G Protein-Coupled Receptor Kinase 2 Is Required for Rhythmic Olfactory Responses in Drosophila

SummaryBackgroundThe Drosophila circadian clock controls rhythms in the amplitude of odor-induced electrophysiological responses that peak during the middle of night. These rhythms are dependent on clocks in olfactory sensory neurons (OSNs), suggesting that odorant receptors (ORs) or OR-dependent processes are under clock control. Because responses to odors are initiated by heteromeric OR complexes that form odor-gated and cyclic-nucleotide-activated cation channels, we tested whether regulators of ORs were under circadian-clock control.ResultsThe levels of G protein-coupled receptor kinase 2 (Gprk2) messenger RNA and protein cycle in a circadian-clock-dependent manner with a peak around the middle of the night in antennae. Gprk2 overexpression in OSNs from wild-type or cyc01 flies elicits constant high-amplitude electroantennogram (EAG) responses to ethyl acetate, whereas Gprk2 mutants produce constant low-amplitude EAG responses. ORs accumulate to high levels in the dendrites of OSNs around the middle of the night, and this dendritic localization of ORs is enhanced by GPRK2 overexpression at times when ORs are primarily localized in the cell body.ConclusionsThese results support a model in which circadian-clock-dependent rhythms in GPRK2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses. The enhancement of OR function by GPRK2 contrasts with the traditional role of GPRKs in desensitizing activated receptors and suggests that GPRK2 functions through a fundamentally different mechanism to modulate OR activity

CLOCK expression identifies developing circadian oscillator neurons in the brains of Drosophila embryos

<p>Abstract</p> <p>Background</p> <p>The <it>Drosophila </it>circadian oscillator is composed of transcriptional feedback loops in which CLOCK-CYCLE (CLK-CYC) heterodimers activate their feedback regulators <it>period </it>(<it>per</it>) and <it>timeless </it>(<it>tim</it>) via E-box mediated transcription. These feedback loop oscillators are present in distinct clusters of dorsal and lateral neurons in the adult brain, but how this pattern of expression is established during development is not known. Since CLK is required to initiate feedback loop function, defining the pattern of CLK expression in embryos and larvae will shed light on oscillator neuron development.</p> <p>Results</p> <p>A novel CLK antiserum is used to show that CLK expression in the larval CNS and adult brain is limited to circadian oscillator cells. CLK is initially expressed in presumptive small ventral lateral neurons (s-LN_vs), dorsal neurons 2 s (DN₂s), and dorsal neuron 1 s (DN₁s) at embryonic stage (ES) 16, and this CLK expression pattern persists through larval development. PER then accumulates in all CLK-expressing cells except presumptive DN₂s during late ES 16 and ES 17, consistent with the delayed accumulation of PER in adult oscillator neurons and antiphase cycling of PER in larval DN₂s. PER is also expressed in non-CLK-expressing cells in the embryonic CNS starting at ES 12. Although PER expression in CLK-negative cells continues in <it>Clk</it>^Jrkembryos, PER expression in cells that co-express PER and CLK is eliminated.</p> <p>Conclusion</p> <p>These data demonstrate that brain oscillator neurons begin development during embryogenesis, that PER expression in non-oscillator cells is CLK-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons. These results define the temporal and spatial coordinates of factors that initiate <it>Clk </it>expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that PER functions in a different capacity before oscillator cell development is initiated.</p

DOUBLETIME Plays a Noncatalytic Role To Mediate CLOCK Phosphorylation and Repress CLOCK-Dependent Transcription within the Drosophila Circadian Clock

Circadian clocks keep time via gene expression feedback loops that are controlled by time-of-day-specific changes in the synthesis, activity, and degradation of transcription factors. Within the Drosophila melanogaster circadian clock, DOUBLETIME (DBT) kinase is necessary for the phosphorylation of PERIOD (PER), a transcriptional repressor, and CLOCK (CLK), a transcriptional activator, as CLK-dependent transcription is being repressed. PER- and DBT-containing protein complexes feed back to repress CLK-dependent transcription, but how DBT promotes PER and CLK phosphorylation and how PER and CLK phosphorylation contributes to transcriptional repression have not been defined. Here, we show that DBT catalytic activity is not required for CLK phosphorylation or transcriptional repression and that PER phosphorylation is dispensable for repressing CLK-dependent transcription. These results support a model in which DBT plays a novel noncatalytic role in recruiting additional kinases that phosphorylate CLK, thereby repressing transcription. A similar mechanism likely operates in mammals, given the conserved activities of PER, DBT, and CLK orthologs

Vertical distribution of microbial and meiofaunal populations in sediments of a natural coastal hydrocarbon seep

We studied the vertical distribution of microbes and meiofauna in natural hydrocarbon seep sediments to determine if there was a relationship between profiles of benthic trophic structure and the unique biogeochemical conditions present at the seep. Three stations in the Santa Barbara Channel represented a gradient of natural petroleum seepage, from very active, to moderate, to none. Seasonal differences were examined by sampling in the three major oceanographic seasons, upwelling (April), mixed (July), and Davidson (December). Densities of microbes and meiofauna were highest in July, and decreased in winter. All population sizes decreased with increasing depth in the sediment. Harpacticoids and Chl a were practically restricted to the surface sediments. Harpacticoids and Chl a were more dense (number per unit volume or strata of sediment) and abundant (number per unit area of sediment or sum of the strata) at the comparison site than at the seep sites. Density and abundance of nematodes, bacteria cell counts, and bacterial biomass were greater at the station with the most active seepage rates. Bacterial biovolumes appeared constant among sediment depths and stations, but cell biovolumes were larger in July. The data are consistent with the hypothesis that organic enrichment via petroleum utilization is responsible for increased abundances of bacteria and nematodes at the seep. There were strong correlations between densities of harpacticoids and microalgae, and densities of nematodes and bacteria. These links indicate that seeping petroleum might have an enhanced effect on the detrital (bacterial based) food web, but a toxic effect on the grazing (microalgal based) food web

Temporal variability and the relationship between benthic meiofaunal and microbial populations of a natural coastal petroleum seep

Previous studies of the Isla Vista petroleum seep in the Santa Barbara Channel found much higher abundances of macrofauna and concentrations of adenosine triphosphate (ATP) in sediments near petroleum seepage compared to those from nonseep areas. To further assess the possible effect of petroleum on organisms at the base of benthic food webs, population abundances of meiobenthos and their suspected microbial food (bacteria and diatoms) were measured biweekly for one year at three stations with differing petroleum exposure. Determinations of suspended particulate matter and the abundance and gut contents of juvenile fishes were also made at seep and nonseep stations. Nematodes and bacteria had higher abundances in areas of active petroleum seepage than in areas of moderate seepage (within 20 m) or no seepage (1.4 km away). Bacterial productivity (based on the frequency of dividing cells) was 340% greater in sediments from areas of active seepage compared to those from a nonseep station. Sediments within the seep, but away from active seepage, had rates of bacterial productivity 15 times greater than a nonseep comparison site. Densities of harpacticoid copepods and their probable principal food, diatoms, were not affected by petroleum seepage. Suspended organic matter caught in settling traps was not different between seep and nonseep stations. In addition, there was no evidence that predation pressure by juvenile fish on meiofauna was different between stations. The higher bacterial biomass and productivity in areas of petroleum seepage are consistent with the hypothesis that petroleum carbon is available for assimilation by sediment bacteria. The enhanced level of microbial carbon associated with the petroleum seep is available for consumption by benthic invertebrates and could explain the higher abundances of macrofauna and meiofauna found there

Proceedings of the scientific visit on crystalline rock repository development.

A scientific visit on Crystalline Rock Repository Development was held in the Czech Republic on September 24-27, 2012. The visit was hosted by the Czech Radioactive Waste Repository Authority (RAWRA), co-hosted by Sandia National Laboratories (SNL), and supported by the International Atomic Energy Agency (IAEA). The purpose of the visit was to promote technical information exchange between participants from countries engaged in the investigation and exploration of crystalline rock for the eventual construction of nuclear waste repositories. The visit was designed especially for participants of countries that have recently commenced (or recommenced) national repository programmes in crystalline host rock formations. Discussion topics included repository programme development, site screening and selection, site characterization, disposal concepts in crystalline host rock, regulatory frameworks, and safety assessment methodology. Interest was surveyed in establishing a %E2%80%9Cclub,%E2%80%9D the mission of which would be to identify and address the various technical challenges that confront the disposal of radioactive waste in crystalline rock environments. The idea of a second scientific visit to be held one year later in another host country received popular support. The visit concluded with a trip to the countryside south of Prague where participants were treated to a tour of the laboratory and underground facilities of the Josef Regional Underground Research Centre

Granite disposal of U.S. high-level radioactive waste.

This report evaluates the feasibility of disposing U.S. high-level radioactive waste in granite several hundred meters below the surface of the earth. The U.S. has many granite formations with positive attributes for permanent disposal. Similar crystalline formations have been extensively studied by international programs, two of which, in Sweden and Finland, are the host rocks of submitted or imminent repository license applications. This report is enabled by the advanced work of the international community to establish functional and operational requirements for disposal of a range of waste forms in granite media. In this report we develop scoping performance analyses, based on the applicable features, events, and processes (FEPs) identified by international investigators, to support generic conclusions regarding post-closure safety. Unlike the safety analyses for disposal in salt, shale/clay, or deep boreholes, the safety analysis for a mined granite repository depends largely on waste package preservation. In crystalline rock, waste packages are preserved by the high mechanical stability of the excavations, the diffusive barrier of the buffer, and favorable chemical conditions. The buffer is preserved by low groundwater fluxes, favorable chemical conditions, backfill, and the rigid confines of the host rock. An added advantage of a mined granite repository is that waste packages would be fairly easy to retrieve, should retrievability be an important objective. The results of the safety analyses performed in this study are consistent with the results of comprehensive safety assessments performed for sites in Sweden, Finland, and Canada. They indicate that a granite repository would satisfy established safety criteria and suggest that a small number of FEPs would largely control the release and transport of radionuclides. In the event the U.S. decides to pursue a potential repository in granite, a detailed evaluation of these FEPs would be needed to inform site selection and safety assessment

A Role for the Adult Fat Body in Drosophila Male Courtship Behavior

Mating behavior in Drosophila depends critically on the sexual identity of specific regions in the brain, but several studies have identified courtship genes that express products only outside the nervous system. Although these genes are each active in a variety of non-neuronal cell types, they are all prominently expressed in the adult fat body, suggesting an important role for this tissue in behavior. To test its role in male courtship, fat body was feminized using the highly specific Larval serum protein promoter. We report here that the specific feminization of this tissue strongly reduces the competence of males to perform courtship. This effect is limited to the fat body of sexually mature adults as the feminization of larval fat body that normally persists in young adults does not affect mating. We propose that feminization of fat body affects the synthesis of male-specific secreted circulating proteins that influence the central nervous system. In support of this idea, we demonstrate that Takeout, a protein known to influence mating, is present in the hemolymph of adult males but not females and acts as a secreted protein