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Effects of Vitamin D and Calcium on Proliferation and Differentiation In Normal Colon Mucosa: a Randomized Clinical Trial
Veronika Fedirko, Roberd M. Bostick, W. Dana Flanders, Qi Long, Eduard Sidelnikov, Aasma Shaukat, Carrie R. Daniel, Robin E. Rutherford and Jill Joelle Woodard
Veronika Fedirko
Roberd M. Bostick
W. Dana Flanders
Qi Long
Eduard Sidelnikov
Aasma Shaukat
Carrie R. Daniel
Robin E. Rutherford
Jill Joelle Woodard
DOI: 10.1158/1055-9965.EPI-09-0239 Published November 2009
To investigate the potential efficacy of calcium and vitamin D in reducing risk for colorectal neoplasms and to develop "treatable" phenotypic biomarkers of risk for colorectal neoplasms, we conducted a pilot, randomized, double-blind, placebo-controlled, 2 × 2 factorial clinical trial to test the effects of these agents on cell cycle markers in the normal colorectal mucosa. Ninety-two men and women with at least one pathology-confirmed colorectal adenoma were treated with 2 g/day calcium and/or 800 IU/day vitamin D3 versus placebo over 6 months. Overall expression and distributions of p21waf1/cip1 (marker of differentiation), MIB-1 (marker of short-term proliferation), and hTERT (marker of long-term proliferation) in colorectal crypts in the normal-appearing rectal mucosa were detected by automated immunohistochemistry and quantified by image analysis. In the calcium, vitamin D, and calcium plus vitamin D groups relative to the placebo, p21 expression increased by 201% (P = 0.03), 242% (P = 0.005), and 25% (P = 0.47), respectively, along the full lengths of colorectal crypts after 6 months of treatment. There were no statistically significant changes in the expression of either MIB-1 or hTERT in the crypts overall; however, the proportion of hTERT, but not MIB-1, expression that extended into the upper 40% of the crypts was reduced by 15% (P = 0.02) in the vitamin D plus calcium group relative to the placebo. These results indicate that calcium and vitamin D promote colorectal epithelial cell differentiation and may "normalize" the colorectal crypt proliferative zone in sporadic adenoma patients, and support further investigation of calcium and vitamin D as chemopreventive agents against colorectal neoplasms. (Cancer Epidemiol Biomarkers Prev 2009;18(11):2933–41)
colonic neoplasms
normal colorectal mucosa
Colorectal cancer is the second leading cause of cancer death in the United States (1). Despite advances in treatment, screening, and prevention, colorectal cancer mortality has declined only modestly in recent years, indicating the need for treatable preneoplastic biomarkers of risk for colorectal neoplasms in humans that could be used for monitoring the efficacy of preventive interventions and for developing chemopreventive agents against colorectal cancer.
Calcium and vitamin D are two plausible and evidentially well-supported dietary potential antineoplastic agents. Proposed, likely complementary, mechanisms of calcium against colorectal cancer include protection of colonocytes against bile acids and fatty acids (2), direct effects on cell proliferation, and modulation of the APC colon carcinogenesis pathway (3). Proposed, likely complementary, mechanisms for vitamin D include effects on regulating cell cycle events; promoting bile acid degradation; influencing growth factor signaling, cell adhesion, inflammation, and immune function; and modulating >200 responsive genes (3, 4). Also, epidemiologic studies found that higher total calcium intakes have been consistently associated with reduced risk for colorectal neoplasms (5, 6), calcium supplementation reduced adenoma recurrence (7), and higher blood 25-OH–vitamin D levels have been associated with reduced risk for colorectal adenoma (5, 8).
Almost all carcinomas of the colon and rectum develop from adenomatous polyps, which are thought to arise from the "susceptible" colorectal epithelium characterized by hyperproliferation, impaired apoptosis, and reduced differentiation. Removal of the polyps does not eliminate risk for adenoma recurrence, suggesting that the normal-appearing colorectal epithelium possesses molecular phenotypic changes that put a person at risk for developing a neoplasm. Reduced differentiation and altered cell cycle control occur during the early stages of colon tumorigenesis; therefore, markers of cell proliferation and differentiation in the colorectal epithelium may serve as phenotypic biomarkers of risk for colorectal neoplasia and may be modifiable by chemopreventive agents.
As described herein, we tested the effects of calcium and vitamin D on a marker of cell differentiation (p21waf1/cip1), and two markers of cell proliferation (MIB-1/Ki-67 and hTERT). p21 is a cyclin-dependent kinase inhibitor that plays an important role in cell differentiation, cell cycle control, apoptosis modulation, and tumorigenesis (9). In colorectal crypts, p21 is expressed only in fully differentiated cells (10), whereas telomerase (as indicated by detection of hTERT, the catalytic subunit of the telomerase) is expressed only in proliferative cells of colon crypts (11); and the proliferation-associated marker MIB-1/Ki-67 is expressed in all cells not in G0 phase of the cell cycle (12). We used telomerase expression as indicated by hTERT in colon crypt cells as a marker of long term proliferative activity, and the S-phase marker MIB-1 as a "snapshot" or short-term proliferative indicator.
Few published human studies tested the effect of calcium and vitamin D supplementation on colorectal epithelial cell proliferation and differentiation (13-16), which may serve as preneoplastic modifiable biomarkers of risk for colorectal neoplasms. To address this, we conducted a pilot, randomized, double-blind, placebo-controlled, 2 × 2 factorial chemoprevention clinical trial of supplemental calcium and vitamin D3, alone and in combination versus placebo over 6 months, to estimate the efficacy of these agents on markers of differentiation and proliferation in the normal colorectal mucosa. We hypothesized that calcium and vitamin D3, alone and in combination, increase colonocyte differentiation, decrease the overall rate of proliferation, and normalize the distribution of proliferating cells in crypts within the normal-appearing colorectal epithelium.
This study was approved by the Emory University Institutional Review Board; written informed consent was obtained from each study participant.
Participant Population
The detailed protocol of study recruitment and procedures with detailed specific exclusions was published previously (17). Briefly, eligible patients, 30 to 75 y of age, in general good health, with a history of at least one pathology-confirmed colorectal adenoma within the past 36 mo, and no contraindications to calcium or vitamin D supplementation or rectal biopsy procedures and no medical conditions, habits, or medication usage that would otherwise interfere with the study treatment or procedures, were recruited from the patient population attending the Digestive Diseases Clinic at the Emory Clinic, Emory University.
Clinical Trial Protocol
Between April 2005 and January 2006, 522 eligible patients were identified after initial screening of electronic medical records, and 224 (43%) patients were sent an introductory letter and contacted by telephone to see if they would be interested and eligible to participate in the study. Potential participants (n = 105; 47%) attended the eligibility visit, during which they were interviewed, signed a consent form, completed questionnaires, and provided a blood sample (17). Diet was assessed with a semiquantitative food frequency questionnaire (18). Medical and pathology records were reviewed. After a 30-d placebo run-in trial, 92 (88%) participants without significant perceived side effects and who had taken at least 80% of their tablets underwent a baseline rectal biopsy and, if still willing to participate, were randomly assigned to the following four treatment groups (n = 23/treatment group) for 6 mo (duration to ensure 25-OH–vitamin D steady-state): a placebo control group, a 2.0-g elemental calcium (as calcium carbonate in equal doses twice daily) supplementation group, an 800-IU vitamin D3 supplementation group (400 IU twice daily), and a calcium plus vitamin D supplementation group taking 2.0 g elemental calcium plus 800 IU of vitamin D3 daily. Additional details on the rationale for the doses and forms of calcium and vitamin D supplements were previously published (17).
Participants attended follow-up visits 2 and 6 mo after randomization and were contacted by telephone monthly between the second and final follow-up visits. Pill-taking adherence was assessed by questionnaire, interview, and pill count. Participants were instructed to remain on their usual diet and not take any nutritional supplements not being taken on entry into the study. At each of the follow-up visits, participants were interviewed and completed questionnaires. At the last visit all participants underwent venipuncture and a rectal biopsy procedure. All visits for a given participant were scheduled at the same time of day to control for possible circadian variability in the outcome measures. Factors hypothesized to be related to the expression of biomarkers in normal colon mucosa (e.g., diet, age) were assessed at baseline and at the final follow-up visit. Participants did not have to be fasting for their visits and did not take a bowel cleansing preparation or enema.
All laboratory assays for serum 25-OH– and 1,25-(OH)2–vitamin D were done by Dr. Bruce Hollis at the Medical University of South Carolina in a blinded manner using a RIA method as previously described (19). The average intraassay coefficient of variation for 25-OH–vitamin D was 2.3%, and for 1,25-(OH)2–vitamin D, 6.2%.
Six sextant 1-mm-thick biopsy specimens were taken from the rectal mucosa 10 cm proximal to the external anal aperture through a rigid sigmoidocsope with a jumbo cup flexible endoscopic forceps mounted on a semiflexible rod. The biopsies were then immediately placed in PBS, reoriented under a dissecting microscope, and transferred to 10% normal buffered formalin followed by transfer to 70% ethanol 24 h after initial placement in formalin. Then, within a week, the biopsies were processed and embedded in paraffin blocks (three biopsies per block).
Five slides with four 3.0-μm-thick section levels each taken 40 μm apart were prepared for each antigen. Heat-mediated antigen retrieval was accomplished by placing the slides in a preheated Pretreatment Module (Lab Vision Corp.) with 100× Citrate Buffer (pH 6.0; DAKO S1699, DAKO Corp.) and steaming them for 40 min. After antigen retrieval, slides were placed in a DAKO Automated Immunostainer and immunohistochemically processed using a labeled streptavidin-biotin method for p21, hTERT, and MIB-1 as summarized in Fig. 1. The slides were not counterstained. After staining, the slides were coverslipped with a Leica CV5000 Coverslipper (Leica Microsystems, Inc.). In each staining batch of slides, positive and negative control slides were included. Tonsil was used as a control tissue for all biomarkers. The negative and the positive control slides were treated identically to the patients' slides except that antibody diluent was used rather than primary antibody on the negative control slide.
Summary of biomarker immunohistochemical protocols and images (at ×200 magnification) of colon crypts immunohistochemically processed for: A. p21waf1/cip1, differentiation marker; B. MIB-1/Ki-67, marker of short-term proliferative activity; C. hTERT, marker of long-term proliferative activity.
Protocol for Quantifying Staining Density of Immunohistochemically Detected Biomarkers in Normal Colon Crypts ("Scoring")
A quantitative image analysis method (scoring) was used to evaluate the expression of the biomarkers in the colon crypts, as described previously (17) and shown in Fig. 2A. Briefly, a "scorable" crypt was defined as an intact crypt extending from the muscularis mucosa to the colon lumen (20). Before analysis, negative and positive control slides were checked for staining adequacy. Standardized settings were used on all equipment and software for the image analysis procedures: Olympus BX40 light microscope (Olympus America, Inc.), Polaroid DMC digital light microscope camera (Polaroid Corporation), computer, digital drawing board, ImagePro Plus image analysis software (Media Cybernetics, Inc.), our custom plug-in software for colorectal crypt analysis, and Microsoft Access (Microsoft Corporation). The technician reviewed slides under the light microscope and selected two of three biopsies with 8 to 10 scorable crypts per biopsy, then created a background correction image for each slide, captured the 16-bit grayscale 1,600 × 1,200 pixel image of the crypt at 200× magnification, and traced the borders of the "hemicrypt" (one half of the crypt). The program then divided the tracing into equally spaced intervals to yield segments with the average widths of normal colonocytes, measured the absorbance of the labeling across the entire hemicrypt as well as within each segment, and saved the resulting data into the database. Then, the technician moved to the next hemicrypt and repeated all the previously described analysis steps.
A quantitative image analysis (A) with an example of resulting distributions of p21 marker expression (staining optical densities) along the normal-appearing colorectal crypts in the calcium (B) and vitamin D (C) groups at baseline and follow-up visits. A. Quantitative image analysis of p21 staining optical densities along the normal colorectal crypts consists of several steps: (i) finding scorable crypts (refer to text for details); (ii) manually tracing half of the crypt ("hemicrypt"), followed by automated division of the outline into segments representing the width of an average colonocyte; (iii) automated background-corrected densitometry of the overall and segment-specific labeling of the biomarker and entry of the resulting data into the database. B. Mean batch-standardized absorbance of p21 staining along normal colorectal crypts in the calcium group at baseline and 6 months of follow-up. C. Mean batch-standardized absorbance of p21 staining along normal colorectal crypts in the vitamin D group at baseline and 6 months of follow-up.
One slide reader analyzed all of the stained slides throughout the study with high intrareader reliability—0.95 for MIB-1, 0.98 for hTERT, and 0.96 for p21.
Treatment groups were assessed for comparability of characteristics at baseline and at final follow-up by the Fisher's exact test for categorical variables and ANOVA for continuous variables. Slide scoring reliability was analyzed using intraclass correlation coefficients.
Several outcome variables were defined to estimate the expression of the markers in the crypts overall as well as how they were distributed within the crypts. The mean optical density of staining for MIB-1, hTERT, and p21 in normal colon crypts was calculated for each patient at baseline and 6-mo follow-up by summing all the densities from all analyzed crypts from the biopsy specimens and dividing by the number of crypts analyzed (this measure indicates the overall rate of proliferation or differentiation of rectal crypt epithelial cells and is further called LI, labeling index ref. 20). The crypt differentiation compartment was defined a priori as the upper 40% of the crypts, and the crypt proliferation compartment as the bottom 60% of the crypts (Fig. 1; refs. 15, 20, 21). Measures of the within-crypt distributions of the proliferation markers (i.e., the ratio of expression in the upper 40% to that in the entire crypts, ϕh) were calculated for each patient by taking the mean of the biomarker densities in the upper 40% of crypts and dividing it by the biomarker densities in the entire crypt. For the proliferation markers, we decided a priori to use the ϕh because it is an indicator of an upward extension of the canonical proliferative zone of the colon crypt and was found previously to be modified by calcium and/or calcium plus vitamin D supplementation (15, 22, 23).
Primary analyses were based on assigned treatment at the time of randomization, regardless of adherence status (intent-to-treat analysis). The three biomarkers were analyzed separately. We transformed biomarker expression density data by dividing each individual measurement by the staining batch's average density to adjust for possible batch effects (batch standardization). At baseline, batch-specific mean staining densities were calculated using the measurements from all treatment groups, whereas for the follow-up visit, only measurements from the placebo group were used. Absolute treatment effects were calculated as the differences in the batch-standardized densities from baseline to the 6-mo follow-up visit between patients in each active treatment group and the placebo group using a MIXED effects model. Interaction between calcium and vitamin D treatments was assessed in the MIXED model by including calcium and vitamin D as factors and interaction term between them. Since optical density is measured in arbitrary units, to provide perspective on the magnitude of the treatment effects, we also calculated relative effects (17, 20), defined as: (treatment group follow-up mean/treatment group baseline mean)/(placebo follow-up mean/placebo baseline mean). The relative effect provides an estimate of the proportional change in the treatment group relative to that in the placebo group, and its interpretation is somewhat analogous to that of an odds ratio (e.g., a relative effect of 2.0 would mean that the relative proportional change in the treatment group was twice as great as that in the placebo group). The Delta method was used to derive the 95% confidence intervals for the relative effects (24). Because the treatment groups were balanced on risk factors at baseline, no adjustment was made for other covariates in the primary intent-to-treat analyses.
Spearman's rank and partial Spearman's rank correlation coefficients were used to compare cell proliferation marker values at baseline and follow-up, respectively.
The distributions of the biomarkers' staining densities were graphically evaluated using the LOESS procedure with smoothing parameter 0.5 and local quadratic fitting. First, the number of sections within a hemicrypt was standardized to 50. Then, the average for each section across all crypts was predicted by the LOESS model separately for each patient, and then for each treatment group by follow-up visit. The results were plotted in the graphs along with smoothing lines.
In sensitivity analyses, we also analyzed data without standardization for batch, as well as by including batch as a covariate and using different transformations. The results from these analyses did not differ materially from those reported. Statistical analyses were done using SAS System software (version 9.1.3; SAS Institute, Inc.). A cutoff level of P ≤ 0.05 (two-sided) was used for assessing statistical significance.
Characteristics of Study Participants
The treatment groups did not differ significantly on participant characteristics measured at baseline (Table 1) or at the end of the study (data not shown). The mean age of the participants was 61 years, 64% were men, 71% were White, and 20% had a family history of colorectal cancer in a first-degree relative. Most participants were nonsmokers, college graduates, and overweight. Biopsy specimens that were scorable were obtained for 87, 90, and 90 participants at baseline, and for 83, 85, and 84 participants at 6-month follow-up for the hTERT, MIB-1, and p21 markers, respectively.
Selected baseline characteristics of the study participants (n = 92)
Adherence to visit attendance averaged 92% and did not differ significantly among the four treatment groups. On average, at least 80% of pills were taken by 93% of participants at the first follow-up visit and 84% at the final follow-up visit. There were no treatment or biopsy complications. Seven people (8%) were lost to follow-up due to perceived drug intolerance (n = 2), unwillingness to continue participation (n = 3), physician's advice (n = 1), and death (n = 1). Dropouts included one person from the vitamin D supplementation group, and two persons from each of the other three groups.
At baseline, there were no significant differences between the four study groups in serum 25-OH–vitamin D or 1,25-(OH)2–vitamin D levels. At the study end, the vitamin D and calcium plus vitamin D groups had significantly higher levels of serum 25-OH–vitamin D (P < 0.001), whereas the placebo and calcium groups had slight nonsignificant decreases in 25-OH–vitamin D levels (17). As expected, serum levels of 1,25-(OH)2–vitamin D at the end of follow-up period did not differ significantly between study groups (17).
Effects of Calcium and/or Vitamin D on p21 Expression in Normal Colorectal Crypts
After 6 months of treatment, p21 expression along the full lengths of crypts increased statistically significantly by 201% (P = 0.03), 242% (P = 0.005), and 25% (P = 0.47) in the calcium, vitamin D, and calcium plus vitamin D groups, respectively, relative to the placebo group (Table 2A). The graphical assessment of changes over 6 months in the distribution of p21 expression along crypts showed that the largest postsupplemental increases in p21 were in the upper 40% of the crypts (Fig. 2B and C), and the numerical findings limited to the upper 40% of the crypts were essentially the same as for the entire crypt (Table 2A). There was a statistically significant antagonistic interaction between calcium and vitamin D3 treatments on p21 expression (Table 2A).
Colorectal expression of p21, MIB-1, and hTERT during the clinical trial
Effects of Calcium and/or Vitamin D on MIB-1 and hTERT Expression in Normal Colorectal Crypts
There were no statistically significant treatment effects on the expression of MIB-1 in the crypts overall or in the proportion of its overall expression that extended into the upper 40% of the crypts (ϕh) in any active treatment group relative to placebo (Table 2B). Also, there were no statistically significant changes in the expression of hTERT in the entire crypt at the end of follow-up; however, the hTERT LI ϕh decreased by 10% (P = 0.13), 3% (P = 0.61), and 15% (P = 0.02) in the calcium, vitamin D, and calcium plus vitamin D groups relative to the placebo, respectively (Table 2C).
Graphical assessments of changes in the distributions of MIB-1 and hTERT, and separate analyses of changes in the expression of these biomarkers in the upper 40% and lower 60% of the crypts over 6 months of treatment indicated that the decrease in the ϕh observed in each active treatment group relative to the placebo at the end of the follow-up, while related to decreases in biomarker expression in the upper 40% of the crypts, was also related, in part, to slight increases in expression in the bottoms of the crypts (data not shown).
A statistically significant positive correlation was found between the baseline expression of MIB-1 and hTERT with Spearman's rank correlation coefficients being 0.35 (P = 0.001) and 0.28 (P = 0.009) for the LI and ϕh, respectively. At the end of follow-up, the MIB-1 and hTERT labeling indices were positively correlated (ρpartial = 0.35, P = 0.001), but not the MIB-1 ϕh with the hTERT ϕh (ρpartial = 0.13, P = 0.24). A weak statistically nonsignificant correlation was noted between the LI and ϕh for each of the cell proliferation biomarkers at both study visits (ρ < |0.15|, P > 0.31).
We also investigated whether VDR genotype, change in 25-OH–vitamin D levels, adherence to treatment, sex, family history of colorectal cancer, and nonsteroidal anti-inflammatory drug use modified the observed associations; however, the sample size was too small for these results to be reliable (data not shown).
These data provide evidence for a substantial increase in cell differentiation, as indicated by increased expression of p21, in the normal colorectal epithelium of sporadic adenoma patients in response to vitamin D3 or calcium supplementation and, thus, are consistent with the hypothesis that increased levels of circulating vitamin D or a higher intake of calcium may reduce risk for colorectal neoplasms. Our data also suggest that vitamin D3 may have a slightly greater effect than calcium on p21 expression, and vitamin D combined with calcium may have a lesser treatment effect than either calcium or vitamin D alone on p21. Furthermore, the data provide no evidence that the overall colorectal epithelial cell proliferation rate, as indicated by the expression of short- and long-term markers of proliferation in the entire colorectal crypt, can be reduced by calcium and vitamin D, alone or in combination. However, our data suggested that calcium combined with vitamin D may shift downwards ("normalize") the distribution of proliferating cells in the colorectal crypts as indicated by the expression of a long-term, but not short-term, marker of cell proliferation.
p21waf1/cip1, a cyclin-dependent kinase inhibitor used in this study as a marker of differentiation, is a potent inducer of differentiation in intestinal colonocytes (10), and its expression is downregulated during the early stages of colon tumorigenesis (10, 25). p21 was also reported to participate in cell cycle regulation (9) and control of DNA methylation (26), and to interact with regulatory proteins, among which is calmodulin (27). As was found in colon cancer cells in vitro (28-32), we hypothesized that vitamin D and calcium would increase p21 expression in the normal human colorectal epithelium in vivo. The plausibility of this hypothesis is supported by the fact that the p21 gene is a primary 1,25-(OH)2–vitamin D3–responsive gene with at least three vitamin D response element–containing regions within its promoter (33); and that calcium, through the calcium-sensing receptor, promotes differentiation in colorectal epithelial cells (31, 32). However, there is little literature regarding direct regulation of p21 by calcium, but there is some evidence that extracellular calcium activates protein kinase C, which is associated with the differential induction of p21 in the intestinal epithelium (3). Also, an intracellular calcium gradient along the colon crypt that coincides with the differentiation compartment may modulate differentiation of the colonocytes, thus, regulating p21 expression (34). As hypothesized, we observed the largest increase in p21 expression in the vitamin D group and, to a lesser extent, in the calcium group; however, we found only a relatively small increase in the calcium plus vitamin D group, and a statistically significant antagonistic interaction between the two treatments. There are several possible explanations for the latter finding, including the possibility that the observed treatment effect in the calcium plus vitamin D group may have been due to chance, or that the two agents may have attenuated the effects of either alone. One animal study (35) found that calcium and vitamin D separately are more potent inhibitors of colon tumorigenesis than when combined. However, several other animal studies that investigated the combined effect of calcium and vitamin D reported stronger effects with vitamin D and calcium combined (36, 37), and the results of a large adenoma recurrence trial suggested that vitamin D enhanced the chemopreventive effect of calcium (38). Thus, the combined effect of calcium and vitamin D on colon crypt epithelial cell differentiation as indicated by p21 expression is not clear and a larger more definitive study is needed to clarify it.
No previous human studies tested the effect of calcium and/or vitamin D supplementation on p21 expression in the normal colorectal mucosa, but three small studies (15, 16, 21) investigated the effects of these agents or low-fat dairy foods on other markers of differentiation (acidic mucins and/or cytokeratin AE1) in the normal colorectal mucosa with inconsistent results. Two small studies found no changes in the normal rectal crypt differentiation markers after supplementation with calcium and vitamin D3 (15), or with calcium or low-fat dairy foods (16), but a third, larger (n = 70), randomized, placebo-controlled trial reported significant changes in differentiation markers after supplementation with low-fat dairy foods, which are rich in calcium and vitamin D, but contain other components that may also exert prodifferentiative effects (21). Taken altogether, the results of the present and past studies combined with the biological evidence suggest that calcium and vitamin D induce differentiation in the normal human colorectal mucosa, and that expression of p21 may be a more suitable biomarker of differentiation than other currently investigated markers.
Unlike other studies, we used two different markers of proliferation, hTERT and MIB-1, detected by immunohistochemical methods. MIB-1/Ki-67 is expressed in all cells not in G0 phase of the cell cycle (12), and hTERT protein, a catalytic subunit of telomerase, which functions to regenerate telomeres on the ends of chromosomes, is expressed in almost all human cancers and some normal proliferative epithelial cells such as in the colorectal crypt base (11, 39, 40). We hypothesized that hTERT expression in colorectal crypts better reflects average, long-term proliferative activity than do "snapshot" proliferative indicators, such as the S-phase marker MIB-1, which show rapid, large responses to short-term physiologic stimuli. Biological evidence supports the growth-restraining actions of calcium and vitamin D on colorectal epithelial cells (3); however, few human studies tested the effect of vitamin D and calcium on cell proliferation in the colon.
There have been two large clinical trials of calcium and colorectal epithelial cell proliferation (13, 14) as well as several smaller trials (reviewed in ref. 22, also refs. 16, 21, 41-43). One of these trials (n = 193) found no evidence for a reduction in the LI, but a marked, statistically significant proportional decrease in the ϕh (13), but the second trial (N = 333; ref. 14), with more methodologic problems (22), found no effect on either measure of cell proliferation. The findings from several smaller controlled studies were inconsistent, with some suggesting decreases in the LI and/or ϕh, and other studies indicating no change or statistically nonsignificant increases in the LI and/or ϕh. The results of the present study for the LI are consistent with those from the previously conducted large clinical trials (13, 14), and for the ϕh, with one large clinical trial (13) and several smaller clinical trials (reviewed in ref. 22, also refs. 42, 43). However, it must be emphasized that the present study was a pilot study with limited statistical power; thus, our findings may have been due to chance. Other possible explanations for our findings may have been the use of an antibody that may have low specificity detecting hTERT (11); that the MIB-1 and/or hTERT markers may not be good biomarkers of cell proliferation in normal colorectal crypts; or that calcium may indeed have no substantial effect on colorectal cell proliferation in sporadic adenoma patients.
No published human studies tested the effect of vitamin D alone or combined with calcium on the hTERT or MIB-1 markers of proliferation, but one small randomized clinical trial (n = 21) found a significantly decreased MIB-1 LI, but not the ϕh, in flat mucosa and resected polypoid tissue after 6 months of supplementation with calcium (1,500 mg/day) plus vitamin D3 (400 IU/day; ref. 15). Contrary to the results of one study (15), we did not find evidence for an effect of vitamin D alone or in combination with calcium on overall MIB-1 or hTERT labeling, but we did find a significant downward shift in hTERT expression in the calcium plus vitamin D group. However, as pointed out above, these findings may be due to chance, nonspecific detection methods, or an insufficient vitamin D3 dose or duration.
Previous studies (44) and our study found that the LI and ϕh are statistically independent variables, and other controlled trials testing calcium or other agents on cell proliferation rates found statistically significant reductions in the ϕh, but not the LI (13, 45-47). Therefore, the LI and ϕh may represent different biological aspects of colon tumorigenesis, and serve as independent markers of risk for colorectal neoplasia.
The present study was conducted to test the joint and separate effects of calcium and vitamin D on the individual components and aggregate profile of a molecular phenotype panel of biomarkers of risk for colorectal cancer, which includes biomarkers of APC and mismatch repair pathways, cell cycle events, and others. We previously reported a statistically significant effect of vitamin D on the proapoptotic marker Bax (17), and analyses for other biomarkers in the panel are currently under way. Taken all together, the present and previously published data (13, 17) suggest that calcium and vitamin D may have stronger effects on cell differentiation and apoptosis than on proliferation, and that even relatively low-dose vitamin D may have greater effects on colorectal epithelial cell differentiation and apoptosis than does high-dose calcium alone or in combination with low dose vitamin D. However, larger, more definitive clinical studies are needed to confirm these results.
This study has several limitations. The most obvious limitation is the small sample size resulting in an increased role for chance in detecting or not detecting a treatment effect. The small size also did not allow us to conduct additional subgroup analyses. Another limitation is that, although human studies have found that cell proliferation rates observed in the rectal mucosa are correlated with those found throughout the colon (48, 49), animal studies found that calcium affects cell proliferation throughout the colon (50, 51), and one intervention trial found that calcium decreases the LI and ϕh in the rectum and sigmoid colon, but not in the descending colon (45), there are insufficient data to assume that the effect of calcium is the same in the distal and proximal parts of the colon in humans. Furthermore, the effects of vitamin D alone or in combination with calcium on proliferation and differentiation in different parts of the colon (other than the rectum) are not clear, as there were no such studies in humans. Also, it is unknown whether vitamin D and/or calcium may affect human normal colon, adenoma, and cancer tissue differently. Another potential limitation of this study is that proliferation and differentiation markers are evidentially well supported, but not proven biomarkers of risk for colorectal neoplasms. Therefore, this study cannot prove that because calcium and vitamin D substantially increase p21 expression and may shrink the proliferative zone in the colorectal crypts, they can reduce risk for colorectal neoplasms. The findings of this study may not be generalizable to other populations. Finally, there may be more specific methods and antibodies to detect telomerase expression in colorectal crypts (11), and MIB-1 and hTERT may not adequately reflect cell proliferation rates in normal-appearing colorectal crypts.
The strengths of this study are that it is, to our knowledge, the first clinical trial of the effects of calcium and vitamin D3, alone and in combination on colorectal epithelial proliferation and differentiation in sporadic adenoma patients; the randomized, double-blind, placebo-controlled trial design; the evaluation of both long- and short-term proliferation markers; the high protocol adherence by study participants; the automated biopsy processing and immunostaining procedures; the use of quantitative image analysis; and the strict quality control and consequent high scoring reliability of rectal biopsies.
In summary, these preliminary results from this pilot clinical trial indicate that calcium and vitamin D increase colorectal epithelial cell differentiation and may have relatively little, if any, effect on overall proliferation rates in the colorectal mucosa, but do not rule out a potential normalization of the proliferative zone in the colorectal crypts. This study suggests that p21 expression may be a treatable biomarker of risk for colorectal neoplasms and supports further investigation of calcium and vitamin D3 as chemopreventive agents against colorectal neoplasms.
We thank Vaunita Cohen and Eileen Veronica Smith for excellent technical support, Dr. Bruce W. Hollis for conducting the blood vitamin D assays, Christopher Farino and Stuart Myerberg for the development of the study database, the physicians of the Emory Clinic for work on biopsy procurement, and all study participants for their time and dedication to the study.
Grant support: National Cancer Institute, NIH grant R01 CA104637 (R.M. Bostick), Georgia Cancer Coalition Distinguished Scholar award (R.M. Bostick), and the Franklin Foundation. The National Cancer Institute, the Georgia Cancer Coalition, and the Franklin Foundation had no influence on the design of the study; the collection, analysis, and interpretation of the data; the decision to submit the manuscript for publication; or the writing of the manuscript.
Received March 13, 2009.
Accepted August 26, 2009.
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. Vitamin D and prevention of colorectal adenoma: a meta-analysis. Cancer Epidemiol Biomarkers Prev 2008;17:2958–69.
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. p21(WAF1/Cip1): more than a break to the cell cycle? Biochim Biophys Acta 2000;1471:M43–56.
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. Human studies of calcium supplementation and colorectal epithelial cell proliferation. Cancer Epidemiol Biomarkers Prev 1997;6:971–80.
Arber N,
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. Colonic epithelial cell proliferation decreases with increasing levels of serum 25-hydroxy vitamin D. Cancer Epidemiol Biomarkers Prev 2002;11:113–9.
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. Linear statistical inference and its applications. 2nd ed. New York (NY): Wiley; 1973.
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. Early alteration of cell-cycle-regulated gene expression in colorectal neoplasia. Am J Pathol 1996;149:381–7.
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. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science 1997;277:1996–2000.
Taules M,
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. Calmodulin binds to p21(Cip1) and is involved in the regulation of its nuclear localization. J Biol Chem 1999;274:24445–8.
Evans SR,
Soldatenkov V,
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. Novel 19-nor-hexafluoride vitamin D3 analog (Ro 25-6760) inhibits human colon cancer in vitro via apoptosis. Int J Oncol 1999;14:979–85.
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. A vitamin D3 analog induces a G1-phase arrest in CaCo-2 cells by inhibiting cdk2 and cdk6: roles of cyclin E, p21Waf1, and p27Kip1. Endocrinology 2000;141:3931–9.
Chakrabarty S,
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. Extracellular calcium and calcium sensing receptor function in human colon carcinomas: promotion of E-cadherin expression and suppression of β-catenin/TCF activation. Cancer Res 2003;63:67–71.
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. Role of calcium and other trace elements in the gastrointestinal physiology. World J Gastroenterol 2006;12:3229–36.
Carlberg C,
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. Controlling the chromatin organization of vitamin D target genes by multiple vitamin D receptor binding sites. J Steroid Biochem Mol Biol 2007;103:338–43.
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. The effect of dietary vitamin D3 on the intracellular calcium gradient in mammalian colonic crypts. Cancer Lett 1998;127:43–53.
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. Inhibition of dietary fat-promoted colon carcinogenesis in rats by supplemental calcium or vitamin D3. Carcinogenesis 1988;9:187–90.
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. Dietary calcium and vitamin D modulate 1,2-dimethylhydrazine-induced colonic carcinogenesis in the rat. Cancer Res 1991;51:5608–13.
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. Influence of dietary calcium and vitamin D on colon epithelial cell proliferation and 1,2-dimethylhydrazine-induced colon carcinogenesis in rats fed high fat diets. J Nutr 1993;123:144–52.
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. Vitamin D, calcium supplementation, and colorectal adenomas: results of a randomized trial. J Natl Cancer Inst 2003;95:1765–71.
Cech TR
. Beginning to understand the end of the chromosome. Cell 2004;116:273–9.
Tahara E,
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. Quantitative reevaluation of telomerase activity in cancerous and noncancerous gastrointestinal tissues. Mol Carcinog 1999;26:312–20.
van Gorkom BA,
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. Calcium or resistant starch does not affect colonic epithelial cell proliferation throughout the colon in adenoma patients: a randomized controlled trial. Nutr Cancer 2002;43:31–8.
Rozen P,
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. Calcium supplements interact significantly with long-term diet while suppressing rectal epithelial proliferation of adenoma patients. Cancer 2001;91:833–40.
van der Meer R,
. Calcium affects biomarkers of colon carcinogenesis after right hemicolectomy. Eur J Clin Invest 2002;32:693–9.
Risio M,
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. Correlations between rectal mucosa cell proliferation and the clinical and pathological features of nonfamilial neoplasia of the large intestine. Cancer Res 1991;51:1917–21.
Cats A,
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. Randomized, double-blinded, placebo-controlled intervention study with supplemental calcium in families with hereditary nonpolyposis colorectal cancer. J Natl Cancer Inst 1995;87:598–603.
Paganelli GM,
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. Effect of vitamin A, C, and E supplementation on rectal cell proliferation in patients with colorectal adenomas. J Natl Cancer Inst 1992;84:47–51.
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. Effect of ω-3 fatty acids on rectal mucosal cell proliferation in subjects at risk for colon cancer. Gastroenterology 1992;103:883–91.
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. Abnormal pattern of cell proliferation in the entire colonic mucosa of patients with colon adenoma or cancer. Gastroenterology 1987;92:704–8.
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. Colonic crypt cell proliferation state assessed by whole crypt microdissection in sporadic neoplasia and familial adenomatous polyposis. Gut 2001;48:41–6.
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. Effect of dietary calcium and cholic acid on the proliferative indices of murine colonic epithelium. Carcinogenesis 1986;7:1657–61.
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Wednesday night through Thursday morning, the National Weather Service has advised temperatures will drop into the upper 30s and low 40s, with even colder wind chill values during nighttime hours.
Do not use charcoal or gas grills inside as they can produce deadly carbon monoxide.
Be careful with space heaters. Keep them away from flammable items such as curtains and upholstery. Inspect the heater and electrical cord before use. Do not use extension cords or heaters with defective power cords.
You may notice a foul smell for a few minutes if you haven't turned on your heat yet this winter. If there is an odor of smoke or smoke visible after turning on the heat, call 911.
Using fuel-fired appliances indoors for heat is dangerous. Read the manufacturer's instructions and follow all directions for use.
Make sure your smoke detector is working.
Check on neighbors, friends and relatives. The elderly and children are especially vulnerable to cold temperatures.
Stay indoors or dress warmly in layers. Wear a hat if you will be outside.
Cover outdoor plants and bring potted plants indoors.
The Salvation Army will expand its sheltering capacity Wednesday night into Thursday morning for those needing shelter from the cold weather. Registration begins at 7 p.m. The Salvation Army is located at 2476 Edison Ave., Fort Myers.
Emergency Management will be opening a cold weather shelter for residents at the Bert J. Harris Agricultural Center Conference Room #1 at 7:00 p.m. Wednesday until 8:00 a.m. Thursday Morning. This shelter is located at 4509 George Blvd., Sebring FL 33875.
The Red Cross and Collier Parks will open a warming station from 9 p.m. Wednesday to 9 a.m. Thursday morning at Immokalee Community Park on 321 North First Street, Immokalee. Residents who plan to stay at a warming center are advised to take their own bedding, food, medications, toiletries, water, and anything else they need to be comfortable.
The Homeless Coalition, 1476 Kenesaw Street, Port Charlotte, will open a shelter to persons needing refuge from the cold. The shelter will be open Wednesday night from 6:30 p.m. to 8 a.m.
The American Red Cross will be supporting the shelter with cots and blankets. For more information contact the Charlotte County Emergency Management Office at 941-833-4000.


Collect book jacket blurbs from Goodreads.
Collect more book jacket blurbs from Goodreads.
Go to the movies again.
Analyze the book jacket blurbs for that magic hook.
Listen again to the NPR interview with the writers of Dallas Buyers Club on how sheer will and tenacity got the movie made.
It's really time to stop procrastinating.
What I'd rather do than work on query letters.


text:
/**
 *  Chapter 9 - Interface - Strategy Pattern
 *  Implements the interface Party
 *  Founding father George Washington is the Federalist.
 *  @author wei.shen@iro.umontreal.ca
 *  @version 1.0
 */

package com.ciaoshen.thinkinjava.chapter9;
import java.util.*;
import java.lang.Math;

class Federalist extends PartyMember {

    /**
     *  PUBLIC PROXY OF CONSTRUCTOR
     */

    /**
     *  PUBLIC METHODS
     */

    /**
     *  PACKAGE ACCESS CONSTRUCTOR
     */
    Federalist(String inputName){
        super(inputName);
    }

    /**
     *  PRIVATE FIELDS
     */

    /**
     *  MAIN
     *  @param args void
     */
    public static void main(String[] args){

    }

}

redpajama_set_name: RedPajamaGithub


Subglandular vs Submuscular Placement: Which Is Better?
Subglandular vs. Submuscular Placement: Which Is Better?
When it comes to breast augmentation, women primarily have two important options from which to choose regarding placement: whether to have implants placed beneath the muscle (sub muscular placement "under") or above the muscle (subglandular placement "over"). Choosing the best option for your body helps to ensure your desired breast augmentation results.
Placing an implant beneath the pectoralis major muscle to cover the implant is what plastic surgeons refer to as a submuscular placement. Many surgeons feel submuscular placement helps reduce the likelihood of capsular contracture, although not all doctors agree. Submuscular placement also can reduce the chances of visible implant ripples after surgery, which may occur in women with less breast tissue for coverage.It has also been felt that may minimize visualization of the breast tissue during a mammogram. Submuscular placement will soften upper pole fullness especially in those patients with minimal breast tissue or in particularly thin patients.
Subglandular placement occurs when the surgeon places the implant beneath the mammary gland but above the pectoral muscles. Women who have a generous amount of natural breast tissue generally are better candidates for subglandular placement. In such instances, subglandular placement can provide a more natural look while making the most of your natural assets. Subglandular placement will optimize and enhance upper breast fullness (if desired) while achieving the greatest "filling" of atrophic (sagging, pseudo-ptotic or "deflated") breasts. Placing implants over the muscle will minimize the distortion seen with muscular contraction when implants are placed under the muscle.
Subfascial placement is accomplished by placing the implants under a thin layer of tissue that intimately covers the muscle. In this location it will be under the fascia but over the muscle. It claims to achieve the advantages of both subglandular and submuscular insertion. In reality, this tissue varies greatly among individuals and is quite difficult to raise as an intact structure. This commonly results in an outcome that is closer to a subglandular result.
Dual plane placement involves dissection both above and below the muscle. The former is typically used to rearrange or redrape the breast tissue especially with constricted (tuberous "tubular") breast deformity, but then the implants are placed under the muscle. Essentially, one operates in both or "dual" planes.
Of course, there are potential drawbacks to each breast implant placement option as well. For example, women who are very active may be less likely to see fully positive results from submuscular placement, while subglandular placement oftentimes produces less natural-looking results in slender, smaller-breasted women.
Making the right choice ultimately rests with the patient, who has a much better sense of what her body needs and can make fully informed decisions after consulting with a skilled breast augmentation specialist.


text:
Spent too much time in the field last season?
Posted by Luxury Leather Gifts | Apr 5, 2017 | Luxury Leather Gifts | 0 |
Yes, there is such a thing. If you've been accused of abandoning your partner to pursue your shooting pleasures you may need to get on their right side again, with some serious luxury grovelling. Splash the cash and get back into favour by booking a stay in one of these glorious royal palace hotels. Book now and all will be forgiven. (And then you can come back and get on with booking next season's shooting.)
13 Royal Palaces You Can Actually Stay In
We're always on the lookout for an incredible place to stay – and these fabulous palace hotels don't disappoint…
1. Gstaad Palace, Switzerland
The magnificent Gstaad Palace is nestled in Switzerland's Bernese Alps, its flag-topped turrets flanked by snow-capped mountains. The building itself is over a century old, an imposing grand dame when viewed from the outside – but its chic interior design keeps it cosy.
In its 1960s heyday, Hollywood stars flocked to this quaint corner of Switzerland, just to stay at the Gstaad. The likes of Louis Armstrong and Marlene Dietrich would spend their days on the ski slopes around the hotel, before letting their hair down in its bars and five-star restaurant. If walls could talk… More information
2. Palais Faraj Suites & Spa, Morocco
This hilltop palace overlooks the 1,200-year-old medina (marketplace) in the city of Fez. It was designed in the 19th century by Jean Baptist Barian, King Hassan II's very own architect and interior designer, complete with suites and a lavish hammam – a good way to soothe those joints bothered by standing for hours in damp cover.
With its antique decor, regal suites and pretty shaded courtyards (a cool spot for a traditional mint tea), this regal property offers an authentic glimpse of old Morocco. More information
3. Four Seasons Hotel Gresham Palace, Hungary
When five-star hotel chain Four Seasons acquired Budapest's Gresham Palace in 1998, it set about restoring this derelict Art Nouveau residence back to its former splendour. Hungary's finest silversmiths, artists, ceramists and mosaicists were recruited for the $110-million project.
Their skill is evident in every corner of this spectacular hotel – in the two million-piece mosaic floor, the sweeping staircase, and soaring stained glass ceilings.
Of course, Hungary is a top shooting destination, so maybe you can discreetly scope something out whilst you're there.
4. Hampton Court Palace, England
OK, so you won't be staying in an actual palace, but The Georgian House – an imposing 18th-century residence which sleeps eight – is in the grounds of Hampton Court Palace. It was originally built to accommodate the kitchens of the Prince of Wales, but has held various uses in the royal household over the years.
This is a good one if you're based in the south of England and you don't want to stray too far from home (and your beloved gundogs).
The best part of staying here? You're free to roam the gardens, courtyards and public rooms of the palace during opening hours as often as you like. More information
5. Narain Niwas Palace Hotel, India
In the 1920s, General Amar Singh – a local ruler – built a lavish palace in Jaipur for his family. His sprawling mansion was bedecked with fine art and furniture, with elaborate murals in typically bold Rajasthani colours.
Much effort has been taken to restore the palace's original character, and (with the exception of a few mod cons) staying here is like taking a trip back to the days of the Raj. More information
6. Château Rhianfa, Wales
When Sir John Hay Williams designed a home for him and his wife in the 1800s, he was inspired by the Loire Valley chateaux they'd seen on their travels. True to his word, he created a little slice of France right in the heart of Wales, though it did take him over a decade to finish it.
This mini castle, set in lovely landscaped gardens with views of Snowdonia, is now a private hotel with suitably grand rooms and wedding reception venue. Our favourite part? The turrets have been converted into showers for some of the suites. More information
7. Pousada Palacio de Estoi, Portugal
This 19th-century Algarve palace is pretty in pink, after a renovation restored it to its original rose-tinted glory.
Now a five-star hotel – complete with gold furniture, luxury spa and elegant landscaped gardens – the regal property is famous for its elaborate Rococo ceilings, which are a work of art in themselves. More information
8. Borthwick Castle, Scotland
Just a few miles from Edinburgh you'll find the former home of Mary Queen of Scots – now transformed into an elaborate private hire venue. With room to sleep up to 85 people, it's perfect for weddings and big parties.
Be sure to bag Mary's bedchamber as your own: it boasts a hand-carved four-poster bed and has views of the hill from which Sir Oliver Cromwell launched his attack on the castle. Make your home in this unique piece of history – for one night, at least. More information
9. Olissippo Lapa Palace Hotel, Portugal
This Lisbon palace dates back to the 1800s, when it was built by the characterful Count of Valenças. While he dressed, he would look out over the high-end neighbourhood from the tower. And his parties – attended by Lisbon's aristocracy – were legendary.
Many of the palace's original features have been preserved, such as the stained glass windows of the Columbano room, and the 170-year-old carpet of the Eça de Queiroz room. It's still a treat to look out over the Lapa District and the Tagus River, though you might want to draw the curtains while dressing… More information
10. Ashford Castle, Ireland
This sprawling estate in west Ireland was once the home of the Guinness family (yes, the brewing dynasty), and has a history that can be traced back to the 1200s.
Dine in 'The Dungeon' for a Medieval banquet-style experience, or quaff your favourite vintages in the cavernous wine tasting tunnels. More modern accoutrements include a 30-seat private cinema and a state-of-the-art spa, with a zip line and tree climbing trail for adventurous kids. More information
11. The Gritti Palace, Italy
Overlooking the Grand Canal and a mere five-minute walk from St Mark's, the Gritti is a really special spot in Venice. It was built by the Doge of Venice, Andrea Gritti, in 1475, and was later used as the residence of the Vatican ambassadors to Venice.
It's still a super-swanky place to stay, and is positively dripping with expensive artwork and furniture. It's hard to miss the eye-popping Murano glass chandeliers, and the precious Rubelli fabrics that decorate the rooms and suites. More information
12. Schönbrunn Palace, Austria
The ludicrously opulent Schönbrunn Palace is Vienna's most popular tourist attraction, a feast of gorgeous imperial decor, gilded furniture and magnificent marble statues at every turn. But few people realise that you can actually stay at the palace, in your very own stately suite.
At over £1,000 per night, the former Emperor's residence is no budget bolthole. However, it comes with a private cook, butler and limousine service – so maybe it's good value after all…
If you need to do some serious damage repair, then this is probably the place to do it. More information
13. Ciragan Palace Kempinski, Turkey
This former Ottoman palace was built on the shores of the Bosphorus – a strategic spot in case of invasion, which just so happened to have incredible views too. It's on the European side of the river, so you can look out over luxury yachts and the Asian side of the city while sipping your Turkish coffee.
If money was no object, you'd find us in the Sultan Suite, which has its very own hammam and Bosphorus-view bathtub. And if getting to Istanbul via budget airline simply won't cut it, this is the only hotel in Istanbul accessible by car, yacht and helicopter. More information
The post Spent too much time in the field last season? appeared first on Luxury Leather Gifts.
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redpajama_set_name: RedPajamaCommonCrawl


The DesignWare® ARC® Processor IP portfolio consists of proven 32-bit CPU and DSP cores, subsystems and software development tools. ARC processors are supported by a broad spectrum of 3rd-party tools, operating systems and middleware from leading industry vendors enrolled in the ARC Access Program, as well as a comprehensive suite of free and open source software available through the embARC Open Software Platform.
Synopsys offers the ASIP Designer tool for automating the design and implementation of application-specific instruction-set processors (ASIPs). ASIP Designer enables designers to create custom processors and programmable hardware accelerators for specialized processing requirements.
Learn more about the legacy ARC 600 and ARC 700 processor families.


During a six-month secondment, Denise will work on the Start-up Europe Partnership.
She will be examining motivations of firms to scale-up and looking at common pathways that are used. This will result in policy conclusions about how stakeholders can better support European firms to scale.
Prior to Nesta, Denise worked for several years at Unknown Group, a global innovation agency focused on unlocking business potential. Her role here focuses on analysing entrepreneurial ecosystems and assessing the presence of necessary conditions for firms to start and scale, and advising governments around the world on how to implement policies to strengthen the ecosystem. She has also been involved in several other initiatives aimed at supporting startups, including Get in the Ring and Dutch Centers for Entrepreneurship.
Denise is from the Netherlands and moved to London four years ago. She holds a postgraduate degree in public policy and administration at the London School of Economics and Political Science and an undergraduate degree in communication and media from the Erasmus University, Rotterdam. Outside of work, she enjoys running, vegetarian cooking, reading novels and travelling.
How can startups raise money in Europe? A practical guide.


Must I translate the internal links «Special:X/Y|X»?
Karsten, I checked if it was merged successfully and yes, it was merged, but in TranslateWiki there is errors yet. I made a Ctrl + Shif + R, but the errors persist.
Oh, I was desperate to check the change hehe, excuse me! Yes, not it is fine and it doesn't detect as fail the correct format. I already translated all the SMW's strings, so tomorrow I will try to begin to review all the Spanish translation, because I have detected errors that have to be fixed, some of them are not very alarming, but there are another that have to be fixed until of the release of SMW 3.0. I am expecting the release, I want to do the best as possible, and what I can, to collaborate and improve SMW :).
That's the spirit! :) Thanks a lot for your involvement!
What just comes to my mind: Never ever change the link [[Special:Types/Date|type]] to [[Especial:Tipos/Fecha|tipo]] because in this case everything breaks for people that use Spanish on a non Spanish wiki. You can only do [[Special:Types/Date|tipo]]. I think that adding an alternate label was possible before so disabling the link check was a pretty bad idea since it will do more harm than good.
Oh, true... Excuse me, Karsten. I will fix it in TranslateWiki. I will notice you. I am busy now, but I will do it and the Twitter issues soon.


text:
seasight ( seasight) wrote in ontd_political,
seasight
UCLA Math Major Fighting in Libya
The Internet's newest fascination is a guy named Chis Jeon, a 21-year-old math major at UCLA, who decided a fun way to finish out his summer vacation would be to fly to Libya and fight with the rebels there. Now we want to know everything about him stateside, so we've done a bit of web stalking to learn more. Perhaps Jeon, who will be a senior this year, is getting a jump on the kind of post-college diversionary position Jennifer 8. Lee wrote about in The New York Times on Thursday.
Kristen Chick, the Christian Science Monitor's Egypt correspondent, was one of the first Western journalists to find Jeon, whom she photographed holding an assault rifle and wearing a blue Los Angeles basketball jersey. He told her, "At spring break I told my friends a 'sick' vacation would be to come here and fight with the rebels." Jeon's story is priceless not just because it's a really weird thing to do -- he flew to Cairo on a one-way ticket, then snuck into Libya and linked up with the rebels, mostly communicating through sign language -- but because Jeon is so darned goofy. His reason for the one-way ticket, as told to The National's Bradley Hope: "If I get captured or something, I don't want to waste another US$800." And Hope's description of him trying to use a gun is priceless: " 'How do you fire this thing?' he asked on Wednesday as a bearded rebel handed him an AK-47. Locating the trigger of the assault rifle and switching off the safety, Mr Jeon fired it in the air in two short bursts."
Jeon's Facebook profile shows a photo of him at what the LA Weekly identified as the Coachella music festival.( Read more...Collapse )
Uh. IDEK what to make of this. I mean, on the one hand, he's very brave? OTOH, it seems like he did this because it would be "sick" and he can go home anytime he wants. (ETA: sorry about the ginormous photo. Should I hide it?)
Tags: libya


text:
Q: Как выполнить проверку Мне нужно выполнить проверку и исключить нажатие, при нажатии клавиши отключить нажатие другой клавиши, то есть, если я иду влево, то я не могу пойти направо

A: Ну как-то так. Если нажата клавиша S и при этом направление змейки любое отличное от "вверх", то выполнится условие
if ((buttonPressed == "S") && (direction != UP))
{

}

Т.е. необходима переменная, которая обозначает направление движения. Она у вас должна быть в любом случае для дальнейшего движения и условия приращения координат.

redpajama_set_name: RedPajamaStackExchange


This series of products displays the main functional types of benthic macroinvertebrates derived from a multivariate analysis of 13 life history traits.
This series of products displays the main functional types of benthic macroinvertebrates derived from a multivariate analysis of 13 life history traits. Three main living modes were identified. The first one comprises species resistant to physical stress in natural conditions through a strong mobility, a short life cycle and a high offspring survival probability. The second one is composed of opportunistic species, also with a relatively short life span, among which many are pioneer species which are not very habitat-specific. The third group is composed of species which require many years or even decades to achieve a minimum of reproductive success. Population survival in the two first groups was shown to be unaffected by bottom trawling, whereas the response of the third species group is generally negative (ICES, 2017). Hence, these products provide information on the benthic ecology, but also on ecosystem vulnerability to human pressures, but it must be noted that vulnerable organisms like corals and sponges, which frequently occur at boreal latitudes are not considered in this product.
One representative species of each group illustrates this functional typology. Urothoe poseidonis is typical of the first group. This is a small crustacean which is very abundant in coarse sediments subjected to high tidal velocities and shear stress, as encountered in the Southern Bight and on the Dogger Bank in the North Sea, and areas of northwestern France and southwestern UK with similar habitat characteristics. It has an annual cycle and releases a few large offspring as miniature-adults which become sexually mature only a few months later.
From the second group, Spiophanes bombyx is a small tubicolous worm which can cope with various environmental conditions, including constraining ones like oxygen depletion and salinity variation.
In the third group, Arctica islandica is a very iconic species, famous for its very long life span that exceeds hundred years and can reach five centuries. It matures and reproduces very late, at a large individual size which enables the production of a high number of small eggs. This large number compensates for the very high early mortality rates. Such a life cycle requires stable environmental conditions with low adult mortality. The very low metabolic rate of the species helps to survive naturally occurring stress from low food for prolonged periods. However, the species is very vulnerable to human disturbance causing direct adult mortality. Recovery from such disturbance is very slow.
Complementary maps in the data product show the underlying data used to delineate the three major types discussed above. Each of the 13 life history traits used in the analysis had different modalities. In total, 62 trait modalities have been documented over the 13 traits. Maps have been produced displaying the scores for each of these 62 trait modalities, both based on absolute and relative organism densities averaged per spatial location.
For example, the maps below show the relative proportion of the 4 burrowing depth modalities: 0 cm, 0 - 5 cm, 5 - 15 cm, > 15 cm.
These maps are available for all traits.
The map of the burrowing depth shows clearly that the burrowing depth in the Baltic is mainly at depths between 0 and 5 cm, this corresponds to the life traits of the resistant living mode (first group), see maps below.
The map below shows the distribution data of the three living modes.
A DIVAnd interpolated map of the relative distribution of the three living modes, indicating that the areas with the highest number of vulnerable species are the Greater North Sea or the French Mediterranean coast.
Firstly, the different occurrence lists were merged, and the validity of taxonomic names was checked with the World Register of Marine Species (WoRMS). Two measures were considered per geographic location: individual organism density and number of taxa, in two separated data frames.
Individual organism counts were summed per combination of location, sampling gear, sampling surface area, year, month and taxon. Sampling effort per location and sampling gear was calculated by summing the sampling surface areas of the different samples (when a location was sampled several times) and divided by the number of sampling times. This enabled the calculation of individual organism densities per sampling gear and per location, and finally per location (expressed in number of individuals per squared meters).
The number of taxa per location was calculated by successive averaging, firstly per combination of sampling gear, sampling surface area, year and month; then, per combination of sampling gear, sampling surface area and year, and so on until finally averaging per location.


Stuart Vevers showed off winter florals at Coach's fall/ winter 19 show.
As New York Fashion Week nears its end for another season, Vevers made sure fashion editors had something to remember with his outstanding outwear offering for the upcoming colder months.
The British designer showed off both men's and women's fashions in Coach's co-ed show on Tuesday (12Feb19), with the boys modelling shearling-lined buffalo plaids and quilted leather jackets, while the girls showed off fringing, furry and shearling styles.
Flowers adorned multiple garments in the collection, seen in deep purples, burnt oranges and blues – perfect shades for fall/ winter.
Vevers also didn't stray too far from the prairie trends he's coveted in recent seasons, seen this time in pointed-toe boots, flowing chiffon and ruffled detailing, but for 2019 they were given a tougher edge.
There was also a nod to the psychedelic, with pops of colour and swirly patterns. And artist Kaffe Fassett served as an inspiration too, with patchwork quilt prints directly inspired by him.
"His style has that kind of West Coast aesthetic authority that I was really drawn to. I was fortunate enough that he was into the idea of working together, so we chose prints directly from his archive; some we kept very close to the original, some we recoloured with him," he told the publication.
Other standout looks include a plaid lumberjack print, seen on jackets, both men's and women's and dresses, faux fur coats and Vevers' new season tailoring.
Coach ambassadors Michael B. Jordan and Chloe Grace Moretz sat front row at the style spectacle, while model of the moment Kaia Gerber lead the catwalk stars.
Marc Jacobs will bring New York Fashion Week to a close on Wednesday, with shows from Michael Kors and Boss too.


The extension of my fingers, that are able to create whatever I see in hair. A lifetime tool.
Before MIZUTANI, I never knew that something that hard, can be so soft.
Peter is a hairstylist with indeed passion for people and making them happy. Besides the love for the daily salon work with a great team, Peter enjoys workshops, shows, platform work worldwide. Peter's humorous and entertaining way to speak to hairdressers is well known and on a remarkable degree of esteem in small in-salon classes as well as in front of a big audience.


text:
VPNTunnel review
Paul Renner December 5, 2020
VPNTunnel is a Seychelles-based provider of anonymous services that, while not offering as many servers as some of its competitors, manages to perform exceptionally well. If you are using a Mac device, you won't find it very user friendly because you have to install third-party software and set up VPNTunnel's servers manually, but once it is up and running, it will show you everything it can do. Although this VPN service is pretty good, even better, and more user-friendly, VPN providers can be discovered among the best VPN services. This is our VPNTunnel review.
VPNTunnel Review: Privacy and logging
VPNTunnel supports a wide range of protocols and combinations, including OpenVPN, PPTP, WireGuard, L2TP, IPsec, and Stunnel+OpenVPN. You will be able to share large files via P2P and torrenting clients securely; as it says itself, "P2P is enabled in many locations (as much as we can) – your peering activity will not be followed by private or government agencies".
The company states that it will never ask you for personal information, nor will it ever "track online activity logs or store private information about individual user activities on our network." It also adds that it never "records IP addresses, times, or other details such as that." This always applies to our servers in Sweden.
When we read this, we began to wonder why the policy only mentions Sweden as it goes further: "With respect to our servers in Sweden and other countries where we do not keep traffic logs, it is impossible to provide details that could, for example, connect a specific user to a particular activity at a particular time, for the simple reason that we do not store this data. In other words, it is not an issue that we should consider when making such a request.
Things become a bit clearer (and a bit alarming) in additional text: "For countries where logbooks need to be kept, we will only provide information when required by law."
So logbooks are indeed kept for those countries where the law requires it, even though the VPNT tunnel is located in the Seychelles, which is all a bit odd. More things are buried deeper into VPNTunnel's privacy policy, which states that if a third party acquires VPNTunnel, users' personal information will be released to that third party, opening up several loopholes for anyone willing to exploit it.
VPNTunnel review: Performance
The website states that VPNTunnel's servers are connected to a 10Gbps uplink and that the company is continually adding the latest hardware to ensure that the network is never overloaded. This should guarantee excellent speeds and our tests yielded satisfactory results.
Specifically, we first let the app randomly connect us to "Any Country," and it chose Japan. Despite the distance, we were not disappointed by the download speeds, which averaged 35Mbps on our 45Mbps test connection. The results were even better when we connected to a server in the Czech Republic at 39Mbps. However, a server in New Jersey, USA, marked as ideal for Netflix, performed slightly worse (but still better than some) – 18Mbps.
VPNTunnel review: Pricing and plans
This seller's services are available through three subscription lengths – 1-month at $9.99/month, 3-month at $6.66/month (billed $19.98 every 3 months), and 12-month at $2.99/month (billed $35.88 annually).
The annual package is on the cheaper side of the industry, although there are certainly less expensive VPNs. Accepted payment methods include credit cards (Visa, Mastercard, American Express, Discover, Diners Club), Mint, Sofort, PayPal, GlobalPayments, WebMoney, Perfect Money, as well as for cryptocurrencies such as Bitcoin, Ethereum, Litecoin, Monero, and many more, if you prefer to remain anonymous while paying. There is no free trial period, but there is a 7-day money-back guarantee.
VPNTunnel review does what it's supposed to do – hide your identity online, unblock Netflix and BBC iPlayer and allow safe turning. However, it only has about 150 servers, the customer support seems a bit sloppy, and the privacy policy has a few loopholes. Given these loopholes, a market leader like ExpressVPN could be a better choice, especially since its privacy policy is relatively straightforward and transparent, there is no room for different interpretations, and customer support is available at all times within seconds.
VPNTunnel provides perfect privacy online, access to torrenting, and popular streaming channels, but has a limited server network, customer support is slow to respond, and the privacy policy leaves room for potential exploitation.
Unblocks Netflix and BBC iPlayer
Wide platform support
Accepts cryptocurrencies
A low number of servers
Loopholes in the privacy policy
No Mac client
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and had a professional jewelry maker fabricate specially for monocircus.
'DIA' makes a pretty present for Mother's Day.
DIA now comes in two variations of sterling silver chains that you can choose from.
and a 62 cm long link chain that hangs near the cleavage.
the item comes wrapped up and placed in a handmade fur pouch in a box.