Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2''-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.     

Role of histidine for charge regulation of unstructured peptides at interfaces and in bulk

Histidine‐rich, unstructured peptides adsorb to charged interfaces such as mineral surfaces and microbial cell membranes. At a molecular level, we investigate the adsorption mechanism as a function of pH, salt, and multivalent ions showing that (1) proton charge fluctuations are—in contrast to the majority of proteins—optimal at neutral pH, promoting electrostatic interactions with anionic surfaces through charge regulation and (2) specific zinc(II)‐histidine binding competes with protons and ensures an unusually constant charge distribution over a broad pH interval. In turn, this further enhances surface adsorption. Our analysis is based on atomistic molecular dynamics simulations, coarse grained Metropolis Monte Carlo, and classical polymer density functional theory. This multiscale modeling provides a consistent picture in good agreement with experimental data on Histatin 5, an antimicrobial salivary peptide. Biological function is discussed and we suggest that charge regulation is a significant driving force for the remarkably robust activity of histidine‐rich antimicrobial peptides. Proteins 2014; 82:657–667. © 2013 Wiley Periodicals, Inc.     

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1^-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1^-/- tp53^-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1^-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.     

Characterization of Early Disease Status in Treatment-Naive Male Paediatric Patients with Fabry Disease Enrolled in a Randomized Clinical Trial

Trial Design

This analysis characterizes the degree of early organ involvement in a cohort of oligo-symptomatic untreated young patients with Fabry disease enrolled in an ongoing randomized, open-label, parallel-group, phase 3B clinical trial.

Methods

Males aged 5–18 years with complete α-galactosidase A deficiency, without symptoms of major organ damage, were enrolled in a phase 3B trial evaluating two doses of agalsidase beta. Baseline disease characteristics of 31 eligible patients (median age 12 years) were studied, including cellular globotriaosylceramide (GL-3) accumulation in skin (n = 31) and kidney biopsy (n = 6; median age 15 years; range 13–17 years), renal function, and glycolipid levels (plasma, urine).

Results

Plasma and urinary GL-3 levels were abnormal in 25 of 30 and 31 of 31 patients, respectively. Plasma lyso-GL-3 was elevated in all patients. GL-3 accumulation was documented in superficial skin capillary endothelial cells (23/31 patients) and deep vessel endothelial cells (23/29 patients). The mean glomerular filtration rate (GFR), measured by plasma disappearance of iohexol, was 118.1 mL/min/1.73 m² (range 90.4–161.0 mL/min/1.73 m²) and the median urinary albumin/creatinine ratio was 10 mg/g (range 4.0–27.0 mg/g). On electron microscopy, renal biopsy revealed GL-3 accumulation in all glomerular cell types (podocytes and parietal, endothelial, and mesangial cells), as well as in peritubular capillary and non-capillary endothelial, interstitial, vascular smooth muscle, and distal tubules/collecting duct cells. Lesions indicative of early Fabry arteriopathy and segmental effacement of podocyte foot processes were found in all 6 patients.

Conclusions

These data reveal that in this small cohort of children with Fabry disease, histological evidence of GL-3 accumulation, and cellular and vascular injury are present in renal tissues at very early stages of the disease, and are noted before onset of microalbuminuria and development of clinically significant renal events (e.g. reduced GFR). These data give additional support to the consideration of early initiation of enzyme replacement therapy, potentially improving long-term outcome.

Trial Registration

ClinicalTrials.gov NCT00701415     

A comparative analysis of the complete mitochondrial genome of the Eurasian otter <Emphasis Type="Italic">Lutra lutra</Emphasis> (Carnivora; Mustelidae)

Otter populations are declining throughout the world and most otter species are considered endangered. Molecular methods are suitable tools for population genetic research on endangered species. In the present study, we analyzed the complete mitochondrial genome (mitogenome) sequence of the Eurasian otter Lutra lutra. The mitochondrial DNA sequence of the Eurasian otter is 16,505 bp in length and consists of 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and a control region (CR). The CR sequence of otters from Europe and Asia showed nearly identical numbers and nucleotide sequences of minisatellites. Phylogenetic analysis of Mustelidae mitogenomes, including individual genes, revealed that Lutrinae and Mustelinae form a clade, and that L. lutra and Enhydra lutris are sister taxa within the Lutrinae. Phylogenetic analyses revealed that of the 13 mitochondrial protein-coding genes, ND5 is the most reliable marker for analysis of phylogenetic relationships within the Mustelidae.     

Pharmacophore-based discovery of triaryl-substituted imidazole as new telomeric G-quadruplex ligand

Discovery of potent and selective ligands for telomeric G-quadruplex DNA is a challenging work. Through a combination approach of pharmacophore model construction, model validation, database virtual screening, chemical synthesis and interaction evaluation, we discovered and confirmed triaryl-substituted imidazole TSIZ01 to be a new telomeric G-quadruplex ligand with potent binding and stabilizing activity to G-quadruplex DNA, as well as a 8.7-fold selectivity towards telomeric G-quadruplex DNA over duplex DNA.     

甘肃兴隆山自然保护区马麝夏季栖息地特征及生境选择格局

2006—2008年夏季(7—8月),对甘肃兴隆山自然保护区马麝(Moschus Sifanicus)的栖息地特征和夏季生境选择格局进行了研究。用Mann-Whitney U检验和χ2检验比较了马麝夏季利用生境样地(n=71)和对照性非利用样地(n=264)间的海拔等17个生态变量的差异,结果表明,兴隆山马麝夏季利用生境的郁闭度[(53.87±3.09)%]和乔木均高[(7.57±0.83)m]均显著大于非利用样地[郁闭度:(49.07±1.66)%,乔木均高:(6.33±0.32)m],喜食植物多度[(12.97±1.80)]株也显著大于非利用样地[(9.61±0.67)株]。此外,与非利用样地相比,兴隆山马麝夏季倾向于选择位于中坡位较陡(67.61%)、距离水源较近(<1000m,77.46%)、隐蔽度(88.73%)和避风性均较好(90%)及距人为干扰较远(>1000m,76.06%)的生境;主成分分析结果表明,前4个因子的累积贡献率达72.45%,由乔木胸径和乔木郁闭度变量组成的乔木因子是决定马麝夏季生境选择的首要因素,此外,海拔因子,由地表植被盖度和食物多度组成的食物因子,由灌木盖度、乔木密度和灌木...     

我国设施黄瓜新育成品种芽期耐盐性评价

用100 mmol/L Mg(NO3)2盐溶液对不同生态类型的143个设施黄瓜新品种进行4 d芽期胁迫处理,调查各种质的发芽势、发芽指数、相对伤害率、发芽率、相对发芽率、第2天和第4天胚根伸长长度、后2 d胚根伸长速度等8个指标,利用各指标隶属函数值对其耐盐性进行聚类分析,并对耐盐强的种质进行恢复出苗初步筛选。研究结果表明,黄瓜萌发期的耐盐性强弱评价结果受多个指标的影响。8个发芽指标的变异系数为10.5%~81.0%,除了发芽率和相对发芽率的变异较小外,其他性状的变异幅度均较大(32.0%~81.0%);各性状指标变异系数在不同生态类型种质中表现不同,华北型和欧洲温室型种质以相对伤害率变异系数为最大,华南型和欧美加工型种质以后2 d胚根伸长速度变异系数最大。从种质总体抗盐结果看,欧美加工型和欧洲温室型>华南型>华北型。各指标隶属函数值聚类分析结果表明,将所有种质划为耐盐强、中、弱3类,其中耐盐强的品种共有54个,占37.8%。54个耐盐品种胁迫后经出苗试验初步筛选出12个恢复能力强的品种,即:J31-1>绿精灵>DRT5>YT24>东农02号>上海农科4号>冬灵102>IVF8>津优35>HGS-2>扬大13>J30-1。