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Inflammatory bowel diseases (IBDs) are chronic, relapsing disorders that affect the gastrointestinal tract of millions of people and continue to increase in incidence each year. While several factors have been associated with development of IBDs, the exact etiology is unknown. Research using animal models of IBDs is beginning to provide insights into how the different factors contribute to disease development. Oral administration of dextran sulfate sodium (DSS) to mice induces a reproducible experimental colitis that models several intestinal lesions associated with IBDs. The murine DSS colitis model can also be adapted to quantify intestinal repair following injury. Understanding the mechanistic basis behind intestinal repair is critical to development of new therapeutics for IBDs because of their chronic relapsing nature.
The murine DSS colitis model was adapted to provide a system enabling the quantification of severe intestinal injury with impaired wound healing or mild intestinal injury with rapid restoration of mucosal integrity, by altering DSS concentrations and including a recovery phase. We showed that through a novel format for presentation of the clinical disease data, the temporal progression of intestinal lesions can be quantified on an individual mouse basis. Additionally, parameters for quantification of DSS-induced alterations in epithelial cell populations are included to provide insights into mechanisms underlying the development of these lesions. For example, the use of the two different model systems showed that toll-like receptor 9, a nucleic acid-sensing pattern recognition receptor, is important for protection only following mild intestinal damage and suggests that this model is superior for identifying proteins necessary for intestinal repair.
We showed that using a murine DSS-induced experimental colitis model system, and presenting data in a longitudinal manner on a per mouse basis, enhanced the usefulness of this model, and provided novel insights into the role of an innate immune receptor in intestinal repair. By elucidating the mechanistic basis of intestinal injury and repair, we can begin to understand the etiology of IBDs, enabling development of novel therapeutics or prophylactics.
Keywords:Dextran sulfate sodium; Inflammatory bowel disease; Intestinal repair; Toll-like receptor 9
Inflammatory bowel diseases (IBDs) are defined as chronic, relapsing, inflammatory disorders that affect the colon and other parts of the gastrointestinal tract [1,2]. The two main types of IBDs are Crohn's disease, which commonly causes transmural ulceration of the small intestine and colon, and ulcerative colitis, which causes non-transmural ulceration of the colon [1,2]. The annual incidence of ulcerative colitis ranges from 24.3 per 100,000 person-years in developed countries to 6.3 per 100,000 person-years in developing countries, while Crohn's disease ranges from 20.2 to 5.0 with increasing prevalence of both IBDs observed over time [3]. IBDs develop following dysregulation of the delicate homeostatic balance maintained between the immune system of the intestine and the resident commensal bacteria [2,4-6]. The exact etiology of IBDs is unknown, but has been proposed to occur through a combination of factors, including genetic predisposition, altered expression of pattern recognition receptors, alteration in commensal microflora, and environmental stressors [2,4-6]. Prevention or treatment of IBDs is of critical importance due to the significantly increased risk of colorectal cancer, greatly diminished quality of life caused by disease symptoms, and the medical cost associated with life-long disease management [1,7,8]. Current therapeutic strategies for management of IBDs involve pharmacological interventions, including anti-inflammatory drugs, immunosuppressives, antibiotics, and biologic agents, with surgical approaches employed in critical or non-responsive patients [1,9,10]. Despite several options for IBD treatment, not all patients are responsive to therapy, and the lack of drug specificity results in several side effects complicating their use [1,5,10,11].
Current IBD treatment options lack specificity, targeting inflammation non-specifically, and lack the ability to stimulate epithelial restitution, which is important for induction of remission [1,5,10,11]. Therefore, new types of therapies are needed that can prevent disease or relapse. Several promising options are currently under evaluation in clinical trials and in different animal models of experimental colitis [1,9,12,13]. IBD-like symptoms can be chemically-induced in several animal models, but the mouse model offers several advantages including multiple methods for inducing experimental colitis, a wide array of biochemical and immunological reagents, a well-defined immune system, and the availability of numerous signaling molecule-deficient mice to evaluate treatment specificity [11,14-16]. Dextran sulfate sodium (DSS)-induced experimental colitis in mice provides a model of acute intestinal injury that mimics several intestinal lesions consistent with IBDs [14,17]. The murine DSS colitis model provides several advantages over other chemically-induced models of ulcerative colitis, including oral delivery via drinking water and induction of consistent colitis levels with a defined disease onset [14,16]. This model allows for investigation of environmental impacts, such as specific bacterial infections (e.g. Campylobacter jejuni), or exposure to probiotics [18,19]. However, most of the research on experimental colitis focuses on understanding the etiology of IBD development by studying the acute phase of intestinal injury and evaluation of methods to prevent the development of IBDs [12,20-23]. Because of the their chronic, relapsing nature and the millions of people currently affected by IBDs [1-3], there also is a need for in vivo models to evaluate therapeutic options that can aid in intestinal repair following IBD-induced injury.
To address the need for different models of intestinal injury and repair, we present methods to enhance the traditional DSS experimental colitis model and modifications to the model system that permit quantification of mild intestinal injury and subsequent repair. Previous studies have used severe intestinal damage models where wild-type mice are unable to repair damage during a recovery period [24-26], delivered different doses of DSS to wild-type and mutant mice in an attempt to achieve similar damage during the acute phase [27], or used intervention strategies during a recovery phase, but did not provide clinical data for the level of damage induced during the acute phase of DSS treatment [28]. One study showed that mice deficient in the negative regulator of WNT signaling had increased basal colonic epithelial cell proliferation and corresponding crypt length, and when treated with low dose DSS recovered faster than wild-type mice [29]. This suggests that using a carefully titrated dose of DSS will induce low-level damage that can be repaired rapidly, allowing comparisons between wild-type and gene-deficient mice.
We show that administration of carefully titrated concentrations of DSS to induce acute damage, and monitoring clinical and histological parameters longitudinally on a per mouse basis presented a valid system for modeling restitution of mucosal integrity. Although severity of DSS-induced colitis varies with genotype and intestinal flora, we chose to focus on C57BL/6 mice due to the availability of genetic mutants relevant to immunity on this background [19,30-34]. We show methods for presentation of clinical parameter data that are more robust than traditional disease activity indices, and describe the inclusion of several intestinal epithelial cell parameters that enhance the clarity of the data and provide insights into the potential mechanisms involved in development of intestinal lesions. Using toll-like receptor 9 (TLR9)-deficient mice, we show that by using mild intestinal damage and recovery, we could identify a novel role for TLR9 in intestinal repair, a role that was not appreciated when severe intestinal damage was induced. This approach would permit identification of genes and pathways that promote normal intestinal restitution and might be new targets for therapeutic intervention in IBD.
Clinical parameters of the two intestinal injury models
Traditional data presentation methods for clinical parameters recorded during DSS-induced colitis involve a disease activity index score or an average clinical score, representing a summation of one or all clinical scores for all mice for the total experimental time [18,21-23,35]. While this value provides easily comparable numbers for identifying potential differences among groups (Table 1), this method fails to detect daily fluctuations in individual mouse parameters. By evaluating, and representing multiple parameters on a daily basis, we gain the ability to perform statistical analysis of ordinal data to identify longitudinal differences based on individual clinical parameters. Figure 1A shows daily occult blood scores for five wild-type mice treated with 3% DSS in their drinking water for seven days, followed by seven days of regular drinking water, and five wild-type (untreated) mice provided regular drinking water for fourteen days. The untreated wild-type mice did not show any signs of occult blood on any day during the course of the experiment (Figure 1A, open symbols on X axis). However, for the 3% DSS-treated wild-type mice, one mouse was Hemoccult® positive (score of 1) on day three, and by day six, all wild-type mice had visible blood in their stool (score of 2). Several wild-type mice continued to show blood in their stool even three days after cessation of DSS treatments, with one mouse testing Hemoccult® positive on day fourteen. Typical disease activity index scores shown for day 7 and day 14 (Table 1) would miss the observation that there is not an immediate resolution of occult blood following cessation of treatment. The Mann–Whitney test for ordinal data demonstrated that differences in occult blood scores between wild-type untreated and 3% DSS treated mice were statistically significant overall (p < 0.01), and more importantly, on a daily basis (p < 0.05; Days 4–10). When we compared day 7 (end of acute phase) and day 14 (recovery phase), 3% DSS-treated mice significantly improved (p < 0.01).
Table 1. Effect of DSS concentrations in wild-type mice
Figure 1. Clinical parameter data presented for individual wild-type mice on a daily time scale. Wild-type mice were treated with 3% (A, C, E) or 1% (B, D, F) DSS (n = 5–6 mice/group) in their drinking water for seven days, then supplied with regular drinking water for seven days (closed symbols). Control mice received normal drinking water throughout (open symbols on the X axis) Note that all 1% DSS mice scored 0 and overlap with the control mice on the X axis. Mice were scored for the presence of occult blood (A, B) and stool consistency (C, D) on a daily basis during the fourteen day experiment as described in the materials and methods. (E, F) Mice were weighed on a daily basis to calculate the percent change in weight for the treatment (days 0–7) and recovery (days 7–14) periods. One of two experiments (5–6 mice per condition) with similar results shown. *** p < 0.001; Student's t-test; n.s., not significant.
Two additional clinical measurements of disease activity are stool consistency and weight loss. As early as 1 day after the start of 3% DSS treatment, one mouse tested positive for loose stool (Figure 1C). By day four, all mice showed signs of loose stool, which persisted, in four out of five mice, for up to seven days after the removal of DSS. Overall (p < 0.001; Mann–Whitney test) and daily (p < 0.05; Days 4–14; Mann–Whitney test) comparisons showed significantly higher stool consistency scores for the 3% DSS treated compared to untreated wild-type mice. However, treated mice on day 14 were not improved compared to day 7 (p > 0.05). Wild-type mice administered 3% DSS lost significantly (p < 0.001; Student's t-test) more weight than the untreated wild-type mice during the seven days of treatment, and showed a non-significant (p = 0.06; Student's t-test) increase in weight during the recovery period (Figure 1E). Collectively, our data presentation method allows for quantitative and qualitative interpretation of the data to identify trends among different experimental groups. This presentation method reveals that a 3% DSS treatment model results in severe disease that is not fully repaired by seven days post-cessation of treatment.
Because IBDs are characterized by recurrent episodes of intestinal injury and repair [2,4], a model that allows for evaluation of mild intestinal injury and subsequent repair is of critical importance. Based on our data showing significant intestinal injury in mice even seven days after removal of 3% DSS (Figures 1A, C) and previous studies correlating DSS concentration with intestinal injury severity [36,37], we evaluated the clinical impact of 1% DSS in our established mouse model. Interestingly, only one of the six mice administered 1% DSS tested Hemoccult® positive on the last day of treatment (Figure 1B). No significant (p > 0.05; Mann–Whitney test) difference in occult blood severity was observed compared to untreated wild-type mice, while severity was significantly (p < 0.01; Mann–Whitney test) reduced compared to 3% DSS-treated wild-type mice (Figures 1A, B). A similar significant (p < 0.001; Mann–Whitney test) reduction in stool consistency scores was observed for the 1% compared to 3% DSS-treated mice (Figures 1C, D). Stool consistency in the 1% DSS-treated mice did not develop until day five, never increased past a score of 1 (loose stool), and with the exception of one mouse, scores returned to pre-treatment levels after removal of DSS (Figure 1D). Quantification of changes in weight during the treatment or recovery periods showed no significant (p > 0.05; Student's t-test) differences between 1% DSS-treated and untreated wild-type mice (Figure 1F). Using traditional methods of disease activity indices, this 1% DSS model would not typically be characterized as disease. However, as we show below, damage is detected on a histologic level, and the significantly milder intestinal injury allows for return to baseline three days after cessation of treatment for clinical parameters, and slightly longer for histologic parameters. Therefore, the 1% DSS model is powerful method for evaluating the repair phase of IBDs.
Histopathologic parameters of the two intestinal injury models
Histopathology and quantification of morphologic and immunologic changes in the colon post-DSS treatment provides another, more sensitive, measure of intestinal injury and repair [20-22,38]. Colons from 3% or 1% DSS-treated wild-type mice were collected on days zero, seven, and fourteen, then paraffin-embedded and stained with Hematoxylin and Eosin (H&E) to visualize intestinal integrity (Figure 2). Untreated wild-type mice (day zero) showed a distinctive, non-disrupted, repeating crypt architecture, with small numbers of leukocytes present in the lamina propria (Figure 2A, B). After seven days of 3% DSS treatment, however, wild-type mice showed mucosal erosions (white arrow) with effacement of the mucosa by lymphocytes, neutrophils, and macrophages, which also infiltrate and expand the submucosa (black arrow; Figure 2C). These changes indicate severe disease, consistent with severe observed clinical disease (Figure 1). Despite the mild clinical intestinal injury observed in the 1% DSS-treated wild-type mice, histopathology showed significant loss of crypts (white arrow, Figure 2D), and expansion of the lamina propria by edema fluid after seven days (black arrow, Figure 2D). However, by seven days after 1% DSS removal (day fourteen), there was complete restitution of normal colonic architecture (Figure 2F). In contrast, and consistent with maintained clinical indices of disease seven days post-cessation of 3% DSS treatment, we observed continued mucosal effacement, with early attempts at repair through neovascularization and surface re-epithelialization (white arrow, Figure 2E), as well as increase submucosal infiltration by lymphocytes and plasma cells (black arrow, Figure 2E). Together, the histopathologic observations indicate that the 1% DSS-treated wild-type mice experienced mild intestinal injury that they were able to repair within seven days post cessation of treatment, while 3% DSS treatment induce extensive intestinal injury that showed markedly delayed repair during a seven day recovery period. However, histopathology is often subjective and not presented in a quantitative, or statistically amenable form.
Figure 2. Differences in histologic parameters between the two intestinal injury models post cessation of DSS treatments. Wild-type mice were treated with 3% (A, C, E) or 1% (B, D, F) DSS as described in Figure 1. Colons were collected from mice on days 0 (A, B), 7 (C, D), and 14 (E, F). Histopathologic changes in individual crypts (20x dry objective, scale bars = 100 μm) are shown in representative H&E-stained sections. Loss of crypt architecture associated with epithelial damage (white arrows) and leukocyte infiltration (black arrows) is observed following DSS treatment.
Quantitative scoring of histopathology by a board-certified veterinary pathologist using a previously described scoring system [39], showed no epithelial damage or leukocyte infiltration in the wild-type mice at day zero (Figure 3). Seven days of treatment with 3 or 1% DSS resulted in significantly (p < 0.01; Mann–Whitney test) increased epithelial damage scores compared to day zero (Figures 3A, B). At day 14, seven days after changing to normal drinking water, the epithelial damage scores for 3% DSS-treated wild-type mice were still significantly (p < 0.01; Mann–Whitney test) increased (Figure 3A). In contrast, epithelial damage scores returned to pre-treatment levels in mice that were treated with 1% DSS (Figure 3B). A significant increase in leukocyte infiltration scores for 3% DSS-treated mice was observed on days seven (p < 0.05; Mann–Whitney test) and fourteen (p < 0.01; Mann–Whitney test) (Figure 3C). This indicated that the inflammatory response to tissue damage was still occurring at this time point. In contrast, mice treated with 1% DSS did not show significantly (p > 0.05; Mann–Whitney test) higher leukocyte infiltration scores on either day seven or fourteen (Figure 3D). Scoring analysis supported the histologic observations that treatment of wild-type mice with 3% DSS induced a severe intestinal injury that failed to repair within one week of cessation of DSS treatment, while 1% DSS treatment induced milder injury that was readily repaired. This milder treatment model, along with the statistical analysis of quantitative histopathologic data, provides a system to evaluate the role of various parameters in intestinal repair.
Figure 3. Quantification of histologic parameters collected from individual wild-type mice before, during, and after DSS treatment. H&E-stained colon sections were collected as described in Figure 2 and scored in a blinded manner by a board-certified veterinary pathologist. (A, B) Epithelial damage was scored as: 0) no damage; 1) discrete focal lymphoepithelial lesions; 2) mucosal erosion; or 3) mucosal erosion with submucosal damage. (C, D) Leukocyte infiltration was scored as: 0) rare inflammatory cells in the lamina propria; 1) inflammatory cells throughout most of the lamina propria; 2) inflammatory cells extending into the submucosa; or 3) transmural extension of inflammatory cells. * p < 0.05, ** p < 0.01; Mann Whitney test; n.s., not significant.
Intestinal epithelial cell parameters of the two intestinal injury models
Intestinal epithelial cells play a critical role in maintenance of intestinal homeostasis following injury [20,40,41]. Therefore, we propose that in addition to quantifying clinical and histologic parameters in the DSS colitis model, changes in intestinal epithelial cell parameters should also be quantified to provide potential insights into the mechanisms underlying responses to intestinal injury and repair. Based on several published works showing differences in intestinal epithelial proliferation following induction of intestinal injury [20,42,43], we quantified the number of Ki-67+ cells present in colon crypts (Figures 4A and 5A). Wild-type mice showed a significant (p < 0.001; ANOVA) increase in Ki-67+ cells after seven days of treatment with 3% or 1% DSS (Figure 5A). Seven days post-DSS treatment, wild-type mice administered 3% DSS still exhibited significantly (p < 0.001; ANOVA) elevated numbers of Ki-67+ cells while 1% DSS-treated wild-type mice returned to pre-treatment numbers. One potential explanation for the increased numbers of Ki-67+ cells following induction of intestinal injury is a compensatory increase in colon crypt length following initiation of repair; therefore we quantified crypt lengths (Figures 4B and 5B). As expected, a significant (p < 0.01; ANOVA) reduction in crypt length was observed for mice treated with 3% DSS for seven days; however, a slight, but non-significant (p > 0.05; ANOVA), decrease was observed for 1% DSS-treated mice (Figure 5B). After recovery (day fourteen), the 3% DSS-treated mice showed a significant (p < 0.05; ANOVA) increase in crypt length (Figure 5B). This correlated with significantly increased numbers of goblet cells present in the crypts (Figures 4B and 5C). While TUNEL + cells per crypt were increased at day 14 for both 3% (p < 0.001; ANOVA), and 1% (p < 0.05; ANOVA), DSS-treated mice, no dramatic increases in apoptosis were observed overall (Figures 4C and 5D). Collectively, the quantitative intestinal epithelial data shows that severe intestinal injury (3% DSS) produces significant and prolonged changes at the level of individual cells, while mild injury (1% DSS) produces only minor changes with a rapid return to a pre-injury state.
Figure 4. Representative quantitative histology images. Images from colonic sections of 3% or 1% DSS-treated, wild-type mice collected on days 0, 7, and 14. (A) Sections were stained for Ki-67 (red) and DAPI (blue) and imaged with a 63x oil objective. (B) Sections were stained with PAS for goblet cells and imaged with a 20x dry objective. (C) Sections were TUNEL-stained (green) for apoptotic cells, counterstained for DAPI (blue), and imaged with a 63x oil objective. All scale bars = 50 μm.
Figure 5. DSS treatment induced quantitative changes in colonic epithelial cell populations. Colonic sections were collected from 3 or 1% DSS-treated, wild-type mice on days 0, 7, and 14. 50 crypts from three mice at each time point for each DSS treatment (150 crypts at each treatment time point) were analyzed for: (A) number of Ki-67+ cells per colonic crypt, (B) colonic crypt length, (C) goblet cells per colonic crypt, and (D) number of TUNEL + cells per colonic crypt. Bars represent mean ± standard error of the means. Values were not significant unless indicated: * p < 0.05, ** p < 0.01, *** p < 0.001; ANOVA.
A role for TLR9 in intestinal repair is only observed in a mild intestinal injury model
Mice lacking MyD88, a critical innate immune signaling molecule [44], exhibited altered severity to DSS-induced colitis indicating an important role for innate immune signaling in intestinal injury [20]. However, the role of TLRs, most of which signal through MyD88 [44], in intestinal repair following injury needs to be evaluated further. By using the two different DSS models of intestinal injury, the mechanistic role of TLRs, for instance TLR9, can be quantified on a clinical, histologic, and epithelial cell level. TLR9-deficient mice treated with 3% DSS for seven days then provided regular drinking water for seven days showed significantly (p < 0.001; Mann–Whitney test) increased occult blood scores compared to untreated TLR9-deficient mice (Figure 6A, control wild-type mice from the same experiment are depicted in Figure 1A). Occult blood scores of 3% DSS-treated TLR9-deficient mice were not significantly different from 3% DSS-treated wild-type mice (p > 0.05; Mann–Whitney test). Furthermore, the development and progression of occult blood was very similar between wild-type and TLR9-deficient mice (Figures 1A and 6A). Similar results were obtained for stool consistency scores. TLR9-deficient mice treated with 3% DSS showed significant increased stool consistency scores (p < 0.001; Mann–Whitney test), but these were not different from 3% DSS-treated wild-type mice (Figures 1C and 6C). As with the wild-type mice (Figure 1E), treatment of TLR9-deficient mice with 3% DSS produced a significant (p < 0.001; Student's t-test) reduction in total body weight during the treatment period, with a significant (p < 0.01; Student's t-test) increase observed during the recovery period (Figure 6E). Using this model, we would conclude that TLR9 plays no role in intestinal damage or repair induced by DSS.
Figure 6. TLR9 is important for protection against mild, but not severe intestinal injury. TLR9-deficient mice were treated with 3% (A, C, E) or 1% (B, D, F) DSS in their drinking water for seven days, then supplied with regular drinking water for seven days. Wild type mice from the same experiment are depicted in Figure 1 for comparison. Mice were scored for the presence of occult blood (A, B) and stool consistency (C, D) on a daily basis during the fourteen day experiment as described in the materials and methods. (E, F) Mice were weighed on a daily basis to calculate the percent change in weight for the treatment (days 0–7) and recovery (days 7–14) periods. One of two experiments (5–6 mice per condition) with similar results shown. *** p < 0.001; Student's t-test; n.s., not significant.
To determine whether a role for TLR9 in intestinal damage and repair was masked by such severe intestinal damage induced by the 3% DSS treatment regimen, we employed the 1% DSS mild intestinal injury model. Interestingly, the TLR9-decifient mice treated with 1% DSS not only showed significantly (p < 0.01; Mann–Whitney test) increased occult blood scores compared to untreated TLR9-deficient mice (Figure 6B), they also scored significantly (p < 0.05; Mann–Whitney test) higher than similarly treated wild-type mice (control wild-type mice from the same experiment depicted in Figure 1B). By using daily analysis, we observed that one 1% DSS-treated TLR9-deficient mouse tested Hemoccult® positive as early as day four after beginning DSS treatment, and remained positive as late as five days after removal of DSS (Figure 6B). These observations would be missed using standard disease indices as described above (Figure 1, Table 1). The stool consistency scores for the 1% DSS-treated TLR9-deficient mice were also significantly (p < 0.001; Mann–Whitney test) increased over both untreated TLR9-deficient mice, and 1% DSS-treated wild-type mice (p < 0.05; Mann–Whitney test) (Figures 1D and 6D). Initial onset of loose stool was noted on day two for one 1% DSS-treated mouse, with several mice showing very loose stool (score = 2) after DSS cessation and continued clinical outcomes until day thirteen (Figure 6D). Again, these subtle differences would not be noted in a traditional disease index score evaluation. The 1% DSS-treated TLR9-deficient mice did not show any significant (p > 0.05; Student's t-test) change in weight compared to untreated TLR9-deficient or 1% DSS-treated wild-type mice during the treatment or recovery periods (Figures 1F and 6F). The increased severity, earlier onset, and delayed disappearance of clinical outcomes in the TLR9-deficient mice, indicate that TLR9 signaling plays an important role in protection against mild intestinal injury and in repair.
While there are several therapeutic options for inducing remission of IBDs [1,9,10], some patients are refractory to treatment or exhibit adverse side effects [1,5,10,11], therefore a critical need exists for new intervention strategies. Current research focuses on elucidating the etiology of IBDs [12,20-23], but due to the chronic, relapsing nature of IBDs and the millions of people currently affected by IBDs [1-3], research aimed at improving our understanding of intestinal repair processes after injury is also necessary. To address this issue, we showed a method for generating two different reproducible models of intestinal lesion development by administering selected concentrations of DSS in mice. Additionally, we provided new methods to analyze clinical parameter data in addition to inclusion of epithelial cell parameter data. We also show the importance of the two different model systems for evaluating the contribution of pattern recognition receptors towards preventing intestinal injury and in stimulating repair. The two different model systems provide methods that will enhance our understanding of the mechanistic basis of IBD development and intestinal repair following injury.
Oral delivery of DSS in mice results in the development of experimental colitis that models several clinical signs and lesions associated with IBDs [14,17]. Based on the proposed epithelial cell toxicity of DSS [45], we showed that increasing concentrations of DSS in the regular drinking water of mice resulted in increasing severity of intestinal injury (Figures 1, 2, 3). The reproducible nature of DSS-induced experimental colitis allowed for optimization of DSS concentrations to provide two model systems consisting of: 1) a model of the severe intestinal lesions and impaired would healing associated with IBD lesions, and 2) a model of mild intestinal injury with subsequent repair. The severe intestinal injury or acute model is important for identifying factors that are involved in the development of IBDs [2,4-6]. The mild intestinal injury and repair model is a complementary approach that may reveal novel functions of proteins that are obscured when severe intestinal damage is induced [1,2]. Utilization of the two different model systems will provide a better understanding of the temporal development of intestinal lesions and resulting clinical outcomes that are hallmarks of IBDs.
Representation of clinical parameters obtained during DSS-induced experimental colitis in mice vary greatly from a single disease activity index score to an average score for each disease parameter based on daily or total experimental time [18,21-23]. Issues with this type of data presentation include obscuring potential differences in disease outcomes, lack of temporal context for progression of the disease, potential outliers within the population, and summation of discrete ordinal data mixed with continuous data hypothesis testing. Separate presentation of each measured clinical parameter allows for more complete understanding of the extent of disease outcome as it relates to different types of intestinal lesions (Figure 1). Coupled with temporal presentation of the individual clinical parameters, the progression of intestinal lesions can be quantified and provide insights into the initiating events that are critical to understanding the development of IBDs. Because most clinical parameters are scored on a graded integer scale [18,21-23], summation of the data for the group of mice can result in a non-integer score that creates confusion as to the exact clinical outcome. Presentation of the individual data points for each mouse avoids the issue and hypothesis testing using non-parametric methods allows for statistical quantification of the data (Figure 1). The new methods we have provided for presentation of clinical data parameters offers a novel format for effectively and quickly conveying information that is relevant to understanding how intestinal lesions develop and progress during intestinal injury and repair.
The intestinal epithelium is composed of different types of epithelial cells that are important for normal host biology and providing protection against development of intestinal diseases [20,40,46]. Therefore, understanding how these cell populations are potentially altered following intestinal injury and repair is of critical importance. We show that after seven days of 3% DSS treatment in wild-type mice, colon crypts were significantly ablated and most of the remaining epithelial cells were actively proliferating to compensate for loss of intestinal barrier integrity (Figures 4 and 5). After removal of DSS, the proliferative cells continued to divide and crypt lengths increased significantly by day 14 to provide more absorptive surface area and the cells needed to repair the lesions [40]. An increase in goblet cell numbers also was observed and correlates with the known role of goblet cell mucins and intestinal trefoil factors in providing protection against the development of experimental colitis and initiation of intestinal repair processes in mice [47-49]. Additionally, an increase in the number of apoptotic cells was observed as expected based on the proposed intestinal epithelial cell toxicity of DSS [45]. Together, the quantified intestinal epithelial cell parameters show that the specific epithelial cell populations are dramatically altered following intestinal injury and induction of repair.
Pattern recognition receptors play a critical role in maintaining intestinal homeostasis, and alteration of receptor expression has been implicated as one of the factors involved in the development of IBDs [50]. To quantify the potential involvement of a nucleic acid sensing pattern recognition receptor, we evaluated TLR9-deficient mice in both intestinal injury models (Figure 6). The results from TLR9-deficient mice treated with 3% DSS indicate that TLR9 does not play a role in protection against severe intestinal injury or a potential contribution is obfuscated by the severity of the intestinal damage. Interestingly, evaluation of TLR9-deficient mice in the mild intestinal injury model (1% DSS) showed that TLR9 signaling is important for protection against injury and is involved in repair. Other TLRs and their adaptor molecules were shown to provide protection against intestinal injury through different mechanisms of action and MyD88, specifically, was shown to contribute to intestinal repair [12,20,42,51]. Notch signaling is required for the differentiation of intestinal epithelial progenitor cells, and NF-kB activation contributes to the initiation of intestinal repair. Both signaling pathways are activated following TLR stimulation [40,52]. Therefore, TLRs, such as TLR9, likely play a critical role in normal host biology and provide protection against chronic intestinal disease through the induction of intestinal repair.
We show that mild DSS-induced experimental colitis in mice permits evaluation of various effectors, including TLR9, in protection from intestinal damage, and for induction of intestinal restitution. We provide a new method for presenting clinical parameter data that highlights individual mouse disease outcomes over the course of the experiment, and that permits statistical analysis of differences on a daily or total experimental time basis. We demonstrate methods for quantification of changes in the intestinal epithelial cell populations, following intestinal injury and repair, to determine the etiologies of observed intestinal lesions. Knowledge of the mechanistic basis resulting in intestinal lesion development associated with IBDs will provide key insights into the development of novel therapeutic strategies. This same information will also provide insights into the normal host intestinal functions in the context of mild inflammation and loss of epithelial barrier function.
C57BL/6 (WT) and TLR9 receptor-deficient mice were 8–12 weeks old and weighed an average of 20 grams. The mice were bred in the Cornell University Transgenic Mouse Core Facility and housed under specific pathogen-free conditions. This animal study was approved by the Cornell University Institutional Animal Care and Use Committee (IACUC).
Colitis induction
Wild type and TLR9-deficient mice, in the same experiment, were administered DSS (36,000-50,000 Da; MP Biomedical, Solon, OH) ad libitum in drinking water at 1% or 3% weight per volume. Fresh 1% or 3% DSS was supplied on days 3 and 5 for treated mice. Control mice received regular drinking water for all fourteen days of the experiment. For the purposes of data analysis, wild type mice are depicted in Figure 1 and TLR9-deficient mice are depicted in Figure 6; however, the mice were from the same experiment.
Clinical scoring
Weight, stool consistency, and presence of occult blood (Hemoccult® SENSA®, Beckman Coulter, Brea, CA) were scored daily as previously described [38]. Briefly, scores for stool consistency were: 1) soft, but still formed; 2) very soft stool; or 3) diarrhea. Scores for occult blood were: 1) a positive Hemoccult® test; 2) visible blood in the stool; or 3) rectal bleeding. Total % weight change during treatment was calculated as: % Weight Change = [(weight at day 7 – weight at day 0)/weight at day 0] × 100 and recovery as: [(day 14 – day 7)/day7] × 100.
Colon collection and histopathology scoring
On days 0, 7, and 14, mice were euthanized and whole colons were excised from the anus to the cecum. The colons were coiled starting at the cecum, then fixed in 10% formalin (Azer Scientific, Morgantown, PA) for 24 hours. The fixed colons were paraffin-embedded and sectioned (5 μm), then H&E or periodic acid-Schiff (PAS) stained at the Cornell University Animal Health Diagnostic Center. Blinded scoring of H&E-stained sections was performed by a board-certified veterinary pathologist as previously described [39].
Quantitative histology of colon sections
Sections from paraffin-embedded colons were deparaffinized with three, two minute washes in xylene (Sigma, St. Louis, MO), then rehydrated with two minute washes in a graded ethanol series (Sigma) of 100, 90, and 75%. For heat-induced epitope retrieval, slides were microwaved at 800 watts for 20 minutes in a 10 mM sodium citrate pH 6.0 solution with 0.05% Tween-20 (Sigma). The In Situ Cell Death Detection kit, Fluorescein (Roche, Indianapolis, IN) was used to identify apoptotic cells in colonic sections. KI-67 positive cells were labeled using a Mouse on Mouse kit (Vector, Burlingame, CA) with a monoclonal Ki-67 (Clone MM1; Leica, Buffalo Grove, IL) primary antibody and a streptavidin-conjugated Alexa Fluor 555 (Life Technologies, Grand Island, NY) detection antibody. Slides were counterstained with Prolong Gold Anti-fade Reagent with DAPI (Life Technologies), then imaged using an Axio Imager M1 microscope (Zeiss, Thornwood, NY) and an Axiocam MRm (Zeiss). To quantify crypt length and goblet cell numbers, H&E (crypt length) or PAS (goblet cells)-stained colonic sections were imaged using an Axio Imager M1 microscope and an Axiocam HRc (Zeiss). Crypt length was quantified by measuring from the basolateral surface of the basal crypt cells to the apical cell surface at the top of intact crypts using the length function in the AxioVision Rel. 4.8 software (Zeiss). A total of 50 measurements were collected for each quantitative outcome.
Statistical analysis
Statistical analyses were performed using Prism software package v5 (Graphpad, La Jolla, CA) as described in the figure legends.
DSS: Dextran sulfate sodium; H&E: Hematoxylin and eosin; IBD: Inflammatory bowel disease; PAS: Periodic acid-Schiff; TLR9: Toll-like receptor 9.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
WR and CL designed the experiments, WR performed the experiments, KS performed pathology analysis and edited the manuscript, and WR and CL interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.
This work was funded in part by the National Institutes of Health (RO1 AI076588 to CL). The authors would like to thank the members of the Leifer lab for helpful discussions and for review of the manuscript.
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Koon HW, Shih DQ, Chen J, Bakirtzi K, Hing TC, Law I, Ho S, Ichikawa R, Zhao D, Xu H, et al.: Cathelicidin signaling via the Toll-like receptor protects against colitis in mice.
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e1851-1853PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Owen KA, Abshire MY, Tilghman RW, Casanova JE, Bouton AH: FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing.
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Zimmerman NP, Vongsa RA, Faherty SL, Salzman NH, Dwinell MB: Targeted intestinal epithelial deletion of the chemokine receptor CXCR4 reveals important roles for extracellular-regulated kinase-1/2 in restitution.
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Williams KL, Fuller CR, Dieleman LA, DaCosta CM, Haldeman KM, Sartor RB, Lund PK: Enhanced survival and mucosal repair after dextran sodium sulfate-induced colitis in transgenic mice that overexpress growth hormone.
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McConnell BB, Kim SS, Bialkowska AB, Yu K, Sitaraman SV, Yang VW: Kruppel-like factor 5 protects against dextran sulfate sodium-induced colonic injury in mice by promoting epithelial repair.
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Heimesaat MM, Fischer A, Siegmund B, Kupz A, Niebergall J, Fuchs D, Jahn HK, Freudenberg M, Loddenkemper C, Batra A, et al.: Shift towards pro-inflammatory intestinal bacteria aggravates acute murine colitis via Toll-like receptors 2 and 4.
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Solomon L, Mansor S, Mallon P, Donnelly E, Hoper M, Loughrey M, Kirk S, Gardiner K: The dextran sulphate sodium (DSS) model of colitis: an overview.
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Shimizu T, Suzuki M, Fujimura J, Hisada K, Yoshikazu O, Obinata K, Yamashiro Y: The relationship between the concentration of dextran sodium sulfate and the degree of induced experimental colitis in weanling rats.
J Pediatr Gastroenterol Nutr 2003, 37(4):481-486. PubMed Abstract | Publisher Full Text
Wirtz S, Neufert C, Weigmann B, Neurath MF: Chemically induced mouse models of intestinal inflammation.
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Jawhara S, Poulain D: Saccharomyces boulardii decreases inflammation and intestinal colonization by Candida albicans in a mouse model of chemically-induced colitis.
Med Mycol 2007, 45(8):691-700. PubMed Abstract | Publisher Full Text
Okamoto R, Watanabe M: Cellular and molecular mechanisms of the epithelial repair in IBD.
Dig Dis Sci 2005, 50(Suppl 1):S34-38. PubMed Abstract
Rakoff-Nahoum S, Hao L, Medzhitov R: Role of toll-like receptors in spontaneous commensal-dependent colitis.
Immunity 2006, 25(2):319-329. PubMed Abstract | Publisher Full Text
Fukata M, Michelsen KS, Eri R, Thomas LS, Hu B, Lukasek K, Nast CC, Lechago J, Xu R, Naiki Y, et al.: Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis.
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Edelblum KL, Washington MK, Koyama T, Robine S, Baccarini M, Polk DB: Raf protects against colitis by promoting mouse colon epithelial cell survival through NF-kappaB.
Gastroenterology 2008, 135(2):539-551. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kawai T, Akira S: Toll-like receptors and their crosstalk with other innate receptors in infection and immunity.
Immunity 2011, 34(5):637-650. PubMed Abstract | Publisher Full Text
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van der Flier LG, Clevers H: Stem cells, self-renewal, and differentiation in the intestinal epithelium.
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Taupin D, Podolsky DK: Trefoil factors: initiators of mucosal healing.
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Cario E: Toll-like receptors in inflammatory bowel diseases: a decade later.
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Am J Physiol Cell Physiol 2003, 285(5):C1028-1035. PubMed Abstract | Publisher Full Text


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The boats of Hanoi, part 2
[ad#intheboatshed-post] I promised a few more of my brother Matt's photos from Hanoi, and here they are. You can see the earlier post here. There's an interesting thread on Vietnamese boats at the Woodenboat Forum and an English language website devoted to the boats of Vietnam here. Don't miss something good! If you'd like to … Continue reading The boats of Hanoi, part 2

subset: commoncrawl


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Make a smooth transition to Word 2011, Microsoft's word processor for the Mac.
Create, format, and print a wide variety of documents in Microsoft Word 2011.
Examines the important features of Word 2008 for Mac, from creating a document to printing and collaborating.
"I've already been in consideration for a position I've recently interviewed for based on the Access training I've taken the last week or so. Awesome!" —David P.
more from our members »


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Loewe Agua de Loewe Él (EDT, 50ml)
£46.00 Product Code 1929887
- Overview
Agua de Loewe Él enhances pleasure and wellbeing with a luxurious and masculine fragrance. Bergamot and fresh grapefruit is blended with pineapple and cassis before descending to a warm woody base of oak moss and musk.
- Details
- Top notes: Japanese yuzu, Italian bergamot, Clementine and pink grapefruit
- Middle notes: Brazilian pineapple, spring water, cassisand green apple
- Base notes: sandalwood, Moroccan atlas cedarwood, holm oak moss and musk
- Eau de toilette, 50ml
- Regrettably, this product is non-returnable and non-exportable
- Delivery & Returns
Delivery Option Duration Price Standard Delivery 3-5 working days, M-F only. £5.95 Next Day Delivery Next working day on all orders placed before 1pm, M-F only. £12 Saturday Delivery Saturday only on orders placed before Friday 1pm £15.95
If it's not right, return shipping is free. Find out more.


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Article ID: 137361 - View products that this article applies to.
This article was previously published under Q137361
On This Page
When you are using Dial-Up Networking, you may experience any of the following symptoms:
This behavior can occur if you are not currently logged on to Windows. If you click Cancel in the logon dialog box that appears when Windows is starting, your password cache list file is not opened, and your passwords cannot be saved.
This behavior can also occur if you have installed Dial-Up Networking but the client for Microsoft networks or the client for NetWare networks has not been installed.
To work around this behavior, use one of the following methods.
Method 1When you start Windows, do not click Cancel in the logon dialog box. If you do not have a password, create a new one by typing it in the Password box, or create a null password by clicking OK. If you create a null password, you are no longer prompted to log on to Windows when Windows starts. Creating a null password does not prevent other people from accessing your computer, but does allow you to save your password when you are using Dial-Up Networking or a shared network resource.
NOTE: Creating a null password does not allow you to save your Dial-Up Networking password if you are using user profiles. Using user profiles prevents the Dial-Up Networking password from being saved.
To restart Windows so that you receive the logon dialog box, use the appropriate method below.
Internet Explorer 4.0 or 4.01 Not InstalledClick Start, click Shut Down, click Close all programs and log on as a different user, and then click OK.
Internet Explorer 4.0 or 4.01 InstalledClick Start, click Log Off Username, and then click Yes.
Method 2Install the client for Microsoft networks or the client for NetWare networks in Network properties. To install one of these clients:
Password cache list files are associated with individuals. If you do not log on to Windows, Windows does not know who you are, and cannot open your password cache list file.
For additional information about this issue, click the article numbers below to view the articles in the Microsoft Knowledge Base:
(https://support.microsoft.com/kb/148925/EN-US/ )Dial-Up Networking Password Is Not Saved
(https://support.microsoft.com/kb/141858/EN-US/ )No Windows or Network Logon Dialog Box at Startup
Article ID: 137361 - Last Review: January 19, 2007 - Revision: 1.3
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In This Section
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Audibility of a CD-Standard A/DA/A Loop Inserted into High-Resolution Audio Playback - September 2007
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Sound Board: Food for Thought, Aesthetics in Orchestra Recording - April 2015
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Reflecting on Reflections - June 2014
4 comments
AES E-Library
A Software-Oriented Digital Audio Transceiver Integrated Circuit
The Motorola DSP56401 is a low cost, digital audio transceiver chip compatible with the AES-EBU and EIAJ CP-340 digital audio transmission standards. When used with a DSP56001 digital signal processor and DSP56351 sigma-delta A/D-D/A converters, it provides a software controlled, minimum chip digital audio system. This paper describes the architecture, software drivers and clock recovery of the DSP56401 integrated circuit.
Click to purchase paper or login as an AES member. If your company or school subscribes to the E-Library then switch to the institutional version. If you are not an AES member and would like to subscribe to the E-Library then Join the AES!
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Originally written on Fox Sports | Last updated 9/18/12
Atlanta Braves shortstop Paul Janish dislocated his left shoulder Tuesday night diving for an infield hit in the ninth inning of their 4-3, 10-inning loss at Miami. X-rays revealed no structural damage, but Janish said he doesn't expect to sidelined for long. ''I've probably done it eight or nine times before,'' he said. ''Usually it just slides right back in. This is the first time I've ever had it get stuck and not be able to get it back in. They popped it back in. It's going to be sore for a few days.'' Janish entered the game in the ninth. He's hitting .186 in 55 games.
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Interested to buy Turkish knitting and crocheting yarns?
Interested to buy in bulk with affordable and small quantity?
BenangKait Online Store is a Turkish Yarns Supplier since 2010 and this is our 5th year supplying Turkish yarns all over the world direct from Turkey to your destinations!
For further information, do contact us at email@example.com
Thank you and best regards,
-Ayza Gursoy
BenangKait Online Store


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Press release from Business Wire
The Votes Are in: Flo Elected to the Madison Avenue Advertising "Walk of Fame"

 The popularity of Progressive's perky insurance clerk shines again

Tuesday, October 02, 2012
The Votes Are in: Flo Elected to the Madison Avenue Advertising "Walk of Fame"09:19 EDT Tuesday, October 02, 2012 MAYFIELD VILLAGE, Ohio (Business Wire) -- The votes have been counted, polls are closed and the nation has elected ad icon "Flo" from Progressive to the Madison Avenue Advertising "Walk of Fame." After a three-week voting period blanketing social media, consumers chose "Flo" for the distinction over a range of other famous icons and characters, covering a century of advertising and marketing. The votes are in: Flo elected to the Madison Avenue Advertising "Walk of Fame" (Photo: Business Wire) "I'd like to thank glitter, gummy bears, and most importantly, all my fans for their support," said Flo. "I'm just a girl with a white apron and a blue headband who tries to make car insurance a little easier to understand. The fact that people took the time to log on and vote for me is absolutely flattering." This isn't the first time that consumers have chosen Flo in an advertising icon battle. In 2011, fans voted Flo the top advertising icon in the country in a head-to-head, Entertainment Weekly competition. But Flo's popularity doesn't end with her victories in contests against other ad icons. She was recently named by Advertising Age as one of the "Top Ten Female Icons of All Time," a list that included Betty Crocker, The Morton Salt Girl and Rosie the Waitress. Flo is the only icon on Ad Age's list from the past 25 years. "While she was born on TV, Flo's iconic presence now extends well beyond the screen to social, mobile, games, print, radio and throughout pop culture," said Jeff Charney, chief marketing officer at Progressive. "After nearly five years in the market and 80-plus commercials, she's on top of her game and more relevant than ever. She's real, confident and current. Flo isn't trying to change who she is, she's trying to be who she is. That's why consumers across the board relate so well to her." About Progressive Celebrating its 75th anniversary in 2012, The Progressive Group of Insurance Companies (collectively, "Progressive Insurance") makes it easy to understand, buy, and use auto insurance. Progressive offers choices so consumers can reach it whenever, wherever, and however it's most convenient—online at progressive.com, by phone at 1-800-PROGRESSIVE, on a mobile device or in-person with a local agent. Progressive offers insurance for personal and commercial autos and trucks, motorcycles, boats, recreational vehicles, and homes. It's the fourth largest auto insurer in the country, the largest seller of motorcycle insurance, and a leader in commercial auto insurance. Progressive also offers car insurance online in Australia at http://www.progressiveonline.com.au. Founded in 1937, Progressive continues its long history of offering shopping tools and services that save customers time and money, like Name Your Price®, the Snapshot Discount®, and a concierge level of claims service. The Common Shares of The Progressive Corporation, the Mayfield Village, Ohio-based holding company, trade publicly at (NYSE:PGR). Photos/Multimedia Gallery Available: http://www.businesswire.com/cgi-bin/mmg.cgi?eid=50427566&lang=en Progressive PRSteve Kaczynski, firstname.lastname@example.com


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GenreDrama, Romance
Release DateTBA 2012
StudioCapitol Films
Cast and Crew
DirectorMichael Radford
ProducerMichele Berk, Anna Chi, Alan Ladd Jr., Julia Palau
ScreenwriterDonald Martin
Inspired by a true story, A DREAM OF RED MANSIONS is an epic tale of love and courage, set against the background of the Chinese cultural revolution. When American photo-journalist Diana Mellors travels to war-torn China in 1949, she is catapulted into a whole new world - a Communist one - just as Mao Zedong assumes control of the nation and declares the People's Republic of China.
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Another week in the WWE and another blog. I was feeling nostalgic this week and dug out a load of my old WWE videos to feed my hunger for some WWE nostalgia. Not dvd's or blue-ray, videos. As Roddy Piper says, "old schools cool".
So i dug them out and decided to watch Judgement Day from 2001. Don't get me wrong there was a plethora of classic matches on the ppv and it was great
Updated 04-22-2013 at 12:34 AM by Frank
© 2011 eWrestlingNews, All Rights Reserved.


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Midwest Dreamin' 2015
- Science & Technology
- 600 East Grand Avenue, Grand Ballroom (far east side of pier), Chicago, IL, 60611
IoT Workshop on AT&T M2X Data Service APIs (Chicago – AFTERNOON)
AT&T Developer Program - DevLab
Thursday, June 26, 2014 from 12:00 PM to 4:00 PM (CDT)
Free AFTERNOON IoT workshop on the RESTful AT&T M2X Data Service APIs
Can't make it in the afternoon? Then come to our evening DevLab.
Are you a developer who is interested in the Internet of Things (IoT)? Would you like to be able to collect and retrieve data from Internet-connected sensors for applications in health, home, government, and industry through network and programming language agnostic RESTful APIs? Join the AT&T Developer Program for an afternoon coding workshop on the M2X Data Service. The M2X Data Service is a cloud-based fully managed data storage service for network connected machine-to-machine (M2M) devices. This will be an afternoon of learning and hands-on coding ("lab time"), including working with an Internet-connected sensor. Bring your laptops and leave with our APIs working from your own M2X account.
This workshop will be taught by AT&T subject matter experts and AT&T Developer Program staff. It is also a great opportunity to network with AT&T and your fellow mobile developers.
The event is free and we will be serving lunch. If you are an app or device developer working independently or with a company planning or already producing mobile applications and/or IoT devices then this is the event for you. Space is limited so register now.
Find out how carrier APIs from the AT&T Developer Program can benefit you and your app and device strategy. We look forward to meeting you!
Does an after-hours workshop better suit your needs? Then come to our evening DevLab training session that goes from 6:30 p.m. to 9:30 p.m. -- same content, just a different time! You can register for it here.
DevLab in NYC
|12:00 p.m. - 1:00 p.m.
|Registration & Lunch
|1:00 p.m. - 4:00 p.m.
|AT&T M2X Data Service API
Overlooking Chicago, The Metropolitan Club, located on the 66th and 67th floors of the old Sears Tower, is the gathering point for elevating personal and professional success. As the Loop has flourished the The Metropolitan Club has become known as the place to conduct business and meet Chicago's "who's who."
Installing Postman and cURL for the Lab Exercises
The lab exercises use Postman and cURL so please install them before the event.
We will use cURL for the M2X API lab exercises but you can also use Postman (a free Chrome add-in) if you prefer. You can download Postman at http://www.getpostman.com and the Chrome browser at http://www.google.com/chrome.
For the lab exercises we will use cURL, a powerful command-line tool for making HTTP calls.
Pre-requisites: Windows 7 or later (cURL is already installed on Mac computers)
Use the following steps to install curl:
set path=%path%;"c:\Program Files\curl"
You should see the following message:
curl: try 'curl –help' or 'curl –message' for more information
This means that curl is installed and the path is correct.
curl https://api.att.com
You should see JSON returned:
"error": "invalid API request"
SSL certification error
If you see an SSL certification error, add a -k flag into your curl command.
https not supported error
If you get an error that says, "Protocol http not supported or disabled in libcurl", then you need a different version of curl. Make sure you have downloaded one that says SSL enabled. If you have downloaded the Win64 version, try the Win32 version instead, even if you are on a 64 bit machine.
"DevLab by AT&T" is a series of free coding workshops that AT&T holds across the US. Our goal is to provide high-quality, hands-on training for application developers.
Join millions of people on Eventbrite.
In order to purchase these tickets in installments, you'll need an Eventbrite account. Log in or sign up for a free account to continue.


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- Home Page
- Plasma & Drilling
- Laser Cutting
- Laser Marking
- Accessories
- Service & Spares
- Contact Us
- News
Precision plasma and oxy-fuel cutting, marking, drilling and tapping to high tolerances, in one set-up. This machine is ideal for multi-processing production parts that require medium sized drilling. The Hercules has provided job shops to OEMs with the competitive edge.
CNC Controller: Farley Laserlab PDF-TS Touch screen controller
Profiling speed: up to 20m/min
Rapid traverse: up to 50m/min
Spindle Motor: Siemens
Max drilling power: 14.6kW (20hp)
Max spindle speed: 4500 rpm
Accuracy: ± 0.15mm
Max drill (insert): 45mm
Repeatability: ± 0.1mm
Max tool diameter: 65mm
Cutting widths: 2.5m to 5m
Max clamping thrust: 8000N
Cutting length: 3m to 50m
Spindle taper: BT40
Material thickness: 6mm- 150mm
Coolant system: High pressure through spindle
Drive system: Twin side drive Helical rack & twin pinion, backlash free gear box
Servo Motros: Siemens
Components: Hypertherm HyDefinition plasma up to 400 amp, 5-axis bevel head, oxy-fuel systems, marking systems, BT40 spindle taper with 4 or 20 tool changer, raise/lower water table and touch screen controller. All Siemens servos and drives.


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Christ Church is the essential place to visit in Oxford. Two of its famous landmarks, Tom Tower, by Christopher Wren, and Oxford's Cathedral spire, contribute to the city's celebrated skyline and mark Christ Church as a unique dual foundation; one of Oxford University's largest Colleges and the Cathedral Church for the Diocese of Oxford.
Stroll through the tranquil landscape of Christ Church Meadow where Longhorn cattle graze at the centre of a bustling city. Enter the majestic Meadow Gate and walk in the footsteps of academics, kings and queens, a little girl called Alice and even a few wizards! Gaze upon the beautiful buildings, elegant quadrangles and vibrant stained glass windows and gain an insight into the rich history of an Oxford college and the life of a community dedicated to learning.
This section of our website contains useful information to help you plan your visit and details about upcoming events that you might like to attend.
Please note that the Hall will be closed until mid-July following structural repairs to the roof. The college and cathedral will remain open during this time and the entry charge will be adjusted accordingly. Please visit the 'opening times and prices' page for information about the Cathedral's opening hours.
The Christ Church Guidebook is on sale now at the Cathedral Shop and the Porters' Lodge. The Guidebook tells the fascinating history of Christ Church in beautiful prose and images. Readers are offered a glimpse into everyday life at the college, including areas which are not accessible to visitors.
"Christ Church has been one of England's prime educational establishments for nearly 500 years, and is now a progressively modern college in a very modern university. But it is much more than that too. It is the seat of a cathedral, for one thing. It is an architectural exhibition. It is a treasure house and a portrait gallery. It is a place of literature and beautiful music. It is a school of statesmen, eminent divines, men and women prominent in all walks of life. It is a legend and a myth" (From the introduction by Jan Morris)
Printable Version
Registered Charity Number: 1143423


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Convergent Design Unveils nanoFlash HD Recorder/Player
Miniature version of the company's Flash XDR recorder
As both a recorder and player, nanoFlash provides HD-SDI I/O as well as an LTC input, RS-232/485 (for remote control) and a power/start/tally-light connector. MPEG-2 video and up to eight channels of embedded audio and timecode can be stored in the MXF format using the FAT32 file system. The nanoFlash records and plays back files across the two CF cards. 32 GB cards are $135 each. Two cards combined provide 140 minutes of 50 Mbps record time or 70 minutes at 100Mbps 4:2:2.
The company said that, unlike FireWire-based hard-disk recorders, which merely copy the native camera video quality, nanoFlash encodes a live camera's "never-compressed" HD-SDI output to a much higher quality. This means higher-bit-rate acquisition with less compression, eliminating most of the common noise and blurring artifacts. The unit also offers 24p pulldown removal, image flip, time-lapse recording and RAID 1 redundant recording capability (automatic backup).
An ASI I/O (MPEG-2 TS) firmware upgrade is available, which allows the nanoFlash to be used for HD ENG/EFP (live news coverage) and video-over-IP applications. Adjustable bit rates from 17.5 to 100 Mbps support microwave and satellite transmissions.
The nanoFlash is priced at $3,495 and ships in September.
For more information, visit www.convergent-design.com.
Categories: Business, Creativity, News, Shooting, Technology


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KEN GORE (American, 1911-1990). Autumn Maples. Oil on canvas laid on board. 20 x 30 inches (50.8 x 76.2 cm). Signed lowe...
2011 May Signature Fine American, European Art & Western Art #5062
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Heritage Auctions - Design District Annex
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Autumn Maples
Oil on canvas laid on board
20 x 30 inches (50.8 x 76.2 cm)
Signed lower right: KEN GORE
Titled verso: "AUTUMN MAPLES"
Doll & Richards, Inc., Boston (label verso).
Condition Report*:Overall very good condition; under UV examination, no evidence of restoration. Framed Dimensions 27 X 36.5 Inches
*Heritage Auctions strives to provide as much information as possible but encourages in-person inspection by bidders. Statements regarding the condition of objects are only for general guidance and should not be relied upon as complete statements of fact, and do not constitute a representation, warranty or assumption of liability by Heritage. Please note that we do not de-frame lots estimated at $1,000 or less and may not be able to provide additional details for lots valued under $500. All lots are sold "AS IS" under the Terms & Conditions of Auction.
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Feature...Join CNU!Be a Part of the Movement»»»
Eye on the Street: Friday, October 28, 2011
Submitted by Ben Schulman on Sat, 09/10/2011 - 6:57pm
CNU's own Matt Berggren points his lens on a Portland, OR street.
"Eye on the Street" is an ongoing series of user-generated photos and commentary submitted by dedicated New Urbanists like yourself. Ever been walking down a busy, well-developed street and thought to yourself "I am walking through a great pic of urban design!" and wished you could share it? Ever been attempting to cross an six-lane road and thought to yourself "I just wanna cross! I wish I could take a picture and share this insanity with everyone"? Well, "Eye on the Street" is your way to do exactly that. Snap-as-you-go and submit your pics to: firstname.lastname@example.com.
Submitted by Ben Schulman on Fri, 10/28/2011 - 11:22am.
Write your comments in the box below and share on your Facebook!


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President Barack Obama
Federal employees and the State of the UnionComment
Davidson interview January 27, 2014
President Obama touting State of the Union policy goals in Wisconsin
President Barack Obama is back on the road Thursday focusing on training workers for in-demand jobs, another theme from his State of the Union address.
At Costco Obama touts his plan to boost wages
President Barack Obama stood alongside Americans on the job Wednesday in a morning-after trip to promote his State of the Union proposals to boost personal finances of low-wage workers.
State of the Union: Republicans respond to Obama's remarks
Congressional Republicans responded swiftly and sharply to President Barack Obama's State of the Union vow to act on his own if lawmakers won't cooperate on "creating new jobs, not creating new crises."
State of the Union: Obama addresses Congress (TEXT included)
Seeking to energize his sluggish second term, President Barack Obama vowed Tuesday night in his State of the Union address to sidestep Congress "whenever and wherever" necessary to narrow economic disparities between rich and poor.
State of the Union full text
Text of President Barack Obama's State of the Union address, as delivered and provided by the White House.
Obama targets college sexual assault epidemic
President Barack Obama is launching an initiative to combat sexual assault, especially on college campuses. Obama plans today to sign a presidential memorandum to create a task force to protect students from sexual assault.
Breaking down NSA reformsComment
Friedman interview January 20, 2014
President Obama celebrates MLK by visiting soup kitchen
President Barack Obama is honoring Martin Luther King Jr.'s legacy of service by helping a soup kitchen prepare its daily meals.
That was the week that wasComment
Burkman, Fowler interview January 17, 2014
Obama: 2014 can be breakthrough year for U.S.
President Barack Obama says he believes 2014 can be a breakthrough year for the country.
Obama urges expanding access to higher education
President Obama called on an assembly of college and university presidents and leaders of nonprofit and other education groups to rally around a goal of widening opportunities for disadvantaged students.
NSA reformsComment
Wingo interview January 15, 2014
Obama's NSA changes cause concern for phone companies
Telephone companies are quietly balking at the idea of changing how they collect and store Americans' phone records to help the National Security Agency's surveillance programs.
Supreme Court recess apointment case scheduled for Monday
President Barack Obama and Senate Republicans are squaring off at the Supreme Court over the president's power to temporarily fill high-level positions.
Obamacare back in GOP crosshairs
President Barack Obama's health care law is back in Republicans' legislative crosshairs as the GOP-led House weighs a bill that would impose new requirements on the administration.
Obama expected to tighten NSA restrictions
Obama could unveil his highly anticipated decisions as early as next week. Ahead of that announcement, he is consulting with lawmakers, privacy advocates and intelligence officials who were invited to White House meetings Wednesday and Thursday.
Robert Gates' memoir critical of Obama, Bush, Biden
The White House is bristling over former Defense Secretary Robert Gates' new memoir accusing President Barack Obama of showing too little enthusiasm for the U.S. war mission in Afghanistan and sharply criticizing Vice President Joe Biden's foreign policy instincts.
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What do you think is the best overall area (theme and attractions) in DCA.
OK, OK, I know Many of you are already thinking "Best" and "DCA" are contradicting terms, an enigma, something like Jumbo Shrimp, but then think of it this way, "What is the least awful area of DCA?" :devil:
Here are the areas and how they are divided
(Please note that I have divided Golden State and
Paradise Pier because of the large sections they cover
and have kept the various attractions within close proximity.
I would have divided Hollywood Backlot too, except
half of that is already closed):
Sunshine Plaza
Hollywood Pictures Backlot
A Bug's Land: Includes the 3d movie and previous farm area
Pacific Warf Area: Includes the 2 factory tours, stage and food area, Golden Dreams, Seasons of the Vine
Condor Flats/GRR: includes Soarin' over California, Redwood Creek Challenge Trail, and Grizzly River Run
Route 66: Mulholland Madness, Orange Stinger, Golden Zephyr, Jumpin Jellyfish, SS Rustworthy
Paradise Pier: California Screamin, Maliboomer, Sun Wheel, King Tritons Carousel, Games of the Boardwalk


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Meristant® 101L
Essential Ingredients
Meristant® is a naturally derived liquid, water resistant agent based on Abietic Acid as extracted from pine rosin. Is proven to increase water resistance in sunscreens, long wear mascara and lip care, and have the additional property of solubilizing Avobenzone and Oxybenzone. It's high refractive index can add gloss to personal care products such as hair care, lipstick and lip gloss.
Specification Sheet
- Meristant® 101L (English)
- MSDS (English)
Essential Ingredients works very hard to help suppliers and their customers achieve success in the cosmetic, personal care and related industries by satisfying their expectations for customer service, technical sales support, timely delivery, great products and first and foremost - innovation.


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Mario Plushies
Mario Plush OMG THEY ARE SO ADORABLE I WANT MORE 3D
Aren't they so adorable? I NEED MORE OF THEM!
Note: Although this image is formmatted with .JPG formatting it was converted from .MPO formatting, if you view it with a Nintendo 3DS it will be in 3D.
- 0


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Outdoor Adventure Living-Learning Center
Hall Information
Program Information
OA-LLC Website
Mr. Jimmy Britven
The Outdoor Adventure Living and Learning Center is one if the unique opportunities at Baylor designed to put students with common interests together in living and learning environments that serve to enhance all areas of their development as college students.
Students in the OA-LLC program will live on campus together in Brooks Flats. As a resident of Brooks Flats, we anticipate your experience to be fun and exciting, inviting and communal, positive and meaningful, and filled with memorable moments.
Location: Brooks Flats
Eligibility: All majors and classifications


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F-Series Hentai Pack : Collection of 25 short hentai flash animations featuring anime and manga heroines25 January 2010, by Oliver AKA The Admin
Fresh off the torrents, here's something highly unusual, a pack of 25 hentai movies in flash format, under the name "F-Series".
Usually I'm no fan of flash format hentai animations, but this time, with a pack of 25 ones, most of them in very decent quality, I thought it worth sharing
A few different costumes are available for each girl, you can click-and-move zones like the tits, and the hardcorest part is a dildo come-and-go.
The pack is 69 MB big, to make yourself an idea before downloading and decide if it is worth downloading, I uploaded one of the anims to megaupload, see for yourselves if you like it
These are just a few illustrations, for the complete list of illustrations, cf. the bottom of this post.
Download the F-Serie Flash Anims Pack in a Zip file
(25 different sets, 69 MB)
(please, is there a chance you can use the depositfiles link ? That will generate a tiny bonus for me, thank you very much if that's possible for you ! :)
A bonus for you, the coupon '07xpy4r3b7eiio5108b0db12dzwmjkxm' gives a 10% discount on Depositfiles 6-months and 1-years accounts. )
Or you can also use the Alternative :
Zip Mirror #1 – or – Zip Mirror #2
UPDATE : to play them, you can either drag and drop them into firefox/IE/Whatever, or open them with Winamp, or download from Adobe's website the freeware macromedia Flash player (an app, not the usual plugin)
Here is the list of the illustrations :
And at this point, before publishing this share, I'm wondering if I should categorize it in the "movies" category, or in the "CG" category… Blast it, I'll put it in both categories
to grab the file you want. You can just pick the stuff you need, it's quick and easy :)
To find more works about a subject ("Dragon Ball" for instance), use the search field in the upper-right corner of the page. When I share enough works by a good artist I create redirection pages with links to all his shared works.
A problem to play videos, or questions like "how to download the zips" or "why does it ask a password, you said there isn't one" ? Read the Help & information page :)
Most fundamental rule : if a download fails once, that can happen : try again ! ;-)
You are welcome to share the links to my uploads - I didn't draw the hentai myself anyway - but please, NOT a word-to-word carbon copy.
Sign up to Gravatar and give here the same email to show your avatar.
You can use these allowed html tags :   ,   ,   ,  (NO trailing slash, NO double quotes) ,   (NO double quotes)
26 Responses to ' F-Series Hentai Pack : Collection of 25 short hentai flash animations featuring anime and manga heroines '
To be honest, I have actually seen these before, though I never thought of downloading them.
Interesting share. Thank you.
I try to play those swf with internet explorer but all I get is a white screen?
I open them with Internet Explorer, but all it shows is a white screen? How do you make those files work?
Sweet! I've been meaning to download these for a while, you made my life that much easier.
thanx a bunch Oliver
its best if you just use Winamp to open them…i did notice something really fucked up with some of the animations….for example "Iroha-F" when her top comes off and the boobs jiggle at that exact point something weird happens…do have a look at it…i was cracking up maybe not much of a deal to anyone else, but i find it quite hilarious check it out!
Hi: you need Macromedia Flash installed in your PC. After you installed it you open'em and you'll enjoy. Good luck
Hi there Oliver. I certainly enjoyed this one by far. I was wondering if you could post more of these ones, they're just sweet!!!!!!!!!!!! Thanks anyway, see ya.
I don't get any sound on mine. Is anyone else getting this problem?
Many thanx Oliver.
Winamp, huh? I'd have never thought of that. Thanks for the suggestion! (And the goods, obviously.)
Super, ça fait une éternité que je cherche certaines de ces animations !
Merci beaucoup =)
For all of you that have some problems playing .flv files, I recommend using VideoLan's VLC Player, with an smooth and kindly interface and capable of run as most of currently video formats. It's free ofc!
I remember sometime in the far past when I discovered this flash animations. Taking that into consideration they were good then but right now they're just mementos. Thank you for the share oliver.
You don't need to download anything to open these flash animations. Just use INTERNET EXPLORER to open them. It should work perfectly.
Though, for some reason firefox doesn't seem to open them… oh well, IE works fine.
You don't need to download anything to open these flash animations. Just use INTERNET EXPLORER to open them. It should work perfectly.
Though, for some reason firefox doesn't seem to open them… oh well, IE works fine.
Good quality flash that I only wish was longer.
I remember there were several attempts to uncencor them.
hmm this can open with "Shockwave Flash" so u need it… get it from Adobe it free anyway… there are 2 option, 1 DL for Firefox user than install it, and 2 DL for IE with ActiveX it will install automaticaly… well u can play that now. for IE just click that file and select IE to open it, and for Firefox just write the address where u save the files… it work anyway if u already install "Shockwave Flash"…:)
for comment… it nice see this type game again… well at last the quality is good nice for eye…:D
weird, I'm getting no sound. And Ive tried using flash player, vlc and winamp. Anyone else have this problem?
These are nice, I like them a lot. The only thing that bugs me is I wish we could make em go… y'know… faster XD
These are nice, I like them a lot. The only thing that bugs me is I wish we could make em go… y'know… faster XD
um i would like to know if there is a way to where i can go to this site on my psp, hit a link, and it will download right into my psp already converted. i am able to do this on real life pron sites and it would be easyer if i could do it here too.
ahhh big loads of cum with rio :DDD
ooohh thanks I love f series *¬*
i posted them all online in case u wanted them http://gofurself.weebly.com/
thank you very much, I was looking for this hentai pack , I hope there is something similar like this for hentai videos, like a hentai clip video pack>
You liked it ? Hated it ? You've got additional information and links ? Or did you see broken links (please report them, I will reupload it) ? Bring more life here, participate, add a comment ;-)
If you report a dead link, please, tell me what is the precise dead hyperlink and the precise error. Never forget to try again !
Show your avatar in the comments »


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1858 P25C Quarter Dollar, Judd-221, Pollock-264, High R.7, PR63 PCGS....
2010 April-May Milwaukee, WI CSNS US Coin Auction #1139
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The Midwest Airlines Center
Paquet-Designed 1858 Quarter, Judd-221, PR631858 P25C Quarter Dollar, Judd-221, Pollock-264, High R.7, PR63 PCGS. The regular Liberty Seated quarter dollar obverse for the year. Anthony Paquet designed the reverse die for this variety, which was also used to produce the 1859 Judd-234 quarter patterns. A seemingly malformed eagle with outstretched wings displays a shield on its breast. The left claw holds three long, slender arrows while the right talons grasp an olive sprig. UNITED STATES OF AMERICA / QUARTER DOLLAR surrounds the periphery. Struck in silver with a reeded edge. The devices are fully struck throughout, and the lightly hairlined surfaces are toned golden with occasional dabs of cobalt-blue at the margins. (PCGS# 11914)
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Sean Avery won't test free agency, still plans to retire
Jun 27, 2012, 1:48 PM EDT
Looks like throwing his skates in the Hudson River will be Sean Avery's last hockey-related act.
According to Katie Strang of ESPN, the ex-Rangers forward won't try to sign with a team come July 1 and still plans on retiring. Avery told Strang he has "no interest" in testing the free agent market which, I would assume, is roughly the same level of interest teams had in Avery.
The 32-year-old torched whatever was left of his relationship with the Rangers via a series of antics in AHL Connecticut. He last played on Jan. 27 and, after being a healthy scratch for 15 straight games, was sent home on Mar. 5.
Avery then appeared on Bravo TV's Watch What Happens Live and said "I am officially retired — I threw my skates in the Hudson."
Avery's agent, Pat Morris, said his client's comments were "not serious" and he hadn't given up on his career. Shortly thereafter, it was learned Avery refused to sign retirement papers sent to him by Rangers GM Glen Sather.
But now it appears one of the most controversial figures in league history has called it a day.
If it is indeed the end, Avery will finish with 247 points in 580 career games — with 1533 penalty minutes.
- govtminion - Jun 27, 2012 at 1:49 PM
And stay out.
- jernster21 - Jun 27, 2012 at 1:52 PM
Slow hockey news day.
- mrpigs - Jun 27, 2012 at 1:54 PM
I'll still wear my blue 16 sweater with pride! Ull be missed jackass!
- ithacamike - Jun 27, 2012 at 2:49 PM
Who's Sean Avery?
- 950003cups - Jun 27, 2012 at 2:52 PM
If he ever has children, will the Devils draft him first round?
- babar61 - Jun 27, 2012 at 3:13 PM
I just re-watched espns "the season" featuring the 2003 red wings…watching all the great players on that team like brett hull and others talk up Avery about how he will be a star. Strange how things turn out sometimes…
- rushledger - Jun 27, 2012 at 3:18 PM
It's funny his cameos on hbo's 24/7 he's having breakfast with Brandon prust and he's asking of the orange juice is free with sunglasses on… Yeah he's a toolbag no questions asked I wanna hear more about his antics in CT with the whale though
- killerpgh - Jun 27, 2012 at 3:53 PM
I think it involved spitting in the coaches face if I am remember correctly in the AHL.
- capsfan19 - Jun 27, 2012 at 3:39 PM
Ugh this guy is a character…. Would be glad to never hear about him again.
- drunkbro - Jun 27, 2012 at 4:17 PM
avery is following his true passion – designing handbags
- jernster21 - Jun 27, 2012 at 7:08 PM
Or being a much larger man's handbag.
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