Hatch to run for reelection to block Romney out.
"He will make a great Senator and worthy successor to @OrrinHatch, and has my full support and endorsement!" Trump tweeted. Trump and Romney have had a complicated relationship. The 2012 GOP presidential nominee was a frequent critic during Trump's own presidential campaign.
These are a few reminders that I've gathered on what Mitt Romney has said about Donald Trump.
Mitt Romney sits down with Kimmel and calls Trump a "phony"...Then goes after him on his tax returns. Again, he doesn't mention defeating Hillary Clinton, not once, just Trump.
Watch: Romney hits Trump within 30 seconds of taking the seat.