Activation of the Hypoglossal to Tongue Musculature Motor Pathway by Remote Control.

Reduced tongue muscle tone precipitates obstructive sleep apnea (OSA), and activation of the tongue musculature can lessen OSA. The hypoglossal motor nucleus (HMN) innervates the tongue muscles but there is no pharmacological agent currently able to selectively manipulate a channel (e.g., Kir2.4) that is highly restricted in its expression to cranial motor pools such as the HMN. To model the effect of manipulating such a restricted target, we introduced a "designer" receptor into the HMN and selectively modulated it with a "designer" drug. We used cre-dependent viral vectors (AAV8-hSyn-DIO-hM3Dq-mCherry) to transduce hypoglossal motoneurons of ChAT-Cre+ mice with hM3Dq (activating) receptors. We measured sleep and breathing in three conditions: (i) sham, (ii) after systemic administration of clozapine-N-oxide (CNO; 1 mg/kg) or (iii) vehicle. CNO activates hM3Dq receptors but is otherwise biologically inert. Systemic administration of CNO caused significant and sustained increases in tongue muscle activity in non-REM (261 ± 33% for 10 hrs) and REM sleep (217 ± 21% for 8 hrs), both P < 0.01 versus controls. Responses were specific and selective for the tongue with no effects on diaphragm or postural muscle activities, or sleep-wake states. These results support targeting a selective and restricted "druggable" target at the HMN (e.g., Kir2.4) to activate tongue motor activity during sleep.

A Standardized DNA Variant Scoring System for Pathogenicity Assessments in Mendelian Disorders.

We developed a rules-based scoring system to classify DNA variants into five categories including pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign. Over 16,500 pathogenicity assessments on 11,894 variants from 338 genes were analyzed for pathogenicity based on prediction tools, population frequency, co-occurrence, segregation, and functional studies collected from internal and external sources. Scores were calculated by trained scientists using a quantitative framework that assigned differential weighting to these five types of data. We performed descriptive and comparative statistics on the dataset and tested interobserver concordance among the trained scientists. Private variants defined as variants found within single families (n = 5,182), were either VUS (80.5%; n = 4,169) or likely pathogenic (19.5%; n = 1,013). The remaining variants (n = 6,712) were VUS (38.4%; n = 2,577) or likely benign/benign (34.7%; n = 2,327) or likely pathogenic/pathogenic (26.9%, n = 1,808). Exact agreement between the trained scientists on the final variant score was 98.5% [95% confidence interval (CI) (98.0, 98.9)] with an interobserver consistency of 97% [95% CI (91.5, 99.4)]. Variant scores were stable and showed increasing odds of being in agreement with new data when re-evaluated periodically. This carefully curated, standardized variant pathogenicity scoring system provides reliable pathogenicity scores for DNA variants encountered in a clinical laboratory setting.

Monoamine Release during Unihemispheric Sleep and Unihemispheric Waking in the Fur Seal.

STUDY OBJECTIVES: Our understanding of the role of neurotransmitters in the control of the electroencephalogram (EEG) has been entirely based on studies of animals with bilateral sleep. The study of animals with unihemispheric sleep presents the opportunity of separating the neurochemical substrates of waking and sleep EEG from the systemic, bilateral correlates of sleep and waking states. METHODS: The release of histamine (HI), norepinephrine (NE), and serotonin (5HT) in cortical and subcortical areas (hypothalamus, thalamus and caudate nucleus) was measured in unrestrained northern fur seals (Callorhinus ursinus) using in vivo microdialysis, in combination with, polygraphic recording of EEG, electrooculogram, and neck electromyogram. RESULTS: The pattern of cortical and subcortical HI, NE, and 5HT release in fur seals is similar during bilaterally symmetrical states: highest in active waking, reduced in quiet waking and bilateral slow wave sleep, and lowest in rapid eye movement (REM) sleep. Cortical and subcortical HI, NE, and 5HT release in seals is highly elevated during certain waking stimuli and behaviors, such as being sprayed with water and feeding. However, in contrast to acetylcholine (ACh), which we have previously studied, the release of HI, NE, and 5HT during unihemispheric sleep is not lateralized in the fur seal. CONCLUSIONS: Among the studied neurotransmitters most strongly implicated in waking control, only ACh release is asymmetric in unihemispheric sleep and waking, being greatly increased on the activated side of the brain. COMMENTARY: A commentary on this article appears in this issue on page 491.

Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye.

PURPOSE: The major challenges of developing an RNAi therapeutic include efficient delivery to and entry into the cell type of interest. Conventional ("naked" and chemically stabilized) small interfering RNAs (siRNAs) have been used in the eye in the past but they demonstrated limited clinical efficacy. Here we investigated a recently developed class of small, hydrophobic, asymmetric RNAi compounds. These compounds, termed "self-delivering rxRNAs" (sd-rxRNA(®)), are extensively modified, have a small duplex region of <15 base pairs, contain a fully phosphorothioated single-stranded tail, and readily enter cells and tissues without the requirement for a delivery vehicle. METHODS: We compared sd-rxRNA compounds with stabilized siRNAs in vitro (in ARPE-19 cells) and in vivo (intravitreal injection in mouse and rabbit eyes). Specifically, we investigated the retinal uptake, distribution, efficacy, and preliminary safety of sd-rxRNAs. RESULTS: Treatment with sd-rxRNAs resulted in uniform cellular uptake and full retina penetration in both animal models while no detectable cellular uptake was observed with stabilized siRNAs either in vitro or in vivo. Further, both in vitro and in vivo delivery (without any transfection reagent or formulation) resulted in a significant reduction of the targeted mRNA levels, which lasted 14-21 days in vivo. Retinal morphology and function were unaltered following a single administration of sd-rxRNAs. CONCLUSION: These data support the potential of developing sd-rxRNAs as a therapeutic for ocular disease.

Human hypocretin and melanin-concentrating hormone levels are linked to emotion and social interaction.

The neurochemical changes underlying human emotions and social behaviour are largely unknown. Here we report on the changes in the levels of two hypothalamic neuropeptides, hypocretin-1 and melanin-concentrating hormone, measured in the human amygdala. We show that hypocretin-1 levels are maximal during positive emotion, social interaction and anger, behaviours that induce cataplexy in human narcoleptics. In contrast, melanin-concentrating hormone levels are minimal during social interaction, but are increased after eating. Both peptides are at minimal levels during periods of postoperative pain despite high levels of arousal. Melanin-concentrating hormone levels increase at sleep onset, consistent with a role in sleep induction, whereas hypocretin-1 levels increase at wake onset, consistent with a role in wake induction. Levels of these two peptides in humans are not simply linked to arousal, but rather to specific emotions and state transitions. Other arousal systems may be similarly emotionally specialized.

Symmetrical serotonin release during asymmetrical slow-wave sleep: implications for the neurochemistry of sleep-waking states.

On land, fur seals predominately display bilaterally synchronized electroencephalogram (EEG) activity during slow-wave sleep (SWS), similar to that observed in all terrestrial mammals. In water, however, fur seals exhibit asymmetric slow-wave sleep (ASWS), resembling the unihemispheric slow-wave sleep of odontocetes (toothed whales). The unique sleeping pattern of fur seals allows us to distinguish neuronal mechanisms mediating EEG changes from those mediating behavioral quiescence. In a prior study we found that cortical acetylcholine release is lateralized during ASWS in the northern fur seal, with greater release in the hemisphere displaying low-voltage (waking) EEG activity, linking acetylcholine release to hemispheric EEG activation (Lapierre et al. 2007). In contrast to acetylcholine, we now report that cortical serotonin release is not lateralized during ASWS. Our data demonstrate that bilaterally symmetric levels of serotonin are compatible with interhemispheric EEG asymmetry in the fur seal. We also find greatly elevated levels during eating and hosing the animals with water, suggesting that serotonin is more closely linked to bilateral variables, such as axial motor and autonomic control, than to the lateralized cortical activation manifested in asymmetrical sleep.

Potent and systematic RNAi mediated silencing with single oligonucleotide compounds.

RNA interference (RNAi) has been established as an important tool for functional genomics studies and has great promise as a therapeutic intervention for human diseases. In mammalian cells, RNAi is conventionally induced by 19-27-bp RNA duplexes generated by hybridization of two complementary oligonucleotide strands (oligos). Here we describe a novel class of RNAi molecules composed of a single 25-28-nucleotide (nt) oligo. The oligo has a 16-nt mRNA targeting region, followed by an additional 8-10 nt to enable self-dimerization into a partially complementary duplex. Analysis of numerous diverse structures demonstrates that molecules composed of two short helices separated by a loop can efficiently enter and activate the RNA-induced silencing complex (RISC). This finding enables the design of highly effective single-oligo compounds for any mRNA target.

Modified dsRNAs that are not processed by Dicer maintain potency and are incorporated into the RISC.

Chemical modification of RNA duplexes can provide practical advantages for RNA interference (RNAi) triggering molecules including increased stability, safety and specificity. The impact of nucleotide modifications on Dicer processing, RISC loading and RNAi-mediated mRNA cleavage was investigated with duplexes >or=25 bp in length. It is known that dsRNAs >or=25 bp are processed by Dicer to create classic 19-bp siRNAs with 3'-end overhangs. We demonstrate that the presence of minimal modification configurations on longer RNA duplexes can block Dicer processing and result in the loading of the full-length guide strand into RISC with resultant mRNA cleavage at a defined site. These longer, modified duplexes can be highly potent gene silencers, with EC50s in the picomolar concentration range, demonstrating that Dicer processing is not required for incorporation into RISC or potent target silencing.

Fur seals display a strong drive for bilateral slow-wave sleep while on land.

Fur seals (pinnipeds of the family Otariidae) display two fundamentally different patterns of sleep: bilaterally symmetrical slow-wave sleep (BSWS) as seen in terrestrial mammals and slow-wave sleep (SWS) with a striking interhemispheric EEG asymmetry (asymmetrical SWS or ASWS) as observed in cetaceans. We examined the effect of preventing fur seals from sleeping in BSWS on their pattern of sleep. Four northern fur seals (Callorhinus ursinus) kept on land were sleep deprived (SD) of BSWS for 3 consecutive days, followed by 1 recovery day. EEG asymmetry was evaluated both visually and by EEG spectral analysis. SD significantly reduced the percentage of high-voltage BSWS (on average to 14% of baseline) and REM sleep (to 60% of baseline) whereas the percentage of low-voltage BSWS was not affected. During the SD period, all seals repeatedly tried to enter BSWS (109-411 attempts per day). SD significantly increased the amount of ASWS in each seal when scored visually (to 116-235% of baseline) and the difference in the EEG slow-wave activity (spectral power in the range of 1.2-4.0 Hz) between the two hemispheres (117-197%) as measured by the asymmetry index. High-voltage BSWS and the amount of SWS in each hemisphere were significantly elevated during the first 4 h of recovery. These data indicate that fur seals display a homeostatic response to the loss of SWS and that alternating SWS in the two hemispheres does not adequately compensate for the absence of BSWS.

Electroencephalogram asymmetry and spectral power during sleep in the northern fur seal.

The fur seal (Callorhinus ursinus), a member of the Pinniped family, displays a highly expressed electroencephalogram (EEG) asymmetry during slow wave sleep (SWS), which is comparable with the unihemispheric sleep in cetaceans. In this study, we investigated the EEG asymmetry in the fur seal using spectral analysis. Four young (2-3 years old) seals were implanted with EEG electrodes for polygraphic sleep recording. In each animal, EEG spectral power in the frequency range of 1.2-16 Hz was computed in symmetrical cortical recordings over two consecutive nights. The degree of EEG asymmetry was measured by using the asymmetry index [AI = (L - R)/(L + R), where L and R are the spectral powers in the left and right hemispheres, respectively]. In fur seals, EEG asymmetry, as measured by the percent of 20-s epochs with absolute AI > 0.3 and >0.6, was expressed in the entire frequency range (1.2-16 Hz). The asymmetry was significantly greater during SWS (25.6-44.2% of all SWS epochs had an absolute AI > 0.3 and 2.1-12.2% of all epochs had AI > 0.6) than during quiet waking (11.0-20.3% and 0-1.9% of all waking epochs, respectively) and REM sleep (4.2-8.9% of all REM sleep epochs and no epochs, respectively). EEG asymmetry was recorded during both low- and high-voltage SWS, and was maximal in the range of 1.2-4 and 12-16 Hz. As shown in this study, the degree of EEG asymmetry and the frequency range in which it is expressed during SWS in fur seals are profoundly different from those of terrestrial mammals and birds.

Cortical acetylcholine release is lateralized during asymmetrical slow-wave sleep in northern fur seals.

Fur seals are unique in that they display both bilateral slow-wave sleep (BSWS), as seen in all terrestrial mammals, and slow-wave sleep with interhemispheric electroencephalogram (EEG) asymmetry, resembling the unihemispheric slow waves of cetaceans. Little is known about the underlying mechanisms of this phenomenon, which is also termed asymmetrical slow wave sleep (ASWS). However, we may begin to understand the expression of ASWS by studying the neurotransmitter systems thought to be involved in the generation and maintenance of sleep-wake states in terrestrial mammals. We examined bilaterally the release of cortical acetylcholine (ACh), a neurotransmitter implicated in the regulation of cortical EEG and behavioral arousal, across the sleep-wake cycle in four juvenile northern fur seals (Callorhinus ursinus). In vivo microdialysis and high-performance liquid chromatography coupled with electrochemical detection were used to measure cortical ACh levels during polygraphically defined behavioral states. Cortical ACh release was state-dependent, showing maximal release during active waking (AW), similar levels during quiet waking (QW), and rapid eye movement (REM) sleep, and minimal release during BSWS. When compared with BSWS, cortical ACh levels increased approximately 300% during AW, and approximately 200% during QW and REM sleep. During these bilaterally symmetrical EEG states, ACh was synchronously released from both hemispheres. However, during ASWS, ACh release was lateralized with greater release in the hemisphere displaying lower voltage activity, at levels approximating those seen in QW. These findings demonstrate that cortical ACh release is tightly linked to hemispheric EEG activation.

Developmental changes in cardiorespiratory patterns associated with terrestrial apnoeas in harbour seal pups.

During the nursing period seals undergo several physiological and behavioural changes. A key component of development is increased cardiorespiratory control, fundamental for breath-holding and thus diving. This study focused on the ontogenetic changes in cardiac responses to respiration in quietly resting, pre-weaned harbour seal pups (Phoca vitulina). During periods of quiet rest, breathing became episodic, eupnoea interspersed with periods of apnoea. Little change was observed in respiration (approximately 35 breaths min(-1)) and eupnoeic heart rate (approximately 160 beats min(-1)) throughout the nursing period. However, apnoea duration increased (from approximately 20 to 40 s), while apnoeic heart rate decreased with age (from approximately 150 to 90 beats min(-1)). The observed decline in apnoeic heart rate resulted from an increase in cardiorespiratory control as pups approached weaning, evident by the ability to maintain a lower heart rate more consistently. Similar changes in cardiorespiratory patterns have been reported for elephant and Weddell seals. Due to the early onset of independent foraging, however, the rate of cardiorespiratory control development was more rapid in harbour seals. Our findings suggest that by 1 month of age, harbour seal pups possess the cardiorespiratory control necessary to sustain long-duration apnoeas, fundamental for proficient diving and successful foraging upon weaning.

The difluoromethylene group as a replacement for the labile oxygen in steroid sulfates: a new approach to steroid sulfatase inhibitors.

Several estrone sulfate and estradiol sulfate analogues, in which the sulfate group was replaced with an alpha,alpha-difluoromethylenesulfonate group or an alpha,alpha-difluoromethylenetetrazole group, were examined as inhibitors of steroid sulfatase (STS). These compounds were 4.5-10.5 times more potent than their non-fluorinated analogues. Moreover, the presence of the fluorines changed the mode of inhibition from mixed to competitive. The inhibitor bearing the alpha,alpha-difluoromethylenetetrazole group exhibited an affinity for STS approaching that of the natural STS substrate, estrone sulfate. Possible reasons for the enhanced affinity of the fluorinated compounds compared to their non-fluorinated counterparts are discussed.