Polycomb regulates circadian rhythms in Drosophila in clock neurons.

Circadian rhythms are essential physiological feature for most living organisms. Previous studies have shown that epigenetic regulation plays a crucial role. There is a knowledge gap in the chromatin state of some key clock neuron clusters. In this study, we show that circadian rhythm is affected by the epigenetic regulator Polycomb (Pc) within the Drosophila clock neurons. To investigate the molecular mechanisms underlying the roles of Pc in these clock neuron clusters, we use targeted DamID (TaDa) to identify genes significantly bound by Pc in the neurons marked by C929-Gal4 (including l-LNvs cluster), R6-Gal4 (including s-LNvs cluster), R18H11-Gal4 (including DN1 cluster), and DVpdf-Gal4, pdf-Gal80 (including LNds cluster). It shows that Pc binds to the genes involved in the circadian rhythm pathways, arguing a direct role for Pc in regulating circadian rhythms through specific clock genes. This study shows the identification of Pc targets in the clock neuron clusters, providing potential resource for understanding the regulatory mechanisms of circadian rhythms by the PcG complex. Thus, this study provided an example for epigenetic regulation of adult behavior.

Analysis of sleep in individual Drosophila melanogaster reveals a self-regulatory role for cuticular hydrocarbons pheromones.

It is well established that pheromones are used by insects to transmit information between individuals. However, research has revealed that individual insects can be both the sender and the receiver of some pheromonal signals. It is therefore interesting to consider whether the pheromonal state of an individual insect can exert an effect on itself. In this study, we monitored the sleep activity of single flies exhibiting a mutation that leads to pheromonal deficiency and found that cuticular hydrocarbons (CHs) exerted self-regulatory effects on the amount of sleep experienced by these flies. To identify the physiological significance of this mechanism, we compared the amounts of sleep in individual young flies and individual old flies (flies are known to sleep less as they get older) and compared this data with young and old flies exhibiting mutations that lead to CH reception defects. The differences in the amount of sleep experienced by young and old mutant flies were significantly lower than those of the control flies. Our data show that hydrocarbon signals produced by the cuticle in Drosophila can be self-perceived and regulate the amount of sleep acquired in a maturation-dependent manner.

PHARMACOKINETICS OF SUBCUTANEOUS ALPHA LIPOIC ACID, A PROPOSED THERAPEUTIC AID FOR DOMOIC ACID INTOXICATION IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

Domoic acid (DA) is a potent neurotoxin produced by certain marine algae that can cause neurologic and cardiac dysfunction by activating glutamate receptors. Glutamate receptor overexcitation results in continuous cellular activation, oxidative damage, and cell death. DA toxicosis causes disorientation and seizures, and antiseizure medications are the primary treatment. Alpha lipoic acid (ALA), a powerful antioxidant and glutathione precursor widely used in humans and dogs, can cross the blood-brain barrier to provide antioxidant availability to brain tissue. Hundreds of stranded California sea lions (CSL; Zalophus californianus) are diagnosed annually with DA toxicosis and thus are an appropriate animal in which to establish ALA dosing recommendations for treatment. The objective of this study was to determine the population pharmacokinetics of a single 10- or 20-mg/kg dose of ALA administered subcutaneously into the interscapular region to healthy rehabilitated CSL. Blood was collected at two time points between 15 min and 24 h after administration. Serum ALA concentrations were measured by liquid chromatography-mass spectrometry, and parameters were evaluated using a nonlinear mixed effects model. ALA was rapidly absorbed for each dose, peaking within 20 to 30 minutes, and t1/2 of 40 and 32 min (10 and 20 mg/kg, respectively), followed by an initial steep distribution phase and prolonged elimination phase. Peak concentration (CMAX) was 1,243 ng/ml (10-mg/ml dose) and 5,010 ng/ml (20-mg/ml dose). Serum from 13 CSLd with DA toxicosis treated with 10 mg/kg ALA for 1 to 9 d had measurable levels, and ALA was also measurable in cerebrospinal fluid from two treated CSLs. Therapeutic effects are noted with a CMAX of 4,000 to 5,000 ng/ml in humans; thus in CSLs, 20 mg/kg administered subcutaneously once daily may be sufficient to achieve a therapeutic level in this species. Determination of efficacy and optimal dosing interval and duration require additional investigation.

Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octß2R cascade.

Sleep homeostasis is crucial for sleep regulation. The role of epigenetic regulation in sleep homeostasis is unestablished. Previous studies showed that octopamine is important for sleep homeostasis. However, the regulatory mechanism of octopamine reception in sleep is unknown. In this study, we identify an epigenetic regulatory cascade (Stuxnet-Polycomb-Octß2R) that modulates the octopamine receptor in Drosophila. We demonstrate that stuxnet positively regulates Octß2R through repression of Polycomb in the ellipsoid body of the adult fly brain and that Octß2R is one of the major receptors mediating octopamine function in sleep homeostasis. In response to octopamine, Octß2R transcription is inhibited as a result of stuxnet downregulation. This feedback through the Stuxnet-Polycomb-Octß2R cascade is crucial for sleep homeostasis regulation. This study demonstrates a Stuxnet-Polycomb-Octß2R-mediated epigenetic regulatory mechanism for octopamine reception, thus providing an example of epigenetic regulation of sleep homeostasis.

Regulatory mechanism of daily sleep by miR-276a.

MiRNAs have attracted more attention in recent years as regulators of sleep and circadian rhythms after their roles in circadian rhythm and sleep were discovered. In this study, we explored the roles of the miR-276a on daily sleep in Drosophila melanogaster, and found a regulatory cycle for the miR-276a pathway, in which miR-276a, regulated by the core CLOCK/CYCLE (CLK/CYC) transcription factor upstream, regulates sleep via suppressing targets TIM and NPFR1. (a) Loss of miR-276a function makes the flies sleep more during both daytime and nighttime, while flies with gain of miR-276a function sleep less; (b) MiR-276a is widely expressed in the mushroom body (MB), the pars intercerebralis (PI) and some clock neurons lateral dorsal neurons (LNds), in which tim neurons is important for sleep regulation; (c) MiR-276a promoter is identified to locate in the 8th fragment (aFrag8) of the pre-miR-276a, and this fragment is directly activated and regulated by CLK/CYC; (4) MiR-276a is rhythmically oscillating in heads of the wild-type w1118 , but this oscillation disappears in the loss of function mutant clkjrk ; (5) The neuropeptide F receptor 1 (npfr1) was found to be a downstream target of miR-276a. These results clarify that the miR-276a is a very important factor for sleep regulation.

Extra sex combs buffers sleep-related stresses through regulating Heat shock proteins.

The impact of global warming on the life of the earth is increasingly concerned. Previous studies indicated that temperature changes have a serious impact on insect sleep. Sleep is critical for animals as it has many important physiological functions. It is of great significance to study the regulation mechanism of temperature-induced sleep changes for understanding the impact of global warming on insects. More importantly, understanding how these pressures regulate sleep can provide insights into improving sleep. In this study, we found that extra sex combs (ESC) are a regulatory factor in this process. Our data showed that ESC was an upstream negative regulatory factor of Heat shock proteins (Hsps), and it could regulate sleep in mushroom and ellipsoid of Drosophila. ESC mutation exaggerates the sleep change caused by temperature, while buffering the shortening of life caused by sleep deprivation. These phenotypes can be rescued by Hsps mutants. Therefore, we concluded that the ESC buffers sleep-related stresses through regulating Hsps.

Regulation of circadian rhythm and sleep by miR-375-timeless interaction in Drosophila.

MicroRNAs are important coordinators of circadian regulation that mediate the fine-tuning of gene expression. Although many studies have shown the effects of individual miRNAs on the circadian clock, the global functional miRNA-mRNA interaction network involved in the circadian system remains poorly understood. Here, we used CLEAR (Covalent Ligation of Endogenous Argonaute-bound RNAs)-CLIP (Cross-Linking and Immuno-Precipitation) to explore the regulatory functions of miRNAs in the circadian system by comparing the miRNA-mRNA interactions between Drosophila wild-type strain W1118 and a mutant of the key circadian transcriptional regulator Clock (Clkjrk ). This experimental approach unambiguously identified tens of thousands of miRNA-mRNA interactions in both the head and body. The miRNA-mRNA interactome showed dramatic changes in the Clkjrk flies. Particularly, among ~300 miRNA-mRNA circadian relevant interactions, multiple interactions involving core clock genes pdp1, tim, and vri displayed distinct changes as a result of the Clk mutation. Based on the CLEAR-CLIP analysis, we found a novel regulation of the circadian rhythm and sleep by the miR-375-timeless interaction. The results indicated that Clk disruption abolished normal rhythmic expression of miR-375 and the functional regulation occurred in the l-LNv neurons, where miR-375 modulated the circadian rhythm and sleep via targeting timeless. This work provides the first global view of miRNA regulation in the circadian rhythm.

Diurnal protein oscillation profiles in Drosophila head.

Circadian clocks control daily rhythms in physiology, metabolism and behaviour in most organisms. Proteome-wide analysis of protein oscillations is still lacking in Drosophila. In this study, the total protein and phosphorylated protein in Drosophila heads in a 24-h daily time-course were assayed by using the isobaric tags for relative and absolute quantitation (iTRAQ) method, and 10 and 7 oscillating proteins as well as 19 and 22 oscillating phosphoproteins in the w1118 control and ClkJrk mutant strains were separately identified. Lastly, we performed a mini screen to investigate the functions of some oscillating proteins in circadian locomotion rhythms. This study provides the first proteomic profiling of diurnally oscillating proteins in fly heads, thereby providing a basis for further mechanistic studies of these proteins in circadian rhythm.

Modeling and simulation approach to support dosing and study design requirements for treating HIV-related neuropsychiatric disease with the NK1-R antagonist aprepitant.

Psychiatric illness is common in HIV-infected patients and underlines the importance for screening not only for cognitive impairment but also for co-morbid mental disease. The rationale for combining immunomodulatory neurokinin- 1 receptor (NK1-R) antagonists with combined antiretroviral therapy (cART) is based on multimodal pharmacologic mechanisms. The NK1-R antagonist aprepitant's potential utility as a drug for depression is complicated by >99.9% protein binding and both enzyme inhibition and induction of CYP3A4. A population-based PK model developed from a pilot Phase 1B trial in 19 HIV-infected patients (125 or 250 mg/d aprepitant for 2 weeks) was modified to account for enzyme induction and impact of an exposure enhancer on CYP3A4 metabolism. Likelihood of clinical success in depression was assessed based on achievement of target trough plasma concentration and evaluated using Monte Carlo simulation. Scenarios were generated for varying daily dose (375, 625, 750 and 875 mg), pharmacokinetic variability, exposure enhancement (EE), duration (2 and 6 months) and sample size (n=12 and 24/arm). Daily dosing of ≥ 625 mg with EE yielded desirable troughs (based on in vitro infectivity experiments) of > 2.65 ug/mL for the majority of virtual patients simulated. Results are dependent on the degree of exposure enhancement and extent of enzyme induction. Actual threshold exposure requirements for aprepitant in HIV-associated depression are unknown though preclinical evidence supports trough levels > 2.65 ug/mL. If 100% NK1r blockage is necessary for efficacy, doses of 875 mg (625 mg with EE) or higher may be required. The benefit of aprepitant on innate immunity(natural killer cells) and absence of negative effects onex vivo neutrophil chemotaxis alleviates concerns regarding drug dependent inhibition (DDI)-mediated infection risk.

Study on the treatment of acute thallium poisoning.

BACKGROUND: Acute thallium poisoning rarely occurs but is a serious and even fatal medical condition. Currently, patients with acute thallium poisoning are usually treated with Prussian blue and blood purification therapy. However, there are few studies about these treatments for acute thallium poisoning. METHODS: Nine patients with acute thallium poisoning from 1 family were treated successfully with Prussian blue and different types of blood purification therapies and analyzed. RESULTS: Prussian blue combined with sequential hemodialysis, hemoperfusion and/or continuous veno-venous hemofiltration were effective for the treatment of patients with acute thallium poisoning, even after delayed diagnosis. CONCLUSIONS: Blood purification therapies help in the clearance of thallium in those with acute thallium poisoning. Prussian blue treatment may do the benefit during this process.

Central mechanisms of menthol-induced analgesia.

Menthol is one of the most commonly used chemicals in our daily life, not only because of its fresh flavor and cooling feeling but also because of its medical benefit. Previous studies have suggested that menthol produces analgesic action in acute and neuropathic pain through peripheral mechanisms. However, the central actions and mechanisms of menthol remain unclear. Here, we report that menthol has direct effects on the spinal cord. Menthol decreased both ipsilateral and contralateral pain hypersensitivity induced by complete Freund's adjuvant in a dose-dependent manner. Menthol also reduced both first and second phases of formalin-induced spontaneous nocifensive behavior. We then identified the potential central mechanisms underlying the analgesic effect of menthol. In cultured dorsal horn neurons, menthol induced inward and outward currents in a dose-dependent manner. The menthol-activated current was mediated by Cl(-) and blocked by bicuculline, suggesting that menthol activates γ-aminobutyric acid type A receptors. In addition, menthol blocked voltage-gated sodium channels and voltage-gated calcium channels in a voltage-, state-, and use-dependent manner. Furthermore, menthol reduced repetitive firing and action potential amplitude, decreased neuronal excitability, and blocked spontaneous synaptic transmission of cultured superficial dorsal horn neurons. Liquid chromatography/tandem mass spectrometry analysis of brain menthol levels indicated that menthol was rapidly concentrated in the brain when administered systemically. Our results indicate that menthol produces its central analgesic action on inflammatory pain probably via the blockage of voltage-gated Na(+) and Ca(2+) channels. These data provide molecular and cellular mechanisms by which menthol decreases neuronal excitability, therefore contributing to menthol-induced central analgesia.

A sensitive and rapid liquid chromatography-tandem mass spectrometry method for the quantification of the novel neurokinin-1 receptor antagonist aprepitant in rhesus macaque plasma, and cerebral spinal fluid, and human plasma with application in translational NeuroAIDs research.

A sensitive and rapid liquid chromatography-tandem mass spectrometry method has been developed for to assess therapeutic exposures of aprepitant in HIV-infected patients and rhesus macaques. The method utilized a simple sample-preparation procedure of protein precipitation with methanol. Chromatographic separation was performed on a reversed phase C(8) column (Hypersil Gold, 50 mm x 2.1 mm, 3 microm) using a mobile phase composed of acetonitrile and water in 0.5% formic acid through gradient elution. Electro-spray ionization in positive mode was incorporated in the tandem mass spectrometric detection. The lower limit of quantitation of aprepitant in plasma of rhesus macaques and human and cerebral spinal fluid of rhesus macaques were 1, 1, and 0.1 ng/mL, respectively. The method has been successfully employed to measure aprepitant in preclinical and clinical samples collected from three SIV-infected rhesus macaques and ten patients with HIV infection. In conclusion, this liquid chromatography-tandem mass spectrometry method is suitable for preclinical-clinical translational research exploring exposure-response relationships with aprepitant as well as therapeutic drug monitoring of aprepitant.

Comparative in vitro metabolism of 14C-ciclesonide in hepatocytes from the mouse, rat, rabbit, dog, and human.

The in vitro metabolism of ciclesonide, a novel inhaled nonhalogenated glucocorticoid for the treatment of asthma, was compared in cryopreserved hepatocytes from mice, rats, rabbits, dogs, and humans. Incubations of C-ciclesonide with individual hepatocyte suspensions revealed similar metabolite profiles in all 5 in vitro systems used. Ciclesonide was rapidly converted to its active metabolite, desisobutyryl-ciclesonide (des-CIC). Des-CIC was then extensively metabolized to pharmacologically inactive metabolites through oxidation and reduction, followed by glucuronidation. A total of 12 groups of metabolites derived from des-CIC were characterized and identified by liquid chromatography/radioactivity monitor/mass spectrometry. Oxidation occurred on both the cyclohexane ring and the steroid moiety. Hippuric acid formation by cleavage of the cyclohexylmethyl moiety of ciclesonide, followed by aromatization of the cyclohexane ring through multiple steps of hydroxylation, dehydration, and conjugation with glycine, was found in rat, rabbit, and human hepatocyte incubations. The results indicated that ciclesonide and its active metabolite, des-CIC, were extensively metabolized in vitro in animal and human hepatocytes and that the metabolite profiles in mouse, rat, rabbit, and dog hepatocytes were similar to the profiles in human hepatocytes.

Reduction of nitriles to amines in positive ion electrospray ionization mass spectrometry.

Some compounds readily form [M+46]+ adduct ions during positive ion electrospray ionization mass spectrometry ((+)ESI-MS) analysis. These [M+46]+ ions were characterized as [M+CH3CH2NH2+H]+ by accurate mass determination. Ethylamine involved in the adduct was proposed to be the reduction product of acetonitrile and this was confirmed using deuterated acetonitrile. Other nitrile-containing compounds tested, including isobutyronitrile and benzonitrile, also formed the adduct ions of the respective amine forms under (+)ESI-MS conditions. Hydrogen/deuterium exchange experiments demonstrated that the reductive hydrogen originated from water. Reduction of nitriles (R-CN) to their respective amines (R-CH2NH2) under (+)ESI-MS conditions expands the ability to identify nitrile-containing chemical unknowns.

Accurate mass filtering of ion chromatograms for metabolite identification using a unit mass resolution liquid chromatography/mass spectrometry system.

Acceleration of liquid chromatography/mass spectrometric (LC/MS) analysis for metabolite identification critically relies on effective data processing since the rate of data acquisition is much faster than the rate of data mining. The rapid and accurate identification of metabolite peaks from complex LC/MS data is a key component to speeding up the process. Current approaches routinely use selected ion chromatograms that can suffer severely from matrix effects. This paper describes a new method to automatically extract and filter metabolite-related information from LC/MS data obtained at unit mass resolution in the presence of complex biological matrices. This approach is illustrated by LC/MS analysis of the metabolites of verapamil from a rat microsome incubation spiked with biological matrix (bile). MS data were acquired in profile mode on a unit mass resolution triple-quadrupole instrument, externally calibrated using a unique procedure that corrects for both mass axis and mass spectral peak shape to facilitate metabolite identification with high mass accuracy. Through the double-filtering effects of accurate mass and isotope profile, conventional extracted ion chromatograms corresponding to the parent drug (verapamil at m/z 455), demethylated verapamil (m/z 441), and dealkylated verapamil (m/z 291), that contained substantial false-positive peaks, were simplified into chromatograms that are substantially free from matrix interferences. These filtered chromatograms approach what would have been obtained by using a radioactivity detector to detect radio-labeled metabolites of interest.

[Improvement of suture technique for arteriovenous fistula].

OBJECTIVE: To investigate the clinical effect of continuous suture technique for closure of surgically created arteriovenous fistulas at different sites. METHOD: A total of 160 patients with chronic renal failure underwent surgery to create arteriovenous fistulas at different sites, which were closed with continuous suture technique. RESULTS: The time for vessel anastomosis was reduced with this suture technique, and the success rate of anastomosis at one time and the overall operative success rate were improved, with also reduction of complications to achieve satisfactory clinical results. CONCLUSION: Continuous suture technique well suits the demand in anastomosis of arteriovenous fistula at different sites, especially in cases with poor vascular condition.

[Transfection of human umbilical vein endothelial cell line ECV-304 with liposome-oligonucleotide complexes under hypothermic and anoxic conditions].

OBJECTIVE: To investigate the transfection efficiency of nuclear factor (NF)-kappaB decoy oligodeoxynucleotides (ODN) mediated by in vivo liposome in human umbilical vascular endothelial cell line ECV-304 under hypothermic and anoxic conditions. METHODS: ECV-304 cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum at 37 degrees Celsius; in the presence of 5% CO2. Liposome-ODN complexes were prepared just before use and added to the cells with a liposome-ODN charge ratio of 2:1. ECV-304 cell monolayers were transfected with liposome-ODN complexes containing 0.50, 0.75, 1.00 and 1.25 micromol/L ODN respectively in Euro-Collins solution (ECs) at 4 degrees celsius; and then stored for 2, 4, 6 and 8 hours respectively under anoxic condition. The ODN without liposome was transfected into ECV-304 cells under identical conditions as the control. The distribution of fluorescein isothiocyanate (FITC)-labeled ODN in ECV-304 cells was observed by fluorescence microscope, and the transfection efficiency and mean fluorescence intensity (MFI) were evaluated by flow cytometry. RESULTS: MFI was enhanced as the storage time extended and ODN concentration increased, reaching the peak level at 6 h (P<0.05). After a 6-hour storage, most of the ODN was found to locate in the cell nuclei, and the transfection efficiency did not vary significantly between the groups. Compared with the control group, however, the differences in transfection efficiency and MFI were significant. CONCLUSION: ODN can be highly efficiently transfected into ECV-304 cells by in vivo liposome in ECs under hypothermic and anoxic conditions, which provides an experimental basis for further study of the donor organ preservation at the level of genetic regulation.

Pyrrolizidine alkaloid clivorine inhibits human normal liver L-02 cells growth and activates p38 mitogen-activated protein kinase in L-02 cells.

Clivorine, a pyrrolizidine alkaloid extracted from Chinese medicinal plant Ligularia hodgsonii Hook significantly inhibited human normal liver L-02 cells proliferation and decreased L-02 cells viability. The results of western blot showed that clivorine strongly evoked phosphorylation of p38 mitogen-activated protein (MAP) Kinase in L-02 cells, but had no effect on extracellular signal-related kinases MAP Kinase phosphorylation. Moreover, another pyrrolizidine alkaloid monocrotaline had no effect on phosphorylation of p38 MAP Kinase in L-02 cells. These studies document the effects of pyrrolizidine alkaloid clivorine on the MAPK cascade and on the growth of human normal liver L-02 cells for the first time, which may be a possible reason for the toxic effects observed in those plants containing pyrrolizidine alkaloids.