Transcriptional regulatory logic of the diurnal cycle in the mouse liver.

Many organisms exhibit temporal rhythms in gene expression that propel diurnal cycles in physiology. In the liver of mammals, these rhythms are controlled by transcription-translation feedback loops of the core circadian clock and by feeding-fasting cycles. To better understand the regulatory interplay between the circadian clock and feeding rhythms, we mapped DNase I hypersensitive sites (DHSs) in the mouse liver during a diurnal cycle. The intensity of DNase I cleavages cycled at a substantial fraction of all DHSs, suggesting that DHSs harbor regulatory elements that control rhythmic transcription. Using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq), we found that hypersensitivity cycled in phase with RNA polymerase II (Pol II) loading and H3K27ac histone marks. We then combined the DHSs with temporal Pol II profiles in wild-type (WT) and Bmal1-/- livers to computationally identify transcription factors through which the core clock and feeding-fasting cycles control diurnal rhythms in transcription. While a similar number of mRNAs accumulated rhythmically in Bmal1-/- compared to WT livers, the amplitudes in Bmal1-/- were generally lower. The residual rhythms in Bmal1-/- reflected transcriptional regulators mediating feeding-fasting responses as well as responses to rhythmic systemic signals. Finally, the analysis of DNase I cuts at nucleotide resolution showed dynamically changing footprints consistent with dynamic binding of CLOCK:BMAL1 complexes. Structural modeling suggested that these footprints are driven by a transient heterotetramer binding configuration at peak activity. Together, our temporal DNase I mappings allowed us to decipher the global regulation of diurnal transcription rhythms in the mouse liver

A Realistic Validation Study of a New Nitrogen Multiple-Breath Washout System

Background For reliable assessment of ventilation inhomogeneity, multiple-breath washout (MBW) systems should be realistically validated. We describe a new lung model for in vitro validation under physiological conditions and the assessment of a new nitrogen (N2)MBW system. Methods The N2MBW setup indirectly measures the N2 fraction (FN2) from main-stream carbon dioxide (CO2) and side-stream oxygen (O2) signals: FN2 = 1−FO2−FCO2−FArgon. For in vitro N2MBW, a double chamber plastic lung model was filled with water, heated to 37°C, and ventilated at various lung volumes, respiratory rates, and FCO2. In vivo N2MBW was undertaken in triplets on two occasions in 30 healthy adults. Primary N2MBW outcome was functional residual capacity (FRC). We assessed in vitro error (√[difference]2) between measured and model FRC (100–4174 mL), and error between tests of in vivo FRC, lung clearance index (LCI), and normalized phase III slope indices (Sacin and Scond). Results The model generated 145 FRCs under BTPS conditions and various breathing patterns. Mean (SD) error was 2.3 (1.7)%. In 500 to 4174 mL FRCs, 121 (98%) of FRCs were within 5%. In 100 to 400 mL FRCs, the error was better than 7%. In vivo FRC error between tests was 10.1 (8.2)%. LCI was the most reproducible ventilation inhomogeneity index. Conclusion The lung model generates lung volumes under the conditions encountered during clinical MBW testing and enables realistic validation of MBW systems. The new N2MBW system reliably measures lung volumes and delivers reproducible LCI values

PER2 Variation is Associated with Diurnal Preference in a Korean Young Population

The PER2 gene has been reported to influence diurnal preference. In this study, we have attempted to characterize the associations between the PER2 gene polymorphisms and diurnal preference in a population of healthy young subjects, controlling for the social and environmental confounding factors. Subjects were 299 students in a college, carefully selected to be mentally and physically healthy. All subjects completed the 13-item composite scale for morningness (CSM). PER2 gene polymorphisms were genotyped by PCR-based methods. Genotype and allele carrier status of a PER2 G3853A polymorphism (rs934945) were associated with CSM scores. Carriers of the 3853G allele showed significantly higher CSM scores (P = 0.004, P = 0.009, and P = 0.001; total, morningness, and activity plan, respectively). There were no significant differences on CSM scores among genotypes and allele status of PER2 rs2304672. This result indicates that rs934945 of PER2 may be associated with diurnal preference in a Korean healthy population

Harmonics of Circadian Gene Transcription in Mammals

The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior. Numerous investigations have addressed the contribution of circadian rhythmicity to cellular, organ, and organismal physiology. We recently developed a method to look at transcriptional oscillations with unprecedented precision and accuracy using high-density time sampling. Here, we report a comparison of oscillating transcription from mouse liver, NIH3T3, and U2OS cells. Several surprising observations resulted from this study, including a 100-fold difference in the number of cycling transcripts in autonomous cellular models of the oscillator versus tissues harvested from intact mice. Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells. Validation experiments show that 12-hour oscillatory transcripts occur in several other peripheral tissues as well including heart, kidney, and lungs. These harmonics are lost ex vivo, as well as under restricted feeding conditions. Taken in sum, these studies illustrate the importance of time sampling with respect to multiple testing, suggest caution in use of autonomous cellular models to study clock output, and demonstrate the existence of harmonics of circadian gene expression in the mouse

Enhanced Food Anticipatory Activity Associated with Enhanced Activation of Extrahypothalamic Neural Pathways in Serotonin2C Receptor Null Mutant Mice

The ability to entrain circadian rhythms to food availability is important for survival. Food-entrained circadian rhythms are characterized by increased locomotor activity in anticipation of food availability (food anticipatory activity). However, the molecular components and neural circuitry underlying the regulation of food anticipatory activity remain unclear. Here we show that serotonin2C receptor (5-HT2CR) null mutant mice subjected to a daytime restricted feeding schedule exhibit enhanced food anticipatory activity compared to wild-type littermates, without phenotypic differences in the impact of restricted feeding on food consumption, body weight loss, or blood glucose levels. Moreover, we show that the enhanced food anticipatory activity in 5-HT2CR null mutant mice develops independent of external light cues and persists during two days of total food deprivation, indicating that food anticipatory activity in 5-HT2CR null mutant mice reflects the locomotor output of a food-entrainable oscillator. Whereas restricted feeding induces c-fos expression to a similar extent in hypothalamic nuclei of wild-type and null mutant animals, it produces enhanced expression in the nucleus accumbens and other extrahypothalamic regions of null mutant mice relative to wild-type subjects. These data suggest that 5-HT2CRs gate food anticipatory activity through mechanisms involving extrahypothalamic neural pathways

On-site data cast doubts on the hypothesis of shifting cultivation in the Late Neolithic (c. 4300-2400 cal. BC): Landscape management as an alternative paradigm

This article brings together in a comprehensive way, and for the first time, on- and off-site palaeoenvironmental data from the area of the Central European lake dwellings (a UNESCO World Cultural Heritage Site since 2011). The types of data considered are as follows: high-resolution off-site pollen cores, including micro-charcoal counts, and on-site data, including botanical macro- and micro-remains, hand-collected animal bones, remains of microfauna, and data on woodland management (dendrotypology). The period considered is the late Neolithic (c. 4300–2400 cal. BC). For this period, especially for its earlier phases, discussions of land-use patterns are contradictory. Based on off-site data, slash-and-burn – as known from tropical regions – is thought to be the only possible way to cultivate the land. On-site data however show a completely different picture: all indications point to the permanent cultivation of cereals (Triticum spp., Hordeum vulgare), pea (Pisum sativum), flax (Linum usitatissimum) and opium-poppy (Papaver somniferum). Cycles of landscape use are traceable, including coppicing and moving around the landscape with animal herds. Archaeobiological studies further indicate also that hunting and gathering were an important component and that the landscape was manipulated accordingly. Late Neolithic land-use systems also included the use of fire as a tool for opening up the landscape. Here we argue that bringing together all the types of palaeoenvironmental proxies in an integrative way allows us to draw a more comprehensive and reliable picture of the land-use systems in the late Neolithic than had been reconstructed previously largely on the basis of off-site data

Cognitive Aging in Zebrafish

BACKGROUND: Age-related impairments in cognitive functions represent a growing clinical and social issue. Genetic and behavioral characterization of animal models can provide critical information on the intrinsic and environmental factors that determine the deterioration or preservation of cognitive abilities throughout life. METHODOLOGY/PRINCIPAL FINDINGS: Behavior of wild-type, mutant and gamma-irradiated zebrafish (Danio rerio) was documented using image-analysis technique. Conditioned responses to spatial, visual and temporal cues were investigated in young, middle-aged and old animals. The results demonstrate that zebrafish aging is associated with changes in cognitive responses to emotionally positive and negative experiences, reduced generalization of adaptive associations, increased stereotypic and reduced exploratory behavior and altered temporal entrainment. Genetic upregulation of cholinergic transmission attenuates cognitive decline in middle-aged achesb55/+ mutants, compared to wild-type siblings. In contrast, the genotoxic stress of gamma-irradiation accelerates the onset of cognitive impairment in young zebrafish. CONCLUSIONS/SIGNIFICANCE: These findings would allow the use of powerful molecular biological resources accumulated in the zebrafish field to address the mechanisms of cognitive senescence, and promote the search for therapeutic strategies which may attenuate age-related cognitive decline