The effect of pseudoknot base pairing on cotranscriptional structural switching of the fluoride riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of Escherichia coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37°C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65°C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

The Effect of Pseudoknot Base Pairing on Cotranscriptional Structural Switching of the Fluoride Riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of E. coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37 °C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65 °C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

Sleep and respiratory abnormalities in adults with developmental and epileptic encephalopathies using polysomnography and video-EEG monitoring.

This study evaluated sleep and respiratory abnormalities, and their relationship with seizures, in adults with developmental and epileptic encephalopathies (DEEs). We studied consecutive adults with DEEs undergoing inpatient video-EEG monitoring and concurrent polysomnography between December 2011 and July 2022. Thirteen patients with DEEs were included (median age: 31 years, range: 20-50; 69.2% female): Lennox-Gastaut syndrome (n = 6), Lennox-Gastaut syndrome-like phenotype (n = 2), Landau-Kleffner syndrome (n = 1), epilepsy with myoclonic-atonic seizures (n = 1), and unclassified DEEs (n = 3). Sleep architecture was often fragmented by epileptiform discharges and seizures resulting in arousals (median arousal index: 29.0 per h, range: 5.1-65.3). Moderate-to-severe obstructive sleep apnea (OSA) was observed in seven patients (53.8%). Three patients (23.1%) had tonic seizures that frequently occurred with central apnea; one met criteria for mild central sleep apnea. Of the patients with tonic seizures, two had other identifiable seizure manifestations, but in one patient, central apnea was commonly the only discernable seizure manifestation. Polysomnography during video-EEG is an effective diagnostic tool in detecting sleep and seizure-related respiratory abnormalities. Clinically significant OSA may increase the risk of comorbid cardiovascular disease and premature mortality. Treatment of epilepsy may improve sleep quality, and conversely, improved sleep, may decrease seizure burden.

Efficacy of botulinum toxin type a in the targeted treatment of sleep bruxism: a double-blind, randomised, placebo-controlled, cross-over study.

Background: Intramuscular injections of botulinum toxin A (BTX-A) have been used in the treatment of sleep bruxism (SB) however controlled trials are limited and the optimal injection strategy and dose is not known. Methods: This double-blind, randomised, placebo-controlled, cross-over study evaluated the efficacy and safety of BTX-A in participants with SB. Average bruxism events per hour of sleep (Bruxism Index, BI) was calculated using surface electromyography. Participants with BI >5 were included and randomised by order of injection (active or placebo with the opposite 20 weeks later) and into one of three differing treatment groups: bilateral masseter (60 units(U)), bilateral masseter and temporalis (90U) and bilateral masseter, temporalis and medial pterygoid muscles (120U). Change in BI and subjective measures of headache, pain, and bruxism at 4 and 12 weeks was calculated following intervention, and differences between treatment groups analysed. Results: 41 participants were recruited, 35 randomised and data from 22 participants (14 female) were analysed. BI was significantly lower at 4 weeks after active treatment when compared with placebo (mean=-1.66, p=0.003), not sustained at 12 weeks. The difference was greater with higher doses injected and among those with greater baseline BI. There was no difference in subjective measures at any time point. Five participants injected had mild, transient side effects. Discussion: Targeted BTX-A injection is a safe and effective treatment for SB. A greater benefit may be achieved by administering BTX-A into more muscles and at higher total doses and among those with higher baseline BI. Trial registration number: ACTRN12618001430224.

Cotranscriptional RNA strand exchange underlies the gene regulation mechanism in a purine-sensing transcriptional riboswitch.

RNA folds cotranscriptionally to traverse out-of-equilibrium intermediate structures that are important for RNA function in the context of gene regulation. To investigate this process, here we study the structure and function of the Bacillus subtilis yxjA purine riboswitch, a transcriptional riboswitch that downregulates a nucleoside transporter in response to binding guanine. Although the aptamer and expression platform domain sequences of the yxjA riboswitch do not completely overlap, we hypothesized that a strand exchange process triggers its structural switching in response to ligand binding. In vivo fluorescence assays, structural chemical probing data and experimentally informed secondary structure modeling suggest the presence of a nascent intermediate central helix. The formation of this central helix in the absence of ligand appears to compete with both the aptamer's P1 helix and the expression platform's transcriptional terminator. All-atom molecular dynamics simulations support the hypothesis that ligand binding stabilizes the aptamer P1 helix against central helix strand invasion, thus allowing the terminator to form. These results present a potential model mechanism to explain how ligand binding can induce downstream conformational changes by influencing local strand displacement processes of intermediate folds that could be at play in multiple riboswitch classes.

Riboswitches have challenged our understanding of biological regulation for almost two decades. The ability of small molecules to bind to RNA and control gene expression offers another layer of regulation and the potential for direct action by compounds in the environment. While some riboswitches have been well studied, we lack a general understanding of how changes in RNA structure switch genetic expression from "On" to "Off". In this study, the authors propose an elegant "strand displacement" model to explain how the RNA structure shifts between "On" and "Off" states as the concentration of small molecule ligand changes. These observations help us to understand how riboswitches enable genetic decision-making. The data provide a possible general mechanism for understanding how the competition between different strand displacement outcomes can influence RNA folding. Understanding RNA folding pathways could advance the successful design of drugs that target RNA.

Sleep-disordered breathing among patients admitted for inpatient video-EEG monitoring.

OBJECTIVE: To examine the prevalence and risk factors of sleep-disordered breathing (SDB) in individuals with epilepsy and psychogenic nonepileptic seizures (PNES). METHODS: We conducted a cross-sectional study of consecutive patients admitted for inpatient video-EEG monitoring at The Royal Melbourne Hospital, Australia, between December 1, 2011, and July 31, 2017. Participants underwent routine clinical investigations during their monitoring period including polysomnography, neurocognitive testing, and screening instruments of daytime somnolence, sleep quality, and quality of life. RESULTS: Our study population consisted of 370 participants who received a diagnosis of epilepsy (n = 255), PNES (n = 93), or both disorders (n = 22). Moderate to severe SDB (defined by an apnea-hypopnea index ≥15) was observed in 26.5% (98/370) of individuals, and did not differ across subgroups: epilepsy 26.3% (67/255), PNES 29.0% (27/93), or both disorders 18.2% (4/22; p = 0.610). Following adjustment for confounders, pathologic daytime sleepiness predicted moderate to severe SDB in epilepsy (odds ratio [OR] 10.35, 95% confidence interval [CI] 2.09-51.39; p = 0.004). In multivariable analysis, independent predictors for moderate to severe SDB in epilepsy were older age (OR 1.07, 95% CI 1.04-1.10; p < 0.001) and higher body mass index (OR 1.06, 95% CI 1.01-1.11; p = 0.029), and in PNES older age (OR 1.10, 95% CI 1.03-1.16; p = 0.002). CONCLUSION: Polysomnography during inpatient video-EEG monitoring identified a substantial number of patients with undiagnosed SDB. This was remarkable in the subgroup with PNES, who were often female and obese. Identification of risk factors may improve management of SDB in these populations. The association with pathologic daytime sleepiness suggests that SDB may be an important contributor to these common and disabling symptoms in patients with epilepsy.

Routine polysomnography in an epilepsy monitoring unit.

Up to 13% of patients with epilepsy have moderate or severe sleep-disordered breathing, in particular obstructive sleep apnea (OSA), a disorder associated with reduced quality of life, worsened seizure control, and increased cardiovascular morbidity and mortality. Combining video-EEG monitoring with polysomnography (VPSG) provides the opportunity to diagnose clinically significant OSA as well as relate the occurrence of seizures and the epilepsy diagnosis to the presence and severity of sleep-disordered breathing. We have established routine VPSG in our inpatient video-EEG monitoring unit and present our findings in 87 patients. Clinically significant sleep-disordered breathing was diagnosed in 19 of 87 (22%) patients. Patients with psychogenic non-epileptic seizures (PNES) had poorer sleep quality compared to patients with epilepsy and those with neither diagnosis, whereas the prevalence of clinically significant sleep-disordered breathing in patients with PNES (29%) did not differ significantly compared to patients with epilepsy (21%) and those with neither diagnosis (22%). The differences in sleep quality are not explained by differences in body mass index (BMI) or anti-epileptic drug (AED) effects.