Hypermethylation of the Inhibin Alpha-subunit Gene in Prostate Carcinoma

Molecular Endocrinology (Baltimore, Md.). Feb, 2002  |  Pubmed ID: 11818495

Inhibin is composed of an alpha- and a beta-subunit. Transgenic studies assigned a tumor-suppressive role to the inhibin alpha-subunit, and in human prostate cancer inhibin alpha-subunit gene expression was down-regulated. This study examined the inhibin alpha-subunit gene promoter and gene locus to determine whether promoter hypermethylation or LOH occurred in DNA from prostate cancer. The 5'-untranslated region of the human inhibin alpha-subunit gene was sequenced and shown to be highly homologous to the bovine, rat, and mouse inhibin alpha-subunit promoter sequences. A 135-bp region of the human promoter sequence that continued a cluster of CpG sites was analyzed for hypermethylation. Significant (P < 0.001) hypermethylation of the inhibin alpha-subunit gene promoter occurred in DNA from Gleason pattern 3, 4, and 5 carcinomas compared with nonmalignant tissue samples. A subset of the carcinomas with a cribriform pattern were unmethylated. LOH at 2q32-36, the chromosomal region harboring the inhibin alpha-subunit gene, was observed in 42% of prostate carcinomas. These data provide the first demonstration that promoter hypermethylation and LOH are associated with the inhibin alpha-subunit gene and gene locus in prostate cancer.

Hypermethylation Trigger of the Glutathione-S-transferase Gene (GSTP1) in Prostate Cancer Cells

Oncogene. Feb, 2002  |  Pubmed ID: 11850822

Understanding what triggers hypermethylation of tumour suppressor genes in cancer cells is critical if we are to discern the role of methylation in the oncogenic process. CpG sites in CpG island promoters, that span most tumour suppressor genes, remain unmethylated in the normal cell, despite the fact that CpG sites are the prime target for de novo methylation by the DNA methyltransferases. The CpG island-associated with the GSTP1 gene is an intriguing example of a CpG rich region which is susceptible to hypermethylation in the majority of prostate tumours and yet is unmethylated in the normal prostate cell. In this study we evaluate a number of factors purported to be involved in hypermethylation to test their role in triggering hypermethylation of GSTP1 in prostate cancer DU145 and LNCaP cells. We find that hypermethylation is not associated with (1) elevated expression of the DNA methyltranferases, or (2) removal of Sp1 transcription factor binding sites in the CpG island or (3) removal of CpG island boundary elements or (4) prior gene silencing. Instead our results support a model that requires a combination of prior gene silencing and random "seeds" of methylation to trigger hypermethylation of the GSTP1 gene in the prostate cancer cell. We propose that the GSTP1 gene is initially silenced in the prostate cancer and random sites of methylation accumulate that result in subsequent hypermethylation and chromatin remodelling.

"Tinkering" with the Components of Health

The Journal of School Health. Jan, 2002  |  Pubmed ID: 11865799

DNA Methylation Analysis in Mammalian Cells

Methods (San Diego, Calif.). Jun, 2002  |  Pubmed ID: 12095265

Identification and Resolution of Artifacts in Bisulfite Sequencing

Methods (San Diego, Calif.). Jun, 2002  |  Pubmed ID: 12095266

Bisulfite sequencing has become the most widely used application to detect 5-methylcytosine (5-MeC) in DNA, and provides a reliable way of detecting any methylated cytosine at single-molecule resolution in any sequence context. The process of bisulfite treatment exploits the different sensitivity of cytosine and 5-MeC to deamination by bisulfite under acidic conditions, in which cytosine undergoes conversion to uracil while 5-MeC remains unreactive. In this article, we address the more commonly encountered experimental artifacts associated with bisulfite sequencing, and provide methods for the detection and elimination of these artifacts. In particular, we focus on conditions that inhibit complete bisulfite-mediated conversion of cytosines in a target sequence, and demonstrate the necessity of complete protein removal from DNA samples prior to bisulfite treatment. We also include a brief summary of the experimental protocol for bisulfite treatment and tips for designing polymerase chain reaction (PCR) primers to amplify from bisulfite-treated DNA.

Methylation Sequencing from Limiting DNA: Embryonic, Fixed, and Microdissected Cells

Methods (San Diego, Calif.). Jun, 2002  |  Pubmed ID: 12095267

It is frequently useful to determine the methylation state of samples containing limited amounts of DNA such as from embryos, or from fixed tissue samples in which DNA is degraded or difficult to isolate. By modification of the standard protocols for DNA preparation and bisulfite treatment, it is possible to obtain DNA methylation sequence data for such samples. We present methods for bisulfite treatment of embryos, fixed sections, and samples obtained by laser capture microdissection, and discuss the additional experimental considerations required when working with small numbers of cells or degraded DNA samples.

Conversion-specific Detection of DNA Methylation Using Real-time Polymerase Chain Reaction (ConLight-MSP) to Avoid False Positives

Methods (San Diego, Calif.). Jun, 2002  |  Pubmed ID: 12095268

Methylated cytosines appear as sequence variations following bisulfite treatment and polymerase chain reaction (PCR) amplification. By using methylation-specific PCR (MSP), it is possible to detect methylated sequences in a background of unmethylated DNA with a high level of sensitivity. MSP is frequently used to identify methylated alleles in carcinogenesis, and may be combined with the TaqMan real-time PCR system, which uses fluorescence-based detection of amplification products during the amplification phase of the PCR and increases the sensitivity of detection (MethyLight). Sequences that have been incompletely converted during the bisulfite treatment are frequently coamplified during MSP, resulting in an overestimation of DNA methylation. The presence of amplified sequences originating from partially unconverted material may be determined by sequencing or by restriction digests or Southern blots of MSPs. Alternately, we have developed a method where the PCR and conversion assay are combined within a single TaqMan reaction by using an additional fluorescent probe directed against unconverted DNA (ConLight-MSP). We recommend that MSP detection always should include a step to detect unconverted DNA to avoid overestimation of the frequency or level of methylated DNA in the sample.

DNA Methylation and Gene Silencing in Cancer: Which is the Guilty Party?

Oncogene. Aug, 2002  |  Pubmed ID: 12154400

The DNA methylation pattern of a cell is exquisitely controlled during early development resulting in distinct methylation patterns. The tight control of DNA methylation is released in the cancer cell characterized by a reversal of methylation states. CpG island associated genes, in particular tumour suppressor or related genes, are often hypermethylated and this is associated with silencing of these genes. Therefore methylation is commonly convicted as a critical causal event in silencing this important class of genes in cancer. In this review, we argue that methylation is not the initial guilty party in triggering gene silencing in cancer, but that methylation of CpG islands is a consequence of prior gene silencing, similar to the role of methylation in maintaining the silencing of CpG island genes on the inactive X chromosome. We propose that gene silencing is the critical precursor in cancer, as it changes the dynamic interplay between de novo methylation and demethylation of the CpG island and tilts the balance to favour hypermethylation and chromatin inactivation.

DNA Methylation Changes in Leukaemia

Seminars in Cancer Biology. Oct, 2002  |  Pubmed ID: 12191634

Leukaemogenesis is a multi-step process whereby a clonal population arises that has undergone successive alterations to the genotype and the phenotype of the cells that make up the clone. Leukaemia has traditionally been viewed as a genetic disease, however epigenetic defects also play an important role. Expression of the DNA methyltransferase enzymes is elevated in leukaemia, and aberrant methylation is common with both a decrease in the total genomic 5-methylcytosine, and a concomitant hypermethylation of CpG island-associated tumour suppressor genes. This review will discuss the multitude of DNA methylation changes in haematopoietic malignancies and the implications they have for diagnosis and treatment.

Confirmation of Phenylbutazone Residues in Bovine Kidney by Liquid Chromatography/mass Spectrometry

Journal of AOAC International. Sep-Oct, 2002  |  Pubmed ID: 12374396

A confirmatory method is described for phenylbutazone (PB) residues in bovine kidney tissue. Ground kidney tissue is diluted with water, and the mixture is made basic with 25% ammonium hydroxide in water; the lipids are extracted with ethyl and petroleum ethers. The ether layer is discarded, and the tissue is acidified with 6N HCl. PB residues are extracted with tetrahydrofuranhexane (1 + 4). The extract is passed through a silica solid-phase extraction column, and the eluate is evaporated to dryness. The residue is dissolved in acidified acetonitrile-water-acetic acid (50 + 49.4 + 0.6). A single quadrupole mass spectrometer coupled to a liquid chromatograph with an electrospray interface is used to confirm the identity of the PB residues in the kidney extract. Negative-ion detection with selected-ion monitoring of 4 ions is used. Sets of control and fortified-control kidney tissues (at 50, 100, and 200 ppb PB) and several kidney tissue field samples were analyzed for method validation. The method was tested further during the course of a survey to determine the incidence of PB residues in bovine kidney samples obtained from slaughterhouses across the country. In addition, the method was tested for use with an ion-trap mass spectrometer coupled to a liquid chromatograph, which allowed confirmation of PB at lower levels (5-10 ppb) in kidney tissue.

A Question-and-answer Approach to Drug Education

The Journal of School Health. Oct, 2002  |  Pubmed ID: 12389376

Interlaboratory Comparison of Methods for the Determination of Incurred Tilmicosin Residues in Bovine Liver

Journal of AOAC International. Nov-Dec, 2002  |  Pubmed ID: 12477187

The objective of this study was to compare 2 methods for the determination of tilmicosin residues in bovine liver samples. Three laboratories participated in the comparison of the 2 methods. The first method was described in a New Animal Drug Application (NADA 140-929), and the second was a modification of that method in which hexane was substituted for carbon tetrachloride in one cleanup step. Each of the 3 laboratories analyzed subsamples of 10 bovine livers containing incurred tilmicosin. Residues ranged from 2.3 to 81 ppm tilmicosin in the 10 liver samples with an 11.8% relative standard deviation obtained by using both methods. In addition, fortified-control liver tissue samples were analyzed concurrently with tissues containing incurred residues by using the modified method in one of the laboratories. The fortification levels ranged from 0.3 to 112 ppm, with recoveries ranging from 76 to 92%. The results from the 3 laboratories were comparable, indicating that the modified method was not only as effective as the original NADA method, but also more desirable because of the change to a less hazardous solvent.

The Biochemistry of Antioxidants Revisited

Nutrition in Clinical Practice : Official Publication of the American Society for Parenteral and Enteral Nutrition. Feb, 2002  |  Pubmed ID: 16214960

Biochemical relationships between oxidative stress, antioxidant nutrients, and chronic diseases are complicated and often conflicting. Basic research supports the concept that reactive oxygen species precipitate changes that result in oxidative damage to lipid, protein, and DNA biomolecules. Oxidative stress is implicated in the development of cancer, cardiovascular disease, diabetes, sepsis, various eye diseases, and neurologic conditions. Supplementation with antioxidant nutrients seems plausible to counter the effects of oxidative stress, but the preferred mode of delivery for these nutrients may be through the patient's diet rather than as supplements to the diet. In fact, evidence supporting consumption of at least 5 servings of fruits and vegetables continues to grow. To better understand the role of antioxidant nutrients in disease promotion or prevention, this review will discuss basic nutritional biochemistry relating to oxidative stress and antioxidant defense systems, followed by a discussion of the metabolism (vitamins E, C, A) and interrelationships of select antioxidant nutrients.

Stepwise Progression of Familial Adenomatous Polyposis-associated Desmoid Precursor Lesions Demonstrated by a Novel CT Scoring System

Diseases of the Colon and Rectum. Apr, 2003  |  Pubmed ID: 12682541

Desmoids are rare, locally aggressive but nonmetastasizing clonal proliferations of fibroblasts that occur both sporadically and in association with familial adenomatous polyposis. Most occur in intra-abdominal sites, where they may lead to major morbidity and mortality. A proposed desmoid precursor lesion occurs in the mesentery of one-third of patients with familial adenomatous polyposis, and postoperative mesenteric fibromatosis has been identified in 20 percent of such patients. True desmoids occur in 10 percent, which suggests a model of development in which the phenotype becomes increasingly severe in a manner analogous to the adenoma-carcinoma sequence. This work aimed to confirm such a progression.

Bisulfite Methylation Analysis of Tumor Suppressor Genes in Prostate Cancer from Fresh and Archival Tissue Samples

Methods in Molecular Medicine. 2003  |  Pubmed ID: 12725123

Health Professionals Helping the Community

The Journal of School Health. Sep, 2003  |  Pubmed ID: 14513632

Alterations in the P16(INK4a) and P53 Tumor Suppressor Genes of HTERT-immortalized Human Fibroblasts

Oncogene. Apr, 2004  |  Pubmed ID: 14743210

Exogenous expression of the catalytic subunit of telomerase, hTERT, in a normal human foreskin fibroblast cell strain resulted in telomerase activity and an extended proliferative lifespan prior to a period of crisis. Three immortalized cell lines with stably maintained telomere lengths were established from cells that escaped crisis. Each of these cultures underwent a significant downregulation of p16(INK4a) expression due to gene deletion events. One cell line also acquired mutations in both alleles of the p53 tumor suppressor gene. Downregulation of p16(INK4a) and loss of wild-type p53 expression occurred after escape from crisis, so these mutations are most likely not required for immortalization of these cells but rather were selected for during continuous growth in vitro. These findings emphasize the need for caution in the use of hTERT-immortalized cells in studies of normal cell biology or in tissue engineering and the need to monitor for genetic instability and the accumulation of mutations in both the p16(INK4a)/pRb and p53 pathways.

Spirituality and Sexuality: Compatible Components for Optimal Health

The Journal of School Health. Jan, 2004  |  Pubmed ID: 15022373

Transcriptional Gene Silencing Promotes DNA Hypermethylation Through a Sequential Change in Chromatin Modifications in Cancer Cells

Cancer Research. Jun, 2004  |  Pubmed ID: 15172996

It is well established that DNA hypermethylation of tumor suppressor and tumor-related genes can occur in cancer cells and that each cancer subtype has specific gene sets that are commonly susceptible to methylation and silencing. Glutathione S-transferase (GSTP1) is one example of a gene that is hypermethylated and inactivated in the majority of prostate cancers. We previously reported that hypermethylation of the GSTP1 CpG island promoter in prostate cancer cells is initiated by a combination of transcriptional gene silencing (by removal of the Sp1 sites) and seeds of methylation that, instead of being constantly removed because of demethylation associated with transcription, acts as a catalyst for the spread of methylation across the CpG island. In this study, we now demonstrate that the seeds of DNA methylation also play an important role in initiating chromatin modification. Our results address a number of central questions about the temporal relationship between gene expression, DNA hypermethylation, and chromatin modification in cancer cells. We find that for the GSTP1 gene, (a). histone acetylation is independent of gene expression, (b). histone deacetylation is triggered by seeds of DNA methylation, (c). the spread of DNA hypermethylation across the island is linked to MBD2 and not MeCP2 binding, and (d). histone methylation occurs after histone deacetylation and is associated with extensive DNA methylation of the CpG island. These findings have important implications for understanding the biochemical events underlying the mechanisms responsible for abnormal hypermethylation of CpG island-associated genes in cancer cells.

Alimentary Tract Surgery in the Nonagenarian: Elective Vs. Emergent Operations

Journal of Gastrointestinal Surgery : Official Journal of the Society for Surgery of the Alimentary Tract. Jul-Aug, 2004  |  Pubmed ID: 15239987

The objective of this study was to compare elective with emergent surgery in patients over the age of 90 years. We retrospectively reviewed the records of patients over 90 years of age who underwent alimentary tract surgery between 1994 and 2002 at a community teaching hospital. Of 100 patients (mean age 92 years; range 90 to 98 years), 82 were women and 18 were men. Seventy-three percent were admitted from private homes or assisted-living facilities, and 27% came from a skilled-nursing facility (SNF). Major comorbid conditions existed in 93%. Procedures included right hemicolectomy (22%), adhesiolysis and/or small bowel resection (19%), cholecystectomy (14%), left-sided or sigmoid colectomy (11%), and perineal proctectomy (8%). Overall morbidity and mortality were 36% and 15%, respectively. Postoperative complications included respiratory failure and pneumonia (11%), arrhythmias (9%), delirium (7%), congestive heart failure and myocardial infarction (6%), and urinary complications (4%). Twenty-eight percent of the operations were elective, and 72% were emergent. Morbidity and mortality were higher in the emergent group (41% and 19%, respectively) than in the elective group (26% and 4%, respectively; P=0.04), especially for patients with an emergent surgical problem who came from a nursing home (22%). Average length of stay was 12 +/- 10 days (range 2 to 69 days) with little difference between elective and emergent cases. Sixty-four percent of patients were discharged to skilled-nursing facilities. Alimentary tract surgery can be performed safely in nonagenarians, and they should not be denied surgical care solely because of age.

High-dose Tamoxifen and Sulindac As First-line Treatment for Desmoid Tumors

Cancer. Aug, 2004  |  Pubmed ID: 15274082

Incidence of Low Free T4 Values in Premature Infants As Determined by Direct Equilibrium Dialysis

Journal of Perinatology : Official Journal of the California Perinatal Association. Oct, 2004  |  Pubmed ID: 15306825

The incidence of transient reductions in serum free T(4) (FT(4)) in premature infants may be overestimated because certain FT(4) analytical methods underestimate FT(4) concentrations. Transient reductions of FT(4) measurements have been reported in the majority of premature newborn infants. Direct equilibrium dialysis (DED) does not underestimate FT(4) concentrations and is the best available technique to measure serum FT(4) in the premature infant.

Evaluation of Decontamination Procedures Used in Primary Care

Nursing Times. Sep 7-13, 2004  |  Pubmed ID: 15478836

This study examined decontamination and infection control procedures in general practice. Four primary care trusts participated with a response rate of 80 per cent. The study showed evidence of poor compliance with national standards and examples of unsafe practice.

All-optical Switching in Rubidium Vapor

Science (New York, N.Y.). Apr, 2005  |  Pubmed ID: 15860622

We report on an all-optical switch that operates at low light levels. It consists of laser beams counterpropagating through a warm rubidium vapor that induce an off-axis optical pattern. A switching laser beam causes this pattern to rotate even when the power in the switching beam is much lower than the power in the pattern. The observed switching energy density is very low, suggesting that the switch might operate at the single-photon level with system optimization. This approach opens the possibility of realizing a single-photon switch for quantum information networks and for improving transparent optical telecommunication networks.

Developing Empathy for Hearing-impaired Students: Can You Hear What I Hear?

The Journal of School Health. Feb, 2005  |  Pubmed ID: 15929597

Confirmation of Sulfamethazine, Sulfathiazole, and Sulfadimethoxine Residues in Condensed Milk and Soft-cheese Products by Liquid Chromatography/tandem Mass Spectrometry

Journal of AOAC International. May-Jun, 2005  |  Pubmed ID: 16001847

A liquid chromatography/tandem mass spectrometry method (LC/MS/MS) is described for the simultaneous detection of 3 sulfonamide drug residues at 1.25 ppb in condensed milk and soft-cheese products. The 3 sulfonamide drugs of interest are sulfathiazole (STZ), sulfamethazine (SMZ), and sulfadimethoxine (SDM). The method includes extraction of the product with phosphate buffer, centrifugation of the diluted product, and application of a portion of the extract onto a polymeric solid-phase extraction cartridge. The cartridge is washed with water, and the sulfonamides are eluted with methanol. After evaporation, the residue is dissolved in 0.1% formic acid solution, and the solution is filtered before analysis by LC/MS/MS. The LC/MS/MS program involved a series of time-scheduled selected-reaction monitoring transitions. The transitions of MH+ to the common product ions at m/z 156, 108, and 92 were monitored for each residue. In addition, SMZ and SDM had a fourth significant and unique product ion transition that could be measured. Validation was performed with control and fortified-control condensed bovine milk with 2.5, 5, and 10 ppb sulfonamides. This method was applied to imported flavored and unflavored condensed milk and cream cheese bars. The presence of STZ and SMZ residues was confirmed in 3 out of 6 products.

Hypermethylation of the Prostacyclin Synthase (PTGIS) Promoter is a Frequent Event in Colorectal Cancer and Associated with Aneuploidy

Oncogene. Nov, 2005  |  Pubmed ID: 16007128

Inactivation of specific tumor suppressor genes by transcriptional silencing associated with hypermethylation of the promoter is a common event in cancer. We have applied the amplification of intermethylated sites (AIMS) technique to a 100 human colorectal cancers and seven cell lines to identify recurrent alterations that may unveil silenced tumor suppressor genes. Bisulfite sequencing was used to confirm differential DNA methylation results. Gene expression analysis was performed by real-time RT-PCR. An AIMS band recurrently displayed in tumors but not in normal tissues was isolated and identified as part of the CpG island of the prostacyclin synthase (PTGIS) gene promoter. PTGIS promoter was hypermethylated in 43 out of 100 colorectal cancers and in all cell lines. Bisulfite sequencing and clonal analysis confirmed the results obtained by AIMS and demonstrated biallelic hypermethylation of PTGIS promoter. Hypermethylation of the PTGIS promoter was associated with diminished gene expression, that was restored after treatment with demethylating and histone deacetylases inhibitor agents. PTGIS hypermethylation was associated with aneuploidy and p53 mutations. In the adjusted model, PTGIS methylation, but not p53 mutation, maintained the association with aneuploidy. We conclude that epigenetic inactivation of the PTGIS gene is a recurrent alteration in colorectal carcinogenesis.

Headloop Suppression PCR and Its Application to Selective Amplification of Methylated DNA Sequences

Nucleic Acids Research. 2005  |  Pubmed ID: 16091627

Selective amplification in PCR is principally determined by the sequence of the primers and the temperature of the annealing step. We have developed a new PCR technique for distinguishing related sequences in which additional selectivity is dependent on sequences within the amplicon. A 5' extension is included in one (or both) primer(s) that corresponds to sequences within one of the related amplicons. After copying and incorporation into the PCR product this sequence is then able to loop back, anneal to the internal sequences and prime to form a hairpin structure-this structure is then refractory to further amplification. Thus, amplification of sequences containing a perfect match to the 5' extension is suppressed while amplification of sequences containing mismatches or lacking the sequence is unaffected. We have applied Headloop PCR to DNA that had been bisulphite-treated for the selective amplification of methylated sequences of the human GSTP1 gene in the presence of up to a 10(5)-fold excess of unmethylated sequences. Headloop PCR has a potential for clinical application in the detection of differently methylated DNAs following bisulphite treatment as well as for selective amplification of sequence variants or mutants in the presence of an excess of closely related DNA sequences.

Permissive Transcriptional Activity at the Centromere Through Pockets of DNA Hypomethylation

PLoS Genetics. Feb, 2006  |  Pubmed ID: 16477312

DNA methylation is a hallmark of transcriptional silencing, yet transcription has been reported at the centromere. To address this apparent paradox, we employed a fully sequence-defined ectopic human centromere (or neocentromere) to investigate the relationship between DNA methylation and transcription. We used sodium bisulfite PCR and sequencing to determine the methylation status of 2,041 CpG dinucleotides distributed across a 6.76-Mbp chromosomal region containing a neocentromere. These CpG dinucleotides were associated with conventional and nonconventional CpG islands. We found an overall hypermethylation of the neocentric DNA at nonconventional CpG islands that we designated as CpG islets and CpG orphans. The observed hypermethylation was consistent with the presence of a presumed transcriptionally silent chromatin state at the neocentromere. Within this neocentric chromatin, specific sites of active transcription and the centromeric chromatin boundary are defined by DNA hypomethylation. Our data demonstrate, for the first time to our knowledge, a correlation between DNA methylation and centromere formation in mammals, and that transcription and "chromatin-boundary activity" are permissible at the centromere through the selective hypomethylation of pockets of sequences without compromising the overall silent chromatin state and function of the centromere.

Epigenetic Remodeling in Colorectal Cancer Results in Coordinate Gene Suppression Across an Entire Chromosome Band

Nature Genetics. May, 2006  |  Pubmed ID: 16642018

We report a new mechanism in carcinogenesis involving coordinate long-range epigenetic gene silencing. Epigenetic silencing in cancer has always been envisaged as a local event silencing discrete genes. However, in this study of silencing in colorectal cancer, we found common repression of the entire 4-Mb band of chromosome 2q.14.2, associated with global methylation of histone H3 Lys9. DNA hypermethylation within the repressed genomic neighborhood was localized to three separate enriched CpG island 'suburbs', with the largest hypermethylated suburb spanning 1 Mb. These data change our understanding of epigenetic gene silencing in cancer cells: namely, epigenetic silencing can span large regions of the chromosome, and both DNA-methylated and neighboring unmethylated genes can be coordinately suppressed by global changes in histone modification. We propose that loss of gene expression can occur through long-range epigenetic silencing, with similar implications as loss of heterozygosity in cancer.

Current Ideas in Desmoid Tumours

Familial Cancer. 2006  |  Pubmed ID: 16998673

Desmoid tumours are rare neoplasms of fibroblastic origin which arise with disproportionate frequency in patients with familial adenomatous polyposis (FAP). They are thought to develop in about 10-25% of FAP patients and may be the leading cause of death amongst those who have undergone colectomy. Risk factors include trauma, having a distal germline APC mutation, having a family history of desmoids, and probably oestrogens. In very high-risk individuals there may be a case for delay of colectomy or chemoprophylaxis at the time of surgery. Desmoids are now known to be true neoplasms but with normal telomere length and telomerase activity. FAP-associated tumours seem to carry biallelic APC mutations, one of which lies in the distal part of the gene. Such loss of wild-type APC seems to occur relatively late in tumour development. It is likely that beta-catenin plays an important role in tumourigenesis. FAP-associated desmoids tend to arise in the abdomen or abdominal wall. CT scanning gives the best information on tumour anatomy whilst T2-weighted MRI indicates likely behaviour. Treatment may simply consist of observation. Otherwise, usual first-line therapy is with sulindac with or without an anti-oestrogen. Cytotoxic chemotherapy is an option in unresectable tumours. Surgery is a reasonable first-line treatment in abdominal wall tumours but is risky for intra-abdominal tumours and may necessitate massive small bowel resection. Desmoids are the greatest remaining challenge in the management of FAP and further research into their aetiology needs to be combined with multicentre clinical trials of new treatments in order to improve management of the disease.

The Effect of Link Nurses on Hospital Readmission Rates

Nursing Times. Oct 17-23, 2006  |  Pubmed ID: 17066881

This study investigates whether visits to a patient early post-discharge by a member of the ward team (locality link nurse) reduces psychiatric hospital readmission rates for older people and improves the physical and psychological state of the patient. This is a summary of the paper the full version can be accessed at nursingtimes.net.

Telomere-driven Karyotypic Complexity Concurs with P16INK4a Inactivation in TP53-competent Immortal Endothelial Cells

Cancer Research. Nov, 2006  |  Pubmed ID: 17108106

Critically short telomeres promote chromosomal fusions, which in TP53-defective cells initiate the formation of cytogenetic aberrations that are typical of human cancer cells. Expression of the enzyme telomerase stabilizes normal and aberrant chromosomes by maintaining telomere length. However, previous investigations, including our own, have shown that overexpression of telomerase reverse transcriptase (hTERT) does not prevent net telomere shortening in human endothelial cells. In the present study, two mass cultures of hTERT-transduced bone marrow endothelial cells (BMhTERT) and 26 clones were employed to further investigate the immortalization process and consequences of telomere shortening. Eighty-five percent (22 of 26) of the clones and both mass cultures were immortalized. However, cytogenetic analyses revealed recurring cytogenetic aberrations in the mass cultures and 12 representative clones. Several of the recurring aberrations, including +5p, +11, -13, +19, and +20, and nonreciprocal translocations involving 17p and 2p were previously implicated in human carcinogenesis. One mass culture and a subset of clones (5 of 12) had complex karyotypes, characterized by cytogenetic heterogeneity and at least five chromosomal abnormalities. p16(INK4a) was silenced exclusively in the five clones and mass culture with complex karyotypes, whereas the p53/p21(cip1) pathway was defective in only one clone. Telomere dysfunction was implicated in the evolution of complex karyotypes by the presence of anaphase bridges, telomere associations, and dicentric chromosomes. These results show that complex karyotypes can evolve in TP53-competent cells and provide evidence that p16(INK4a) functions as a gatekeeper to prevent telomere-driven cytogenetic evolution. These investigations provide new insight to the role of p16(INK4a) as a tumor suppressor.

Exercise in the Heat: Effect of Fluid Ingestion on Blood-brain Barrier Permeability

Medicine and Science in Sports and Exercise. Dec, 2006  |  Pubmed ID: 17146318

This study examined changes in serum S100beta concentration, a peripheral marker of BBB permeability, in response to exercise in the heat with and without fluid ingestion.

DNA Methylation: Bisulphite Modification and Analysis

Nature Protocols. 2006  |  Pubmed ID: 17406479

DNA methylation is an important epigenetic modification of DNA in mammalian genomes. DNA methylation patterns are established early in development, modulated during tissue-specific differentiation and disrupted in many disease states, including cancer. To understand further the biological functions of these changes, accurate and reproducible methods are required to fully analyze the DNA methylation sequence. Here, we describe the 'gold-standard' bisulphite conversion protocol that can be used to re-sequence DNA from mammalian cells in order to determine and quantify the methylation state of a gene or genomic region at single-nucleotide resolution. The process of bisulphite treatment exploits the different sensitivities of cytosine and 5-methylcytosine (5-MeC) to deamination by bisulphite under acidic conditions--in which cytosine undergoes conversion to uracil, whereas 5-MeC remains unreactive. Bisulphite conversion of DNA, in either single tubes or in a 96-well format, can be performed in a minimum of 8 h and a maximum of 18 h, depending on the amount and quality of starting DNA.

Bisulphite Differential Denaturation PCR for Analysis of DNA Methylation

Epigenetics : Official Journal of the DNA Methylation Society. Apr-Jun, 2006  |  Pubmed ID: 17998815

Differential denaturation during PCR can be used to selectively amplify unmethylated DNA from a methylated DNA background. The use of differential denaturation in PCR is particularly suited to amplification of undermethylated sequences following treatment with bisulphite, since bisulphite selectively converts cytosines to uracil while methylated cytosines remain unreactive. Thus amplicons derived from unmethylated DNA retain fewer cytosines and their lower G + C content allows for their amplification at the lower melting temperatures, while limiting amplification of the corresponding methylated amplicons (Bisulphite Differential Denaturation PCR, BDD-PCR). Selective amplification of unmethylated DNA of four human genomic regions from three genes, GSTP1, BRCA1 and MAGE-A1, is demonstrated with selectivity observed at a ratio of down to one unmethylated molecule in 10(5) methylated molecules. BDD-PCR has the potential to be used to selectively amplify and detect aberrantly demethylated genes, such as oncogenes, in cancers. Additionally BDD-PCR can be effectively utilized in improving the specificity of methylation specific PCR (MSP) by limiting amplification of DNA that is not fully converted, thus preventing misinterpretation of the methylation versus non-conversion.

Concordant Epigenetic Silencing of Transforming Growth Factor-beta Signaling Pathway Genes Occurs Early in Breast Carcinogenesis

Cancer Research. Dec, 2007  |  Pubmed ID: 18089780

Human mammary epithelial cells (HMEC) grown under standard cell culture conditions enter a growth phase referred to as selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection or variant HMECs, may be derived from progenitor cells found in normal mammary epithelium that subsequently acquire premalignant lesions, including p16(INK4A) promoter hypermethylation. Epigenetic silencing of tumor suppressor genes through DNA methylation and histone modification is an early event in tumorigenesis. A major challenge is to find genes or gene pathways that are commonly silenced to provide early epigenetic diagnostic and therapeutic cancer targets. To identify very early epigenetic events that occur in breast cancer, we used microarrays to screen for gene pathways that were suppressed in post-selection HMECs but reactivated after treatment with the demethylation agent 5-aza-2'-deoxycytidine. We found that several members of the transforming growth factor beta (TGF-beta) signaling pathway were consistently down-regulated in the post-selection HMEC populations, and this was associated with a marked decrease in Smad4 nuclear staining. Gene suppression was not associated with DNA methylation but with chromatin remodeling, involving a decrease in histone H3 lysine 27 trimethylation and an increase in histone H3 lysine 9 dimethylation and deacetylation. These results show for the first time that TGF-beta2, its receptors TGF-beta R1 and TGF-beta R2, and activator thrombospondin-1 are concordantly suppressed early in breast carcinogenesis by histone modifications and indicate that the TGF-beta signaling pathway is a novel target for gene activation by epigenetic therapy.

The Impact of Technical Factors on Outcome of Restorative Proctocolectomy for Familial Adenomatous Polyposis

Diseases of the Colon and Rectum. Jul, 2007  |  Pubmed ID: 17464542

This study was designed to assess the impact of technical factors on functional outcomes and complications in patients undergoing restorative proctocolectomy for familial adenomatous polyposis.

The Effect of Restorative Proctocolectomy on Sexual Function, Urinary Function, Fertility, Pregnancy and Delivery: a Systematic Review

Diseases of the Colon and Rectum. Aug, 2007  |  Pubmed ID: 17588223

This study was designed to evaluate the effect of restorative proctocolectomy on sexual function, urinary function, fertility, pregnancy, and delivery in patients with ulcerative colitis.

Action at a Distance: Epigenetic Silencing of Large Chromosomal Regions in Carcinogenesis

Human Molecular Genetics. Apr, 2007  |  Pubmed ID: 17613553

Despite the completion of the Human Genome Project, we are still far from understanding the molecular events underlying epigenetic change in cancer. Cancer is a disease of the DNA with both genetic and epigenetic changes contributing to changes in gene expression. Epigenetics involves the interplay between DNA methylation, histone modifications and expression of non-coding RNAs in the regulation of gene transcription. We now know that tumour suppressor genes, with CpG island-associated promoters, are commonly hypermethylated and silenced in cancer, but we do not understood what triggers this process or when it occurs during carcinogenesis. Epigenetic gene silencing has always been envisaged as a local event silencing discrete genes, but recent data now indicates that large regions of chromosomes can be co-coordinately suppressed; a process termed long range epigenetic silencing (LRES). LRES can span megabases of DNA and involves broad heterochromatin formation accompanied by hypermethylation of clusters of contiguous CpG islands within the region. It is not clear if LRES is initiated by one critical gene target that spreads and conscripts innocent bystanders, analogous to large genetic deletions or if coordinate silencing of multiple genes is important in carcinogenesis? Over the next decade with the exciting new genomic approaches to epigenome analysis and the initiation of a Human Epigenome Project, we will understand more about the interplay between DNA methylation and chromatin modifications and the expression of non-coding RNAs, promising a new range of molecular diagnostic cancer markers and molecular targets for cancer epigenetic therapy.

Quantum Computers Based on Electron Spins Controlled by Ultrafast Off-resonant Single Optical Pulses

Physical Review Letters. Jul, 2007  |  Pubmed ID: 17678343

We describe a fast quantum computer based on optically controlled electron spins in charged quantum dots that are coupled to microcavities. This scheme uses broadband optical pulses to rotate electron spins and provide the clock signal to the system. Nonlocal two-qubit gates are performed by phase shifts induced by electron spins on laser pulses propagating along a shared waveguide. Numerical simulations of this scheme demonstrate high-fidelity single-qubit and two-qubit gates with operation times comparable to the inverse Zeeman frequency.

Genomic Profiling of CpG Methylation and Allelic Specificity Using Quantitative High-throughput Mass Spectrometry: Critical Evaluation and Improvements

Nucleic Acids Research. 2007  |  Pubmed ID: 17855397

CpG methylation is a key component of the epigenome architecture that is associated with changes in gene expression without a change to the DNA sequence. Since the first reports on deregulation of DNA methylation, in diseases such as cancer, and the initiation of the Human Epigenome Project, an increasing need has arisen for a detailed, high-throughput and quantitative method of analysis to discover and validate normal and aberrant DNA methylation profiles in large sample cohorts. Here we present an improved protocol using base-specific fragmentation and MALDI-TOF mass spectrometry that enables a sensitive and high-throughput method of DNA methylation analysis, quantitative to 5% methylation for each informative CpG residue. We have determined the accuracy, variability and sensitivity of the protocol, implemented critical improvements in experimental design and interpretation of the data and developed a new formula to accurately measure CpG methylation. Key innovations now permit determination of differential and allele-specific methylation, such as in cancer and imprinting. The new protocol is ideally suitable for detailed DNA methylation analysis of multiple genomic regions and large sample cohorts that is critical for comprehensive profiling of normal and diseased human epigenomes.

Epigenetic Inactivation of a Cluster of Genes Flanking MLH1 in Microsatellite-unstable Colorectal Cancer

Cancer Research. Oct, 2007  |  Pubmed ID: 17909015

Biallelic promoter methylation and transcriptional silencing of the MLH1 gene occurs in the majority of sporadic colorectal cancers exhibiting microsatellite instability due to defective DNA mismatch repair. Long-range epigenetic silencing of contiguous genes has been found on chromosome 2q14 in colorectal cancer. We hypothesized that epigenetic silencing of MLH1 could occur on a regional scale affecting additional genes within 3p22, rather than as a focal event. We studied the levels of CpG island methylation and expression of multiple contiguous genes across a 4 Mb segment of 3p22 including MLH1 in microsatellite-unstable and -stable cancers, and their paired normal colonic mucosa. We found concordant CpG island hypermethylation, H3-K9 dimethylation and transcriptional silencing of MLH1 and multiple flanking genes spanning up to 2.4 Mb in microsatellite-unstable colorectal cancers. This region was interspersed with unmethylated genes, which were also transcriptionally repressed. Expression of both methylated and unmethylated genes was reactivated by methyltransferase and histone deacetylase inhibitors in a microsatellite-unstable colorectal carcinoma cell line. Two genes at the telomeric end of the region were also hypermethylated in microsatellite-stable cancers, adenomas, and at low levels in normal colonic mucosa from older individuals. Thus, the cluster of genes flanking MLH1 that was specifically methylated in the microsatellite-unstable group of cancers extended across 1.1 Mb. Our results show that coordinate epigenetic silencing extends across a large chromosomal region encompassing MLH1 in microsatellite-unstable colorectal cancers. Simultaneous epigenetic silencing of this cluster of 3p22 genes may contribute to the development or progression of this type of cancer.

Enhancing Health Literacy Through Accessing Health Information, Products, and Services: an Exercise for Children and Adolescents

The Journal of School Health. Nov, 2007  |  Pubmed ID: 17970869

Homozygous PMS2 Deletion Causes a Severe Colorectal Cancer and Multiple Adenoma Phenotype Without Extraintestinal Cancer

Gastroenterology. Feb, 2007  |  Pubmed ID: 17258725

We report a patient of Indian descent with parental consanguinity, who developed 10 carcinomas and 35 adenomatous polyps at age 23 and duodenal adenocarcinoma at age 25. He also had dysmorphic features, mental retardation, and café-au-lait spots but no brain tumor. We aimed to establish his molecular diagnosis.

Perception of Voice in the Transgender Client

Journal of Voice : Official Journal of the Voice Foundation. Nov, 2008  |  Pubmed ID: 17400427

Fundamental frequency (F(0)) of speech is used to measure the success of voice therapy in male-to-female transgender clients. This study evaluates the relationship between F(0) and patients' happiness with their voice. The study design used was a cross-sectional evaluation of client satisfaction questionnaires and voice recordings from transgender clients. This study was a comparative evaluation of voice recordings by voice professionals and lay observers. Twelve male-to-female transgender participants completed visual analogue scales (VASs), rating happiness with self-perceived femininity of their voice. Fifteen speech and language therapists (SLTs) and 40 naïve observers evaluated the anonymized recordings, using the same rating system. The correlation between mean F(0) and participant happiness was established. Relationships between participant happiness and rater opinions were explored. A significant relationship between F(0) and participant happiness could not be demonstrated (r=0.32, P=0.32). There was a moderately strong positive correlation between self-perception of vocal femininity and perception of femininity by SLTs and naïve observers (r=0.76 and 0.68, P=0.003 and P=0.01, respectively). This study demonstrates that happiness with voice in male-to-female transgender clients is not directly related to F(0). Clients can assess femininity of their voice in the form of perceived pitch. This may not affect happiness scores. Voice satisfaction may not correlate with perceptions of supervising voice professionals. However, professionals can reliably evaluate how the voice will be received by the lay public. Subjective measures of patient satisfaction, including VASs, are reliable and valid tools in evaluating therapeutic success.

DNA Methylation Changes in Ovarian Cancer: Implications for Early Diagnosis, Prognosis and Treatment

Gynecologic Oncology. Apr, 2008  |  Pubmed ID: 18234305

To review epigenetic changes identified in ovarian cancer, focusing on their potential as clinical markers for detection, monitoring of disease progression and as markers of therapeutic response.

Iron Deficiency Anemia

Nutrition in Clinical Practice : Official Publication of the American Society for Parenteral and Enteral Nutrition. Apr-May, 2008  |  Pubmed ID: 18390780

The most severe consequence of iron depletion is iron deficiency anemia (IDA), and it is still considered the most common nutrition deficiency worldwide. Although the etiology of IDA is multifaceted, it generally results when the iron demands by the body are not met by iron absorption, regardless of the reason. Individuals with IDA have inadequate intake, impaired absorption or transport, physiologic losses associated with chronological or reproductive age, or chronic blood loss secondary to disease. In adults, IDA can result in a wide variety of adverse outcomes including diminished work or exercise capacity, impaired thermoregulation, immune dysfunction, GI disturbances, and neurocognitive impairment. In addition, IDA concomitant with chronic kidney disease or congestive heart failure can worsen the outcome of both conditions. In this review, the prevalence of IDA related to confounding medical conditions will be described along with its diverse etiologies. Distinguishing IDA from anemia of chronic disease using hematologic measures is reviewed as well. In addition, current diagnostic strategies that are inclusive of clinical presentation, biochemical tests, and differential diagnosis will be outlined, followed by a discussion of treatment modalities and future research recommendations.

This is Your Future: a Case Study Approach to Foster Health Literacy

The Journal of School Health. Jun, 2008  |  Pubmed ID: 18489469

Determination and Confirmation of Melamine Residues in Catfish, Trout, Tilapia, Salmon, and Shrimp by Liquid Chromatography with Tandem Mass Spectrometry

Journal of Agricultural and Food Chemistry. Jun, 2008  |  Pubmed ID: 18494486

Pet and food animal (hogs, chicken, and fish) feeds were recently found to be contaminated with melamine (MEL). A quantitative and confirmatory method is presented to determine MEL residues in edible tissues from fish fed this contaminant. Edible tissues were extracted with acidic acetonitrile, defatted with dichloromethane, and cleaned up using mixed-mode cation exchange solid-phase extraction cartridges. Extracts were analyzed by liquid chromatography with tandem mass spectrometry with hydrophilic interaction chromatography and electrospray ionization in positive ion mode. Fish and shrimp tissues were fortified with 10-500 microg/kg (ppb) of MEL with an average recovery of 63.8% (21.5% relative standard deviation, n = 121). Incurred fish tissues were generated by feeding fish up to 400 mg/kg of MEL or a combination of MEL and the related triazine cyanuric acid (CYA). MEL and CYA are known to form an insoluble complex in the kidneys, which may lead to renal failure. Fifty-five treated catfish, trout, tilapia, and salmon were analyzed after withdrawal times of 1-14 days. MEL residues were found in edible tissues from all of the fish with concentrations ranging from 0.011 to 210 mg/kg (ppm). Incurred shrimp and a survey of market seafood products were also analyzed as part of this study.

Epigenetic Markers of Ovarian Cancer

Advances in Experimental Medicine and Biology. 2008  |  Pubmed ID: 18546617

Persistent Perineal Sinus After Ileoanal Pouch Excision in Inflammatory Bowel Diseases: Incidence, Risk Factors, and Clinical Course

Diseases of the Colon and Rectum. Dec, 2008  |  Pubmed ID: 18626712

This study was designed to determine the incidence of persistent perineal sinus after pouch excision in inflammatory bowel disease, risk factors, and long-term clinical course.

The Risk of Oral Contraceptives in the Etiology of Inflammatory Bowel Disease: a Meta-analysis

The American Journal of Gastroenterology. Sep, 2008  |  Pubmed ID: 18684177

Several environmental and genetic factors have been implicated to date in the development of Crohn's disease (CD) and ulcerative colitis (UC). The aim of this study was to provide a quantification of the risk of oral contraceptive pill (OCP) use in the etiology of inflammatory bowel disease.

Breast Cancer Epigenetics: Normal Human Mammary Epithelial Cells As a Model System

Journal of Molecular Medicine (Berlin, Germany). Dec, 2008  |  Pubmed ID: 18716754

DNA hypermethylation and histone modifications are two critical players involved in epigenetic regulation and together play an important role in silencing tumor-suppressor genes in all cancers, including breast cancer. One of the major challenges facing breast cancer researchers is the problem of how to identify critical genes that are epigenetically silenced early in cancer initiation as these genes provide potential early diagnostic and/or therapeutic targets for breast cancer management. This review will focus on compelling evidence that normal Human Mammary Epithelial Cells (HMECs) that escape senescence in culture mimic genetic and epigenetic events occurring in early breast cancer, and provide a valuable system to delineate the early steps in epigenetic deregulation that often occur during transition of a normal breast cell to a premalignant cell. In particular, this model system has been used to investigate the relationship between gene silencing, DNA methylation, histone modifications, and polycomb association that may occur early in oncogenic transformation.

Climate Warming, Marine Protected Areas and the Ocean-scale Integrity of Coral Reef Ecosystems

PloS One. 2008  |  Pubmed ID: 18728776

Coral reefs have emerged as one of the ecosystems most vulnerable to climate variation and change. While the contribution of a warming climate to the loss of live coral cover has been well documented across large spatial and temporal scales, the associated effects on fish have not. Here, we respond to recent and repeated calls to assess the importance of local management in conserving coral reefs in the context of global climate change. Such information is important, as coral reef fish assemblages are the most species dense vertebrate communities on earth, contributing critical ecosystem functions and providing crucial ecosystem services to human societies in tropical countries. Our assessment of the impacts of the 1998 mass bleaching event on coral cover, reef structural complexity, and reef associated fishes spans 7 countries, 66 sites and 26 degrees of latitude in the Indian Ocean. Using Bayesian meta-analysis we show that changes in the size structure, diversity and trophic composition of the reef fish community have followed coral declines. Although the ocean scale integrity of these coral reef ecosystems has been lost, it is positive to see the effects are spatially variable at multiple scales, with impacts and vulnerability affected by geography but not management regime. Existing no-take marine protected areas still support high biomass of fish, however they had no positive affect on the ecosystem response to large-scale disturbance. This suggests a need for future conservation and management efforts to identify and protect regional refugia, which should be integrated into existing management frameworks and combined with policies to improve system-wide resilience to climate variation and change.

Putative Direct and Indirect Wnt Targets Identified Through Consistent Gene Expression Changes in APC-mutant Intestinal Adenomas from Humans and Mice

Human Molecular Genetics. Dec, 2008  |  Pubmed ID: 18782851

In order to identify new genes with differential expression in early intestinal tumours, we performed mRNA (messenger ribonucleic acid) expression profiling of 16 human and 63 mouse adenomas. All individuals had germline APC mutations to ensure that tumorigenesis was driven by 'second hits' at APC. Using stringent filtering to identify changes consistent between humans and mice, we identified 60 genes up-regulated and 151 down-regulated in tumours. For 22 selected genes--including known Wnt targets--expression differences were confirmed by qRT-PCR (quantitative reverse transcription polymerase chain reaction). Most, but not all, differences were also present in colorectal carcinomas. In situ analysis showed a complex picture. Expression of up-regulated genes in adenomas was usually uniform/diffuse (e.g. ITGA6) or prominent in the tumour core (e.g. LGR5); in normal tissue, these genes were expressed at crypt bases or the transit amplifying zone. Down-regulated genes were often undetectable in adenomas, but in normal tissue were expressed in mesenchyme (e.g. GREM1/2) or differentiated cells towards crypt tops (e.g. SGK1). In silico analysis of TCF4-binding motifs showed that some of our genes were probably direct Wnt targets. Previous studies, mostly focused on human tumours, showed partial overlap with our 'expression signature', but 37 genes were unique to our study, including TACSTD2, SEMA3F, HOXA9 and IER3 (up-regulated), and TAGLN, GREM1, GREM2, MAB21L2 and RARRES2 (down-regulated). Combined analysis of our and published human data identified additional genes differentially expressed in adenomas, including decreased BMPs (bone morphogenetic proteins) and increased BUB1/BUB1B. Several of the newly identified, differentially expressed genes represent potential diagnostic or therapeutic targets for intestinal tumours.

Epigenetic and Phenotypic Changes Result from a Continuous Pre and Post Natal Dietary Exposure to Phytoestrogens in an Experimental Population of Mice

BMC Physiology. 2008  |  Pubmed ID: 18793434

Developmental effects of exposure to endocrine disruptors can influence adult characters in mammals, but could also have evolutionary consequences. The aim of this study was to simulate an environmental exposure of an experimental population of mice to high amounts of nutritional phytoestrogens and to evaluate parameters of relevance for evolutionary change in the offspring. The effect of a continuous pre- and post-natal exposure to high levels of dietary isoflavones was evaluated on sexual maturity, morphometric parameters and DNA methylation status in mice. Adult mice male/female couples were fed ad libitum either with control diet (standard laboratory chow) or ISF diet (control diet plus a soy isoflavone extract at 2% (w/w) that contained the phytoestrogens genistein and daidzein). In the offspring we measured: i) the onset of vaginal opening (sexual maturation) in females, ii) weight and size in all pups at 7, 14, 21 and 42 days post-natal (dpn) and iii) DNA methylation patterns in skeletal alpha-actin (Acta1), estrogen receptor-alpha and c-fos in adults (42 dpn).

The Comparative Incidence of Reported Concussions Presenting for Follow-up Management in South African Rugby Union

Clinical Journal of Sport Medicine : Official Journal of the Canadian Academy of Sport Medicine. Sep, 2008  |  Pubmed ID: 18806547

The objective of this study was to compare the seasonal concussion incidence for school, university, club and provincial level Rugby Union players in South Africa.

Low O6-methylguanine-DNA Methyltransferase (MGMT) Expression and Response to Temozolomide in Aggressive Pituitary Tumours

Clinical Endocrinology. Aug, 2009  |  Pubmed ID: 19067722

Recent case reports detail the successful use of temozolomide in the management of aggressive pituitary tumours. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that counteracts the effect of temozolomide.

Guide to Endoscopy of the Ileo-anal Pouch Following Restorative Proctocolectomy with Ileal Pouch-anal Anastomosis; Indications, Technique, and Management of Common Findings

Inflammatory Bowel Diseases. Aug, 2009  |  Pubmed ID: 19180580

Restorative proctocolectomy (RPC) with ileal pouch-anal anastomosis is the surgical procedure of choice for patients with ulcerative colitis (UC). It is also performed in selected patients with familial adenomatous polyposis (FAP). A significant proportion of patients will develop pouch dysfunction. Flexible pouchoscopy is the most important initial investigation in patients with dysfunction. It is also important in UC and FAP surveillance. The aim is to provide gastroenterologists with a clear understanding of the technique, indications, and diagnostic pitfalls when investigating RPC patients with flexible pouchoscopy. Flexible pouchoscopy for the investigation of RPC patients with pouch dysfunction has a high diagnostic yield, with most causes of pouch dysfunction identifiable during this procedure. The risk of developing dysplasia following RPC is low. Surveillance pouchoscopy is only recommended in those with FAP, those with a previous history of dysplasia or carcinoma, primary sclerosing cholangitis, those with a retained rectal cuff, and those with Type C histological changes. Flexible pouchoscopy is a useful first-line investigation in patients with pouch dysfunction. It can be performed without sedation and has a high diagnostic yield; it is also important as part of surveillance in FAP and selected UC patients.

Iron Deficiency Anemia: Diagnosis and Management

Current Opinion in Gastroenterology. Mar, 2009  |  Pubmed ID: 19262200

Iron deficiency anemia (IDA) still remains universally problematic worldwide. The primary focus of this review is to critique articles published over the past 18 months that describe strategies for the diagnosis and management of this prevalent condition.

Features of Duodenal Cancer in Patients with Familial Adenomatous Polyposis

Clinical Gastroenterology and Hepatology : the Official Clinical Practice Journal of the American Gastroenterological Association. Jun, 2009  |  Pubmed ID: 19281862

Most patients with familial adenomatous polyposis (FAP) develop duodenal adenomas; duodenal cancer is a major cause of mortality in this patient group. We reviewed cases of duodenal cancer in patients with FAP to identify factors that determine long-term cancer risk.

Multiresidue Method for the Triphenylmethane Dyes in Fish: Malachite Green, Crystal (gentian) Violet, and Brilliant Green

Analytica Chimica Acta. Apr, 2009  |  Pubmed ID: 19286041

Liquid chromatographic methods are presented for the quantitative and confirmatory determination of crystal violet (CV; also known as gentian violet), leucocrystal violet (LCV), brilliant green (BG), and leucobrilliant green (LBG) in catfish. LCV and LBG were oxidized to the chromic CV and BG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and residues were measured as the combined CV+/-LCV and BG+/-LBG. These methods are extensions of published methods for malachite green (MG) analysis to allow simultaneous determination of MG, CV, and BG. Residues were extracted from muscle with ammonium acetate buffer and acetonitrile, and extracts cleaned up using dichloromethane partitioning and solid-phase extraction. Extracts were analyzed by liquid chromatography with visible detection (LC-VIS). The method was validated for catfish fortified with LCV over the range 0.25-10 ngg(-1) and CV at 2 ngg(-1). Average recoveries were 90.6% (+/-8.1% R.S.D., n=45) for LCV and 84.4% (+/-4.2% R.S.D., n=6) for CV. The average recovery for samples fortified with BG or LBG over the range 0.5-10 ngg(-1) was 67.2% (+/-14.8% R.S.D., n=31). CV and BG were confirmed in fish extracts by ion trap LC-mass spectrometry (LC-MS(n)) with no discharge-atmospheric pressure chemical ionization. Average LC-MS(n) recoveries were 96.5, 96.6, and 70.2% for samples fortified with CV, LCV, and BG or LBG. The limits of detection for CV, BG, and MG were in the range of 0.07-0.24 ngg(-1) (ppb) for the two different instrumental methods. This methodology was applied to the analysis of catfish treated with CV and BG.

Fecal Coliform Testing to Identify Effective Antibiotic Therapies for Patients with Antibiotic-resistant Pouchitis

Clinical Gastroenterology and Hepatology : the Official Clinical Practice Journal of the American Gastroenterological Association. May, 2009  |  Pubmed ID: 19418603

Empiric antibiotic therapy (eg, a combination of ciprofloxacin and metronidazole) is effective in treating the majority of patients with inflammation of the ileal reservoir (pouchitis). Unfortunately, up to 20% of patients develop refractory or rapidly relapsing disease. We developed a fecal sensitivity analysis to determine which antibiotics are most likely to be effective in patients who do not respond to empiric antibiotic therapy or have relapsed after long-term therapy.

Aberrant De Novo Methylation of the P16INK4A CpG Island is Initiated Post Gene Silencing in Association with Chromatin Remodelling and Mimics Nucleosome Positioning

Human Molecular Genetics. Aug, 2009  |  Pubmed ID: 19477956

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly enriched in CpG island associated promoter regions. DNA hypermethylation and histone repression correlate with gene silencing, however, the dynamics of this process are still largely unclear. The tumour suppressor gene p16(INK4A) is inactivated in association with CpG island methylation during neoplastic progression in a variety of cancers, including breast cancer. Here, we investigated the temporal progression of DNA methylation and histone remodelling in the p16(INK4A) CpG island in primary human mammary epithelial cell (HMEC) strains during selection, as a model for early breast cancer. Silencing of p16(INK4A) has been previously shown to be necessary before HMECs can escape from selection. Here, we demonstrate that gene silencing occurs prior to de novo methylation and histone remodelling. An increase in DNA methylation was associated with a rapid loss of both histone H3K27 trimethylation and H3K9 acetylation and a gradual gain of H3K9 dimethylation. Interestingly, we found that regional-specific 'seeding' methylation occurs early after post-selection and that the de novo methylation pattern observed in HMECs correlates with the apparent footprint of nucleosomes across the p16(INK4A) CpG island. Our results demonstrate for the first time that p16(INK4A) gene silencing is a precursor to epigenetic suppression and that subsequent de novo methylation initially occurs in nucleosome-free regions across the p16(INK4A) CpG island and this is associated with a dynamic change in histone modifications.

Symptomatic Polyposis in a 4-year-old: the Exception Proves the Rule

Journal of Paediatrics and Child Health. May, 2009  |  Pubmed ID: 19493128

Incidence and Short-term Implications of Prepouch Ileitis Following Restorative Proctocolectomy with Ileal Pouch-anal Anastomosis for Ulcerative Colitis

Diseases of the Colon and Rectum. May, 2009  |  Pubmed ID: 19502851

Pouchitis following restorative proctocolectomy is common. Inflammation proximal to the pouch, prepouch ileitis (PPI) has recently been described. Its incidence and implications are unknown. The aim of this study was to identify the incidence of PPI at pouchoscopy and correlate this with symptoms, diagnosis, and outcome.

A Mitotic Recombination Map Proximal to the APC Locus on Chromosome 5q and Assessment of Influences on Colorectal Cancer Risk

BMC Medical Genetics. 2009  |  Pubmed ID: 19515250

Mitotic recombination is important for inactivating tumour suppressor genes by copy-neutral loss of heterozygosity (LOH). Although meiotic recombination maps are plentiful, little is known about mitotic recombination. The APC gene (chr5q21) is mutated in most colorectal tumours and its usual mode of LOH is mitotic recombination.

Ultrafast Optical Spin Echo for Electron Spins in Semiconductors

Physical Review Letters. Jun, 2009  |  Pubmed ID: 19659047

Spin-based quantum computing and magnetic resonance techniques rely on the ability to measure the coherence time T(2) of a spin system. We report on the experimental implementation of all-optical spin echo to determine the T(2) time of a semiconductor electron-spin system. We use three ultrafast optical pulses to rotate spins an arbitrary angle and measure an echo signal as the time between pulses is lengthened. Unlike previous spin-echo techniques using microwaves, ultrafast optical pulses allow clean T(2) measurements of systems with dephasing times (T_{2};{*}) fast in comparison to the time scale for microwave control. This demonstration provides a step toward ultrafast optical dynamic decoupling of spin-based qubits.

The Role of Pouch Compliance Measurement in the Management of Pouch Dysfunction

International Journal of Colorectal Disease. Apr, 2010  |  Pubmed ID: 19924421

Ileal pouch anal anastomosis is an established option for patients who require total proctocolectomy and restoration of bowel continuity. However, the functional results are not always good and low pouch compliance has been suggested as one possible cause. We aimed to review the results of pouch compliance tests over 11 years to assess whether measuring pouch compliance is a useful diagnostic tool to guide management of pouch dysfunction.

Consolidation of the Cancer Genome into Domains of Repressive Chromatin by Long-range Epigenetic Silencing (LRES) Reduces Transcriptional Plasticity

Nature Cell Biology. Mar, 2010  |  Pubmed ID: 20173741

Silencing of individual genes can occur by genetic and epigenetic processes during carcinogenesis, but the underlying mechanisms remain unclear. By creating an integrated prostate cancer epigenome map using tiling arrays, we show that contiguous regions of gene suppression commonly occur through long-range epigenetic silencing (LRES). We identified 47 LRES regions in prostate cancer, typically spanning about 2 Mb and harbouring approximately 12 genes, with a prevalence of tumour suppressor and miRNA genes. Our data reveal that LRES is associated with regional histone deacetylation combined with subdomains of different epigenetic remodelling patterns, which include re-enforcement, gain or exchange of repressive histone, and DNA methylation marks. The transcriptional and epigenetic state of genes in normal prostate epithelial and human embryonic stem cells can play a critical part in defining the mode of cancer-associated epigenetic remodelling. We propose that consolidation or effective reduction of the cancer genome commonly occurs in domains through a combination of LRES and LOH or genomic deletion, resulting in reduced transcriptional plasticity within these regions.

A Review of Weight Loss Following Roux-en-Y Gastric Bypass Vs Restrictive Bariatric Surgery: Impact on Adiponectin and Insulin

Obesity Surgery. May, 2010  |  Pubmed ID: 20177811

Bariatric surgery is a common procedure often used to ameliorate comorbidities associated with obesity, including type 2 diabetes. Substantial weight loss leads to alterations in inflammation and insulin sensitivity as well as numerous metabolic and physiologic pathways. Several inflammatory markers have been evaluated, yet adiponectin, an anti-inflammatory adipokine, has not been fully investigated. Adiponectin may play a key role as a mediator between obesity and inflammation, as lower blood levels are more commonly associated with obesity and type 2 diabetes and because adiponectin lessens insulin resistance. This review evaluates outcome variables from patients who underwent Roux-en-Y gastric bypass (RYGB) or restrictive bariatric surgery to compare and contrast any differential surgical impacts on weight loss, adiponectin, and insulin.

Eicosapentaenoic Acid Reduces Rectal Polyp Number and Size in Familial Adenomatous Polyposis

Gut. Jul, 2010  |  Pubmed ID: 20348368

The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) has anticolorectal cancer activity in vitro and in preclinical models. The present study tested whether a novel, enteric-coated formulation of EPA, as the free fatty acid (EPA-FFA), has chemopreventative efficacy in patients with familial adenomatous polyposis (FAP), in a randomised, double-blind, placebo-controlled trial.

Familial Adenomatous Polyposis (FAP) and Gender. Does Gender Influence the Genetic Transmission of FAP?

Familial Cancer. Sep, 2010  |  Pubmed ID: 20411341

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome with a penetrance close to 100% at the age of 40 years. The incidence is thought to be equal among both sexes, but we noticed an excess of males undergoing primary surgery for FAP at our institution. The aim of the study is to investigate the hypothesis that FAP patients produce an excess of affected male offspring. We identified all families with known APC mutation in the polyposis registry at St Mark's from its foundation until October 2009. We analysed their pedigrees with respect to gender of the affected individuals with progeny and to the gender and mutation status of their offspring. Only individuals with complete data regarding their offspring (gender and mutation status) were included. We identified 666 (324 males and 342 females) affected individuals with progeny. We analysed the progeny of 368 (182 males, 186 females) affected individuals with complete data on all offspring: 235 (27.5%) affected males, 212 (24.8%) affected females, 207 (24.3%) unaffected males and 200 (23.4%) unaffected females. The overall ratio of affected/unaffected and male/female offspring did not differ from the expected 50%. Further sub-analysis by gender of parents did not show any statistically significant difference in gender and mutation status of offspring. In addition the mean number of children per affected parent did not depend on gender (males 2.34; females 2.30). This study shows that gender does not influence the genetic transmission of FAP. The excess of males undergoing primary surgery at our institution is probably a result of referral bias.

Repitools: an R Package for the Analysis of Enrichment-based Epigenomic Data

Bioinformatics (Oxford, England). Jul, 2010  |  Pubmed ID: 20457667

Epigenetics, the study of heritable somatic phenotypic changes not related to DNA sequence, has emerged as a critical component of the landscape of gene regulation. The epigenetic layers, such as DNA methylation, histone modifications and nuclear architecture are now being extensively studied in many cell types and disease settings. Few software tools exist to summarize and interpret these datasets. We have created a toolbox of procedures to interrogate and visualize epigenomic data (both array- and sequencing-based) and make available a software package for the cross-platform R language.

Severe Polyposis in Apc(1322T) Mice is Associated with Submaximal Wnt Signalling and Increased Expression of the Stem Cell Marker Lgr5

Gut. Dec, 2010  |  Pubmed ID: 20926645

Adenomatous polyposis coli (APC) is a tumour suppressor gene mutated in the germline of patients with familial adenomatous polyposis (FAP) and somatically in most colorectal cancers. APC mutations impair β-catenin degradation, resulting in increased Wnt signalling. The most frequent APC mutation is a codon 1309 truncation that is associated with severe FAP. A previous study compared two mouse models of intestinal tumorigenesis, Apc(R850X) (Min) and Apc(1322T) (1322T), the latter a model of human codon 1309 changes. 1322T mice had more severe polyposis but, surprisingly, these tumours had lower levels of nuclear β-catenin than Min tumours. The consequences of these different β-catenin levels were investigated.

Evaluation of Affinity-based Genome-wide DNA Methylation Data: Effects of CpG Density, Amplification Bias, and Copy Number Variation

Genome Research. Dec, 2010  |  Pubmed ID: 21045081

DNA methylation is an essential epigenetic modification that plays a key role associated with the regulation of gene expression during differentiation, but in disease states such as cancer, the DNA methylation landscape is often deregulated. There are now numerous technologies available to interrogate the DNA methylation status of CpG sites in a targeted or genome-wide fashion, but each method, due to intrinsic biases, potentially interrogates different fractions of the genome. In this study, we compare the affinity-purification of methylated DNA between two popular genome-wide techniques, methylated DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain-based capture (MBDCap), and show that each technique operates in a different domain of the CpG density landscape. We explored the effect of whole-genome amplification and illustrate that it can reduce sensitivity for detecting DNA methylation in GC-rich regions of the genome. By using MBDCap, we compare and contrast microarray- and sequencing-based readouts and highlight the impact that copy number variation (CNV) can make in differential comparisons of methylomes. These studies reveal that the analysis of DNA methylation data and genome coverage is highly dependent on the method employed, and consideration must be made in light of the GC content, the extent of DNA amplification, and the copy number.

Osteoporosis in Patients over 50 Years of Age Following Restorative Proctocolectomy for Ulcerative Colitis: is DXA Screening Warranted?

Inflammatory Bowel Diseases. Feb, 2010  |  Pubmed ID: 19591132

Ulcerative colitis (UC) and increasing age are associated with an increased risk of osteoporosis. Screening of postmenopausal women and men older than 50 years with ulcerative colitis for osteoporosis is recommended. The prevalence of osteoporosis in restorative proctocolectomy (RPC) patients more than 50 years old is not known.

The Bacteriology of Pouchitis: a Molecular Phylogenetic Analysis Using 16S RRNA Gene Cloning and Sequencing

Annals of Surgery. Jul, 2010  |  Pubmed ID: 20562611

To identify, compare, and contrast the microbiota in patients with and without pouchitis after restorative proctocolectomy (RPC) for ulcerative colitis (UC) and familial adenomatous polyposis (FAP).

A Prospective Study of MR Enterography Versus Capsule Endoscopy for the Surveillance of Adult Patients with Peutz-Jeghers Syndrome

AJR. American Journal of Roentgenology. Jul, 2010  |  Pubmed ID: 20566803

The objective of our study was to assess the utility of MR enterography compared with capsule endoscopy for the detection of small-bowel polyps in patients with Peutz-Jeghers syndrome (PJS).

Defining Responsibilities of Simulated Patients in Medical Education

Simulation in Healthcare : Journal of the Society for Simulation in Healthcare. Jun, 2010  |  Pubmed ID: 20651478

Simulated patients (SPs) play a critical role in medical education. The development of SP methodology has resulted in wide ranging responsibilities. For SPs to work effectively, we believed it was important to clearly articulate their responsibilities, and that this would be best achieved by consultation with all stakeholders-SPs, students, tutors, and administrators.

Prognostic Significance of Myocardial Fibrosis in Hypertrophic Cardiomyopathy

Journal of the American College of Cardiology. Sep, 2010  |  Pubmed ID: 20688032

We investigated the significance of fibrosis detected by late gadolinium enhancement cardiovascular magnetic resonance for the prediction of major clinical events in hypertrophic cardiomyopathy (HCM).

Epigenetic Architecture and MiRNA: Reciprocal Regulators

Epigenomics. Dec, 2010  |  Pubmed ID: 22122085

Deregulation of epigenetic and miRNA pathways are emerging as key events in carcinogenesis. miRNA genes can be epigenetically regulated and miRNAs can themselves repress key enzymes that drive epigenetic remodeling. Epigenetic and miRNA functions are thus tightly interconnected and crucial for maintaining correct local and global genomic architecture as well as gene-expression patterns, yet the underlying molecular mechanisms and their widespread effects remain poorly understood. Owing to the tissue specificity, versatility and relative stability of miRNAs, these small ncRNAs are considered especially promising in clinical applications, and their biogenesis and function is subject of active research. In this article, the current status of epigenetic miRNA regulation is summarized and future therapeutic prospects in the field are discussed with a focus on cancer.

Protocol Matters: Which Methylome Are You Actually Studying?

Epigenomics. Aug, 2010  |  Pubmed ID: 21566704

The field of epigenetics is now capitalizing on the vast number of emerging technologies, largely based on second-generation sequencing, which interrogate DNA methylation status and histone modifications genome-wide. However, getting an exhaustive and unbiased view of a methylome at a reasonable cost is proving to be a significant challenge. In this article, we take a closer look at the impact of the DNA sequence and bias effects introduced to datasets by genome-wide DNA methylation technologies and where possible, explore the bioinformatics tools that deconvolve them. There remains much to be learned about the performance of genome-wide technologies, the data we mine from these assays and how it reflects the actual biology. While there are several methods to interrogate the DNA methylation status genome-wide, our opinion is that no single technique suitably covers the minimum criteria of high coverage and, high resolution at a reasonable cost. In fact, the fraction of the methylome that is studied currently depends entirely on the inherent biases of the protocol employed. There is promise for this to change, as the third generation of sequencing technologies is expected to again 'revolutionize' the way that we study genomes and epigenomes.

Epigenetics in Prostate Cancer: Biologic and Clinical Relevance

European Urology. Oct, 2011  |  Pubmed ID: 21719191

Prostate cancer (PCa) is one of the most common human malignancies and arises through genetic and epigenetic alterations. Epigenetic modifications include DNA methylation, histone modifications, and microRNAs (miRNA) and produce heritable changes in gene expression without altering the DNA coding sequence.

MicroRNA Alterations and Associated Aberrant DNA Methylation Patterns Across Multiple Sample Types in Oral Squamous Cell Carcinoma

PloS One. 2011  |  Pubmed ID: 22132151

MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.

Integrative Genome-wide Expression and Promoter DNA Methylation Profiling Identifies a Potential Novel Panel of Ovarian Cancer Epigenetic Biomarkers

Cancer Letters. Dec, 2011  |  Pubmed ID: 22155104

To identify epigenetic-based biomarkers for diagnosis of ovarian cancer we performed MeDIP-Chip in A2780 and CaOV3 ovarian cancer cell lines. Validation by Sequenom massARRAY methylation analysis confirmed a panel of six gene promoters (ARMCX1, ICAM4, LOC134466, PEG3, PYCARD & SGNE1) where hypermethylation discriminated 27 serous ovarian cancer clinical samples versus 12 normal ovarian surface epithelial cells (OSE) (ROC of 0.98). Notably, CpG sites across the transcription start site of a potential long-intergenic non-coding RNA (lincRNA) gene (LOC134466), was shown to be hypermethylated in 81% of serous EOC and could differentiate tumours from OSE (p<0.05). We propose that this potential biomarker panel holds great promise as a diagnostic test for high-grade (Type II) serous ovarian cancer.

Comparison of Methyl-DNA Immunoprecipitation (MeDIP) and Methyl-CpG Binding Domain (MBD) Protein Capture for Genome-wide DNA Methylation Analysis Reveal CpG Sequence Coverage Bias

Epigenetics : Official Journal of the DNA Methylation Society. Jan, 2011  |  Pubmed ID: 20818161

DNA methylation primarily occurs at CpG dinucleotides in mammals and is a common epigenetic mark that plays a critical role in the regulation of gene expression. Profiling DNA methylation patterns across the genome is vital to understand DNA methylation changes that occur during development and in disease phenotype. In this study, we compared two commonly used approaches to enrich for methylated DNA regions of the genome, namely methyl-DNA immunoprecipitation (MeDIP) that is based on enrichment with antibodies specific for 5'-methylcytosine (5MeC), and capture of methylated DNA using a methyl-CpG binding domain-based (MBD) protein to discover differentially methylated regions (DMRs) in cancer. The enriched methylated DNA fractions were interrogated on Affymetrix promoter tiling arrays and differentially methylated regions were identified. A detailed validation study of 42 regions was performed using Sequenom MassCLEAVE technique. This detailed analysis revealed that both enrichment techniques are sensitive for detecting DMRs and preferentially identified different CpG rich regions of the prostate cancer genome, with MeDIP commonly enriching for methylated regions with a low CpG density, while MBD capture favors regions of higher CpG density and identifies the greatest proportion of CpG islands. This is the first detailed validation report comparing different methylated DNA enrichment techniques for identifying regions of differential DNA methylation. Our study highlights the importance of understanding the nuances of the methods used for DNA genome-wide methylation analyses so that accurate interpretation of the biology is not overlooked.

Lineage Specific Methylation of the Elf5 Promoter in Mammary Epithelial Cells

Stem Cells (Dayton, Ohio). Oct, 2011  |  Pubmed ID: 21823211

Recent characterization of mammary stem and progenitor cells has improved our understanding of the transcriptional network that coordinates mammary development; however, little is known about the mechanisms that enforce lineage commitment and prevent transdifferentiation in the mammary gland. The E-twenty six transcription factor Elf5 forces the differentiation of mammary luminal progenitor cells to establish the milk producing alveolar lineage. Methylation of the Elf5 promoter has been proposed to act as a lineage gatekeeper during embryonic development. We used bisulphite sequencing to investigate in detail whether Elf5 promoter methylation plays a role in lineage commitment during mammary development. An increase in Elf5 expression was associated with decreasing Elf5 promoter methylation in differentiating HC11 mammary cells. Similarly, purified mammary epithelial cells from mice had increased Elf5 expression and decreased promoter methylation during pregnancy. Finally, analysis of epithelial subpopulations revealed that the Elf5 promoter is methylated and silenced in the basal, stem cell-containing population relative to luminal cells. These results demonstrate that Elf5 promoter methylation is lineage-specific and developmentally regulated in the mammary gland in vivo, and suggest that loss of Elf5 methylation specifies the mammary luminal lineage, while continued Elf5 methylation maintains the stem cell and myoepithelial lineages.

Dynamics of Bivalent Chromatin Domains Upon Drug Induced Reactivation and Resilencing in Cancer Cells

Epigenetics : Official Journal of the DNA Methylation Society. Sep, 2011  |  Pubmed ID: 21852760

Epigenetic deregulation revealed by altered profiles of DNA methylation and histone modifications is a frequent event in cancer cells and results in abnormal patterns of gene expression. Cancer silenced genes constitute prime therapeutic targets and considerable progress has been made in the epigenetic characterization of the chromatin scenarios associated with their inactivation and drug induced reactivation. Despite these advances, the mechanisms involved in the maintenance or resetting of epigenetic states in both physiological and pharmacological situations are poorly known. To get insights into the dynamics of chromatin regulation upon drug-induced reactivation, we have investigated the epigenetic profiles of two chromosomal regions undergoing long range epigenetic silencing in colon cancer cells in time-course settings after exposure of cells to chromatin reactivating agents. The DNA methylation states and the balance between histone H3K4 methylation and H3K27 methylation marks clearly define groups of genes with alternative responses to therapy. Drug reactivated cancer silenced genes exhibit dominant bivalent chromatin states that overcome the treatment and restore the transcriptional silencing approximately four weeks after drug exposure. The interplay between DNA methylation and bivalent histone marks appears to configure a plastic but stable chromatin scenario that is fully restored in silenced genes after drug withdrawal.

Nurse Practitioner Management of Chronic Musculoskeletal Pain: a Chart Review

The Nurse Practitioner. Sep, 2011  |  Pubmed ID: 21857215

This study explored NPs' management of chronic musculoskeletal pain via a record review of 50 uninsured patients. The findings include a summary of the utilization of services and treatments along with demographic data. Recommendations for education, practice, and further research are discussed.

Key Challenges in Simulated Patient Programs: an International Comparative Case Study

BMC Medical Education. 2011  |  Pubmed ID: 21943295

The literature on simulated or standardized patient (SP) methodology is expanding. However, at the level of the program, there are several gaps in the literature. We seek to fill this gap through documenting experiences from four programs in Australia, Canada, Switzerland and the United Kingdom. We focused on challenges in SP methodology, faculty, organisational structure and quality assurance.

MiRNA-dependent Gene Silencing Involving Ago2-mediated Cleavage of a Circular Antisense RNA

The EMBO Journal. Nov, 2011  |  Pubmed ID: 21964070

MicroRNAs (miRNAs) are ∼22 nt non-coding RNAs that typically bind to the 3' UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non-coding antisense transcripts in human cells. Specifically, we show that miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non-coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.

Human Gut-specific Homeostatic Dendritic Cells Are Generated from Blood Precursors by the Gut Microenvironment

Inflammatory Bowel Diseases. Oct, 2011  |  Pubmed ID: 21987473

BACKGROUND: Dendritic cells (DC) dictate not only the type of T-cell immunity, but also homing patterns of T cells in mice. In humans, we characterized normal human gut DC and tested whether gut-specific homeostatic DC could be generated from blood precursors by factors in the gut microenvironment. METHODS: We characterized the phenotype and function of healthy human gut DC compared with blood and skin DC, and studied whether conditioning of blood DC in the presence of colonic biopsy supernatants (Bx-SN) induced gut-like phenotype and functions. RESULTS: Blood DC mostly expressed both gut and skin homing markers, indicating potential to migrate to both major immune surface organs, and induced multi-homing T cells. However, DC within gut or skin did not demonstrate this multi-homing phenotype, were tissue-specific, and induced tissue-specific T cells. Human gut DC were less stimulatory for allogeneic T cells than their dermal and blood counterparts. Human blood DC cultured in vitro lost homing marker expression. Conditioning of human enriched blood DC with colonic Bx-SN from healthy controls induced a gut-homing phenotype and a homeostatic profile. Moreover, Bx-SN-conditioned DC demonstrated a restricted T-cell stimulatory capacity and preferentially induced gut-specific T cells. Retinoic acid and transforming growth factor beta (TGF-β) mediated the acquisition of the gut-homing and homeostatic properties, respectively, induced by colonic Bx-SN on blood enriched DC. CONCLUSIONS: Tissue-specific factors manipulate immunity via modulating characteristics of DC and may provide tools to generate tissue-specific immunotherapy. (Inflamm Bowel Dis 2011;).

Impact of the Genome on the Epigenome is Manifested in DNA Methylation Patterns of Imprinted Regions in Monozygotic and Dizygotic Twins

PloS One. 2011  |  Pubmed ID: 21991322

One of the best studied read-outs of epigenetic change is the differential expression of imprinted genes, controlled by differential methylation of imprinted control regions (ICRs). To address the impact of genotype on the epigenome, we performed a detailed study in 128 pairs of monozygotic (MZ) and 128 pairs of dizygotic (DZ) twins, interrogating the DNA methylation status of the ICRs of IGF2, H19, KCNQ1, GNAS and the non-imprinted gene RUNX1. While we found a similar overall pattern of methylation between MZ and DZ twins, we also observed a high degree of variability in individual CpG methylation levels, notably at the H19/IGF2 loci. A degree of methylation plasticity independent of the genome sequence was observed, with both local and regional CpG methylation changes, discordant between MZ and DZ individual pairs. However, concordant gains or losses of methylation, within individual twin pairs were more common in MZ than DZ twin pairs, indicating that de novo and/or maintenance methylation is influenced by the underlying DNA sequence. Specifically, for the first time we showed that the rs10732516 [A] polymorphism, located in a critical CTCF binding site in the H19 ICR locus, is strongly associated with increased hypermethylation of specific CpG sites in the maternal H19 allele. Together, our results highlight the impact of the genome on the epigenome and demonstrate that while DNA methylation states are tightly maintained between genetically identical and related individuals, there remains considerable epigenetic variation that may contribute to disease susceptibility.

The Development of a Social Morbidity Score in Patients with Chronic Ulcerative Colitis As a Potential Guide to Treatment

Colorectal Disease : the Official Journal of the Association of Coloproctology of Great Britain and Ireland. Nov, 2011  |  Pubmed ID: 22053990

Aim:  Present quality of life (QoL) instruments for inflammatory bowel disease (IBD) do not evaluate many social aspects of patients' lives that are potentially important in clinical decision making. We have developed a new Social Impact of Chronic Conditions - IBD questionnaire (SICC-IBD) to assess these areas. Method:  A 34-item questionnaire was piloted to determine quality of life relating to education, personal relationships, employment, independence and finance. It was compared with the SF-36v2 and Inflammatory Bowel Disease (IBDQ) questionnaires in 150 patients with chronic ulcerative colitis on an endoscopic surveillance register who had never had surgery. Results:  Reliability and validity testing enabled the questionnaire to be shortened to only 8 items. There was a high level of reliability (Cronbach's alpha = 0.72). The questionnaire correlated well with the social functioning domain of the SF-36 (r(s) =0.56) and was able to distinguish clinical severity of disease. Conclusion:  The SICC-IBD is a new tool for assessment of patients with ulcerative colitis, which has identified new aspects of social disability for further study and for potential use as an additional tool in therapy decisions.

Colorectal Cancer: No Longer the Issue in Familial Adenomatous Polyposis?

Familial Cancer. Mar, 2011  |  Pubmed ID: 21052851

Familial adenomatous polyposis (FAP) is a dominantly inherited colorectal cancer (CRC) syndrome with an untreated lifetime prevalence of CRC close to 100% and extracolonic manifestations (ECM) of increasing clinical significance. This study examined the effect of systematic callup and prophylactic colectomy on FAP survival. Patients diagnosed, treated and followed-up at our institution were analysed. 'Callups' were those identified via the callup system; 'probands' were those identified by other means. Proportions were analysed by Chi-squared or Fischer's exact test. Mortality rates were indirectly standardised to the UK population. Survival curves from birth were estimated by Kaplan-Meier. A total of 439 patients (293 callups, 146 probands) were analysed. Crude mortality rates (CMRs) of callups and probands were 4.85 per 1,000 person years (PY) and 9.71 per 1,000 PY, respectively-a rate ratio of 0.50 (95% CI 0.34-0.72, P = 0.0001). The standardised mortality ratio (SMR) of callups was non-significantly lower than probands (4.12 vs. 4.70). Callups experienced non-significantly lower age-band specific SMR up to 45 years. More probands died of CRC (42.4 vs. 22.5%, P = 0.025), whereas more callups died of ECM (30.6 vs. 13.4%, P = 0.027). Median survival was 64 years for callups and 60 years for probands; survival curves did not differ significantly (P = 0.253). The crude mortality rate of callups is approximately half that of probands. As fewer callups die of CRC, a greater proportion die of ECMs. Callups experienced non-significantly reduced mortality up to 45 years. Whilst the FAP callup system reduces CRC risk, mortality attributable to ECMs needs to be addressed.

Epigenetic Deregulation Across Chromosome 2q14.2 Differentiates Normal from Prostate Cancer and Provides a Regional Panel of Novel DNA Methylation Cancer Biomarkers

Cancer Epidemiology, Biomarkers & Prevention : a Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology. Jan, 2011  |  Pubmed ID: 21098650

Previously, we showed that gene suppression commonly occurs across chromosome 2q14.2 in colorectal cancer, through a process of long-range epigenetic silencing (LRES), involving a combination of DNA methylation and repressive histone modifications. We now investigate whether LRES also occurs in prostate cancer across this 4-Mb region and whether differential DNA methylation of 2q14.2 genes could provide a regional panel of prostate cancer biomarkers.

Mucosectomy with Handsewn Anastomosis Reduces the Risk of Adenoma Formation in the Anorectal Segment After Restorative Proctocolectomy for Familial Adenomatous Polyposis

Annals of Surgery. Feb, 2011  |  Pubmed ID: 21173697

The study compared the risk of adenoma or carcinoma formation in the anorectal segment after either mucosectomy with manual anastomosis or stapled ileoanal anastomosis (IAA) following restorative proctocolectomy (RPC) for familial adenomatous polyposis (FAP).

Discovery Pipeline for Epigenetically Deregulated MiRNAs in Cancer: Integration of Primary MiRNA Transcription

BMC Genomics. 2011  |  Pubmed ID: 21255435

Cancer is commonly associated with widespread disruption of DNA methylation, chromatin modification and miRNA expression. In this study, we established a robust discovery pipeline to identify epigenetically deregulated miRNAs in cancer.

Analysis of Veterinary Drugs and Metabolites in Milk Using Quadrupole Time-of-flight Liquid Chromatography-mass Spectrometry

Journal of Agricultural and Food Chemistry. Jul, 2011  |  Pubmed ID: 21284383

A quadrupole time-of-flight (Q-TOF) liquid chromatography-mass spectrometry (LC-MS) method was developed to analyze veterinary drug residues in milk. Milk samples were extracted with acetonitrile. A molecular weight cutoff filter was the only cleanup step in the procedure. Initially, a set of target compounds (including representative sulfonamides, tetracyclines, β-lactams, and macrolides) was used for validation. Screening of residues was accomplished by collecting TOF (MS(1)) data and comparing the accurate mass and retention times of found compounds to a database containing information for veterinary drugs. The residues included in the study could be detected in samples fortified at the levels of concern with this procedure 97% of the time. Although the method was intended to be qualitative, an evaluation of the MS data indicated a linear response and acceptable recoveries for a majority of target compounds. In addition, MS/MS data were also generated for the [M + H](+) ions. Product ions for each compound were identified, and their mass accuracy was compared to theoretical values. Finally, incurred milk samples from cows dosed with veterinary drugs, including sulfamethazine, flunixin, cephapirin, or enrofloxacin, were analyzed with Q-TOF LC-MS. In addition to monitoring for the parent residues, several metabolites were detected in these samples by TOF. Proposed identification of these residues could be made by evaluating the MS and MS/MS data. For example, several plausible metabolites of enrofloxacin, some not previously observed in milk, are reported in this study.

CD154-CD40 Interactions in the Control of Murine B Cell Hematopoiesis

Journal of Leukocyte Biology. May, 2011  |  Pubmed ID: 21330346

Interactions between CD40 and CD154 play a very important role in control of immune responses, including the delivery of T cell help to B cells and other APCs. Thus far, there has been no role postulated for CD40-CD154 interactions in hematopoiesis. We show here that CD40 is expressed on murine pro-B cells and that its ligation enhances pro-B cell proliferation in vitro and in vivo. In addition, CD154 mRNA is present in the BM. Moreover, we show that a deficiency in CD154 expression has effects on B cell hematopoiesis. Aged, CD154-deficient mice have significantly lower levels of B hematopoietic subsets downstream of pro-B cells in the BM. In addition, B lineage cells reconstitute more slowly following BMT into CD154-deficient recipients. We hypothesize that CD154 is expressed by radio-resistant cells in the BM and plays a role in fine-tuning B cell hematopoiesis.

College and University Environmental Programs As a Policy Problem (part 2): Strategies for Improvement

Environmental Management. May, 2011  |  Pubmed ID: 21359524

Environmental studies and environmental sciences programs in American and Canadian colleges and universities seek to ameliorate environmental problems through empirical enquiry and analytic judgment. In a companion article (Part 1) we describe the environmental program movement (EPM) and discuss factors that have hindered its performance. Here, we complete our analysis by proposing strategies for improvement. We recommend that environmental programs re-organize around three principles. First, adopt as an overriding goal the concept of human dignity-defined as freedom and social justice in healthy, sustainable environments. This clear higher-order goal captures the human and environmental aspirations of the EPM and would provide a more coherent direction for the efforts of diverse participants. Second, employ an explicit, genuinely interdisciplinary analytical framework that facilitates the use of multiple methods to investigate and address environmental and social problems in context. Third, develop educational programs and applied experiences that provide students with the technical knowledge, powers of observation, critical thinking skills and management acumen required for them to become effective professionals and leaders. Organizing around these three principles would build unity in the EPM while at the same time capitalizing on the strengths of the many disciplines and diverse local conditions involved.

College and University Environmental Programs As a Policy Problem (part 1): Integrating Knowledge, Education, and Action for a Better World?

Environmental Management. May, 2011  |  Pubmed ID: 21359525

The environmental sciences/studies movement, with more than 1000 programs at colleges and universities in the United States and Canada, is unified by a common interest-ameliorating environmental problems through empirical enquiry and analytic judgment. Unfortunately, environmental programs have struggled in their efforts to integrate knowledge across disciplines and educate students to become sound problem solvers and leaders. We examine the environmental program movement as a policy problem, looking at overall goals, mapping trends in relation to those goals, identifying the underlying factors contributing to trends, and projecting the future. We argue that despite its shared common interest, the environmental program movement is disparate and fragmented by goal ambiguity, positivistic disciplinary approaches, and poorly rationalized curricula, pedagogies, and educational philosophies. We discuss these challenges and the nature of the changes that are needed in order to overcome them. In a subsequent article (Part 2) we propose specific strategies for improvement.

Optimization and Validation of a Multiclass Screening and Confirmation Method for Drug Residues in Milk Using High-performance Liquid Chromatography/tandem Mass Spectrometry

Journal of AOAC International. Mar-Apr, 2011  |  Pubmed ID: 21563671

The further optimization and validation of a multiresidue veterinary drug screening method for milk is described. The drug residues of regulatory interest in milk include beta-lactams, sulfonamides, tetracyclines, fluoroquinolones, and macrolides. A previously published procedure has been modified to incorporate new compounds and to collect both screening and confirmatory ion transitions in one acquisition method. Milk samples were extracted with an equal volume of acetonitrile. The samples were then subjected to cleanup with a bonded SPE cartridge and a MW cutoff filter. The SPE protocol was modified to effectively recover a metabolite of flunixin. Established tolerance levels are set for most of these drugs in milk; thus, the screening procedure was semiquantitative, using positive controls for comparison. The positive controls, consisting of extracts from milk fortified with the drugs at their tolerance or safe level, were used to set statistically valid minimum response criteria for unknown samples. This updated method was validated with fortified milk, as well as with milk samples from animals administered veterinary drugs.

Coordinated Epigenetic Repression of the MiR-200 Family and MiR-205 in Invasive Bladder Cancer

International Journal of Cancer. Journal International Du Cancer. Mar, 2011  |  Pubmed ID: 20473948

MicroRNAs (miRNA) are small noncoding RNAs commonly deregulated in cancer. The miR-200 family (miR-200a, -200b, -200c, -141 and -429) and miR-205 are frequently silenced in advanced cancer and have been implicated in epithelial to mesenchymal transition (EMT) and tumor invasion by targeting the transcriptional repressors of E-cadherin, ZEB1 and ZEB2. ZEB1 is also known to repress miR-200c-141 transcription in a negative feedback loop, but otherwise little is known about the transcriptional regulation of the miR-200 family and miR-205. Recently, miR-200 silencing was also reported in cancer stem cells, implying that miR-200 deregulation is a key event in multiple levels of tumor biology. However, what prevents miR-200 expression remains largely unanswered. Here we report concerted transcriptional regulation of the miR-200 and miR-205 loci in bladder tumors and bladder cell lines. Using a combination of miRNA expression arrays, qPCR assays and mass spectrometry DNA methylation analyses, we show that the miR-200 and miR-205 loci are specifically silenced and gain promoter hypermethylation and repressive chromatin marks in muscle invasive bladder tumors and undifferentiated bladder cell lines. Moreover, we report that miR-200c expression is significantly correlated with early stage T1 bladder tumor progression, and propose miR-200 and miR-205 silencing and DNA hypermethylation as possible prognostic markers in bladder cancer. In addition, we observe that the mesoderm transcription factor TWIST1 and miR-200 expression are inversely correlated in bladder tumor samples and cell lines. TWIST1 associates directly with the miR-200 and miR-205 promoters, and may act as a repressor of miR-200 and miR-205 expression.

Prognostic and Diagnostic Significance of DNA Methylation Patterns in High Grade Serous Ovarian Cancer

Gynecologic Oncology. Mar, 2012  |  Pubmed ID: 22115852

Altered DNA methylation patterns hold promise as cancer biomarkers. In this study we selected a panel of genes which are commonly methylated in a variety of cancers to evaluate their potential application as biomarkers for prognosis and diagnosis in high grade serous ovarian carcinoma (HGSOC); the most common and lethal subtype of ovarian cancer.

A Basal Gradient of Wnt and Stem-cell Number Influences Regional Tumour Distribution in Human and Mouse Intestinal Tracts

Gut. Jan, 2012  |  Pubmed ID: 22287596

ObjectiveWnt signalling is critical for normal intestinal development and homeostasis. Wnt dysregulation occurs in almost all human and murine intestinal tumours and an optimal but not excessive level of Wnt activation is considered favourable for tumourigenesis. The authors assessed effects of pan-intestinal Wnt activation on tissue homeostasis, taking into account underlying physiological Wnt activity and stem-cell number in each region of the bowel.DesignThe authors generated mice that expressed temporally controlled, stabilised β-catenin along the crypt-villus axis throughout the intestines. Physiological Wnt target gene activity was assessed in different regions of normal mouse and human tissue. Human intestinal tumour mutation spectra were analysed.ResultsIn the mouse, β-catenin stabilisation resulted in a graduated neoplastic response, ranging from dysplastic transformation of the entire epithelium in the proximal small bowel to slightly enlarged crypts of non-dysplastic morphology in the colorectum. In contrast, stem and proliferating cell numbers were increased in all intestinal regions. In the normal mouse and human intestines, stem-cell and Wnt gradients were non-identical, but higher in the small bowel than large bowel in both species. There was also variation in the expression of some Wnt modulators. Human tumour analysis confirmed that different APC mutation spectra are selected in different regions of the bowel.ConclusionsThere are variable gradients in stem-cell number, physiological Wnt activity and response to pathologically increased Wnt signalling along the crypt-villus axis and throughout the length of the intestinal tract. The authors propose that this variation influences regional mutation spectra, tumour susceptibility and lesion distribution in mice and humans.

Acetylation of H2A.Z is a Key Epigenetic Modification Associated with Gene Deregulation and Epigenetic Remodeling in Cancer

Genome Research. Feb, 2012  |  Pubmed ID: 21788347

Histone H2A.Z (H2A.Z) is an evolutionarily conserved H2A variant implicated in the regulation of gene expression; however, its role in transcriptional deregulation in cancer remains poorly understood. Using genome-wide studies, we investigated the role of promoter-associated H2A.Z and acetylated H2A.Z (acH2A.Z) in gene deregulation and its relationship with DNA methylation and H3K27me3 in prostate cancer. Our results reconcile the conflicting reports of positive and negative roles for histone H2A.Z and gene expression states. We find that H2A.Z is enriched in a bimodal distribution at nucleosomes, surrounding the transcription start sites (TSSs) of both active and poised gene promoters. In addition, H2A.Z spreads across the entire promoter of inactive genes in a deacetylated state. In contrast, acH2A.Z is only localized at the TSSs of active genes. Gene deregulation in cancer is also associated with a reorganization of acH2A.Z and H2A.Z nucleosome occupancy across the promoter region and TSS of genes. Notably, in cancer cells we find that a gain of acH2A.Z at the TSS occurs with an overall decrease of H2A.Z levels, in concert with oncogene activation. Furthermore, deacetylation of H2A.Z at TSSs is increased with silencing of tumor suppressor genes. We also demonstrate that acH2A.Z anti-correlates with promoter H3K27me3 and DNA methylation. We show for the first time, that acetylation of H2A.Z is a key modification associated with gene activity in normal cells and epigenetic gene deregulation in tumorigenesis.