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Background and aims: There are epidemiological, morphological, and molecular differences between normal mucosa as well as between adenocarcinomas of the right and left side of the large bowel. The aim of this study was to investigate differences in gene expression.
Methods: Oligonucleotide microarrays (GeneChip) were used to compare gene expression in 45 single samples from normal mucosa and sporadic colorectal carcinomas (Dukes' B and C) of the caecum compared with the sigmoid and rectosigmoid. Findings were validated by real time polymerase chain reaction.
Results: Fifty eight genes were found to be differentially expressed between the normal mucosa of the caecum and the sigmoid and rectosigmoid (p<0.01), including pS2, S100P, and a sialyltransferase, all being expressed at higher levels in the caecum. A total of 118 and 186 genes were differentially expressed between normal and right or left sided tumours of the colon, showing more pronounced differences in Dukes' C than B tumours. Thirty genes differentially expressed in tumour tissue were common to adenocarcinomas of both sides, including known tumour markers such as the matrix metalloproteinases. Keratins 8, 19, and 20 as well as carbonic anhydrases (II, IV, VII) showed side specific expression and were downregulated in left sided tumours whereas teratocarcinoma growth factor and cyclooxygenase 2 (COX-2) were upregulated in left sided adenocarcinomas. Immunohistochemical analysis confirmed differences in side specific expression for cytokeratin 20 and COX-2.
Conclusions: Differences in gene expression between normal mucosa as well as between adenocarcinomas of the caecum and sigmoid or rectosigmoid exist and should be taken into account when examining new targeted therapeutic regimens.
- LCC, left sided colon cancer
- RCC, right sided colon cancer
- UG cluster, UniGene cluster
- EGFR, epidermal growth factor receptor
- PCNA, proliferating cell nuclear antigen
- DPP IV, dipeptidylpeptidase IV
- RT-PCR, reverse transcription-polymerase chain reaction
- COX-2, cyclooxygenase 2
- MSS, microsatellite stable
- CA, carbonic anhydrase
- MMP, matrix metalloproteinase
- gene expression
- colon cancer
- caecum
- sigmoid
- rectosigmoid
- large bowel
- microarray
- biomarkers
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- LCC, left sided colon cancer
- RCC, right sided colon cancer
- UG cluster, UniGene cluster
- EGFR, epidermal growth factor receptor
- PCNA, proliferating cell nuclear antigen
- DPP IV, dipeptidylpeptidase IV
- RT-PCR, reverse transcription-polymerase chain reaction
- COX-2, cyclooxygenase 2
- MSS, microsatellite stable
- CA, carbonic anhydrase
- MMP, matrix metalloproteinase
Multiple differences between right sided (RCC) and left sided (LCC) sporadic colon adenocarcinomas with regard to epidemiological, morphological, and molecular characteristics suggest that the mechanisms of sporadic colorectal carcinogenesis may differ according to tumour location.1 Cancers of the right and left colon may form different but related groups of tumours because of their different embryological origin (midgut and hindgut, respectively) and different exposure to bowel content. Colon cancer has a different prevalence at varying ages, in high and low incidence nations, and in men and women. RCCs are more common in females, LCCs in males.2 There is also a difference in clinical presentation, in prognosis, and possibly in genetic and environmental epidemiology (see review by Iacopetta3). Furthermore, it has been suggested that a mechanism exists that promotes the progression of mucosal lesions to invasive cancers in the left colon and rectum whereas a de novo pathway from depressed type lesions may be implicated in cancers of the right colon.4 No difference has been found in the distribution of Dukes' stages or in operative mortality between right and left sided sporadic colon cancers. Despite their higher tumour diameter and twofold higher rate of undifferentiated carcinomas, the prognosis of right sided tumours is relatively better than that of left sided tumours, and it has been hypothesised that this could be due to the better blood and lymph supply providing more efficient local tumour defence.5 Recurrence and survival are similar between RCC and LCC6 whereas response to 5-fluorouracil treatment is significantly better in RCC.7
Two studies suggest that molecular differences in gene expression exist between right and left sided colon cancers. Kapiteijn et al showed significantly higher expression of nuclear β-catenin and p53 in rectal cancers compared with proximal cancers.8 Fric et al showed significantly higher expression of cytoplasmic c-erbB2, epidermal growth factor receptor (EGFR), proliferating cell nuclear antigen (PCNA), and dipeptidylpeptidase IV (DPP IV) in right sided sporadic colon cancers compared with left sided cancers.9 Distal tumours display a higher frequency of 17p and 18q allelic loss, p53 accumulation, c-myc expression, and aneuploidy than proximal tumours. Recently, Glebov et al distinguished proximal from distal normal colon mucosa based on gene expression analysis.10
To the best of our knowledge there are no expression data available on differences between adenocarcinomas originating from the proximal or distal part of the colon. As this could have a strong impact on molecularly targeted cancer treatment, we wished to elucidate this aspect and gain insight into differential expression of approximately 7000 human genes of right sided and left sided Dukes' stage B and C adenocarcinomas as well as normal colon mucosa.
Tissue samples, patient information, and RNA isolation
Tissue samples, patient information, and RNA isolation are provided in detail as supplementary data (these data can be viewed on the Gut website at http://www.gut.com/supplemental). Samples from the caecum and rectosigmoid or sigmoid were obtained fresh from surgery and immediately transferred to a solution containing sodium dodecyl sulphate and guanidinium isothiocyanate, snap frozen in liquid nitrogen, and stored at −80°C.
Samples consisted of biopsies from the superficial non-necrotic part of tumours and/or normal mucosa biopsies taken from the oral resection margin. All tumour samples were staged as either Dukes' B (eight from the left colon, five from the caecum) or Dukes' C (seven from the left colon, five from the caecum).
Supplementary table 1 shows detailed clinicopathological information—for example, location of samples in the colon and TNM status (see the Gut website at http://www.gut.com/supplemental). All 15 left sided and 9/10 right sided tumours (90%) were invasive adenocarcinomas; one was an invasive mucinous adenocarcinoma. Six of 10 right sided tumours (60%) were moderately differentiated, 3/10 (30%) were poorly differentiated, and 1/10 (10%) was well differentiated. Ten of 15 left sided tumours (67%) were moderately differentiated, 4/15 (27%) were poorly differentiated, and 1/15 (6%) was well differentiated. The approximate percentages of the volume fractions of tumour cells and stromal cells were semi quantitatively estimated using paraffin embedded diagnostic tissue sections. More than half of the tumour samples showed more than 70% malignant cells. We hypothesise that the percentage of tumour cells is probably higher in the arrayed samples than in the screened paraffin embedded diagnostic histological tissue sections, as the latter represents the whole invasive tumour in the bowel wall. Informed consent was obtained from all patients. All tumours were sporadic. The local scientific ethics commission approved the project.
Total RNA was isolated from approximately 50 mg of single tissue samples using a Polytron homogeniser followed by treatment with Trizol (Invitrogen, Carlsbad, California, USA) according to the manufacturer's instructions. GeneChip (Affymetrix Inc., Santa Clara, California, USA) analysis of single samples was carried out on 10 samples from the caecum (65B, 66B, 73B, 120B, 137B, 90C, 126C, 145C, 138C, and 162C), five Dukes' stage B (median age 76 years) and five Dukes' stage C (median age 66 years). Each of the tumours was accompanied by a corresponding matched normal mucosa sample at the same location from the same patient (median age 70 years). Matched samples were given the same sample number, differentiated by "N" for normal and "B" or "C" for Dukes' B or Dukes' C tumours. Left sided colon samples comprised eight Dukes' stage B (median age 76 years), seven Dukes' stage C (median age 68 years), and 10 "normal mucosa" samples (median age 69 years). Five of these tumours (201C, 202B, 203B, 204C, and 208C) were accompanied by a corresponding matched normal sample at the same location from the same patient. The remaining five normal mucosa samples (157N, 161N, 179N, 195N, and 205N) and 10 tumour samples (16B, 237B, 239B, 54B, 127B, 58C, 74C, 85C, 91C, and 96C) were obtained from an independent set of samples of individual patients who underwent resection of the sigmoid or rectosigmoid colon.
cRNA preparation, array hybridisation and scanning, and RT-PCR
cRNA preparation, array hybridisation, and scanning are provided in detail as supplementary data, including supplementary tables 1–7 (see the Gut website at http://www.gut.com/supplemental).
Data analysis and selection of genes
Data analysis and selection of genes is provided in detail as supplementary data (see the Gut website at http://www.gut.com/supplemental). Comparison analysis was done using Microarray Suite 5.0 (MAS 5.0), MicroDB 3.0 (MDB 3.0), and Datamining Tool 3.0 (DMT 3.0) (Affymetrix) applying the Affymetrix specific software "Statistical Expression Algorithms". Five different comparison groups (A–E) were established and a schematic overview in given in fig 1 and described in detail in the supplementary data (see the Gut website at http://www.gut.com/supplemental).
For all comparisons, several filterings were made to obtain solid and consistent data. To exclude genes with minor or only individual importance, genes were excluded if more than 80% (comparison A) or 70% (B and C) of all datasets were accompanied by a "detection" call of "absent". Genes were included if more than 80% (B and C) or 70% (D) of the comparisons were accompanied by a "change" call of increased or decreased. For statistical analysis, an Affymetrix software integrated Mann-Whitney U test was applied to the signal data of the groups compared with each other. Significance was set at a p value of p⩽0.05.
Real time PCR, normalisation of RT-PCR data, and microsatellite analysis
Real time PCR, normalisation of RT-PCR data, and microsatellite analysis are described in detail as supplementary data (see the Gut website at http://www.gut.com/supplemental).
Formalin fixed paraffin embedded sections from the normal mucosa and matched tumour tissue were stained with monoclonal mouse antihuman cyclooxygenase 2 (COX-2) (cat No 35-8200; Zymed, AH-diagnostisk, Denmark), diluted 1:300, or monoclonal mouse antihuman cytokeratin 20 (cat. No M7019; Dako Cytomation, Denmark), diluted 1:100, as described in detail in the supplementary data (see the Gut website at http://www.gut.com/supplemental).
Using Affymetrix GeneChip oligonucleotide microarrays, we analysed gene expression of 45 colonic samples. The expression profile of 10 sporadic adenocarcinomas of Dukes' B and C from the right side and 15 from the left side were compared with 20 normal colon mucosa samples, 10 matched samples from the right and 10 partly matched samples from the left side. Gene expression differences were determined between: (A) normal mucosa of the right and left side; (B) normal mucosa and Dukes' B or C adenocarcinomas of the right side; (C) normal mucosa and Dukes' B or C adenocarcinomas of the left side; (D) Dukes' B or C adenocarcinomas of the right and left side; and finally (E), differentially expressed genes in the right sided colon from comparison B with those in the left sided colon from comparison C (fig 1).
Comparison A: normal caecum versus sigmoid/rectosigmoid
By comparing normal mucosa samples from 20 different patients—namely, 10 right sided from the caecum to 10 left sided from the sigmoid or rectosigmoid—we identified 160 genes showing site specific differential gene expression, being increased or decreased more than 1.5 fold (p<0.05, Mann-Whitney U test). Fifty eight genes with a p value of <0.01 are shown in table 1; 12 of these genes with fold changes more than threefold the median signal are labelled with an asterisk.
The gene encoding the pS2 protein, maintaining the mucosal surface barrier and stimulating repair processes, showed 7.7-fold higher expression in the left than in the right colon. Other differentially expressed genes with a consistent difference were calcium binding protein S100P (7.6-fold), homeodomain protein HOXB13 (7.2-fold), defensin 5 (6.1-fold), Gal-beta (1-3/1-4) GlcNAc alpha-2.3-sialyltransferase (5.6-fold), 3-beta-hydroxysteroid dehydrogenase gene (5.4-fold), and HE4 extracellular proteinase inhibitor homologue (5.3-fold). Also, the beta subunit of creatine kinase-B, fibrinogen A alpha polypeptide alt. splice 3 E (3.7-fold), cathepsin E, and protein tyrosine phosphatase were among the genes showing more than threefold significantly different expression between the two groups.
Comparison B: normal versus tumour caecum
By comparing normal mucosa of the caecum to matching caecum adenocarcinomas staged as Dukes' B or C and derived from the same patient, we identified 118 genes significantly up or downregulated more than 2.8-fold (p<0.05, Mann-Whitney U test) in adenocarcinomas compared with normal mucosa (see supplementary table on the Gut website at http://www.gut.com/supplemental). Seventy three showed fold changes of more than fourfold, and of these, 22 genes with a p value of <0.01 are shown in table 2.
A characteristic finding was that most genes (n = 15) were downregulated in carcinomas compared with normal mucosa, and only a few were upregulated (n = 7). Several matrix metalloproteinases, such as MMP1, MMP3, and MMP10, located in the extracellular space and involved in proteolysis and peptidolysis were highly upregulated in carcinomas, as well as E1A enhancer binding protein (E1A-F) (fourfold) and calcium binding protein S100P. TRPM-2 protein (fivefold), complement protein component C7 (fivefold), and NAD+ dependent 15 hydroxyprostaglandin dehydrogenase (PGDH; 16-fold) showed decreased expression.
Comparison C: normal versus tumour sigmoid/rectosigmoid
We compared normal mucosa from the left side of the colon to matching adenocarcinomas of Dukes' B and C from the same patient in five cases, and in those 10 cases where a matching normal sample was not present, we compared each of the 10 tumours to each of five single normal samples (for details see material and methods). We identified 186 genes significantly differentially expressed more than 2.8 fold (p<0.05, Mann-Whitney U test) from the normal mucosa to Dukes' B or Dukes' C tumours (see supplementary table 3 on the Gut website at http://www.gut.com/supplemental). The majority confirmed our recently published findings made on pools of colorectal cancer samples11; for example, downregulation of nuclear encoded mitochondrial genes such as TST thiosulfate sulfurtransferase (rhodanese) (4.5-fold) and the SCAD gene 5′ UTR exon 1 and 2 (sevenfold). The 42 most important genes with a fold change ⩾4 (p<0.01) in both Dukes' B and Dukes' C are shown in table 3. Other genes (for example, osteopontin) which showed changes have been omitted here because changes were found to be ⩾4 fold in either Dukes' C or Dukes' B but not in both.
Thirty genes were found to be downregulated in cancer, such as GCAP-II (33-fold), carbonic anhydrase IV (33-fold), and DTD sulfate transporter gene (20-fold). Only 12 genes were upregulated, among these microsomal dipeptidase (MDP4, MDP7; 20-fold) and interleukin 8/MDNCF (15-fold). As a novel finding we found that carbonic anhydrase VII (CA VII) was decreased more than fourfold from normal to Dukes' B and C adenocarcinomas.
Comparison D: tumours from the caecum versus sigmoid/rectosigmoid
Within each of the Dukes' B and C stages, we compared all adenocarcinomas from the left side with all of those from the right side of the colon. We identified five genes in Dukes' B, 39 in Dukes' C, and five genes in both B and C, that showed significant differences in expression levels (p<0.05) with an average fold change of 2.8, corresponding to a total of 44 genes differentially expressed in left and right sided tumours (see supplementary table 4 on the Gut website at http://www.gut.com/supplemental). Among these 44 genes, 16 showed more than threefold upregulation or more than fourfold downregulation (table 4).
Differential gene expression was more common in Dukes' C than in Dukes' B, and among the genes were caldesmon 1, involved in cellular mitosis and receptor capping, modulator recognition factor 2 (a DNA binding factor), ARHB, involved in signal transduction, transgelin 11 (SM22-alpha), and D component of complement (adipsin, involved in proteolysis and peptidolysis), all five showing higher expression in left sided carcinomas. In contrast, homeobox A5 protein, a sequence specific transcription factor, was more strongly expressed in Dukes' C adenocarcinomas of the right side of the colon.
Comparison E: comparison to identify genes in common or differentially expressed in right sided versus left sided tumours
A total of 186 genes previously identified to be differentially expressed from normal mucosa to tumour in the left side of the colon were compared with 118 genes identified in the right colon. This resulted in 30 common cancer genes being significantly differentially expressed more than threefold (accompanied by a p value of <0.05) in at least one of the Dukes' in both right sided as well as left sided tumours. These may make ideal colonic tumour markers (table 5).
Validation of the results by real time PCR applied to aminopeptidase N/CD13, SCAD, and PCK1 is shown in fig 2 where single GeneChip analyses were compared with real time PCR analyses. Additionally, we identified cancer genes being characteristic for one side of the colon only. Eighty eight genes shown in supplementary table 5 (on the Gut website at http://www.gut.com/supplemental) were significantly differentially expressed exclusively in right sided tumours, such as factor XIII subunit a and calcium binding protein S100P (fig 2), suggesting a more crucial role in caecal adenocarcinomas. A total of 156 genes shown in supplementary table 6 (on the Gut website at http://www.gut.com/supplemental) were significantly differentially expressed only in left sided tumours. Among these were MDP4/MDP7 and the interferon inducible protein "9-27". Differences in expression in most of the growth factors were seen in the left colon such as upregulation of teratocarcinoma derived growth factor (>7 fold). Furthermore, the COX-2 gene was more than sixfold higher in Dukes' C tumours of the left colon, and did not show a significant difference in right sided tumours. Most strikingly, expression of keratins 8, 19, and 20 was severely reduced in the left colon but did not show significant differences in the caecum.
Microsatellite analysis
Microsatellite analysis was performed on microdissected tumour tissue, as described in materials and methods in the supplementary data (on the Gut website at http://www.gut.com/supplemental). Of 10 samples, where the amount of tissue allowed microdissection, only one sample (No 120B) was found to be highly microsatellite instable, the other nine samples being microsatellite stable (MSS), as listed in supplementary table 1 (on the Gut website at http://www.gut.com/supplemental). The fact that all except one of the tumours were stable with regard to microsatellites BAT25 and BAT26 (MSS) strongly supports the conclusion that the differences described here do not result from differences in microsatellite stability but have to be regarded as differences characterising the function and behaviour of tumours originating from the caecum or sigmoid and rectosigmoid.
Immunohistochemical analysis
Immunostaining was applied to paraffin embedded specimen from eight of the 10 right sided and 11 of the 15 left sided tumours where snap frozen material had been previously analysed on microarrays to enable a comparison of RNA and protein expression. The 19 tumours where selected based on the availability of their matching normal mucosa from the oral resection edge.
Figure 3 (A, B) shows five right and five left sided tumours with their matching normal mucosa stained with COX-2. In the right colon, COX-2 was moderately to strongly expressed in normal mucosa, mostly throughout the entire epithelium as well as in right sided tumours. Comparing normal tissue with tumour, we detected upregulation (in one of eight tissue sections), downregulation (1/8), or about equal expression in normal tissue and tumour (6/8). In the left side of the colon, COX-2 was not or only very weakly expressed in normal mucosa and was upregulated from normal mucosa to tumour. Comparing normal mucosa to tumour, we observed strong upregulation in more than 50% of cells (3/11), moderate upregulation in more than 50% of cells (5/11), and very strong upregulation in single cell groups corresponding to less than 10% of cells (3/11).
Figure 3 (C, D) shows five right and five left sided tumours with their matching normal mucosa stained with cytokeratin 20 (KRT20). KRT20 was strongly expressed in the luminal epithelium of normal mucosa of both sides. Comparing normal tissue to tumour of the right side, we detected strong upregulation with staining of more than 50% of cells (2/8), downregulation (4/8) with staining of less than 10% of cells, or about equal expression in normal and tumour with staining of approximately 30–40% of tumour cells (2/8). Comparing normal mucosa to tumour on the left side, we observed upregulation with staining of more than 50% of cells (2/11), downregulation with staining of less than 10% of cells (7/11), or about equal expression in normal mucosa and tumour with staining of approximately 30–40% of tumour cells (2/11). Staining of tumour cells was very heterogeneous in most of the tumours.
While published data on right sided versus left sided colon cancers are lacking, colon cancers per se have previously been compared with normal mucosa. In this study, we identified differences in gene expression in the colon that characterised left and right sided normal mucosa and adenocarcinomas. Using statistical algorithms provided by the Affymetrix software, we identified sets of genes differentially expressed, as well as genes in common, between right sided and left sided adenocarcinomas.
In this study, we analysed a total of 45 samples (20 normal and 25 tumour samples). The complexity of our study is comparable with colon cancer expression analyses previously described by Alon et al, analysing 22 normal and 40 tumour samples, and by Notterman et al, analysing 18 adenocarcinomas and four adenomas with paired normal tissue, both using Affymetrix GeneChips, as previously discussed.12,13 The reliability of our data (for example, with regard to comparisons of left sided normal mucosa to Dukes' B and C tumours) is supported by the fact that we confirmed identification of various genes previously identified by other techniques. Metallothionein, fibronectin, and SPARC, for example, had previously been shown to be differentially expressed in normal tissue and tumour by Zhang et al, using the SAGE technique on two normal and two tumour samples.14 Furthermore, we confirmed differential expression of more than 70% of genes previously identified by GeneChip analyses of pooled samples of the left side of the colon.11 In addition, these results were also highly comparable with data previously published by Notterman et al (for example, upregulation of MGSA from normal to tumour tissue and downregulation of guanylin or chromogranin A).13 Reliability of the results with regard to differences between the right and left colon was further supported by expression analysis using RT-PCR showing high reproducibility of expression levels detected by the arrays. Previous studies have, in most cases, not taken into account the Dukes' stage or location within the colon where the samples originated. Obviously, adding more subclasses to the material inevitably leads to fewer samples per class and the main findings of this paper should be repeated on larger material.
A grouped Mann-Whitney test intrinsic to the Affymetrix software DMT 3.0 was used for statistical analyses. As some of the data were paired (different tissues from the same patient) a Wilcoxon matched pairs test may have been more appropriate for these cases and this may have been a limitation of our statistical analyses. On the other hand, some of the samples were grouped (tissue from different patients) and a Mann-Whitney test had to be applied. However, such "breaking of matching" is more likely to make the results more, rather than less, comparable between tumour and normal tissue, and so this limitation is not of major importance as it is not likely to explain any of the observed differences.
We focused the analysis on adenocarcinomas of Dukes' stages B and C as these are the most challenging stages in colon cancer, with the possibility of curative treatment. Most of the factors that may influence gene expression were taken into account but for array analysis it was not possible to match all samples with their normal mucosa (as could be achieved for immunostainings) or to match samples with regard to sex, as most of our right sided colon cancer patients were female. In general, colon cancer affects males and females equally but some studies indicate that right sided colon cancer affects more women than men.2 Comparison of right versus left sided normal mucosa showed 58 genes differentially expressed, 12 with fold changes more than threefold and none located on the Y chromosome. A comparison of right and left sided adenocarcinomas showed two genes located on the Y chromosome and significantly higher expressed in the group of left sided Dukes' C but not Dukes' B. RPS4Y and SMCY show high fold changes of 23- and 12-fold but SMCY increases only up to a signal of 50, which is close to the detection level. From these data there is no evidence that the imbalance between males and females influences the results profoundly.
Genes such as β-catenin, c-erbB2, EGFR, PCNA, or DPP IV, previously shown to be differentially expressed in right and left colon cancers,9 were not identified as significant in this study but did show side differences when less stringent selection criteria were applied. There are many factors affecting gene expression analysis, such as ischaemic delay, defined as the period of time from clamping of blood vessels to snap freezing, ratio of tumour versus non-tumour cells, RNA extraction method and quality of RNA (28s/18s ratio), type of array used (c-DNA arrays, nylon membrane, oligonucleotide arrays), amplification, labelling (Cy3/Cy5 or d-UTP-Biotin/SAPE) and labelling efficiency, sensitivity and detection threshold, software used for analysis, and statistical significance criteria.
The most predominant differences between normal left and right colon mucosa were higher expression in left sided mucosa of genes such as pS2 protein, calcium binding protein S100P, HOXB13, SIAT4C, and WFDC2. This agrees with previous findings of a 7.7-fold higher expression of pS2 protein15 and approximately fourfold higher expression of HOXB13 and S100P10 in the left colon. This agreement is remarkable because different platforms have been used for analysis and two thirds of the samples in the study of Glebov et al were HNPCC samples. Homeobox proteins such as HOXB13 or HOXA5 encode transcription factors and upregulate tumour suppressor p53 and may therefore be involved in side specific tumorigenesis.
Defensin 5 was found to be expressed sixfold higher in right sided mucosa which matches the proposal that the right colon provides more efficient local tumour defence, maintaining the mucosal barrier.5,16 We hypothesise that the right sided colon mucosa provides protection against carcinogens by defensin 5 expression, leading to less frequent carcinogenesis compared with the left side. Remarkably, the site on chromosome 8p housing the defensin gene is frequently lost in liver metastases from primary colon cancers.15
The majority of genes found to be differentially expressed from normal mucosa to Dukes' B or C of the left side confirmed our recently published findings performed on pooled samples.11 Genes such as MDP4/MDP7 and interleukin 8/MDNCF were strongly upregulated, and several nuclear encoded mitochondrial proteins such as rhodanese or SCAD were strongly downregulated in tumours. We also identified reduced levels of several carbonic anhydrases (CA) such as CAVII or CAIV which have not previously been described indepth in colon cancer. CAIV, downregulated by up to 33-fold in left sided tumours, is responsible for maintenance of pH and ion equilibrium. Takenawa et al showed that low level expression of CAIV and aquaporin 1 in renal cell carcinomas was associated with poor survival.17
Notterman et al analysed differential gene expression between the normal colon and tumour, without discriminating between the right and left side.13 In terms of expression differences between normal mucosa and tumour of the left colon, our study is highly comparable with that of Notterman et al. In both studies, prior to analyses samples were defined with regard to Dukes' stage, snap frozen bulk tissue samples yielded high quality RNA, identical labelling and GeneChips were used, and the data were analysed using the Mann-Whitney U test. Notterman et al identified CAIV as being downregulated by 38-fold from normal colon to tumour, which is identical to our results. In summary, this strongly supports the hypothesis that a decrease in CAIV expression is linked to carcinogenesis and colon cancer progression.
Expression of genes such as COX-2, caldesmon 1, adipsin, transgelin 11, and ARHB was found to be higher in left sided compared with right sided adenocarcinomas. A previous study showed a better effect of chemoprevention with non-steroidal anti-inflammatory drugs on right sided than on left sided adenocarcinomas,3 and the sixfold higher expression of COX-2 may explain failure to prevent this, as a higher dose may be needed to inhibit the high levels of this molecule in left sided malignant lesions. Loss of transgelin gene expression may be an important early event in tumour progression as a consequence of deregulation of RAS gene expression through RAF independent pathways.18 Interestingly, ARHB (RhoB), located on chromosome 2pter-p12, is one of three RAS homologue gene family members and is known as an oncogene.19
From normal mucosa to Dukes' B and C of the caecum, we found that TRPM-2 (clusterin) and PGDH were strongly downregulated whereas several matrix metalloproteinases such as MMP1, MMP3, and MMP10 were upregulated, as seen previously in left sided tumours. MMPs are enzymes responsible for extracellular matrix degradation, playing a role in cancer progression and metastatic spreading. MMP1 expression is associated with a poor prognosis in colorectal cancer.20 One possible therapeutic approach for patients with colon cancer, mainly Dukes' C, could therefore be administration of specific MMP inhibitors to prevent distant metastases and prolong survival,21 as has been shown by inhibition of MMP2 expression in mouse xenograft experiments.22 Circulating proenzymes of MMPs have been described as possible serum markers, and proMMP-9, but not proMMP-2 identified here, was found to be significantly higher in cancer sera versus normal sera.23 From a clinical approach, we suggest analysis of sera levels of the MMPs identified here, as these molecules seem to be ideal general colonic tumour markers reflecting the presence of both left and right sided colonic tumours.
In conclusion, the 30 genes identified in adenocarcinomas of both sides have to be regarded as general tumour markers. The present data and our previously published LOH analyses11 strongly support the hypothesis that genes such as aminopeptidase N (CD 13), sulfate transporter DTD, SCAD, or PCK1 should be regarded as potential new tumour suppressors requiring further investigation.
Paraffin embedded tissue sections from Dukes' B and C tumours and their matching normal mucosa were subjected to immunohistochemical analysis for COX-2 and cytokeratin 20 (KRT20). Microarray analysis showed a significant decrease in KRT20 from normal mucosa to tumour in the left side of the colon. Immunostaining confirmed the difference seen between the two sides of the colon as KRT20 was strongly downregulated in 80% of left sided tumours compared with 50% of right sided tumours. In general, KRT20 staining was found to be very heterogeneous within the tumours.
COX-2 microarray data showed that COX-2 was upregulated from normal to Dukes' C in right as well as left sided tumours, but the increase was significant only for left side (p<0.005). Immunostainings support the microarray based findings to date, that COX-2 is not or very weakly expressed in left sided mucosa but upregulated in matching tumours. In contrast, COX-2 is expressed with the same intensity in right sided normal mucosa compared with matching tumours. COX-2 is heterogeneously expressed within a tumour, as only some groups of cells within a tumour are stained. In conclusion, the microarray based findings were confirmed by immunohistochemistry but an absolute quantitative comparison between RNA expression on microarrays and protein expression on tissue specimen is not possible for KRT20 and COX-2 due to their heterogeneous staining patterns.
The existence of a side specific expression difference for COX-2, having been identified by microarray analysis and confirmed by immunohistochemistry in this study, has recently been reported by Nasir and colleagues.24 Immunohistochemical staining applying a COX-2 polyclonal antibody on 18 right sided versus 18 left sided adenocarcinomas showed that COX-2 positivity was significantly higher for left compared with right sided tumours.
We conclude that differences in gene expression between normal mucosa as well as adenocarcinomas of the caecum and sigmoid and rectosigmoid colon clearly exist, and we hypothesise that the difference in gene expression could be related to differences in tumour development and the prognosis of patients.
The emerging treatments directed towards specific molecular targets should emphasise the differences seen in right and left sided tumours of the colon. We suggest that some of the highly expressed molecules that are in both left and right sided colonic adenocarcinomas may be promising new potential serum markers and therapy targets.
We thank Bente Devantier, Ing Lis Thorsens, and Annette B. Nielsen for their technical assistance, and also as project-nurse Edith Kirkedahl Nielsen at Aarhus Sygehus for collection of colon tissue samples. The study was supported by funds from the Karen Elise Jensen Foundation, the Danish Research Council, AROS Applied Biotechnology Aps, Aarhus, the University and County of Aarhus, the Nordic Cancer Union, and the European Union's 5th frameprogram (European Community, No QLG2-CT-2001-01861).
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Conflict of interest: None declared.


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Join the Globcal International Diplomatic Corps
Represent yourself appropriately and officially as an ambassador internationally to promote your organization and personal interests to help improve the world with your ideals by joining our international non-governmental organization to become a civil service officer, international goodwill ambassador or change advocate.
You can join us as a social media advocate for the United Nations Sustainable Development Goals and also become qualified to participate by attending events or going on international missions as an international civil service officer. Globcal International offers its members a wide-range of opportunities to develop their public relations skills and abilities to work with foreign service objectives through our non-governmental organization.
A Cooperative International Non-Governmental Organization (CiNGO)
We began in 2008 as a Model United Nations objective online to emulate an interactive forum for public diplomacy using the social media. In 2009 we began to formalize our objectives when we presented our program at the Summit of the Americas in Trinidad as Globcal International. By 2013 we began to understand to become recognized in multiple jurisdictions we needed to hybridize several organization types in order to become understood and enjoy the privilege of participation as equal partners without any jurisdictional borders leading to our development of the CiNGO.
The Globcal International membership program was created to develop an integral institutional presence, to demonstrate a functional model of the cooperative business structure and to promote mutual social benefit sharing understanding the ideals of equality and fair-trade. As a cooperative our services and work product technically benefits our members and their constituents, both through our unique development and the recent reformation of our purposes benefits are extended to the general public transparently. The organization welcomes anyone who qualifies to become a member in some capacity (with similar needs that the cooperative can serve) to benefit from our emergent philosophy of social responsibility to create sustainable communities and foster peaceful societies world-wide under our formation, administration, and management.
Globcal International membership is unlike that of most societies, non-profit organizations, fraternal orders, civic associations, and international non-governmental organizations. This is because we are a cooperative (which means that our members are the owners and stakeholders). Our greatest difference from others is that we are developed as a horizontal cooperative society which provides public service, advocacy and mutual benefit projects, legally speaking we are the first "Cooperative International Non-Governmental Organization" or (CiNGO) in formation that has become understood.
Commonwealth Principles
In many ways we are essentially an independent cooperative organization that organizes and develops other cooperatives, cooperative foundations and communities that are owned and controlled democratically by people based on the commonwealth principles of communities, collectives, and cooperatives. In the cooperative structure universal equality is commonplace based on the democratic one member, one vote system that we engage.
Our organization is developed to accommodate a maximum of 1,000 equal fully qualified and invested members who host an inordinate number of initiatives, missions, projects and programs which can each also have their own independent cooperative memberships. We consider and view our organization as a tool for highly enabled leaders of civil, environmental, humanitarian, and indigenous peoples community objectives.
Understanding the Cooperative
Cooperatives are based on ownership, investment and mutual benefit. With ownership its members hold absolute rights under Admiralty Maritime and Private International Law (also known as the conflict of laws). It is the aim of all cooperatives that the funds invested by its members remain, are invested for mutual benefit and that their investments retain their value.
Understanding:To be a genuine cooperative membership organization the members of the organization must belong to the organization as much as the organization belongs to them. Organizations that offer free membership are not responsible for their members actions and in essence are only considered to be honorary, volunteer or non-voting members that are acting on their own personal behalf rather than that of the organization. Cooperatives take this one step further as organizations that are owned and controlled by their members.
Globcal International and its parent organization Ecology Crossroads Cooperative Foundation understand this well and offer different levels of membership to better accommodate participation from the public to allow informal and non-voting members to choose their level of participation and investment. Members that are not fully invested cannot represent the organization in an official capacity, receive personal benefits or do business as the organization, but they can become members. The organization as a cooperative and private foundation does not discriminate based on religion, race, gender, age, or national origin. We do restrict membership based on a person's ability to qualify to join our organization, their educational level, ethical values, and ability to present themselves professionally. Those who are fully invested receive all credentials, can call themselves employees, receive mutual benefits, and can be nominated or capacitated as our officers, commissioners, and trustees.
On Membership:There is no such thing as free membership in a legal sense unless an actual pre-existing relationship exists between the member and the organization that is integral, considered based on work in the formation, and responsible in commerce and official representation. Globcal International offers a range of membership opportunities that are integral for its officials as well as memberships that are informal for non-voting members.
-Founders of Globcal International
Goodwill Ambassadors
Our organization is founded as a fellowship of goodwill ambassadors from around the world, which online via the social media from organizations that have all voluntarily come together to establish a professional support network for their individual works and to share their ideals publicly online. Intentionally through the cooperative alliance, our organization has formed, which performs research, develops substantial collective efforts, and promotes the careers of our peers. Dynamically our union is creating much more by extending ourselves as world-wide missionaries of goodwill and peace.
Currently our members and co-founders include change advocates, goodwill ambassadors, former employees, and volunteers from the United Nations, Red Cross, Kentucky Colonels, Amnesty International, Transparency International, and volunteers from a great number of other organizations and universities interested in promoting the ideals of universalism and international human rights to bring the world to a standard under the laws of a civil society in democratic harmony.
Become an Goodwill Ambassador
For many joining our organization the idea is to become a goodwill ambassador, advocate a cause, develop programs for charities, and actually go on goodwill missions to other countries. Globcal International fosters the development of such individuals who show promise and determination by assisting them in creating new opportunities, acquiring credentials and receive training development. Generally ambassadors that are better informed of their abilities, trained, and knowledgeable of their rights when traveling abroad are 1000% more successful.
Become a Member, Advocate, Supporter or Friend
Our cooperative involves the promotion of social charity, philanthropy, and a sustainable human cultural philosophy through extended networks in the social media that place our collective active charitable endeavors and projects in direct competition with personal leisure and consumerism because it is equally or even more satisfying.
The development of Globcal International is based on the ongoing study and conformance of multiple cooperative ideals involving western philosophies and indigenous traditional knowledge systems in order to create a single internationally accepted cooperative (corporate model) that can be operated transparently to do business anywhere in the world in order to facilitate these ideals.
Membership Classes and Categories
There are different classes of membership and different benefits available depending on your involvement with the cooperative. Cooperatives serve and are established by their members, the members can be owners, associates, sponsors, supporters, volunteers, or friends.
A Real Functional and Self-Sustaining Cooperative
Cooperators (shareholders/stakeholders) can enter the co-op by making a simple investment based on their desired participation level. Generally there can be up to six classes of membership in cooperative societies, the general membership types are known as follows: 1) Original or Regular Member; 2) Co-Member or Associate Member; 3) Nominal Member; 4) Sympathizer Member; 5) Minor or Community Member; and 6) Institutional Corporate or Non-Person Member.
For all intents and purposes from the 6 membership types cooperatively speaking there are only (2) two that actually matter, voting and non-voting. The names of the types of membership we have decided to offer are diversified below including one-time shareholder investments, monthly apportioning and annual fees which are necessary in order to operate successfully, as well as get members off to a good start (this ensures mutual benefits for every member). We deviated a bit to accommodate our special hybrid structure as a non-profit charity, a cooperative and as a decentralized autonomous non-governmental organization which defines our organization much better than many others.
Voting Members
Membership and becoming an 'Cooperator' involves working directly with the organization in an official in a recognized capacity as an advocate or ambassador that derives income for the cooperative which in turn compensates its member | owners who further invest those funds in their own non-profit projects and local charities. Members can create events through Globcal International as the organizer of such programs or projects under the umbrella of the organization. All active members are expected to work as a cooperative team member, individually in their personal familiar and/or online social networks to reach, in a collective capacity certain goals with other cooperators. Generally this involves a minimum of 210-270 hours per year online (approximately 3-6 hours each each week, or 2 hours every 2-3 days Monday-Friday).
Lifetime Active Participatory Membership
Charter and Founding Members (Lifetime Members) that have joined prior to June 2020; they do not pay cooperative maintenance fees or membership dues for the first three years.
Fees may be increased at anytime with a 30 day notice to new members based on the increased value of our cooperatively owned assets. As our project portfolio grows so does the overall asset base value of the organization as a whole. Currently a cooperative membership base share (par value) is valuated at US $4,850.
Founding Member - $10,000 (Equitable 1/1000th Share & Trusteeship)
Charter Membership - $5,000 (Equitable 1/1000th Share & Officership)
Those who join as charter or founding members receive special recognition during the charter formation and development through mentions and inclusion in press releases, treaty documents, the constitutional charter, and can be nominated for special awards (recognitions); plus based on their professional curriculum vitae and active cooperative involvement during their first year are invited to become board members, commissioners, directors or officers. Founding, charter and regular member voting privileges are all equal in the cooperative (corporate) infrastructure. See the list of membership benefits below relative to personal development.
Participatory Active/Cooperative Memberships
All active members pay dues of $15 per month, $45 quarterly or $180 per year if paid a full-year in advance. Regular and striking members cannot become board members, directors or commissioners until they have a minimum of six months service and have successfully developed a 1000 person follower base in the social media. Those who already have an active follower base need to complete a training and assimilation program to fully represent the organization as an official.
Regular Member - $3,750 (Paid Once and the First Year's Dues are Waived.)
Striking Member - $4,500 (Subscription Membership is Billed at $250 per Month over 18 Months with Lifetime Privileges after the 1st 3 Months)
Associate Membership (Working Membership)
The Associate Membership is an alternate membership category for those living in developing, under-developed and third-world nations; this special membership category is less than 50% the cost of traditional regular membership. It is also a membership category that is designed to create responsibility on the part of new members through providing their services to the cooperative, demonstrating their credibility and establishing a long term relationship. It is open to others residing in developed nations based on approval understanding the limits of their term of service and active involvement as a cause advocate. Associate members are qualified to work with the organization like members but are not permitted to vote until they become full-members (Lifetime) after making 5 consecutive annual membership payments. This type of membership was formally promoted as a scholarship until October 2015.
Associate members demonstrate their abilities during the course of their first two years by taking on a role in the organization that can be practically utilized to build the organization, they are also required to perform work online and document their work in their own community using the resources provided to them. Associate members are also responsible for paying dues of $15 per month which includes basic cooperative services.
Associate Membership - Sponsored $1,500 Must be paid in advance, all at once by subject's 3rd Party Sponsor (Only available to 140 qualified UN SDGs Target Countries)
Associate Membership - Self-pay $429 initially including administration fee of $179 plus $250 for the first year, then pay $250 per year for five more years before being recognized as an achieved lifetime member. (Available in all countries).
Non-Voting Members
Volunteer Members (Legacy Members)
Volunteer and legacy membership is a special membership class that allows people in third world, underdeveloped, and developing nations to become part of the organization while they earn their way to becoming a full fledged member by taking on the responsibilities and roles to assist active members to earn credits, while they assimilate skills as regular members through an apprenticeship program that is mastered online through social media activity and personal appearances. These types of membership allow people interested in supporting the ideals of the cooperative without becoming a personally responsible member, to generally support the cooperative, our community efforts, new crowdfunding projects, charitable activities, special programs, become volunteer invitees, and even perform work on a seasonal basis as a cooperator, intern or as a student. All volunteer and legacy members must use the title of 'Volunteer' on their profiles in affiliation with the organization. Volunteers must be nominated and qualified by a Lifetime Member and approved by our Board of Commissioners. Volunteers are eligible to have a website and social media page if they pay monthly dues and have at least 6 months of active service working online.
Benefits of Membership
Those who support our capital campaign and initiatives will be invited and afforded the great opportunity to have active input into the formation and decisions in direction of the cooperative as well as the sense of being part of something in which they believe in and are actual shareholders therein. Members receive a wide range of services and benefits shared among members when traveling to a host country or on official business. Including letters of presentation, introduction to the government, awards nominations, news media development, and membership support from a peer-network of fellow goodwill ambassadors.
Complete assisted biographical development portfolio online.
Placement as a Globcal International ambassador in your local news media.
Legal name on our principle page and in statutory documents.
A co-managed and integrated social media presence on Facebook as a public figure with a verified business page.
Private email address and IDaaS for official intrastate diplomatic correspondence.
Closed secure network communications workplace.
Published nomination recommendations for deeds to well-known titles.
Notification to your national president or prime minister of your international representation.
Letters patent for presentation of your international mission (domestic or foreign).
Secure encrypted online homologation file for use with the blockchain.
Complete well developed website focused on member's five year agenda.
Upon becoming recognized as lifetime member a feature story is written about the member, their accomplishments and activities.
Other Membership Types
Honorary Memberships
Sponsoring Member - $1,000-25,000 (based on project or activity being sponsored or promoted)
Honorary members in their own name or in the name of an organization may designate a person from their staff or organization as a goodwill ambassador with full-privileges or designate recognition to their president with an honorary recognition award.
Regular Patronage Memberships (Business Members)
These members can individually join for the year to work with us in special projects that we are developing with them or align themselves or their businesses through the sponsorship of individual ambassadors working on particular projects. The cooperative appreciates them as endorsers and sponsors of the organization.
Sole Proprietorship - $500 per year
Small Business - $250 plus $10 per employee
Corporation (10 or more employees) - $1000 per year
Non-Profit Organization - $250-$2500 per year
International Supporting Memberships
Special memberships that were developed to propel our crowdfunding initiatives, the United Nations SDGs and international ecological sustainability demonstration projects.
Supporting Member - $500 per year (cooperative supporter's certificate, special gift, bulletins and newsletter updates)
Friendship Member - $50 per year (bulletins and newsletter)
Become a Member Now!
You can make donations for honorary and payments for membership or pay regular membership dues online anytime using our Donations Page, Payments Page or PayPal using the links above. If you are buying into the cooperative to invest in the organization as an employee/owner or to work in person with a related project send your payment directly using the appropriate link provided above to the Treasurer at email@example.com using PayPal as our payment processor, you are not required to have a PayPal account to use the links, all of them include the option of using a credit card.
If you wish to make a "tax-deductible" contribution, gift, personal donation or are sending an open sponsorship of US $250 or more please use our Donation Page or send your check or money order directly to the our parent organization, who will direct the fund to the Globcal International Foundation. Make your check payable to 'Ecology Crossroads' using the regular postal mail service to the following address (please allow 15-30 days for processing when using postal mail):
Ecology Crossroads Cooperative Foundation
c/o Nicholas Wright, Treasurer
302 General Smith Drive
Richmond, Kentucky 40475 USA


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- 1 cup plain flour
- 100g unsalted butter
- 1 teaspoon salt
- 250ml cold water
- 3 free-range eggs plus 1 additional egg yolk
- 1 free-range egg, beaten, for the egg wash
- 1¼ cups milk
- ½ teaspoon vanilla extract
- ½ cup caster sugar
- ⅓ cup plain flour, sifted
- 3 free-range eggs plus 1 additional egg yolk
- 1 teaspoon butter
- 20g white chocolate
- 150g a1 sugar
- 20ml water
- 1 tablespoon glucose syrup
- 24 small fondant flowers or butterflies
- 2 baking trays, greased
- silicone sheet
Preparation Method
Preheat the oven to 190°C. Sift the flour into a bowl. Put the butter, salt and water in a heavy-based pan and bring to the boil over a medium heat. As the water comes to the boil, remove from the heat and dump in the flour in one go. Immediately stir briskly with a wooden spoon until the flour and liquid are evenly combined.
Place the pan back on a lower heat. Beat continuously for a minute until you have a coherent mass that comes away easily from the side. Remove from the heat and leave to cool for about 3 minutes. (If you add the eggs immediately, the mixture may curdle.)
Beat the first 3 eggs into the mixture, one at a time, making sure each egg is completely incorporated before adding the next. The mixture should be smooth and glossy. If it is too stiff, trickle in some of the additional egg yolk, beating well.
Leave to cool to room temperature. Fit a piping bag with an 18mm round nozzle and fill with the choux mixture. On a greased baking tray pipe 9 balls around the size of a walnut. Repeat with a second tray. Press the tops down with a wet finger. Brush the tops with egg wash. Bake for 15 minutes at 190°C, then lower the temperature to 160°C for a further 10 minutes until golden brown and puffy. Remove from the oven and make a small slit in the base of each ball. Turn off the oven. Return to the oven to dry out completely.
To make the custard cream, place the milk and vanilla extract in a medium saucepan and gently heat through. Combine the sugar, flour, eggs and yolk in the bowl of a freestanding electric mixer, and beat well until light and fluffy. Reduce the speed and add the warm milk in a steady stream. Once the milk has been added, return the mixture to the pan and whisk over medium heat until the sauce thickens. Remove from the heat and immediately whisk in the butter and white chocolate. Cool.
To make the toffee, combine the sugar and water in a small pan and bring to the boil. Add the glucose and lower the heat. Cook until the mixture is a warm caramel colour.
When ready to assemble, pipe a little custard cream through the base slit of each choux ball. Carefully dip each ball into the hot toffee and allow to set on a silicone sheet. Garnish while still sticky with a small fondant flower or butterfly.


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We have implemented the wishes of many of our users when developing a new version of the Hamster Free Video converter including full support for all Apple products and download process for the converted video through iTunes. Improved new design and increased the speed of video conversion!
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Welcome to Season 2 of Total Reroll, our Dungeons and Dragons podcast! Adam has been playing some some good old fashioned D&D with some friends in London, except it isn't old fashioned because it's 5th edition. After finishing 'Hoard of the Dragon Queen', we've moved on with the same characters to 'Rise of Tiamat', the second part of the Tyranny of Dragons storyline. You can find a mega-post of all of the Season 1 content here.
We start off this week joining Red, Ravoprax, Nausicaa, Eldo, Artin, Dariah and Carnicula a few days after having captured the floating castle of Skyreach. The group receive an urgent message from Leosin, their Harper contact, about a dire situation in the Whalebone Islands, so we set off to investigate.
Download this episode (right click and save)
If you have any feedback email us at email@example.com, tweet us @lost_lighthouse or get in touch via our Facebook! Subscribe to the podcast feed or on itunes, and feel free to share or retweet too! Fancy supporting Total Reroll and The Lost Lighthouse? Check out our completely optional, pay-what-you-want Paypal donation page!
Our sexy new music is 'Acid Splash' by Rich Thomson, and our "adverts" are provided by Anthony Walsh.


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People often come to me at the 2nd (Reconsideration) Stage, or the 3rd (Merit Systems Protection Board) Stage, and ask that I correct the mistakes made in the initial filing. Most mistakes can be corrected. Of course, it would have been better if the Applicant had done it properly the first time, for once the Office of Personnel Management views something which should not have been submitted, it cannot be easily retracted — only further explained.
There are, moreover, certain mistakes which cannot be "explained away" — such as deliberate omissions or deceptions. Thus, if the Office of Personnel Management gets the idea that there is an element of deceptiveness in a disability retirement application — either through omission or deliberate avoidance of an issue — then it becomes a difficult case to win. Honesty is always the best policy, and no Disability Retirement applicant should ever engage in any act of covering up any information. This is conceptually different from emphasizing the elements in a disability retirement application which favor an approval, as opposed to de-emphasizing those elements which tend to obscure the primary elements of an application. Such artful emphasis/de-emphasis should always be a part of every disability retirement application, coordinating the Applicant's Statement of Disability with supporting medical documentation, to convey a consistent "whole" to the Office of Personnel Management.
Robert R. McGill, Esquire
Filed under: OPM Disability Process, Pre-Application Considerations, When the OPM Application Is Denied | Tagged: appealing OPM's denial to the Board, Applicant's Statement of Disability for CSRS, Applicant's Statement of Disability for FERS, avoiding deception during the federal disability application, being truthful but being careful in what you say, de-emphasizing but not hiding issues in postal medical retirement, disability retirement opm, disability retirement packet, federal disability form, federal disability law blog, federal employee disability benefits, federal employee lawyer, federal employees retirement system disability, federal workers comp application forms, federal workers medical compensation, FERS disability retirement regulations, filing a csrs disability claim, filing fers disability application, filing for OPM disability retirement, filling opm disability retirement forms, honesty always pays off, injured federal workers, legal help after first application denial, misstatements as basis for denial, MSPB Third Stage, omission or deliberate avoidance of an issue, OPM Denial Letter, OPM disability application tips, opm disability help forms, OPM medical disability retirement claims and appeals, OPM Reconsideration Stage, post office application for disability retirement, postal service disability retirement, postal service employee light duty forms, rules for filing medical disability with the USPS, Second Step OPM Appeal, SF 3112A, statements from friends to support opm medical documentation, strategists for a good opm disability application, tips for federal workers filing for disability, tips for us government employees on disability retirement, us federal employees filing for disability, usps disability filing, USPS disability retirement, usps workers compensation, what issues to avoid emphasizing during an opm claim, what to do in the OPM Reconsideration Stage, what to say during document filing and preparation |


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Coalition huddles as forces inch towards Mosul
QARAQOSH, Iraq (UPDATED) – Iraqi forces were inching to within striking distance of eastern Mosul Tuesday, October 25, as defense chiefs from the US-led coalition met in Paris to review the offensive on the jihadist bastion.
With the Mosul battle in its second week, French President Francois Hollande called for the coalition against the Islamic State group to prepare for the aftermath and the next stages of the war against ISIS.
Forces from the elite counter-terrorism service (CTS) retook areas close to the eastern outskirts of Mosul, ISIS's last major urban stronghold in Iraq.
"On our front, we have advanced to within five or six kilometers (three to four miles) of Mosul," their commander, General Abdelghani al-Assadi, told AFP.
"We must now coordinate with forces on other fronts to launch a coordinated" attack on Mosul, he said, speaking from the Christian town of Bartalla.
Kurdish peshmerga forces are making gains on the northeastern front, but federal forces advancing from the south have some way to go before reaching the outskirts.
Meanwhile, thousands of men from the Hashed al-Shaabi paramilitary umbrella group dominated by Tehran-backed Shiite militias were preparing for a push to the west of Mosul.
The Hashed leadership has ordered "us to assume the mission of liberating the Tal Afar district", said Jawwad al-Tulaibawi, spokesman for the Asaib Ahl al-Haq militia, referring to an area west of the mainly Sunni city.
The Hashed's mission will be to "cut off and prevent the escape of (ISIS) towards Syria and fully isolate Mosul from Syria", Tulaibawi told AFP.
Turkey threat
"We expect that it will be a difficult and fierce battle," he said.
Iraqi Kurds and Sunni Arab politicians have opposed the Hashed's participation in the operation, as has Turkey, which has a military presence east of Mosul despite repeated demands by Baghdad to withdraw its forces.
Tensions have risen between Baghdad and Ankara, whose foreign minister, Mevlut Cavusoglu, warned Tuesday that if there is a threat to Turkey, "we are ready to use all our resources including a ground operation".
In Paris, French Defense Minister Jean-Yves Le Drian was meeting coalition counterparts, including Pentagon chief Ashton Carter, to review the war on IS after more than two years of air strikes, training and on-the-ground military advisers.
Besides coordinating their support for the forces closing in on Mosul, ministers will also attempt to iron out differences over priorities in the campaign.
France is keen to tackle the jihadists' Syria bastion Raqa, where a large number of French foreign fighters in ISIS ranks are stationed.
As the ministers met, Hollande warned that "the recapture is not an end in itself. We must already anticipate the consequences of the fall of Mosul."
"What is at stake is the political future of the city, the region and Iraq," Hollande said, calling for "all ethnic and religious groups" to have a say in the future running of Mosul.
Looking to Raqa
He also called on the ministers to set out "the stages of the next operations" against ISIS, namely retaking Raqa.
While the jihadists in Mosul are outnumbered about one to 10, there are insufficient forces currently available to take on the estimated 3,000-4,000 ISIS fighters in Raqa.
Seeking to draw attention away from the Mosul campaign, ISIS has staged attacks in the northern city Kirkuk and western town of Rutba in recent days.
Jihadists seized two neighborhoods in Rutba, but officials said that as of Tuesday it was fully back in government hands.
Senior Iraqi and US military officials have reported that ISIS leaders are already trying to leave Mosul to reach the Syrian side of their "caliphate".
But a senior official close to Le Drian said a few hundred ISIS fighters recently moved in the opposite direction, reinforcing the estimated 3,000 to 5,000 jihadists defending Mosul.
ISIS had shown increasing pragmatism in recent battles, tending to fall back in the face of superior firepower.
But with its claim to run a "caliphate" losing credibility with every new loss of territory, ISIS has mounted a fierce and well-organised resistance in the fight for Mosul.
Mosul is where ISIS supremo Abu Bakr al-Baghdadi proclaimed the cross-border "caliphate" in June 2014, and its loss could spell the end of the group's days as a land-holding force in the Iraqi part.
In Moscow Tuesday, Russian Foreign Minister Sergei Lavrov equated US support for Iraqi efforts to recapture Mosul with Russian backing of a Syrian government offensive to seize rebel-held eastern Aleppo. – Rappler.com


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***Please fill out all fields before submitting. "Return Date" is applicable only for Round-Trip inquiries. Please indicate in the "Messages?" box any questions or concerns that we may be able to help you with. Thank you.
Our highly trained and experienced charter coordinators operate luxurious jets and turbo prop aircraft all over the United States, Canada, and Caribbean. With the highest dedication to customer service, we provide the highest standards of safety, efficiency and discretion in air travel. Our expert flight coordinators are ready to accommodate your every need – from initial booking to final passport stamp, we ensure your trip is flawless.
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Beechjet 400A | Hawker 400XP | CitationJet | Citation CJ1 | Citation CJ1+ | Citation CJ2 | Citation CJ2+ | Citation CJ3 | Citation Bravo | Citation Ultra | Citation Encore | Citation Encore+ | Learjet 31A | Premier I| Premier IA | Nextant 400XTi Phenom 300
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***Please fill out all fields before submitting. "Return Date" is applicable only for Round-Trip inquiries. Please indicate in the "Messages?" box any questions or concerns that we may be able to help you with. Thank you.


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A review of Abel Ferrara's eschatological study 4:44 Last Day On Earth, for MSN Movies. Using the above song, in either the featured Galaxie 500 version or the Young Marble Giants' original, might have spruced the picture up. But no.
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Nothing sells like the Apocalypse, which must explain why this Ferrara 21st c. narrative feature, and not e.g. GO-GO TALES or MARY, gets something like a release to theaters. To me, it's amusing that this is a "LAST DAY" that seems to resemble, right down to the exact time o' the telos, Don McKellar's underappreciated, eponymously darker quirkfest LAST NIGHT, a film whose rewards include one of the my favorite closing shots in all of cinema. Personally, I find AF the survivor a far more interesting filmmaker than AF the libertine, as much as he went there indelibly in his infamous oeuvre.
And along the lines of walking that walk...living in the lower Hudson Valley as I do, I had occasion to meet in a local establishment one of AF's crew members who dates back to the Peekskill-born Ferrara's earliest days. I remarked en passant about Vincent Gallo's miserably onanistic, self-penned profile for Grand Royal in the 90's, where he suggested it took remarkable willpower for AF to pull himself away from his crack tail and his "filthy trailer" in order to direct VG and other lesser personalities in THE FUNERAL. This crew member smiled with great satisfaction and replied somewhat brilliantly: "Abel didn't have a trailer on THE FUNERAL". Aha. So there.
I would elaborate on this further, but I already shot my wad on Wells' site when he posted a trailer for that Steve Carell apocalypse movie.
Glad to see some appreciation for the little-seen LAST NIGHT. David Cronenberg's final scene was especially chilling and memorable.
Anyone for MIRACLE MILE?
I love LAST NIGHT. The last shot makes the film.
Me for MIRACLE MILE, featuring two of my favorite neglected (sic) actresses, Mare Winningham (à la all-too-rare Miou-Miou mode) and Denise Crosby -- something tells me not to sit on a hot stove waiting for Hollywood to come up with a comparable gag like the latter reading the Cliff Notes for GRAVITY'S RAINBOW. Also, the great John Agar representing for MM's vulgar, fast-beating B-movie heart.
What was the last good movie Ferrara did? THE FUNERAL? I dunno. I haven't seen MARY, GO GO TALES, or the Chelsea Hotel Doc. I 'd like to.
I think I'll just wait for the Werner Herzog remake.
Petey: Herzog's already been there, with La Soufière.
Graig: I've been dying to see recent Ferrara as well, and have heard only praise for Go Go Tales (partly a function of whom I don't bother to read, no question). But I thought 'R Xmas was pretty fine, even if it ramped up the moralizing while reducing the narrative to a series of frayed, deliberately anticlimactic gestures. Check it out if you haven't.
Ferrara's long time writing partner Nicholas St. John worked with him on a lot (maybe all, except for 'Bad Lieutenant' where St. John is not credited) of Ferrara's best films from 'Driller Killer' through 'The Funeral.' St. John and Ferrara haven't worked together since 'The Funeral,' which has surely had an impact on the quality of Ferrara's output. I don't know why they stopped working together.
I'll rep for 'R Xmas, The Blackout and New Rose Hotel. None of 'em touch Bad Lieutenant or King of New York (or even The Addiction, another one I think is worth seeing, just for the way Christopher Walken says the line "Have you read William Burroughs?"), but each has individual scenes that make the whole thing worthwhile. Hell, The Blackout makes me wish Ferrara had been hired to direct The Hangover.
"St. John and Ferrara haven't worked together since 'The Funeral,' which has surely had an impact on the quality of Ferrara's output."
This is also my reading of the situation.
FWIW, I thought Herzog did a nice homage to Christopher Walken's wonderful dancing in King of New York with Nic Cage in his Ferrara remake. I would've thought that alone should have moderated Ferrara's reaction.
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wind carries
the scent of heather
while we wait
where peace resides
where silence sings
© Xenia Tran
With love from Eivor, Pearl and Xenia xxx
Photographs by Xenia Tran, edited in lr.
Camera: Panasonic Lumix FZ200, setting: iA, 1/200 s, ISO Auto.
Daily Post Weekly Photo Challenge: Waiting
Carpe Diem #1250 – Autumn Colors
Forgiving Fridays: Walking My Own Talk


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Advantages of horse ownership discussed at Ranch Management University - North Texas e-News
By Blair Fannin, Texas A&M
Oct 30, 2011
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COLLEGE STATION – Most ranch owners don't realize the significant impact horse ownership and its contributions have on the Texas economy, according to an equine specialist.
The Texas horse industry has a statewide economic impact of more than $5.2 billion a year, said Dr. Clay Cavinder, an assistant professor of equine science at Texas A&M University.
Cavinder was one of several speakers at the recent Ranch Management University at Texas A&M.
Dr. Clay Cavinder
Dr. Clay Cavinder, assistant professor of equine science at Texas A&M University, discusses horse ownership at the recent Ranch Management University program. (Texas AgriLife Extension Service photo by Blair Fannin)
"Ranch Management University provides a foundation of knowledge for those who are new to owning land in Texas," said Dr. Larry Redmon, workshop coordinator and Texas AgriLife Extension Service state forage specialist.
The program features numerous experts in specific disciplines from AgriLife Extension, Texas AgriLife Research and faculty from the department of animal science at Texas A&M.
"Horses are valued at $4.2 billion just in the state of Texas," Cavinder said. "In terms of comparison with other industries and their effect on the GDP, we are on the same level as the motion picture industry, apparel manufacturing and tobacco industry. So, the horse industry brings a lot to the table."
Annual expenses are roughly $2,300 for the horse owner when calculating feed, health and other requirements, Cavinder said.
Trail riding, one of the more popular activities, has many benefits with regards to physical fitness, family participation, as well as an emotional outlet for disabled individuals.
Cavinder said individuals also enjoy 'horse chores' such as cleaning stalls and use it as an emotional outlet.
A majority of horses now are owned for recreational purposes; most horse owners are not from a rural background.
"In the 1930s, the horse population declined due to the advent of the automobile," Cavinder said. "Today, the horse population is going back up. People are owning horses for the sheer fun of it. There are a lot of 20-year-olds that grew up having horses, but their parents weren't that involved with it. Their grandparents were likely more active in it."
Horse ownership carries with it benefits, as well as responsibilities, he said. Food, water and shelter are three basic responsibilities of horse ownership, according to Cavinder. Nutrition is the largest annual maintenance expense for the horse and is also one of the most neglected aspects of horse care.
The digestive system of a horse is that of an animal that was created to run and move and eat roughage.
"Colic is the No. 1 killer of a horse," Cavinder said. "Because it can't vomit, it creates a different set of problems here (that can lead to colic). We do not want to create a digestive upset.
"Because we're feeding horses concentrate diets, we do have to consider a few special things like distances and time between feeding. If you feed at seven in the morning, feed at seven at night. If you look at the statistics, there's millions of dollars spent on treating colic."
Cavinder said a set, routine feeding time each day will help prevent digestive problems.
Horses don't have gall bladders, which in humans emulsify fat, so they can't be fed low quality forage, Cavinder said.
"This is why we have to feed nice, high quality feed or hay. A horse doesn't have the ability to break down and utilize roughage that is very high in structural carbohydrates."
Cavinder advises rotating grazing of pastures to allow plants to grow before grazing.
"Wait until the forage reaches 3 to 4 inches, then clip the pasture for growth," he said. "If a pasture gets to the point where there isn't forage, then throw hay out there."
Cavinder also advises monitoring horses in the barn and observing their behavior for signs of sickness.
"I always tell students when walking through the barn, don't neglect to look at other horses," he said. "When they are sick, they look depressed. They'll have their ears drooped back and head down."
Registration is already being taken for the next Ranch Management University program scheduled April 9-13. For information, visit https://agriliferegister.tamu.edu/ and enter "Ranch Management" as the keywords.


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This site has been created for fun. I had a personal website while I was a graduate student at the University of Washington, which served to disseminate information about my research activities and as a one-stop source for prospective employers. I have carried several pages from there directly to this website. I think I will continue to use this site as a one-stop information source about me. Feel free to exlpore around and write to me if you desire to.
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Our barristers are involved in the full gamut of occupiers liability cases from everyday slips and trips in supermarkets and restaurants to catastrophic injuries occurring at swimming pools, beaches, schools and leisure centres.
There is a breadth of experience in dealing with claims brought against individual householders, and against all sizes of businesses and large public bodies. Chambers has been at the forefront of the law in this area with two of our members appearing for the Defendant in Yates v National Trust [2014] EWHC 222 where the Claimant unsuccessfully tried to extend the duty of the occupier under the Occupiers' Liability 1957 to the acts of an independent contractor. Members have also appeared in important cases such as Baldacchino v West Wittering Estate Plc [2008] EWHC 3386 (QB) and Geary v JD Wetherspoon [2011] EWHC 1506 (QB) where the courts did not find for severely injured claimants in circumstances as there was nothing wrong with the respective premises and the claimants had consented to the risk of the activities they were undertaking.
Members have also recently been involved in claims for injuries suffered from falling through a transparent weather curtain in a pub's smoking area, from leaping unsuccessfully off a climbing wall to reach a nearby buttress, and from falling whilst ornate lion-clambering outside Portsmouth Guildhall.
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November 16, 2023 @ 9:00 am - 10:00 am MST
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An event every month that begins at 9:00 am on day Third of the month, repeating indefinitely
House and commission reps operating in the MPIBA region are invited to join us for a casual chat about MPIBA activities, industry trends and news, and event updates. The Zoom link is sent to member reps prior to each chat, send us an email if you'd like to be added to that email list. (We do these calls in the afternoon on the first Thursday most months, and in the morning, on the third Thursday most months.)


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From Brock to The Rock, no game can claim as much slobberknockin' action as Smackdown, the wrestler that doesn't just demand you Shut Your Mouth, it knocks the taste out of it with everything from choke slams and chairs, to ladders and steel steps. Smackdown struts the aisle with over 60 wrestlers, and IGN's guide covers 'em all, from Scotty and his Worm to Triple H and his sick tastes for necrophilia... well the guide doesn't go that deep, as luckily for the gaming public, Triple H's little incident with the corpse didn't happen until after the game was finalized, but there's always next year. In the mean time, learn every move for every wrestler, strategies to win the various match types, and how to unlock a variety of secrets Mr. McMahon and his crew at THQ have hidden throughout their championship Smackdown simulator.
Guide by: Jon Robinson
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Grand Theft Auto III Cheats
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Gran Turismo 4 Cheats
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On Monday, October 22, 2018, Indiana State Police Detectives arrested a Seymour, Indiana man on a level one felony count of child molesting after an alleged incident that occurred in rural Jackson County.
In the evening of October 21st, the Seymour Police Department began investigating an alleged child molesting although the investigation was referred to the Indiana State Police due to the offense occurring outside of the Seymour city limits.
Detective Andrew Mitchell, Indiana State Police-Versailles, conducted the investigation with the assistance of the Seymour Police Department, Indiana Department of Child Services, and the Indiana Child Advocacy Center.
Through the investigation, it was determined that Reynaldo E. Reyes-Perdomo, age 30, Seymour, Indiana allegedly took a 13 year old female to a rural location near State Road 11 and I-65 in northern Jackson County and engaged in sexual contact with the juvenile.
A second incident was reported to have occurred on a different date at a Seymour residence involving the same victim.
Reyes-Perdomo was located at a Seymour residence. He was taken into custody without incident.
He was transported to the Jackson County Jail where he was initially incarcerated on a level one felony count of Child Molesting, pending his initial appearance in the Jackson County Circuit Court.


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A Royal Australian Air Force (RAAF) C-17A Globemaster III aircraft will conduct low-level and instrument flying from Sunday 14 – Thursday 18 April 2019 over New Zealand.
Personnel and the aircraft from No. 36 Squadron at RAAF Base Amberley will be based out of RNZAF Base Ohakea near Palmertson North, and will fly over the east coast of the North Island and both coastlines of the South Island.
The ability to fly over different terrains including valleys and mountains, is essential to RAAF C-17A operations and cannot be trained for in Australia.
RAAF air mobility crews maintain close ties with the New Zealand Defence Force, evidenced by the recent cooperation in the Solomon Islands for that country's General Election.
Noise reduction and the environment are vital considerations in the planning and conduct of military activity, and Air Force appreciates the ongoing support of the New Zealand community and Defence Force.
Information on aircraft noise and current flying activities is available at http://www.defence.gov.au/aircraftnoise, or by calling 1300 DEFENCE (1300 333 362)


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Scott Pilgrim just met the girl of his dreams…literally. But in order for them to date, he must defeat her 7 evil exes. From the genre-smashing director of Hot Fuzz and Shaun of the Dead.
The cast of this movie, boy howdy, so many big names in some absolutely fantastic performances. I love the feel of the movie, how it's both a small movie in scope (dude wants to date a girl, but he's already dating someone!) but so large as well (seven deadly exes want to fight!).
It's on Netflix right now.
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My first experience seeing celebrities immortalised in wax was waaaaay back in my childhood when Madame Tussauds came to Melbourne for a brief time. In the decades since that first experience, I have hoped that Madame Tussauds would make a return to Australian shores. You can imagine my delight when I heard that the famous celebrity wax museum had finally opened here in Sydney. James and I made an afternoon of it this Saturday, oohing and aahing at the sheer realism of the waxworks, not to mention surprise at the height (or lack thereof) of some of the world's biggest stars. Things started off a little political...
...Until we realised some pretty big name sportspeople needed a hand:
But when you're such a big name yourself, the talk shows are going to beckon. We scheduled a couple of interviews...
...Then stopped off for a late breakfast:
James wondered who on earth this fella was and what he was doing wearing his underwear:
Next up: I had a lesson to teach one of the amateurs...
While James showed the casting directors that a thick head of hair might serve him well if they choose to reprise Die Hard:
Johnny gets it.
Meanwhile, I discovered I wasn't the only lass whose undergarments were visible through her clothing:
I also discovered that it was best not to pose with the supermodels. No amount of hours in the gym would trim and lengthen me to that extent:
Lesson learned (the hard way), I let Maz alone for her pic.
We saw dozens more VERY famous faces in Madame Tussauds that I haven't uploaded here. I figured some of the best bits you might like to see for yourself. You can book tickets and find out more here. I reckon a visit to the 'waxworks' would be a great way to kick off a Saturday night. Afterwards, you can stop off at King Street Wharf or The Star for some dinner and drinks as you giggle over all the pictures you've taken. Who knows who you'll get snapped with?


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Jeff Schiller: > I may be out of my league here or missing something, but the SVG nodes > implement the SVGElement interface (http://www.w3.org/TR/SVG11/idl.html) > which extends the DOM2 Element interface ( > http://www.w3.org/TR/DOM-Level-2-Core/idl-definitions.html). And no node of type DOCUMENT_NODE is inserted into the tree, like with IE's XML data islands. -- Cameron McCormack ≝ http://mcc.id.au/Received on Thursday, 24 July 2008 00:10:21 UTC
This archive was generated by hypermail 2.4.0 : Friday, 17 January 2020 22:54:19 UTC


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Sunday, December 11, 2016
The First Step in Fighting Fascists Is Not to Be One
This is a moment in which fear entrepreneurs are fanning the flames of tribalism all around the world.
The first thing we need to do in the coming fight with fascism is not to become tribalists ourselves.
The great principles of democracy, pluralism, and America are enough to make a decent and happy society with.
These principles are also our greatest weapons against a narrowing and fearful xenophobia.
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