An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure.

The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis

Piecing Together the Puzzle: Nanopore Technology in Detection and Quantification of Cancer Biomarkers

Cancer is the result of a multistep process, including various genetic and epigenetic alterations, such as structural variants, transcriptional factors, telomere length, DNA methylation, histone–DNA modification, and aberrant expression of miRNAs. These changes cause gene defects in one of two ways: (1) gain in function which shows enhanced expression or activation of oncogenes, or (2) loss of function which shows repression or inactivation of tumor-suppressor genes. However, most conventional methods for screening and diagnosing cancers require highly trained experts, intensive labor, large counter space (footprint) and extensive capital costs. Consequently, current approaches for cancer detection are still considered highly novel and are not yet practically applicable for clinical usage. Nanopore-based technology has grown rapidly in recent years, which have seen the wide application of biosensing research to a number of life sciences. In this review paper, we present a comprehensive outline of various genetic and epigenetic causal factors of cancer at the molecular level, as well as the use of nanopore technology in the detection and study of those specific factors. With the ability to detect both genetic and epigenetic alterations, nanopore technology would offer a cost-efficient, labor-free and highly practical approach to diagnosing pre-cancerous stages and early-staged tumors in both clinical and laboratory settings

Moving beyond DNA sequence to improve plant stress responses

Plants offer a habitat for a range of interactions to occur among different stress factors. Epigenetics has become the most promising functional genomics tool, with huge potential for improving plant adaptation to biotic and abiotic stresses. Advances in plant molecular biology have dramatically changed our understanding of the molecular mechanisms that control these interactions, and plant epigenetics has attracted great interest in this context. Accumulating literature substantiates the crucial role of epigenetics in the diversity of plant responses that can be harnessed to accelerate the progress of crop improvement. However, harnessing epigenetics to its full potential will require a thorough understanding of the epigenetic modifications and assessing the functional relevance of these variants. The modern technologies of profiling and engineering plants at genome-wide scale provide new horizons to elucidate how epigenetic modifications occur in plants in response to stress conditions. This review summarizes recent progress on understanding the epigenetic regulation of plant stress responses, methods to detect genome-wide epigenetic modifications, and disentangling their contributions to plant phenotypes from other sources of variations. Key epigenetic mechanisms underlying stress memory are highlighted. Linking plant response with the patterns of epigenetic variations would help devise breeding strategies for improving crop performance under stressed scenarios

A Protective Role Of Autophagy In A Drosophila Model Of Friedreich\u27s Ataxia (frda)

Friedreich’s ataxia (FRDA) is an inherited autosomal recessive neurodegenerative disease. It affects 1 in every 50,000 people in central Europe and North America. FRDA is caused by deficiency of Frataxin, an essential mitochondrial iron chaperone protein, and the associated oxidative stress damages. Autophagy, a housekeeping process responsible for the bulk degradation and turnover of long half-life proteins and organelles, is featured by the formation of double-membrane vacuoles and lysosomal degradation. Previous researches indicate that Danon’s disease, the inherited neural disorder disease that shares similar symptoms with FRDA, is due to the malfunction of autophagy. Based on this, we raise the question whether the autophagy activity is modified and what is its role in FRDA. Study has shown that oxidative stress may play a major role in the progression of neurodegenerative diseases by attacking the cytoplasmic molecules and organelles, and autophagy is the major pathway in reducing oxidative stress and removal of malfunctioned organelles. Additionally, autophagy has been closely related to cell apoptosis and organism remodeling. Mitochondrial Autophagy (mitophagy) is also the major turnover pathway for damaged mitochondria. Therefore, the dysfunctional autophagy in removing the malfunctioned mitochondria in FRDA may responsible for its pathogenesis. Since the mechanism of autophagy in the development of FRDA is still largely unknown, a systematic analysis of the status and function of autophagy pathway is needed. My thesis is targeting at four goals: (1) to construct FRDA fly model and characterize autophagy expression pattern in each stage; (2) to determine the effect of autophagy modification on the symptoms of the FRDA flies; (3) to identify the potential downstream events of autophagy; (4) to explore the possible upstream activities by examine whether the AMPK or SAPK stress response pathway is involved in FRDA flies. Our hypothesis is the up-regulation of autophagy occurs in the early stage of FRDA and may induce apoptosis or mitophagy. We identified that autophagy level is up-regulated in FRDA flies at both transcriptional and translational levels. Moreover, the overexpression of Frataxin also increases autophagy activity. The comparable Atg5 mRNA level in both Frataxin deficiency and overexpression flies indicates this induction of autophagy in FRDA flies is Atg5 independent. Autophagy inducer Methylene blue and rapamycin could partially prolong the longevity and restore the fertility of FRDA flies, but could not rescue the pupae lethal phenotype. When treated with the autophagy inhibitor chloroquine, FRDA KD flies showed reduced longevity and locomotor activity, implying the beneficial effect of autophagy in certain development stages. The FRDA KD flies also showed up-regulated caspases-3 and cytochrome C level, indicating enhanced apoptosis in cells with reduced Frataxin. We also attempt to apply the heart pacing assay to evaluate the FRDA Drosophila cardiac function. Although the results are inconclusive, the heart pacing assay appears to be a valuable tool for future research

Toward precision medicine with nanopore technology

Currently, when patients are diagnosed with cancer, they often receive a treatment based on the type and stage of the tumor. However, different patients may respond to the same treatment differently, due to the variation in their genomic alteration profile. Thus, it is essential to understand the effect of genomic alterations on cancer drug efficiency and engineer devices to monitor these changes for therapeutic response prediction. Nanopore-based detection technology features devices containing a nanometer-scale pore embedded in a thin membrane that can be utilized for DNA sequencing, biosensing, and detection of biological or chemical modifications on single molecules. Overall, this project aims to evaluate the capability of the biological nanopore, alpha-hemolysin, as a biosensor for genetic and epigenetic biomarkers of cancer. Specifically, we utilized the nanopore to (1) study the effect of point mutations on C-kit1 G-quadruplex formation and its response to CX-5461 cancer drug; (2) evaluate the nanopore\u27s ability to detect cytosine methylation in label-dependent and label-independent manners; and (3) detect circulating-tumor DNA collected from lung cancer patients\u27 plasma for disease detection and treatment response monitoring. Compared to conventional techniques, nanopore assays offer increased flexibility and much shorter processing time

Epigenetic Mechanisms in Developmental Alcohol-Induced Neurobehavioral Deficits

Alcohol consumption during pregnancy and its damaging consequences on the developing infant brain are significant public health, social, and economic issues. The major distinctive features of prenatal alcohol exposure in humans are cognitive and behavioral dysfunction due to damage to the central nervous system (CNS), which results in a continuum of disarray that is collectively called fetal alcohol spectrum disorder (FASD). Many rodent models have been developed to understand the mechanisms of and to reproduce the human FASD phenotypes. These animal FASD studies have provided several molecular pathways that are likely responsible for the neurobehavioral abnormalities that are associated with prenatal alcohol exposure of the developing CNS. Recently, many laboratories have identified several immediate, as well as long-lasting, epigenetic modifications of DNA methylation, DNA-associated histone proteins and microRNA (miRNA) biogenesis by using a variety of epigenetic approaches in rodent FASD models. Because DNA methylation patterns, DNA-associated histone protein modifications and miRNA-regulated gene expression are crucial for synaptic plasticity and learning and memory, they can therefore offer an answer to many of the neurobehavioral abnormalities that are found in FASD. In this review, we briefly discuss the current literature of DNA methylation, DNA-associated histone proteins modification and miRNA and review recent developments concerning epigenetic changes in FASD

Non-Alcoholic Fatty Liver Disease : disease burden and development of novel fibrosis diagnostic and prognostic signatures

PhD ThesisBackground This thesis investigates novel diagnostic and prognostic disease biomarkers in NAFLD and explores both the quality of life (QoL) and economic burden associated with NAFLD. Methods To estimate HRQL burden, 147 patients completed validated QoL assessments within 6 months of diagnostic liver biopsy. NAFLD out-patient service utilisation was evaluated and micro-costed over a 12-month period. The clinical utility of serum collagen neo-epitope biomarkers to identify advanced fibrosis was established. DNA methylation was evaluated in circulating cell free DNA as a diagnostic biomarker in NAFLD using pyrosequencing and evaluated by whole genome bisulfide sequencing (WGBS) from paired liver biopsy tissue to characterise NAFLD prognostic signatures. Results HRQL Burden: Grade of lobular inflammation influenced CLDQ scores and FIS scores. One way ANCOVA analyses showed that CLDQ scores were influenced by fibrosis stage (F=1.910, p=0.014, effect size 0.814) Economic Burden: Multivariate regression analysis established the main cost drivers to be the number of clinic appointments (p=0.042) and the presence of advanced disease (p=0.001). Collagen Neo-epitope biomarkers the novel “FIBC3” diagnostic panel including PROC3 exhibited improved accuracy and outperformed other fibrosis indices for the detection of advanced fibrosis DNA methylation fibrosis biomarkers PPARγ CpG methylation displayed uniform hypermethylation at each CpG site between the liver fibrosis cohorts relative to uniform hypomethylation irrespective of liver disease aetiology DNA methylation prognostic signature; > 657 novel methylation signatures to distinguish low and high risk disease were identified. Conclusion Multiple factors negatively impact on reported HRQL, notably fatigue and lobular inflammation. The direct medical costs associated with NAFLD are substantial and increase with the presence of advanced disease. The ‘FIBC3’ panel is an accurate tool with a single threshold value that maintains both sensitivity and specificity for the identification of advanced fibrosis (F≥3). The first methylome map of low versus high risk disease in NAFLD suggest that high and low risk NAFLD while interrelated, may be biologically distinct from disease onset. Extending this towards clinical utility, uniform hypermethylation at the PPARγ gene promoter confirms this as a potential methylation signature for fibrosis progression in chronic liver disease.EPoS (Elucidating Pathways of Steatohepatitis) consortium, funded by the Horizon 2020 Framework Program of the European Union, Rosetrees Trust and Abbvie

A Hypomethylated population of Brassica rapa for forward and reverse Epi-genetics

Extent: 17p.Background: Epigenetic marks superimposed on the DNA sequence of eukaryote chromosomes provide agility and plasticity in terms of modulating gene expression, ontology, and response to the environment. Modulating the methylation status of cytosine can generate epialleles, which have been detected and characterised at specific loci in several plant systems, and have the potential to generate novel and relatively stable phenotypes. There have been no systematic attempts to explore and utilise epiallelic variation, and so extend the range of phenotypes available for selection in crop improvement. We developed an approach for generating novel epialleles by perturbation of the DNA methylation status. 5- Azacytidine (5-AzaC) provides selective targeting of 5mCG, which in plants is associated with exonic DNA. Targeted chemical intervention using 5-AzaC has advantages over transgenic or mutant modulation of methyltransferases, allowing stochastic generation of epialleles across the genome. Results: We demonstrate the potential of stochastic chemically-induced hypomethylation to generate novel and valuable variation for crop improvement. Systematic analysis of dose–response to 5-AzaC in B. rapa guided generation of a selfed stochastically hypomethylated population, used for forward screening of several agronomic traits. Dose–response was sigmoidal for several traits, similar to that observed for chemical mutagens such as EMS. We demonstrated transgenerational inheritance of some phenotypes. BraRoAZ is a unique hypomethylated population of 1000 E2 sib lines. When compared to untreated controls, 5-Aza C-treated lines exhibited reduced immuno-staining of 5mC on pachytene chromosomes, and Methylation Sensitive Amplified Polymorphism (MSAP) profiles that were both divergent and more variable. There was coincident phenotypic variation among these lines for a range of seed yield and composition traits, including increased seed protein content and decreased oil content, as well as decreased erucic acid and corresponding increases in linoleic and/or palmitic acid. Each 5-AzaC-treated line represents a unique combination of hypomethylated epialleles. Conclusions: The approach and populations developed are available for forward and reverse screening of epiallelic variation and subsequent functional and inheritance studies. The generation of stochastically hypomethylated populations has utility in epiallele discovery for a wide range of crop plants, and has considerable potential as an intervention strategy for crop improvement.Stephen Amoah, Smita Kurup, Carlos Marcelino Rodriguez Lopez, Sue J Welham, Stephen J Powers, Clare J Hopkins, Michael J Wilkinson and Graham J Kin