Rett syndrome caused by mutations in methyl-CpG-binding protein 2 (Mecp2) gene shows abnormalities in autonomic functions in which brain stem norepinephrinergic systems play an important role. Here we present systematic comparisons of intrinsic membrane properties of locus coeruleus (LC) neurons between Mecp2(-/Y) and wild-type (WT) mice. Whole cell current clamp was performed in brain slices of 3- to 4-wk-old mice. Mecp2(-/Y) neurons showed stronger inward rectification and had shorter time constant than WT cells. The former was likely due to overexpression of inward rectifier K(+) (K(ir))4.1 channel, and the latter was attributable to the smaller cell surface area. The action potential duration was prolonged in Mecp2(-/Y) cells with an extended rise time. This was associated with a significant reduction in the voltage-activated Na(+) current density. After action potentials, >60% Mecp2(-/Y) neurons displayed fast and medium afterhyperpolarizations (fAHP and mAHP), while nearly 90% WT neurons showed only mAHP. The mAHP amplitude was smaller in Mecp2(-/Y) neurons. The firing frequency was higher in neurons with mAHP, and the frequency variation was greater in cells with both fAHP and mAHP in Mecp2(-/Y) mice. Small but significant differences in spike frequency adaptation and delayed excitation were found in Mecp2(-/Y) neurons. These results indicate that there are several electrophysiological abnormalities in LC neurons of Mecp2(-/Y) mice, which may contribute to the dysfunction of the norepinephrine system in Rett syndrome.

Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAFmutant and NRASmutant DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAFmutant and NRASmutant ctDNA were determined using droplet digital PCR (ddPCR) assays. Among patients with samples available prior to treatment initiation ctDNA and LDH levels were elevated in 12/15 (80%) and 6/20 (30%) (p=0.006) patients respectively. In patients with RECIST scores <5cm prior to treatment initiation, ctDNA levels were elevated in 5/7 (71%) patients compared to LDH which was elevated in 1/13 (8%) patients (p=0.007). Among all disease progression events the modified bootstrapped sensitivities for ctDNA and LDH were 82% and 40% respectively, with a median difference in sensitivity of 42% (95% confidence interval, 27% to 58%; P<0.001). In addition, ctDNA levels were elevated in 13/16 (81%) instances of non-RECIST disease progression, including 10/12 (83%) instances of new brain metastases. In comparison LDH was elevated 8/16 (50%) instances of non-RECIST disease progression, including 6/12 (50%) instances of new brain metastases. Overall, ctDNA had a higher sensitivity than LDH to detect disease progression, including non-RECIST progression events. ctDNA has the potential to be a useful biomarker for monitoring melanoma disease activity.

gic neurons in the locus coeruleus (LC) play a role in the ventilatory response to hypercapnia. Here, we show evidence for the involvement of transient receptor potential (TRP) channels. We found that the input resistance was reduced during an exposure to 8% CO 2 in ϳ35% LC neurons in mouse brain slices, accompanied by depolarization and higher firing activity. The neuronal responses suggest the opening of Na ϩ or nonselective cationic channels instead of the closure of K ϩ channels. As a major group of cationic channels, the TRP channels are expressed in the brain, some of which are activated by acidic pH. We therefore screened all representative TRP channels using the quantitative real-time PCR analysis. High levels of mRNA expression of TRPC5, TRPM2, and TRPM7 were found in the LC tissue. Of them, the TRPC5 transcript was the most abundant. The TRPC5 channel was activated by extracellular acidification when expressed in human embryonic kidney (HEK) cells. The TRPC5 currents started to be activated at pH 7.4 with pKa 6.9. The TRPC5 currents were also activated by isohydric hypercapnic and intracellular acidosis in a Ca 2ϩ -dependent manner. Consistently, the LC neurons were stimulated by both extra-and intracellular acidosis. The stimulatory effect of hypercapnia on LC neurons was eliminated by selective TRPC inhibitor SKF-96365 with and without the blockade of synaptic transmission. Single-cell PCR analysis indicated that TRPC5 mRNAs existed in the LC neurons. Thus these results strongly suggest that the TRP channels are likely to play a role in the CO 2 chemosensitivity of LC neurons, especially TRPC5. breathing; norepinephrine; brain stem; transient receptor potential channel; TRPC5 BREATHING ACTIVITY IS AUTOMATICALLY regulated by central and peripheral chemoreceptors providing feedback to the brain stem respiratory neuronal networks with the information of PCO 2 and PO 2 levels in the blood stream.

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes. We analyzed paired primary and metastatic tumors from 60 patients and multiple metastatic tumors from 39 patients whose primary tumors were unavailable (n ¼ 271 tumors). We used a combination of multiplex SNaPshot assays, Sanger sequencing, mutation-specific PCR, or droplet digital PCR to determine the presence of BRAF V600 , NRAS Q61 , TERT e124C>T , and TERT e146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERT mutant discordant tumors; seven of these had a single tumor with both TERT e124C>T and TERT e146C>T mutations present at different allele frequencies. Two patients had both BRAF and NRAS mutations; one had different tumors and the other had a single tumor with both mutations. One patient with a BRAF mutant primary lacked mutant BRAF in at least one of their metastases. Overall, we identified mutational heterogeneity in 18 of 99 patients (18%). These results suggest that some primary melanomas may be composed of subclones with differing mutational profiles. Such heterogeneity may be relevant to treatment responses and survival outcomes.