In this report, we describe a novel method for the rapid quantification of CpG methylation on the basis of direct bisulfite-PCR sequencing method. According to the principles of bisulfite-PCR, converting unmethylated cytosines to thymine while leaving methylated cytosines unchanged, we regard the CpG site as a SNP and estimate the methylation status of cytosines in the given CG dinucleotides by measuring the ratio

of the cytosine peak height to the sum of cytosine and thymine peak heights in automated DNA sequencing traces. Furthermore, we take several effective measures to break through the ‘bottleneck’ problems that render the routine bisulfite sequencing method unsuitable for quantitative methylation. In comparison with pyrosequencing and bisulfite-cloning sequencing, our method is confirmed to be a simple, high-throughput and cost-effective technology for determining the see more methylation buy SHP099 status of specific genes. Accordingly, this novel method is anticipated to be an efficient and economical alternative tool for rapid quantification of methylation patterns in screening large numbers of clinical samples across multiple genes. Laboratory Investigation (2010) 90, 282-290; doi:10.1038/labinvest.2009.132; published online 14 December 2009″
“The pre-Botzinger complex (pre-BotC), a functionally defined subregion in the ventrolateral medulla oblongata, is a presumed kernel of normal respiratory

rhythmogenesis. However, less is known about the pre-BotC’s contribution to respiratory neuroplasticity. The most frequently studied model for respiratory neuroplasticity is episodic hypoxia-induced phrenic long-term facilitation, which is 5-HT2A receptors (5-HT2AR)-dependent. We hypothesized that preconditioning with chronic intermittent hypoxic (CIH) would activate the 5-HT/5-HT2AR system and the downstream protein kinase C (PKC) pathway in the pre-BotC. Animals were

exposed to alternating 5 min Buspirone HCl of hypobaric hypoxia and 5 min of normoxia for 10 h/day for 7 days. Hypobaric hypoxia was achieved by continuous air evacuation to reach a pressure of 210-220 mm Hg, corresponding to an altitude of 9000-10000 m. In contrast to the CIH model, a group of animals were pretreated with chronic sustaining hypoxia (CSH), a protocol of continuous hypobaric hypoxia at 360 mm Hg, corresponding to an altitude of about 6000 m, for 10 h/day for 7 days. Immunoreactivity of 5-HT and 5-HT2AR was examined in the pre-BotC, identified by the presence of neurokinin-1 receptor (NK1R). We found that 15.5% of 5-HT-immunoreactive (ir) terminals were in contact with NK1R-ir neurons. Asymmetric synapses could be identified between them. 38.7% of NK1R-ir dendrites were also immunoreactive for 5-HT2AR, which was distributed along the inner surface of the plasma membrane in control animals. CIH challenge increased the expressions of 5-HT and 5-HT2AR in the pre-BotC, an increase in the expressed 5-HT2AR that was not detected in this region in CSH animals.