A Strategy for Studying Epigenetic Diversity in Natural Populations: Proof of Concept in Poplar and Oak.

These last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulphite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions (DMRs) as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, considering the structural complexity of the genomes with their size and their content in unique as coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a representative subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by Sequencing Capture Bisulphite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.

On quantifying the climate of the nonautonomous Lorenz-63 model.

The Lorenz-63 model has been frequently used to inform our understanding of the Earth's climate and provide insight for numerical weather and climate prediction. Most studies have focused on the autonomous (time invariant) model behaviour in which the model's parameters are constants. Here, we investigate the properties of the model under time-varying parameters, providing a closer parallel to the challenges of climate prediction, in which climate forcing varies with time. Initial condition (IC) ensembles are used to construct frequency distributions of model variables, and we interpret these distributions as the time-dependent climate of the model. Results are presented that demonstrate the impact of ICs on the transient behaviour of the model climate. The location in state space from which an IC ensemble is initiated is shown to significantly impact the time it takes for ensembles to converge. The implication for climate prediction is that the climate may-in parallel with weather forecasting-have states from which its future behaviour is more, or less, predictable in distribution. Evidence of resonant behaviour and path dependence is found in model distributions under time varying parameters, demonstrating that prediction in nonautonomous nonlinear systems can be sensitive to the details of time-dependent forcing/parameter variations. Single model realisations are shown to be unable to reliably represent the model's climate; a result which has implications for how real-world climatic timeseries from observation are interpreted. The results have significant implications for the design and interpretation of Global Climate Model experiments.

Protein-coding and microRNA biomarkers of recurrence of prostate cancer following radical prostatectomy.

An important challenge in prostate cancer research is to develop effective predictors of tumor recurrence following surgery to determine whether immediate adjuvant therapy is warranted. To identify biomarkers predictive of biochemical recurrence, we isolated the RNA from 70 formalin-fixed, paraffin-embedded radical prostatectomy specimens with known long-term outcomes to perform DASL expression profiling with a custom panel that we designed of 522 prostate cancer-relevant genes. We identified a panel of 10 protein-coding genes and two miRNA genes (RAD23B, FBP1, TNFRSF1A, CCNG2, NOTCH3, ETV1, BID, SIM2, LETMD1, ANXA1, miR-519d, and miR-647) that could be used to separate patients with and without biochemical recurrence (P < 0.001), as well as for the subset of 42 Gleason score 7 patients (P < 0.001). We performed an independent validation analysis on 40 samples and found that the biomarker panel was also significant at prediction of biochemical recurrence for all cases (P = 0.013) and for a subset of 19 Gleason score 7 cases (P = 0.010), both of which were adjusted for relevant clinical information including T-stage, prostate-specific antigen, and Gleason score. Importantly, these biomarkers could significantly predict clinical recurrence for Gleason score 7 patients. These biomarkers may increase the accuracy of prognostication following radical prostatectomy using formalin-fixed specimens.

RERE deficiency leads to decreased expression of GATA4 and the development of ventricular septal defects.

Deletions of chromosome 1p36 are associated with a high incidence of congenital heart defects (CHDs). The arginine-glutamic acid dipeptide repeats gene (RERE) is located in a critical region for CHD on chromosome 1p36 and encodes a cardiac-expressed nuclear receptor co-regulator. Mutations affecting RERE cause atrial and ventricular septal defects (VSDs) in humans, and RERE-deficient mice also develop VSDs. During cardiac development, mesenchymal cells destined to form part of the atrioventricular (AV) septum are generated when endocardial cells in the AV canal undergo epithelial-to-mesenchymal transition (EMT) and migrate into the space between the endocardium and the myocardium. These newly generated mesenchymal cells then proliferate to fill the developing AV endocardial cushions. Here, we demonstrate that RERE-deficient mouse embryos have reduced numbers of mesenchymal cells in their AV endocardial cushions owing to decreased levels of EMT and mesenchymal cell proliferation. In the endocardium, RERE colocalizes with GATA4, a transcription factor required for normal levels of EMT and mesenchymal cell proliferation. Using a combination of in vivo and in vitro studies, we show that Rere and Gata4 interact genetically in the development of CHDs, RERE positively regulates transcription from the Gata4 promoter and GATA4 levels are reduced in the AV canals of RERE-deficient embryos. Tissue-specific ablation of Rere in the endocardium leads to hypocellularity of the AV endocardial cushions, defective EMT and VSDs, but does not result in decreased GATA4 expression. We conclude that RERE functions in the AV canal to positively regulate the expression of GATA4, and that deficiency of RERE leads to the development of VSDs through its effects on EMT and mesenchymal cell proliferation. However, the cell-autonomous role of RERE in promoting EMT in the endocardium must be mediated by its effects on the expression of proteins other than GATA4.This article has an associated First Person interview with the first author of the paper.

Nottingham-defined mitotic score: comparison with visual and image cytometric phosphohistone H3 labeling indices and correlation with Oncotype DX recurrence score.

Prognosis of breast cancer patients has been determined traditionally by lymph node status, tumor size, and histologic grade. In recent years the Oncotype DX recurrence score (RS) assay has emerged as an expensive adjunct prognostic tool. Markers of proliferation play a large role in determination of RS, and we have shown previously that immunohistochemical expression of proliferation markers Ki-67 and phosphohistone H3 (PPH3) correlates with RS. Our current goal is comparison of the hematoxylin and eosin (H&E) mitotic score, defined by the Nottingham grading system, with anti-PPH3 mitotic figure labeling assessed by both visual and automated image analysis and correlation of mitotic score results with RS. Estrogen receptor-positive breast carcinomas from 137 patients with Oncotype DX testing were selected. A representative H&E-stained tumor section was evaluated. Mitoses were counted per 10 high-power fields and tumors graded using the Nottingham criteria by 1 pathologist in accordance with College of American Pathologists-recommended mitotic count cutoffs for a field diameter of 0.55 mm. An additional section was immunostained with PPH3 antibody. PPH3 mitotic scores were determined visually and by automated imaging system. Statistical analysis was performed using univariate tests and Spearman coefficient. There was a statistically significant positive correlation among the 3 methods of mitotic score assessment. Specifically, correlation of tumor grades obtained using visual and automated methods of assessment of mitotic activity with PPH3 stain was the strongest and most statistically significant (weighted κ value 0.84, P<0.001; Spearman coefficient 0.89, P<0.001). There was a statistically significant positive correlation between H&E mitosis score and RS (P<0.001, Spearman coefficient 0.30) and between visual PPH3 mitotic score and RS (P<0.001, Spearman coefficient 0.28). In conclusion, mitotic score by any of the 3 methods studied may be useful in assessing tumor grade, proliferation, and prognosis.

Proliferation (Ki-67 and phosphohistone H3) and oncotype DX recurrence score in estrogen receptor-positive breast cancer.

The Oncotype DX Recurrence Score (RS) is often used in lymph node-negative, estrogen receptor-positive breast cancer to refine prognosis and direct therapy. Its utility is limited by its cost, proprietary nature, and turnaround time. Markers of proliferation factor heavily into determination of RS. Our aim is to correlate expression of proliferation markers Ki-67 and phosphohistone H3 (PPH3) with RS and other prognostic indicators. Estrogen receptor-positive invasive breast carcinomas from 133 patients with Oncotype DX testing were selected. Representative tumor sections were stained with MIB1, a monoclonal antibody that reacts against Ki-67, and antibody to PPH3. Nuclear staining was quantitated through an automated imaging system. The percentage of positive cells was scored as low (<10%), intermediate (10% to 20%), or high (>20%) for Ki-67, and low (<2%), intermediate (2% to 5%), or high (>5%) for PPH3. Expression of both markers was compared with RS and clinicopathologic parameters including grade, tumor size, lymph node metastasis, and angiolymphatic invasion. Ki-67 and PPH3 expression were both significantly associated with RS (P=0.02 and P=0.027, respectively) and grade (P<0.001 and P=0.002, respectively). Ki-67 expression correlated with angiolymphatic invasion (P=0.01) but not with tumor size or lymph node metastasis; PPH3 expression showed no association with any of these 3 parameters. Expression of proliferation markers Ki-67 and PPH3 by immunohistochemistry is significantly correlated with RS and tumor grade. This observation suggests that immunohistochemical assessment of markers of proliferation may provide useful prognostic information, at lower cost than RS testing.

Triple-negative breast carcinoma in women from Vietnam and the United States: characterization of differential marker expression by tissue microarray.

Triple-negative breast carcinoma accounts for approximately 15% of all breast cancers. It is characterized by an aggressive clinical history, high rate of local relapse, and association with the basal epithelial-like subtype. Variations in breast cancer subtype and clinical outcome often exist across racial and ethnic lines. Therefore, the aim of this study was to compare the immunohistochemical and clinicopathologic characteristics of triple-negative breast carcinoma in women living in Vietnam with those from the United States. Invasive triple-negative breast carcinoma of patients from the 2 populations was characterized by tissue microarray for the expression of basal cytokeratins (CK5/6, CK7, CK14), luminal cytokeratins (CK8, CK18, CK19), and markers associated with the basal phenotype (cKit, epithelial growth factor receptor, P-cadherin, p53, and p63). Significant differences in expression between the 2 populations were not observed for the basal cytokeratins. However, epithelial growth factor receptor and P-cadherin, markers associated with the basal phenotype, were underexpressed in Vietnamese patients. Of the luminal cytokeratins, CK8 was overexpressed and CK18 was underexpressed in the Vietnamese women. Significant differences were also observed regarding the clinicopathologic characteristics. Triple-negative breast carcinoma in Vietnamese women was smaller and less likely to be grade III. In addition, it was more frequently of ductal histologic type and less often medullary or metaplastic. These differences in histology and marker expression suggest that triple-negative breast carcinoma has unique biological characteristics in women from Vietnam and the United States, and may follow a unique clinical course in each of the 2 populations.