Sequence Determinants of Circadian Gene Expression Phase in Cyanobacteria

The cyanobacterium Synechococcus elongatus PCC 7942 exhibits global biphasic circadian oscillations in gene expression under constant-light conditions. Class I genes are maximally expressed in the subjective dusk, whereas class II genes are maximally expressed in the subjective dawn. Here, we identify sequence features that encode the phase of circadian gene expression. We find that, for multiple genes, an ∼70-nucleotide promoter fragment is sufficient to specify class I or II phase. We demonstrate that the gene expression phase can be changed by random mutagenesis and that a single-nucleotide substitution is sufficient to change the phase. Our study provides insight into how the gene expression phase is encoded in the cyanobacterial genome.     

Differential Expression Analysis for Pathways

Life science technologies generate a deluge of data that hold the keys to unlocking the secrets of important biological functions and disease mechanisms. We present DEAP, Differential Expression Analysis for Pathways, which capitalizes on information about biological pathways to identify important regulatory patterns from differential expression data. DEAP makes significant improvements over existing approaches by including information about pathway structure and discovering the most differentially expressed portion of the pathway. On simulated data, DEAP significantly outperformed traditional methods: with high differential expression, DEAP increased power by two orders of magnitude; with very low differential expression, DEAP doubled the power. DEAP performance was illustrated on two different gene and protein expression studies. DEAP discovered fourteen important pathways related to chronic obstructive pulmonary disease and interferon treatment that existing approaches omitted. On the interferon study, DEAP guided focus towards a four protein path within the 26 protein Notch signalling pathway.     

Association Between Phase Shifts,Expression Levels,and Amplitudes in Peripheral Circadian Clocks

Data analyses examining the relationship between circadian phase shifts and amplitudes are scarce. The aim of this data analysis was to explore the association between the phase shifts of gene expression, their amplitudes, and daily levels as a result of a given treatment under ad libitum or restricted feeding (RF) conditions. Two hundred forty data sets of gene expression (clock and metabolic genes) from various tissues and treatments were statistically analyzed. The data revealed a significant association between phase delays and increased amplitudes. Moreover, upon subgroup analyses, separating the RF from the non-RF groups, phase delays were significantly correlated with increased amplitudes and phase advances with decreased amplitudes. This picture was also achieved when clock genes, but not metabolic genes, were analyzed. In contrast, under RF, increased amplitude of metabolic genes correlated with phase advances. Moreover, phase advances under RF led to increased average daily levels in clock genes, but not in metabolic genes. In summary, these data demonstrated statistically significant association between phase shifts, daily levels, and amplitudes in circadian gene expression in peripheral tissues under timed versus ad libitum feeding conditions. (Author correspondence: oren.froy@mail.huji.ac.il)     

Genetically Altering the Thermodynamics and Kinetics of Hepatitis B Virus Capsid Assembly Has Profound Effects on Virus Replication in Cell Culture

Capsid (core) assembly is essential for hepatitis B virus (HBV) replication. We hypothesize that assembly kinetics and stability are tuned for optimal viral replication, not maximal assembly. Assembly effectors (AEfs) are small molecules proposed to disrupt this balance by inappropriately enhancing core assembly. Guided by the structure of an AEf-bound core, we designed a structural mimic of AEf-bound core protein, the V124W mutant. In biochemical studies, the V124W mutant recapitulated the effects of AEfs, with fast assembly kinetics and a strong protein-protein association energy. Also, the mutant was resistant to exogenous AEfs. In cell culture, the V124W mutant behaved like a potent AEf: expression of HBV carrying the V124W mutant was defective for genome replication. Critically, the V124W mutant interfered with replication of wild-type HBV in a dose-dependent manner, mimicking AEf activity. In addition, the V124W mutant was shown to adopt a more compact conformation than that of the wild type, confirming the allosteric regulation in capsid assembly. These studies show that the heteroaryldihydropyrimidine (HAP) binding pocket is a promiscuous target for inducing assembly. Suppression of viral replication by the V124W mutant suggests that mutations that fill the HAP site are not a path for HBV to escape from AEfs.     

Circadian Phase Shifting Associated with Routine Cage Maintenance: A Retrospective Analysis

Stimuli that evoke behavioral activation can phase-shift free-running circadian activity rhythms in Syrian hamsters. Activation-induced phase shifting is characterized by a phase-response curve (PRC) that is dissimilar to the PRC for photic phase shifting, and recent studies indicate that complex interactions may occur between photic and non-photic phase shifting. Since animals in the laboratory may be exposed to both photic and behaviorally activating stimulation during routine cage maintenance procedures, we performed a retrospective analysis of possible phase shifts associated with cage cleaning in individually housed hamsters maintained in either constant darkness (DD) or dim red light (RR) during the course of an ongoing study of drug-induced phase shifting. All cage cleanings were conducted under RR and were separated from drug treatments by at least one week. The results indicated that both photic and non-photic phase shifts could be induced by routine cage maintenance procedures, depending on the circadian timing of the procedure, on lighting conditions, and on the degree of evoked activity.     

Circadian Phase Shifting Associated with Routine Cage Maintenance: A Retrospective Analysis

Stimuli that evoke behavioral activation can phase-shift free-running circadian activity rhythms in Syrian hamsters. Activation-induced phase shifting is characterized by a phase-response curve (PRC) that is dissimilar to the PRC for photic phase shifting, and recent studies indicate that complex interactions may occur between photic and non-photic phase shifting. Since animals in the laboratory may be exposed to both photic and behaviorally activating stimulation during routine cage maintenance procedures, we performed a retrospective analysis of possible phase shifts associated with cage cleaning in individually housed hamsters maintained in either constant darkness (DD) or dim red light (RR) during the course of an ongoing study of drug-induced phase shifting. All cage cleanings were conducted under RR and were separated from drug treatments by at least one week. The results indicated that both photic and non-photic phase shifts could be induced by routine cage maintenance procedures, depending on the circadian timing of the procedure, on lighting conditions, and on the degree of evoked activity.     

Effects of Membrane ATPase Inhibitors on Light-Induced Phase Shifting of the Circadian Clock in Neurospora crassa

Effects of several membrane ATPase inhibitors on light-induced phase shifting of the circadian conidiation rhythm in Neurospora crassa were examined using mycelial discs in liquid culture. Suppression of phase shifting by the inhibitors was strongly dependent on the pH of the liquid medium in which the discs were cultured during the time from light-dark transition (beginning of free-run) to light irradiation. When discs were cultured in pH 6.7 medium, azide, the inhibitors of plasma membrane ATPase (diethylstilbestrol and N, N′-dicyclohexylcarbodiimide), and ethanol completely suppressed the effect of light on the clock. In contrast, mycelial discs cultured in pH 5.7 medium were fully phase-shifted by light in the presence of the same and even higher concentrations of the chemicals. However, sensitivity to light of the discs cultured in relatively acidic medium was eight times higher than that of the discs cultured at neutral pH. Oligomycin and venturicidin, inhibitors of mitochondrial ATPase, did not suppress phase shifting by light at either pH.     

Nitric Oxide Has Differential Effects on Currents in Different Subsets of Manduca sexta Antennal Lobe Neurons

Nitric oxide has been shown to regulate many biological systems including olfaction. In the moth olfactory system nitric oxide is produced in the antennal lobe in response to odor stimulation and has complex effects on the activity of both projection neurons and local interneurons. To examine the cell autonomous effects of nitric oxide on these cells, we used patch-clamp recording in conjunction with pharmacological manipulation of nitric oxide to test the hypothesis that nitric oxide differentially regulates the channel properties of these different antennal lobe neuron subsets. We found that nitric oxide caused increasing inward currents in a subset of projection neurons while the effects on local neurons were variable but consistent within identifiable morphological subtypes.     

Circadian Phase Variation in Bipolar I Disorder

Abnormalities in circadian rhythms are prominent features of bipolar I disorder (BD1). To investigate circadian variation in BD1, we evaluated morningness–eveningness (M/E), a stable trait reflecting circadian phase, using the composite scale (CS) among BD1 patients (DSM IV criteria; n=75), unscreened controls (n=349), and patients with schizophrenia (SZ) or schizoaffective disorder (SZA) (n=81). Our analyses showed that CS scores correlated significantly with age but did not differ by gender among the controls. BD1 patients differed significantly from controls and from SZ/SZA patients when age was considered. CS scores were distributed bi‐modally among BD1 cases. There are several possible reasons for the observed heterogeneity. Younger BD1 patients, and those with rapid mood swings, were significantly more likely to have lower CS scores (i.e., to score in the ‘evening’ range and to have later circadian phase). CS scores were also positively correlated with the age at onset and the duration of the most severe depressive episodes. These relationships were not observed among the SZ/SZA groups. Thus, distinct patterns of M/E were noted among BD1 patients and among BD1 subgroups. The impact of medication, mood state, and chronicity on CS scores needs to be considered.     

Circadian Phase in Adults of Contrasting Ages

There is evidence that aging may impair phase‐shifting responses to light synchronizers, which could lead to disturbed or malsynchronized circadian rhythms. To explore this hypothesis, 62 elder participants (age, 58 to 84 years) and 25 young adults (age, 19 to 40 years) were studied, first with baseline 1‐wk wrist actigraphy at home and then by 72 h in‐laboratory study using an ultra‐short sleep‐wake cycle. Subjects were awake for 60 minutes in 50 lux followed by 30 minutes of darkness for sleep. Saliva samples were collected for melatonin, and urine samples were collected for aMT6s (a urinary metabolite of melatonin) and free cortisol every 90 minutes. Oral temperatures were also measured every 90 minutes. The timing of the circadian rhythms was not significantly more variable among the elders. The times of lights‐out and wake‐up at home and urinary free cortisol occurred earlier among elders, but the acrophases (cosinor analysis‐derived peak time) of the circadian rhythm of salivary melatonin, urinary aMT6s, and oral temperature were not significantly phase‐advanced among elders. The estimated duration of melatonin secretion was 9.9 h among elders and 8.4 h among young adults (p<0.025), though the estimated half‐life of blood melatonin was shorter among elders (p<0.025), and young adults had higher saliva melatonin and urinary aMT6s levels. In summary, there was no evidence for circadian desynchronization associated with aging, but there was evidence of some rearrangement of the internal phase‐angles among the studied circadian rhythms.     

Comparative Circadian Metabolomics Reveal Differential Effects of Nutritional Challenge in the Serum and Liver

Diagnosis and therapeutic interventions in pathological conditions rely upon clinical monitoring of key metabolites in the serum. Recent studies show that a wide range of metabolic pathways are controlled by circadian rhythms whose oscillation is affected by nutritional challenges, underscoring the importance of assessing a temporal window for clinical testing and thereby questioning the accuracy of the reading of critical pathological markers in circulation. We have been interested in studying the communication between peripheral tissues under metabolic homeostasis perturbation. Here we present a comparative circadian metabolomic analysis on serum and liver in mice under high fat diet. Our data reveal that the nutritional challenge induces a loss of serum metabolite rhythmicity compared with liver, indicating a circadian misalignment between the tissues analyzed. Importantly, our results show that the levels of serum metabolites do not reflect the circadian liver metabolic signature or the effect of nutritional challenge. This notion reveals the possibility that misleading reads of metabolites in circulation may result in misdiagnosis and improper treatments. Our findings also demonstrate a tissue-specific and time-dependent disruption of metabolic homeostasis in response to altered nutrition.     

Circadian Phase Dependent Pharmacokinetics of Disopyramide in Mice

The focus of the reported work is investigation of disopyramide chronopharmacokinetics in the mouse. Different groups of male NMRI mice maintained under controlled environmental conditions (LD: 0600-1800) received a single intraperitoneal injection of disopyramide (30mg per kg of body weight) at one of four different fixed time points of a 24-h period, i.e. 1000, 1600, 2200 or 0400. Blood samples were taken 0.5,1,2,3,4 and 6 hr after drug administration and total and free plasma levels of disopyramide were measured by an immunoenzymatic method.

Our data showed statistically significant circadian rhythms in the following pharmacokinetic parameters: highest volume of distribution = 3.91 ± 0.211kg^-1 at 2200 (circadian amplitude, half the peak-to-trough difference relative to the 24-hr mean multiplied by 100, is 34%); highest area under concentration curves = 16.06 ± 1.03μgml^-1hr^-1 at 0400 (circadian amplitude = 43%) and highest clearance = 3.04 ± 0.191hr“kg”¹ at 2200 (circadian amplitude = 21%). Protein binding of the drug was shown to ^he circadian time dependent. Alpha and beta phase elimination half-lives were not found to be significantly circadian phase-dependent. Thus circadian changes in disopyramide clearance may represent circadian changes in the drug's volume of distribution.