Site-specific RNA modification via initiation of in vitro transcription reactions with m6A and isomorphic emissive adenosine analogs.

The templated enzymatic incorporation of adenosine and its analogs, including m6A, thA and tzA into RNA transcripts, has been explored. Enforced transcription initiation with excess free nucleosides and the native triphosphates generates 5'-end modified transcripts, which can be 5'-phosphorylated and ligated to provide full length, singly modified RNA oligomers. To explore structural integrity, functionality and utility of the resulting non-canonical purine-containing RNA constructs, a MazF RNA hairpin substrate has been synthesized and analyzed for its susceptibility to this endonuclease. Additionally, RNA substrates, containing a singly incorporated isomorphic emissive nucleoside, can be used to monitor the enzymatic reactions in real-time by steady state fluorescence spectroscopy.

The Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans.

BACKGROUND: Neurovascular coupling (NVC) is a key process in cerebral blood flow regulation. NVC ensures adequate brain perfusion to changes in local metabolic demands. Neuronal nitric oxide synthase (nNOS) is suspected to be involved in NVC; however, this has not been tested in humans. Our objective was to investigate the effects of nNOS inhibition on NVC in humans. METHODS: We performed a 3-visit partially randomized, double-blinded, placebo-controlled, crossover study in 12 healthy subjects. On each visit, subjects received an intravenous infusion of either S-methyl-L-thiocitrulline (a selective nNOS-inhibitor), 0.9% saline (placebo control), or phenylephrine (pressor control). The NVC assessment involved eliciting posterior circulation hyperemia through visual stimulation while measuring posterior and middle cerebral arteries blood velocity. RESULTS: nNOS inhibition blunted the rapidity of the NVC response versus pressor control, evidenced by a reduced initial rise in mean posterior cerebral artery velocity (-3.3% [-6.5, -0.01], P=0.049), and a reduced rate of increase (ie, acceleration) in posterior cerebral artery velocity (slope reduced -4.3% [-8.5, -0.1], P=0.045). The overall magnitude of posterior cerebral artery response relative to placebo control or pressor control was not affected. Changes in BP parameters were well-matched between the S-methyl-L-thiocitrulline and pressor control arms. CONCLUSIONS: Neuronal NOS plays a role in dynamic cerebral blood flow control in healthy adults, particularly the rapidity of the NVC response to visual stimulation. This work opens the way to further investigation of the role of nNOS in conditions of impaired NVC, potentially revealing a therapeutic target.

Visual task complexity and eye movement patterns influence measures of human neurovascular coupling.

Human neurovascular coupling research conventionally aims to selectively activate the posterior circulation using a visual task. Different research groups use divergent visual tasks ranging in complexity and eye movement patterns with potential confounding effects. To understand the role of task complexity and eye movement patterns in neurovascular coupling, we performed a series of experiments with visual tasks ranging in complexity, eye speed, and eye movement amplitude. Greater task complexity significantly reduced selectivity for the posterior circulation. Greater task amplitude (i.e. larger eye movements) increased selectivity. These findings are important when interpreting and designing neurovascular coupling investigations.