Dual Hypocretin Receptor Antagonism Is More Effective for Sleep Promotion than Antagonism of Either Receptor Alone

The hypocretin (orexin) system is involved in sleep/wake regulation, and antagonists of both hypocretin receptor type 1 (HCRTR1) and/or HCRTR2 are considered to be potential hypnotic medications. It is currently unclear whether blockade of either or both receptors is more effective for promoting sleep with minimal side effects. Accordingly, we compared the properties of selective HCRTR1 (SB-408124 and SB-334867) and HCRTR2 (EMPA) antagonists with that of the dual HCRTR1/R2 antagonist almorexant in the rat. All 4 antagonists bound to their respective receptors with high affinity and selectivity in vitro. Since in vivo pharmacokinetic experiments revealed poor brain penetration for SB-408124, SB-334867 was selected for subsequent in vivo studies. When injected in the mid-active phase, SB-334867 produced small increases in rapid-eye-movement (REM) and non-REM (NR) sleep. EMPA produced a significant increase in NR only at the highest dose studied. In contrast, almorexant decreased NR latency and increased both NR and REM proportionally throughout the subsequent 6 h without rebound wakefulness. The increased NR was due to a greater number of NR bouts; NR bout duration was unchanged. At the highest dose tested (100 mg/kg), almorexant fragmented sleep architecture by increasing the number of waking and REM bouts. No evidence of cataplexy was observed. HCRTR1 occupancy by almorexant declined 4–6 h post-administration while HCRTR2 occupancy was still elevated after 12 h, revealing a complex relationship between occupancy of HCRT receptors and sleep promotion. We conclude that dual HCRTR1/R2 blockade is more effective in promoting sleep than blockade of either HCRTR alone. In contrast to GABA receptor agonists which induce sleep by generalized inhibition, HCRTR antagonists seem to facilitate sleep by reducing waking “drive”

Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

Telomere length is a risk factor in disease and the dynamics of telomere length are crucial to our understanding of cell replication and vitality. The proliferation of whole genome sequencing represents an unprecedented opportunity to glean new insights into telomere biology on a previously unimaginable scale. To this end, a number of approaches for estimating telomere length from whole-genome sequencing data have been proposed. Here we present Telomerecat, a novel approach to the estimation of telomere length. Previous methods have been dependent on the number of telomeres present in a cell being known, which may be problematic when analysing aneuploid cancer data and non-human samples. Telomerecat is designed to be agnostic to the number of telomeres present, making it suited for the purpose of estimating telomere length in cancer studies. Telomerecat also accounts for interstitial telomeric reads and presents a novel approach to dealing with sequencing errors. We show that Telomerecat performs well at telomere length estimation when compared to leading experimental and computational methods. Furthermore, we show that it detects expected patterns in longitudinal data, repeated measurements, and cross-species comparisons. We also apply the method to a cancer cell data, uncovering an interesting relationship with the underlying telomerase genotype

Publisher Correction: Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

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In search for seasonal genes: implication of the KiSS-1/ GPR54 system in the seasonal control of reproduction

Anticipation and adaptation of individuals to the upcomming daily and seasonal variations are a prerequisite for species survival, particulary mammals. Life has developed the ability to measure and manage time, using light as the most reliable environmental time cue. These timing processes involve a photoneuroendocrine axis comprising the retina, various hypothalamic structures of which is the suprachiasmatic nucleus (SCN - seat of the master circadian clock), and the pineal gland which rhythmically releases the hormone melatonin to distribute timing information to the whole organism. Recently, major advances have been made in the understanding of the molecular events underlying circadian rhythms, with the discovery of specific "clock genes" working in double positive and negative transcriptional feedback loops. In contrast, much less is known about the molecular mechanisms operating on a seasonal time-scale and regulating yearly functions such as reproduction, hibernation, weight regulation, or seasonal pathologies in humans. This project initially aimed at identify genes underpinning the seasonal adaptations of animals for which several functions are regulated by photoperiod (i.e. day length). The approach involved two steps: 1) To identify unknown genes which display photoperiodic or melatonin-dependent changes of expression within the photoneuroendocrine system; 2) To determine how these genes and their products may regulate the seasonal functions. For this research, we have used a well defined photoperiodic model, the Syrian hamster (Mesocricetus auratus), in which photoperiod greatly influences physiology and behaviours, in particular the reproductive activity. Hamsters maintained under long summer days (LD) are sexually active, while reproduction is dramatically inhibited after a few weeks in hamsters kept under short winter days (SD)

Kinetic parameters for the association and dissociation of [³H]almorexant in rHCRTR2-HEK293 cell membranes at 37°C.

<p>The K_on (calculated on rate), K_off (observed off rate), t_1/2 (half-maximal binding) and K_d (apparent dissociation constant) values are ± SEM, calculated from three independent experiments (each performed in quadruplicate) as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039131#s2" target="_blank">Materials and Methods</a>”.</p

Potencies of almorexant, SB-408124 and SB-334867 antagonists in inhibition of [³H]almorexant binding to the membrane preparations from HEK293 cells transiently expressing rHCRTR1 and rHCRTR2.

<p>[³H]almorexant was used at a concentration equal to its K_d values of 3.4 nM and 0.5 nM at rHcrtR1 and rHcrtR2, respectively, in these competition binding experiments. K_i values for [³H]almorexant binding inhibition by various antagonists were calculated as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039131#s2" target="_blank">Materials and Methods</a>”. Values are ± SEM of the K_i calculated from three independent experiments, each performed in duplicate.</p

Binding characteristics of [³H]almorexant to rHCRTR1 and rHCRTR2 cell membranes.

<p>(<b>A,B</b>) Saturation binding curves of [³H]almorexant binding to membranes from HEK293 cells transiently transfected with rHCRTR1 (<b>A</b>) or rHCRTR2 (<b>B</b>). Each data point represents the mean±SEM of three independent experiments performed in triplicate. The data were analyzed by nonlinear regression analysis using GraphPad Prism 4.0 software and a single-site binding model. (<b>C,D</b>) Time course for the association (<b>C</b>) and dissociation (<b>D</b>) of [³H]almorexant binding to rHCRTR2 membranes.</p

Average hourly LMA and relative T_core. LMA and relative T_core for 6 h prior to and 18 h after administration of SB-334867 (A), EMPA (B), and almorexant (C) as compared to zolpidem (ZOL) and vehicle.

<p>Shaded area represents the dark phase; vertical dotted line in each panel indicates the time of injection. (A) Average hourly LMA following SB-334867. (A’) The average hourly T_core following SB-334867. (B) The average hourly LMA following EMPA. (B’) The average hourly T_core following EMPA. (C) The average hourly LMA following almorexant. (C’) The average hourly T_core following almorexant. Data represent the mean±SEM (n = 8 rats per group). *, <i>p</i><0.05. For detailed statistical results see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039131#pone.0039131.s009" target="_blank">Text S1</a>.</p