Epigenetic paradigm of DNA methylation for understanding the pathophysiology, diagnostics, and therapeutics in sarcomas.

Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

The nucleosome remodeling and deacetylation (NuRD) complex is required for modulating the transcription of essential pluripotency genes in ESC self-renewal. MBD3 is considered a key player in the formation of a functional NuRD complex. The recruitment of MBD3 to methylated promoters may require other prerequisite factors. We show that cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), an essential gene for early embryonic development, plays a role in pluripotency of ESC by engaging MBD3 to the promoter region of Wnt signaling genes. The occupancy of MBD3 on several promoters of Wnt genes was significantly lost in the absence of CDK2AP1, resulting in hyperactivation of Wnt. We propose that the transcriptional modulation of the Wnt pathway mediated by NuRD requires the presence of essential auxiliary components such as CDK2AP1, which may aid the association of the complex with specific focal regions of the target promoters.

Developmental Study of Fragile X Syndrome Using Human Embryonic Stem Cells Derived from Preimplantation Genetically Diagnosed Embryos

Colorectal cancer is the third most common cancer in Australia and is responsible for the death of over four thousand Australians each year. There are two overarching molecular pathways leading to colorectal cancer. The conventional pathway, which is responsible for ~75% of colorectal cancer diagnoses, occurs in a step-wise manner and is the consequence of a series of genetic alterations including mutations of tumour suppressor genes and gross chromosomal abnormalities. This pathway has been extensively studied over the past three decades.The serrated neoplasia pathway is responsible for the remaining colorectal cancers. This pathway is triggered by oncogenic BRAF mutation and these cancers accumulate epigenetic alterations while progressing to invasive cancer. DNA methylation is important in serrated neoplasia, however the extent and role of DNA methylation on the initiation and progression of serrated lesions is not clear. DNA methylation accumulates in tissues with age, and advanced serrated lesions and cancers occur almost exclusively in elderly patients. How this methylation affects serrated lesions is unknown. In this thesis I set out to address three key research questions related to DNA methylation, age and serrated colorectal neoplasia. First, what is the extent of DNA methylation in colorectal cancers?; Second, Does age-related hypermethylation, and namely that occurring at the loci encoding tumour suppressor genes, increase the risk of serrated colorectal neoplasia?; and if true, how can we reconcile this with the existence of early onset serrated colorectal cancer?In the first chapter of this thesis, I examine the DNA methylation and transcriptional architecture of 216 colorectal cancer samples collected consecutively at the Royal Brisbane and Women’s hospital. Clustering analysis of DNA methylation data revealed five distinct subtypes of colorectal cancers, including two clusters with high levels of DNA methylation, two with intermediate levels of DNA methylation, and one devoid of DNA methylation alterations. This study highlighted striking associations between DNA methylation and age, gender and tumour location. Oncogenic BRAF mutations were common in CIMP-H1, but rare in CIMP-H2. The inverse was true for CIMP-H2. These analyses were robustly verified using data from TCGA. Using exome capture sequencing data from TCGA, we assessed the frequency of mutations in genes encoding epigenetic regulators in cancers by methylation subtype. The frequency of mutation in epigenetic regulators increased concomitant with increasing genomic hypermethylation, and the spectrum of mutations differed by methylation subtype. Mutations in epigenetic regulators may provide insight into the epigenetic aberrancies observed in these highly methylated cancers.In the second chapter of this thesis, I investigate the role of age and age-associated DNA methylation and the associated transcriptional changes, in the risk of serrated neoplastic transformation using a murine model of serrated neoplasia driven by oncogenic Braf. First, the DNA methylation landscape of wild type animals was assessed over the lifespan of the animals via reduced representation bisulphite sequencing. This revealed pervasive age-associated DNA methylation, with a tendency for methylation to accumulate at the promoters of genes encoding WNT signalling regulators. Activating the Braf oncogene at wean induces a remarkable acceleration of DNA methylation accumulation, with a stronger still enrichment at WNT signalling loci. Lastly, we activated the Braf mutation in mice at wean for five months, and at nine months of age for the same period. We observe a 10.5 fold relative risk of advanced serrated lesions in animals induced at nine months compared with the younger animals, despite the same period of oncogenic exposure. These data directly implicate the aging process in determining the malignant threat of Braf induced neoplasia. Methylation analysis of these animals revealed differences in loci identified as age-associated, and those accelerated by oncogenic Braf. This study strongly implicates age and DNA methylation as a risk factors for serrated neoplastic progression and may have implications for patient surveillance.In the third chapter of this thesis, I attempt to elucidate the role of mutations in WNT signalling genes in serrated colorectal cancers, hypothesising that genetic alterations of WNT regulators will underpin early onset BRAF mutant cancers. Large exome sequencing data sets from earlier studies were collated to establish a cohort of 199 BRAF mutant colorectal cancers with somatic mutation data. This study reveals a mosaic of WNT pathway mutations, including several potential novel driver genes. APC mutations in the setting of BRAF mutant cancers were associated with poor patient outcome, and importantly, younger age at diagnosis. These data indicates that mutation of APC could provide an alternate avenue for progressing to invasive cancer, abrogating the need for excessive DNA methylation alterations.To conclude, this thesis has comprehensively evaluated the role of DNA methylation and age in the risk of serrated colorectal neoplasia. These data improve our understanding of the role of DNA methylation in colorectal carcinogenesis and may provide an impetus for investigating patient-centric surveillance regimens according to the age of patients at index colonoscopy.

Epigenetics in health and disease: heralding the EWAS era

DNA (cytosine-5) methylation represents one of the most widely used mechanisms of enduring cellular memory. Stable patterns of DNA methylation are established during development, resulting in creation of persisting cellular phenotypes. There is growing evidence that the nervous system has co-opted a number of cellular mechanisms used during development to subserve the formation of long term memory. In this study, we examined the role DNA (cytosine-5) methyltransferase (DNMT) activity might play in regulating the induction of synaptic plasticity. We found that the DNA within promoters for reelin and brain-derived neurotrophic factor, genes implicated in the induction of synaptic plasticity in the adult hippocampus, exhibited rapid and dramatic changes in cytosine methylation when DNMT activity was inhibited. Moreover, zebularine and 5-aza-2-deoxycytidine, inhibitors of DNMT activity, blocked the induction of long term potentiation at Schaffer collateral synapses. Activation of protein kinase C in the hippocampus decreased reelin promoter methylation and increased DNMT3A gene expression. Interestingly, DNMT activity is required for protein kinase C-induced increases in histone H3 acetylation. Considered together, these results suggest that DNMT activity is dynamically regulated in the adult nervous system and that DNMT may play a role in regulating the induction of synaptic plasticity in the mature CNS.

Decoding the Epigenetic Language of Neuronal Plasticity

Acute myocardial infarction (AMI) is a common cause of death for which effective treatments are available provided that diagnosis is rapid. The current diagnostic gold standards are circulating cardiac troponins I and T. However, their slow release delays diagnosis, and their persistence limits their utility in the identification of reinfarction. The aim was to identify candidate biomarkers of AMI. Isolated mouse hearts were perfused with oxygenated protein-free buffer, and coronary effluent was collected after ischemia or during matched normoxic perfusion. Effluents were analyzed using proteomics approaches based on one- or two-dimensional initial separation. Of the 459 proteins identified after ischemia with one-dimensional separation, 320 were not detected in the control coronary effluent. Among these were all classic existing biomarkers of AMI. We also identified the cardiac isoform of myosin-binding protein C in its full-length form and as a 40-kDa degradation product. This protein was not detected in the other murine organs examined, increased markedly with even trivial myocardial infarction, and could be detected in the plasma after myocardial infarction in vivo, a profile compatible with a biomarker of AMI. Two-dimensional fluorescence DIGE of ischemic and control coronary effluents identified more than 200 asymmetric spots verified by swapping dyes. Once again existing biomarkers of injury were confirmed as well as posttranslational modifications of antioxidant proteins such as peroxiredoxins. Perfusing hearts with protein-free buffers provides a platform of graded ischemic injury that allows detailed analysis of protein release and identification of candidate cardiac biomarkers like myosin-binding protein C.

Pointing Users Toward Citation Searching: Using Google Scholar and Web of Science Robert Schroeder (bio) Much has been written about citation indexing since Eugene Garfield's seminal article in 1955, "Citation Indexes for Science: A New Dimension in Documentation through Association of Ideas."1 In the 1960s, the discussion was about Garfield's Science Citation Index and later its successor, Web of Science. For almost half a century, these were the only available tools for tracing scholarly discourse forward in time. Now there are two new tools. SCOPUS is a commercial product from Elsevier and was launched in November 2004. Google Scholar also arrived on the scene in November 2004. Because Google Scholar is freely accessible from the Google site, students and faculty are finding and using it. They are beginning to ask librarians for their professional opinions of its efficacy. Practicing reference and instruction librarians need to understand the strengths of both Google Scholar and Web of Science so that they can appropriately recommend them for use by their patrons—whether they are undergraduates, graduate students, or faculty. Since April 2005, 10 studies have been published that directly compare the citation features of Google Scholar to those of Web of Science. In an effort to better understand the advantages and disadvantages of these tools, the author undertook an investigation of this research. Although the parameters of the studies varied greatly with respect to the disciplines, dates, and sample sizes, analysis of these preliminary studies can help us gain an initial—albeit sketchy—impression of the relative strength and weakness of each service. The results of this effort are reported here as an informal meta-analysis, followed by some recommendations for utilizing these tools across a range of needs. The coverage of each database is both overlapping and complementary. Web of Science is comprised of a known list of highly prestigious journals. The extent to which disciplines are covered is well known, but it is limited to these journal holdings. Although Google Scholar overlaps with Web of Science in some of its coverage, it also includes conference proceedings, books, preprints, and a variety of versions of articles available in open access databases and institutional repositories. Search results do, however, include many "false hits"—non-scholarly sources or titles that are similar to, but not [End Page 243] exact matches for, the target citation. These spurious links cause the researcher to spend much more time analyzing and evaluating the sources. Click for larger view View full resolution Table 1. Overview of the scope of the 10 studies Date coverage is also complementary, with Web of Science consistently providing older articles. Google Scholar often returns more current results due to its ability to access early versions of works in progress and open access articles available on the Internet. Although the controlled vocabulary in Web of Science is less than perfect, it wins hands-down over Google Scholar, which is totally lacking in any of the finer points of indexing. [End Page 244] Click for larger view View full resolution Table 2. Summary of positive and negative characteristics of Web of Science and Google Scholar from the 10 studies The findings from these initial studies suggest a variety of uses for both of the databases' citation features in academic reference and instruction. For instructing undergraduate students in the use of the "cited by" features, Google Scholar's lack of advanced search functions may actually be a boon. Most students today are familiar with the look and feel of the "one box searching" of Google. Having a similar uncluttered look, Google Scholar appears less intimidating to novice users, allowing them to focus on the concepts involved in citation searching and analysis. Google Scholar acts as a bridge from the known quantity of Google to more advanced instruction required for Web of Science. Discussing the strengths and weaknesses of both databases is a good way to begin a dialog about when Google Scholar might be an appropriate tool [End Page 245] for research and also allow librarians to make a stronger case for why other databases such as Web of Science need to be used as well. For many of these same reasons, Google Scholar...

DNA (De)Methylation: The Passive Route to Naïvety?

The Use Of Real-Time Quantitative Polymerase Chain Reaction To Detect Hypermethylation Of The Cpg Islands In The Promoter Region Flanking The Gstp1 Gene To Diagnose Prostate Carcinoma

Background: Breast cancer is one of the most common cancers among females worldwide, influenced by genetic, environmental, and dietary factors. Choline, an essential nutrient, plays a crucial role in cell membrane integrity and DNA methylation, which are significant for maintaining genomic stability.Objective: This review aims to explore the anti-carcinogenic effects of choline in limiting the progression of breast cancer.Methods: A narrative review was conducted using electronic databases, including PubMed, Google Scholar, and ScienceDirect, to identify relevant studies. Data were extracted from observational studies, clinical trials, and meta-analyses that focused on choline intake and breast cancer risk. Studies were appraised using established quality assessment tools.Results: Evidence suggests that adequate choline intake is linked to reduced breast cancer risk through its role in DNA methylation and gene regulation. However, findings are inconsistent due to genetic variations and methodological differences.Conclusion: While choline shows potential as a protective nutrient, further research is needed to clarify its role in breast cancer prevention, considering genetic and dietary variations

Promoter-targeted siRNAs Induce Gene Silencing of Simian Immunodeficiency Virus (SIV) Infection In Vitro

The discovery of RNA interference has revealed complex roles for small RNAs in regulating gene expression and cellular physiology. Small RNAs have been demonstrated to be involved in post-transcriptional suppression of translation, targeted degradation of messenger RNAs, and transcriptional suppression via epigenetic modifications of histones and DNA. In fission yeast, RNAi mediates suppression of centromeric transcripts, whereas in plants, transcriptional gene silencing appears to be primarily an antiviral mechanism. In mammals, the well annotated functional role of RNAi is primarily post-transcriptional, but there is increasing evidence that this mechanism can also work to suppress or modulate gene transcription, although it is not clear what primary function this serves. We overview, compare, and contrast the transcriptional silencing pathways in yeast, plants, and mammals in this article. This minireview is intended to provide the reader with a framework of how the RNAi machinery appears to be universally involved in various aspects of transcriptional regulation with discussions of similarities and differences in the components and mechanisms of achieving transcriptional silencing.

Patient-reported outcomes (PROs) as a routine measure for cancer inpatients: the final missing piece of the puzzle?