Epigenetic subgroups of esophageal and gastric adenocarcinoma with differential GATA5 DNA methylation associated with clinical and lifestyle factors

Background

Adenocarcinomas located near the gastroesophageal junction have unclear etiology and are difficult to classify. We used DNA methylation analysis to identify subtype-specific markers and new subgroups of gastroesophageal adenocarcinomas, and studied their association with epidemiological risk factors and clinical outcomes.

Methodology/Principal Findings

We used logistic regression models and unsupervised hierarchical cluster analysis of 74 DNA methylation markers on 45 tumor samples (44 patients) of esophageal and gastric adenocarcinomas obtained from a population-based case-control study to uncover epigenetic markers and cluster groups of gastroesophageal adenocarcinomas. No distinct epigenetic differences were evident between subtypes of gastric and esophageal cancers. However, we identified two gastroesophageal adenocarcinoma subclusters based on DNA methylation profiles. Group membership was best predicted by GATA5 DNA methylation status. We analyzed the associations between these two epigenetic groups and exposure using logistic regression, and the associations with survival time using Cox regression in a larger set of 317 tumor samples (278 patients). There were more males with esophageal and gastric cardia cancers in Cluster Group 1 characterized by higher GATA5 DNA methylation values (all p<0.05). This group also showed associations of borderline statistical significance with having ever smoked (p-value = 0.07), high body mass index (p-value = 0.06), and symptoms of gastroesophageal reflux (p-value = 0.07). Subjects in cluster Group 1 showed better survival than those in Group 2 after adjusting for tumor differentiation grade, but this was not found to be independent of tumor stage.

Conclusions/Significance

DNA methylation profiling can be used in population-based studies to identify epigenetic subclasses of gastroesophageal adenocarcinomas and class-specific DNA methylation markers that can be linked to epidemiological data and clinical outcome. Two new epigenetic subgroups of gastroesophageal adenocarcinomas were identified that differ to some extent in their survival rates, risk factors of exposure, and GATA5 DNA methylation.     

Targeted overexpression of G protein-coupled receptor kinase-2 in osteoblasts promotes bone loss

To investigate the role of G protein-coupled receptor kinases (GRKs) in regulating bone formation in vivo, we overexpressed the potent G protein-coupled receptor (GPCR) regulator GRK2 in osteoblasts, using the osteocalcin gene-2 promoter to target expression to osteoblastic cells. Using the parathyroid hormone (PTH) receptor as a model system, we found that overexpression of GRK2 in osteoblasts attenuated PTH-induced cAMP generation by mouse calvaria ex vivo. This decrease in GPCR responsiveness was associated with a reduction in bone mineral density (BMD) in transgenic (TG) mice compared with non-TG littermate controls. The decrease in BMD was most prominent in trabecular-rich lumbar spine and was not observed in cortical bone of the femoral shaft. Quantitative computed tomography indicated that the loss of trabecular bone was due to a decrease in trabecular thickness, with little change in trabecular number. Histomorphometric analyses confirmed the decrease in trabecular bone volume and demonstrated reduced bone remodeling, as evidenced by a decrease in osteoblast numbers and osteoblast-mediated bone formation. Osteoclastic activity also appeared to be reduced because urinary excretion of the osteoclastic activity marker deoxypyridinoline was decreased in TG mice compared with control animals. Consistent with reduced coupling of osteoblast-mediated bone formation to osteoclastic bone resorption, mRNA levels of both osteoprotegrin and receptor activator of NF-kappaB ligand were altered in calvaria of TG mice in a pattern that would promote a low rate of bone remodeling. Taken together, these data suggest that enhancing GRK2 activity and consequently reducing GPCR activity in osteoblasts produces a low bone-turnover state that reduces bone mass.     

2-Cyano-4-fluoro-1-thiovalylpyrrolidine analogues as potent inhibitors of DPP-IV

We report the synthesis and biological activity of a series of 2-cyano-4-fluoro-1-thiovalylpyrrolidine inhibitors of DPP-IV. Within this series, compound 19 provided a potent, selective, and orally active DPP-IV inhibitor which demonstrated a very long duration of action in both rat and dog.     

A confocal microscopic study of solitary pulmonary neuroendocrine cells in human airway epithelium

Background

Pulmonary neuroendocrine cells (PNEC) are specialized epithelial cells that are thought to play important roles in lung development and airway function. PNEC occur either singly or in clusters called neuroepithelial bodies. Our aim was to characterize the three dimensional morphology of PNEC, their distribution, and their relationship to the epithelial nerves in whole mounts of adult human bronchi using confocal microscopy.

Methods

Bronchi were resected from non-diseased portions of a lobe of human lung obtained from 8 thoracotomy patients (Table ​(Table1)1) undergoing surgery for the removal of lung tumors. Whole mounts were stained with antibodies to reveal all nerves (PGP 9.5), sensory nerves (calcitonin gene related peptide, CGRP), and PNEC (PGP 9.5, CGRP and gastrin releasing peptide, GRP). The analysis and rendition of the resulting three-dimensional data sets, including side-projections, was performed using NIH-Image software. Images were colorized and super-imposed using Adobe Photoshop.

Table 1

Patient Demographic Data

Exploring the GluR2 ligand-binding core in complex with the bicyclical AMPA analogue (S)-4-AHCP

The X-ray structure of the ionotropic GluR2 ligand-binding core (GluR2-S1S2J) in complex with the bicyclical AMPA analogue (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]-4-isoxazolyl)propionic acid [(S)-4-AHCP] has been determined, as well as the binding pharmacology of this construct and of the full-length GluR2 receptor. (S)-4-AHCP binds with a glutamate-like binding mode and the ligand adopts two different conformations. The K(i) of (S)-4-AHCP at GluR2-S1S2J was determined to be 185 +/- 29 nM and at full-length GluR2(R)o it was 175 +/- 8 nM. (S)-4-AHCP appears to elicit partial agonism at GluR2 by inducing an intermediate degree of domain closure (17 degrees). Also, functionally (S)-4-AHCP has an efficacy of 0.38 at GluR2(Q)i, relative to (S)-glutamate. The proximity of bound (S)-4-AHCP to domain D2 prevents full D1-D2 domain closure, which is limited by steric repulsion, especially between Leu704 and the ligand.     

The genetic architecture of response to long-term artificial selection for oil concentration in the maize kernel

In one of the longest-running experiments in biology, researchers at the University of Illinois have selected for altered composition of the maize kernel since 1896. Here we use an association study to infer the genetic basis of dramatic changes that occurred in response to selection for changes in oil concentration. The study population was produced by a cross between the high- and low-selection lines at generation 70, followed by 10 generations of random mating and the derivation of 500 lines by selfing. These lines were genotyped for 488 genetic markers and the oil concentration was evaluated in replicated field trials. Three methods of analysis were tested in simulations for ability to detect quantitative trait loci (QTL). The most effective method was model selection in multiple regression. This method detected approximately 50 QTL accounting for approximately 50% of the genetic variance, suggesting that >50 QTL are involved. The QTL effect estimates are small and largely additive. About 20% of the QTL have negative effects (i.e., not predicted by the parental difference), which is consistent with hitchhiking and small population size during selection. The large number of QTL detected accounts for the smooth and sustained response to selection throughout the twentieth century.     

Enhanced toxicity and cellular binding of a modified amyloid beta peptide with a methionine to valine substitution

The amyloid beta peptide (Abeta) is toxic to neuronal cells, and it is probable that this toxicity is responsible for the progressive cognitive decline associated with Alzheimer's disease. However, the nature of the toxic Abeta species and its precise mechanism of action remain to be determined. It has been reported that the methionine residue at position 35 has a pivotal role to play in the toxicity of Abeta. We examined the effect of mutating the methionine to valine in Abeta42 (AbetaM35V). The neurotoxic activity of AbetaM35V on primary mouse neuronal cortical cells was enhanced, and this diminished cell viability occurred at an accelerated rate compared with Abeta42. AbetaM35V binds Cu2+ and produces similar amounts of H2O2 as Abeta42 in vitro, and the neurotoxic activity was attenuated by the H2O2 scavenger catalase. The increased toxicity of AbetaM35V was associated with increased binding of this mutated peptide to cortical cells. The M35V mutation altered the interaction between Abeta and copper in a lipid environment as shown by EPR analysis, which indicated that the valine substitution made the peptide less rigid in the bilayer region with a resulting higher affinity for the bilayer. Circular dichroism spectroscopy showed that both Abeta42 and AbetaM35V displayed a mixture of alpha-helical and beta-sheet conformations. These findings provide further evidence that the toxicity of Abeta is regulated by binding to neuronal cells.     

Response to water deficit and high temperature of transgenic peas (Pisum sativum L.) containing a seed-specific alpha-amylase inhibitor and the subsequent effects on pea weevil (Bruchus pisorum L.) survival

The effects of water deficit and high temperature on the production of alpha-amylase inhibitor 1 (alpha-AI-1) were studied in transgenic peas (Pisum sativum L.) that were developed to control the seed-feeding pea weevil (Bruchus pisorum L., Coleoptera: Bruchidae). Transgenic and non-transgenic plants were subjected to water-deficit and high-temperature treatments under controlled conditions in the glasshouse and growth cabinet, beginning 1 week after the first pods were formed. In the water-deficit treatments, the peas were either adequately watered (control) or water was withheld after first pod formation. The high-temperature experiments were performed in two growth cabinets, one maintained at 27/22 degrees C (control) and one at 32/27 degrees C day/night temperatures, with the vapour pressure deficit maintained at 1.3 kPa. The plants exposure to high temperatures and water deficit produced 27% and 79% fewer seeds, respectively, than the controls. In the transgenic peas the level of alpha-AI-1 as a percentage of total protein was not influenced by water stress, but was reduced on average by 36.3% (the range in two experiments was 11-50%) in the high-temperature treatment. Transgenic and non-transgenic pods of plants grown at 27/22 degrees C and 32/27 degrees C were inoculated with pea weevil eggs to evaluate whether the reduction in level of alpha-AI-1 in the transgenic pea seeds affected pea weevil development and survival. At the higher temperatures, 39% of adult pea weevil emerged, compared to 1.2% in the transgenic peas grown at the lower temperatures, indicating that high temperature reduced the protective capacity of the transgenic peas.     

黑斑羚粪便中碳同位素揭示的食性变化

利用稳定碳同位素数据(δ13C)分析了南非克鲁格国家公园混食性黑斑羚(Aepyceros melampus)时间和空间尺度上的食性变化,验证了两个假说,即有蹄类食性变化是由生境中木本植物与草本植物的相对配比导致;降雨控制有蹄类生态。结果表明:黑斑羚的食性涵盖了精食者-粗食者采食谱系,且食性中木本与草本比例在不同月间、季节、年度和区域间存在很大变化。栖息于开放性热带稀树草原和草原中的黑斑羚通常采食比生境中更高比例的草本,但在时间尺度上并不恒定。在克鲁格北部的一个区域(Punda Maria) ,黑斑羚采食的草本比克鲁格国家公园中其它任何区域都多。与其它生境相比,在河边的黑斑羚采食草本数量更少,尤其是在食性空间变化更为明显的旱季。因此,我们的数据不支持有蹄类食性组成变化是由生境中木本与草本比例不同造成的假说,食性与降雨量间也无明显的关系。我们的结果支持草本中蛋白含量增加引起黑斑羚采食比例的增加这一模型。粪便中氮含量在时间和空间上的变化很小,揭示在可利用食物中,无论木本还是草本,黑斑羚进行选择采食以保证最好的食物质量。基于这些结果,我们认为更具体的食物选择和可利用性最适采食理论能够更好地解释这种生态学变化。