Building cross-scale models of epigenetic mechanisms

Epigenetic changes correspond to heritable modifications of the chromosome structure, which do not involve alteration of the DNA sequence but do affect gene expression. These mechanisms play an important role in normal cell differentiation, but aberration is associated also with several diseases, including cancer and neural disorders. In consequence, despite intensive studies in recent years, the contribution of modifications remains largely unquantified due to overall system complexity and insufficient data. Computational models can provide powerful auxiliary tools to experimentation, not least as scales from the sub-cellular through cell populations (or to networks of genes) can be spanned. In this paper, the challenges to development, of realistic cross-scale models, are discussed and illustrated with respect to current work.

Gamma tocotrienol targets tyrosine phosphatase SHP2 in mammospheres resulting in cell death through RAS/ERK pathway

Background There is increasing evidence supporting the concept of cancer stem cells (CSCs), which are responsible for the initiation, growth and metastasis of tumors. CSCs are thus considered the target for future cancer therapies. To achieve this goal, identifying potential therapeutic targets for CSCs is essential. Methods We used a natural product of vitamin E, gamma tocotrienol (gamma-T3), to treat mammospheres and spheres from colon and cervical cancers. Western blotting and real-time RT-PCR were employed to identify the gene and protein targets of gamma-T3 in mammospheres. Results We found that mammosphere growth was inhibited in a dose dependent manner, with total inhibition at high doses. Gamma-T3 also inhibited sphere growth in two other human epithelial cancers, colon and cervix. Our results suggested that both Src homology 2 domain-containing phosphatase 1 (SHP1) and 2 (SHP2) were affected by gamma-T3 which was accompanied by a decrease in K- and H-Ras gene expression and phosphorylated ERK protein levels in a dose dependent way. In contrast, expression of self-renewal genes TGF-beta and LIF, as well as ESR signal pathways were not affected by the treatment. These results suggest that gamma-T3 specifically targets SHP2 and the RAS/ERK signaling pathway. Conclusions SHP1 and SHP2 are potential therapeutic targets for breast CSCs and gamma-T3 is a promising natural drug for future breast cancer therapy.

Submarine record of volcanic island construction and collapse in the Lesser Antilles arc: First scientific drilling of submarine volcanic island landslides by IODP Expedition 340

IODP Expedition 340 successfully drilled a series of sites offshore Montserrat, Martinique and Dominica in the Lesser Antilles from March to April 2012. These are among the few drill sites gathered around volcanic islands, and the first scientific drilling of large and likely tsunamigenic volcanic island-arc landslide deposits. These cores provide evidence and tests of previous hypotheses for the composition and origin of those deposits. Sites U1394, U1399, and U1400 that penetrated landslide deposits recovered exclusively seafloor sediment, comprising mainly turbidites and hemipelagic deposits, and lacked debris avalanche deposits. This supports the concepts that i/ volcanic debris avalanches tend to stop at the slope break, and ii/ widespread and voluminous failures of preexisting low-gradient seafloor sediment can be triggered by initial emplacement of material from the volcano. Offshore Martinique (U1399 and 1400), the landslide deposits comprised blocks of parallel strata that were tilted or microfaulted, sometimes separated by intervals of homogenized sediment (intense shearing), while Site U1394 offshore Montserrat penetrated a flat-lying block of intact strata. The most likely mechanism for generating these large-scale seafloor sediment failures appears to be propagation of a decollement from proximal areas loaded and incised by a volcanic debris avalanche. These results have implications for the magnitude of tsunami generation. Under some conditions, volcanic island landslide deposits composed of mainly seafloor sediment will tend to form smaller magnitude tsunamis than equivalent volumes of subaerial block-rich mass flows rapidly entering water. Expedition 340 also successfully drilled sites to access the undisturbed record of eruption fallout layers intercalated with marine sediment which provide an outstanding high-resolution data set to analyze eruption and landslides cycles, improve understanding of magmatic evolution as well as offshore sedimentation processes.

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2-ASK1 apoptosis pathways and oxidative stress is mitigated by Srx-Nrf2 anti-oxidant system

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

Association between the interleukin 23 receptor and ankylosing spondylitis is confirmed by a new UK case-control study and meta-analysis of published series

Objectives. It has been shown previously that IL-23R variants are associated with AS. We conducted an extended analysis in the UK population and a meta-analysis with the previously published studies, in order to refine these IL-23R associations with AS. Methods. The UK case-control study included 730 new cases and 1331 healthy controls. In the extended study, the 730 cases were combined with 1088 published cases. Allelic associations were analysed using contingency tables. In the meta-analysis, 3482 cases and 3150 controls from four different published studies and the new UK cases were combined. DerSimonian-Laird test was used to calculate random effects pooled odds ratios (ORs). Results. In the UK case-control study with new cases, four of the eight SNPs showed significant associations, whereas in the extended UK study, seven of the eight IL-23R SNPs showed significant associations (P < 0.05) with AS, maximal with rs11209032 (P < 10-5, OR 1.3), when cases with IBD and/or psoriasis were excluded. The meta-analysis showed significant associations with all eight SNPs; the strongest associations were again seen not only with rs11209032 (P = 4.06 × 10-9, OR ∼1.2) but also with rs11209026 (P < 10-10, OR ∼0.6). Conclusions. IL-23R polymorphisms are clearly associated with AS, but the primary causal association(s) is(are) still not established. These polymorphisms could contribute either increased or decreased susceptibility to AS; functional studies will be required for their full evaluation. Additionally, observed stronger associations with SNPs rs11209026 and rs11465804 upon exclusion of IBD and/or psoriasis cases may represent an independent association with AS. © The Author 2009. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.

Cloning and gastrointestinal expression of rat hephaestin: Relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

Colonoscopy preparation in children: Safety, efficacy, and tolerance of high- versus low-volume cleansing methods

Background: The use of large-volume electrolyte balanced solutions as preparation for colonoscopy often results in poor patient compliance and acceptance. The tolerance, safety, and efficacy of high-versus low volume colon-cleansing methods as preparation for colonoscopy in children were compared by randomized operator-blinded trial. Methods: Twenty-nine children ages 3.6-14.6 years had either high-volume nasogastric balanced polyethylene glycol electrolyte lavage (20 ml/kg/h) until the effluent was clear (n = 15), or two oral doses of sodium phosphate solution (22.5-45 ml) separated by oral fluid intake (n = 14). Results: Both preparations were equally effective. The low-volume preparation was better tolerated and caused less discomfort that the high-volume preparation, judging by serial nurse observations. The incidence of abdominal symptoms, diarrhea, sleep disturbance, and vomiting was not significantly different between the two groups. Both groups had a small reduction in mean hematocrit and serum calcium levels. The sodium phosphate preparation caused increases in mean serum sodium concentrations from 140 to 145 mmol/L and serum phosphate concentrations from 1.41 to 2.53 mmol/L. Ten hours after the commencement of the preanesthetic fast, these concentrations had returned to normal. Conclusions: There are advantages in terms of tolerance, discomfort, and case of administration with acceptable colonic cleansing with the use of the less-invasive oral sodium phosphate low-volume colon-cleansing preparation in children. Safe use requires ensuring an adequate oral fluid intake during the preparation time and avoidance of use in patients with renal insufficiency.

Preparation and in vitro release of drug-loaded microparticles for oral delivery using wholegrain sorghum kafirin protein

Kafirin microparticles have been proposed as an oral nutraceutical and drug delivery system. This study investigates microparticles formed with kafirin extracted from white and raw versus cooked red sorghum grains as an oral delivery system. Targeted delivery to the colon would be beneficial for medication such as prednisolone, which is used in the management of inflammatory bowel disease. Therefore, prednisolone was loaded into microparticles of kafirin from the different sources using phase separation. Differences were observed in the protein content, in vitro protein digestibility, and protein electrophoretic profile of the various sources of sorghum grains, kafirin extracts, and kafirin microparticles. For all of the formulations, the majority of the loaded prednisolone was not released in in vitro conditions simulating the upper gastrointestinal tract, indicating that most of the encapsulated drug could reach the target area of the lower gastrointestinal tract. This suggests that these kafirin microparticles may have potential as a colon-targeted nutraceutical and drug delivery system.

Formulation and characterization of drug-loaded microparticles using distillers dried grain kafirin

Kafirin, a protein extracted from sorghum grain, has been formulated into microparticles and proposed for use as a delivery system owing to the resistance of kafirin to upper gastrointestinal digestion. However, extracting kafirin from sorghum distillers dried grains with solubles (DDGS) may be more efficient, because the carbohydrate component has been removed by fermentation. This study investigated the properties and use of kafirin extracted from DDGS to formulate microparticles. Prednisolone, an anti-inflammatory drug that could benefit from a delayed and targeted delivery system to the colon, was loaded into DDGS kafirin microparticles by phase separation with sodium chloride. Scanning electron micrographs revealed that the empty and prednisolone-loaded microparticles were round in shape and varied in size. Surface binding studies indicated prednisolone was loaded within the microparticles rather than being solely bound on the surface. These findings demonstrate DDGS kafirin can be formulated into microparticles and loaded with medication. Future studies could investigate the potential applications of DDGS kafirin microparticles as an orally administered targeted drug-delivery system.

Clinical Tips: Crohn's and Colitis

Inflammatory bowel disease (IBD) describes a group of chronic relapsing inflammatory conditions of the gastrointestinal tract (GIT), with Crohn’s disease and ulcerative colitis being the two most common. Ulcerative colitis affects the colon, with the inflammation limited to the colonic mucosal layers. In contrast, the full thickness of the gut wall can be inflamed in Crohn’s disease, and any part of the GIT can be affected – from the mouth to the anus, though the small and large intestine are most commonly involved...

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.

Downregulation of microRNA-498 in colorectal cancers and its cellular effects

miR-498 is a non-coding RNA located intergenically in 19q13.41. Due to its predicted targeting of several genes involved in control of cellular growth, we examined the expression of miR-498 in colon cancer cell lines and a large cohort of patients with colorectal adenocarcinoma. Two colon cancer cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The expression of miR-498 was tested in these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). Tissues from 80 patients with surgical resection of colorectum (60 adenocarcinomas and 20 non-neoplastic tissues) were tested for miR-498 expression by qRT-PCR. In addition, an exogenous miR-498 (mimic) was used to detect the miRNA׳s effects on cell proliferation and cell cycle events in SW480 using MTT calorimetric assay and flow cytometry respectively. The colon cancer cell lines showed reduced expression of miR-498 compared to a normal colonic epithelial cell line. Mimic driven over expression of miR-498 in the SW480 cell line resulted in reduced cell proliferation and increased proportions of G2-M phase cells. In tissues, miR-498 expression was too low to be detected in all colorectal adenocarcinoma compared to non-neoplastic tissues. This suggests that the down regulation of miR-498 in colorectal cancer tissues and the direct suppressive cellular effect noted in cancer cell lines implies that miR-498 has some direct or indirect role in the pathogenesis of colorectal adenocarcinomas.

Modulatory roles of microRNAs in the regulation of different signalling pathways in large bowel cancer stem cells

There are emerging data to suggest that microRNAs (miRNAs) have significant roles in regulating the function of normal cells and cancer stem cells (CSCs). This review aims to analyse the roles of miRNAs in the regulation of colon CSCs through their interaction with various signalling pathways. Studies showed a large number of miRNAs that are reported to be deregulated in colon CSCs. However, few of the studies available were able to outline the function of miRNAs in colon CSCs and uncover their signalling pathways. From those miRNAs, which are better described, miR-21 followed by miR-34, miR-200 and miR-215 are the most reported miRNAs to have roles in colon CSC regulation. In particular, miRNAs have been reported to regulate the stemness features of colon CSCs mainly via Wnt/B-catenin and Notch signalling pathways. Additionally, miRNAs have been reported to act on processes involving CSCs through cell cycle regulation genes and epithelial-mesenchymal transition. The relative paucity of data available on the significance of miRNAs in CSCs means that new studies will be of great importance to determine their roles and to identify the signalling pathways through which they operate. Such studies may in future guide further research to target these genes for more effective cancer treatment. miRNAs were shown to regulate the function of cancer stem cells in large bowel cancer by targeting a few key signalling pathways in cells.

Deregulation of miR-126 expression in colorectal cancer pathogenesis and its clinical significance

In this study, we investigated the expression profiles and clinicopathological significance of miR-126 in large cohort of patients with colorectal cancers as well the cellular repercussions of miR-126 in colon cancer cells along with its targets in-vitro. Down regulation of miR-126 expression was associated with histological subtypes, peri-neural tumour infiltration, microsatellite instability and pathological staging of colorectal cancers (p<0.05). Low miR-126 expression was also associated with poorer survival in patients with colorectal cancer. Analysis of matched tissues from the same patient revealed that approximately 70% of the tested patients had similar levels of expression of miR-126 in primary cancer and cancer metastases in both lymph node and distant metastases. In addition, induced overexpression of miR-126 showed reduced cell proliferation, increased apoptosis and decreased accumulation of cells in the G0-G1 phase of the colon cancer cells. Furthermore, SW480(+miR-126) cells showed reduced BCL-2 and increased P53 protein expression. To conclude, deregulation of miR-126 in colorectal cancer at the tissue and cellular levels as well as its correlation with various clinicopathological parameters confirm the cancer suppressive role of miR-126 in colorectal cancer.