The Application of 3base™ Technology to Diagnose Eight of the Most Clinically Important Gastrointestinal Protozoan Infections.

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are spp.

Semi-Quantitative, Duplexed qPCR Assay for the Detection of spp. Using Bisulphite Conversion Technology.

Leishmaniasis is caused by the flagellated protozoan and is a neglected tropical disease (NTD), as defined by the World Health Organisation (WHO). Bisulphite conversion technology converts all genomic material to a simplified form during the lysis step of the nucleic acid extraction process, and increases the efficiency of multiplex quantitative polymerase chain reaction (qPCR) reactions. Through utilization of qPCR real-time probes, in conjunction with bisulphite conversion, a new duplex assay targeting the 18S rDNA gene region was designed to detect all species.

Improved detection of gastrointestinal pathogens using generalised sample processing and amplification panels.

We aimed to streamline the diagnosis of gastrointestinal disease by producing multiplexed real time polymerase chain reaction (PCR) panels employing universal sample processing for DNA and RNA containing pathogens. A total of 487 stored, previously characterised stool samples comprising bacterial, viral, protozoan and Clostridium difficile positive samples were tested using four multiplexed real time PCR panels. A further 81 pre-selected clinical samples from a teaching hospital were included to provide an independent validation of assay performance.

Aberrant de novo methylation of the p16INK4A CpG island is initiated post gene silencing in association with chromatin remodelling and mimics nucleosome positioning.

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.

Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum.

Culture of mesenchymal stem cells (MSCs) under conditions promoting proliferation and differentiation, while supporting genomic and epigenetic stability, is essential for therapeutic use. We report here the extent of genome-wide DNA gains and losses and of DNA methylation instability on 170 cancer-related promoters in bone marrow (BM) MSCs during culture to late passage in medium containing fetal bovine serum (FBS) or autologous serum (AS). Comparative genomic hybridization indicates that expansion of BMMSCs elicits primarily telomeric deletions in a subpopulation of cells, the extent of which varies between donors.

Comparison of a novel HPV test with the Hybrid Capture II (hcII) and a reference PCR method shows high specificity and positive predictive value for 13 high-risk human papillomavirus infections.

Background: It is well established that human papillomavirus (HPV) infection is highly related to the development of precursor lesions of cervical cancer and uterine cancers. However, for a pre-cancerous lesion to develop, a persistent infection with a high-risk type HPV is necessary. The Digene Hybrid Capture II (hcII) assay is the only FDA approved method used in conjunction with cytology for HPV screening of women older than 30.

Concordant epigenetic silencing of transforming growth factor-beta signaling pathway genes occurs early in breast carcinogenesis.

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.

Alterations in the p16(INK4a) and p53 tumor suppressor genes of hTERT-immortalized human fibroblasts.

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.

DNA methylation changes in leukaemia.

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.

DNA methylation and gene silencing in cancer: which is the guilty party?

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.

Methylation sequencing from limiting DNA: embryonic, fixed, and microdissected cells.

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.

Identification and resolution of artifacts in bisulfite sequencing.

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.

Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells.

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.