Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR

We report a novel method for rapid quantification of the degree of DNA methylation of a specific gene. Our method combined bisulfite-mediated PCR and quantification of deoxyribonucleoside monophosphate (dNMP) contents in the PCR product through capillary electrophoresis. A specific bisulfite-PCR product was enzymatically hydrolyzed to dNMP monomers which were quantitatively analyzed through subsequent capillary electrophoresis. PCR following bisulfite treatment converts unmethylated cytosines to thymines while leaving methyl-cytosines unchanged. Then the ratio of cytosine to thymine determined by capillary electrophoresis represents the ratio of methyl-cytosine to cytosine in genomic locus of interest. Pure oligonucleotides with known sequences were processed in parallel as standards for normalization of dNMP peaks in capillary electrophoresis. Sources of quantification uncertainty such as carryovers of dNTPs or primers and incomplete hydrolysis were examined and ruled out. When the method was applied to samples with known methylation levels (by bisulfite-mediated sequencing) as a validation, deviations were within ±5%. After bisulfite-PCR, the analytical procedure can be completed within 1.5 h

The anti-aging gene KLOTHO is a novel target for epigenetic silencing in human cervical carcinoma

<p>Abstract</p> <p>Background</p> <p><it>Klotho </it>was originally characterized as an anti-aging gene that predisposed Klotho-deficient mice to a premature aging-like syndrome. Recently, KLOTHO was reported to function as a secreted Wnt antagonist and as a tumor suppressor. Epigenetic gene silencing of secreted Wnt antagonists is considered a common event in a wide range of human malignancies. Abnormal activation of the canonical Wnt pathway due to epigenetic deregulation of Wnt antagonists is thought to play a crucial role in cervical tumorigenesis. In this study, we examined epigenetic silencing of <it>KLOTHO </it>in human cervical carcinoma.</p> <p>Results</p> <p>Loss of <it>KLOTHO </it>mRNA was observed in several cervical cancer cell lines and in invasive carcinoma samples, but not during the early, preinvasive phase of primary cervical tumorigenesis. <it>KLOTHO </it>mRNA was restored after treatment with either the DNA demethylating agent 2'-deoxy-5-azacytidine or histone deacetylase inhibitor trichostatin A. Methylation-specific PCR and bisulfite genomic sequencing analysis of the promoter region of <it>KLOTHO </it>revealed CpG hypermethylation in non-<it>KLOTHO</it>-expressing cervical cancer cell lines and in 41% (9/22) of invasive carcinoma cases. Histone deacetylation was also found to be the major epigenetic silencing mechanism for <it>KLOTHO </it>in the SiHa cell line. Ectopic expression of the secreted form of KLOTHO restored anti-Wnt signaling and anti-clonogenic activity in the CaSki cell line including decreased active β-catenin levels, suppression of T-cell factor/β-catenin target genes, such as <it>c-MYC </it>and <it>CCND1</it>, and inhibition of colony growth.</p> <p>Conclusions</p> <p>Epigenetic silencing of <it>KLOTHO </it>may occur during the late phase of cervical tumorigenesis, and consequent functional loss of KLOTHO as the secreted Wnt antagonist may contribute to aberrant activation of the canonical Wnt pathway in cervical carcinoma.</p

Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, −2.1%, and −13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA

Gradient-Based Time-Extended Potential Field Method for Real-Time Path Planning in Infrastructure-Based Cooperative Driving Systems

This study proposes a real-time path generation method called the Gradient-based Time-extended Potential Field (GT-PF) for cooperative autonomous driving environments. The proposed approach models the road environment and dynamic obstacles as a time-variant potential field and generates safe and feasible paths by tracing the negative gradient of the field, which corresponds to the direction of steepest descent. In contrast to conventional sampling-based or optimization-based methods, the proposed PF framework enables lightweight computation and continuous trajectory generation in spatiotemporal domains. Furthermore, a velocity-oriented bias is introduced in the PF formulation to ensure that the generated paths satisfy the vehicle&rsquo;s kinematic constraints and desired cruising behavior. The effectiveness of the proposed method is verified through comparative simulations against a sampling-based Rapidly exploring Random Tree (RRT) planner. Results demonstrate that the GT-PF approach exhibits superior performance in terms of runtime efficiency and safety. The system is particularly suitable for RSU (Roadside Unit)-based infrastructure control in real-time traffic environments. Future work includes the extension to complex urban scenarios, integration with multi-agent planning frameworks, and deployment in sensor-fused cooperative perception systems

Modeling Framework to Analyze Effect of Multiple Traffic Information Service Providers on Traffic Network Performance

Along with the increasing popularity of mobile smart devices such as smartphones and tablet personal computers, traffic information is coming to drivers through many intelligent traffic applications. Many countries have multiple traffic information service providers (ISPs), who make every effort to improve their service quality to influence more people to subscribe to their services. Because such a commercial environment is developing, a study of the effect of multiple ISPs on road network performance is necessary. Therefore, a modeling framework of day-to-day dynamics in which multiple ISPs compete and cooperate with each other to enhance their subscriber service quality is developed in this study. A realistic information acquisition and learning mechanism that ensures consistent updating of individually perceived day-by-day travel times is incorporated for driver behavior in this framework. A bounded rational behavior model was adopted for route choice decisions. The framework is capable of investigating the effects of any potential competition or cooperation of multiple ISPs in the traffic information market in terms of their information-sharing strategies. Numerical experiments on a real network were conducted to analyze the impact of such interactions on the network performance. The results showed that a cooperative system was not necessarily the best for network performance and that there was an optimal level of market penetration of traffic information services in transportation networks beyond which the benefits no longer increased or even worsened. </jats:p