Circadian locomotor behaviour is controlled by a pacemaker circuit composed of clock-containing neurons. To interrogate the mechanistic relationship between the molecular clockwork and network communication critical to the operation of the Drosophila circadian pacemaker circuit, we established new fluorescent circadian reporters that permit single-cell recording of transcriptional and post-transcriptional rhythms in brain explants and cultured neurons. Live-imaging experiments combined with pharmacological and genetic manipulations demonstrate that the neuropeptide pigment-dispersing factor (PDF) amplifies the molecular rhythms via time-of-day- and activity-dependent upregulation of transcription from E-box-containing clock gene promoters within key pacemaker neurons. The effect of PDF on clock gene transcription and the known role of PDF in enhancing PER/TIM stability occur via independent pathways downstream of the PDF receptor, the former through a cAMP-independent mechanism and the latter through a cAMP-PKA dependent mechanism. These results confirm and extend the mechanistic understanding of the role of PDF in controlling the synchrony of the pacemaker neurons. More broadly, our results establish the utility of the new live-imaging tools for the study of molecular-neural interactions important for the operation of the circadian pacemaker circuit.
Orchestrated action of genetic programs and neuronal activity determines the emergent output of a neural circuit. The control of circadian behavioural rhythms is a prime example of this orchestration. Networks of dedicated pacemaker neurons, each containing a molecular clock, produce synchronized daily rhythms in gene expression and electrical activity, thereby driving coherent rhythmic behaviour. Owing to its numerically simpler nervous system and a battery of genetic tools, Drosophila offers an unusually powerful model to dissect the molecular and neural mechanisms of circadian behaviour.
The molecular clock of Drosophila consists of interlocked transcriptional-translational feedback loops, as in vertebrates. In the core feedback loop of the Drosophila clock, transcription factors CLOCK (CLK) and CYCLE (CYC) heterodimerize and activate transcription of period (per) and timeless (tim) through binding to their upstream and intronic E-boxes. Following the translation of per and tim mRNAs, PER and TIM translocate into the nucleus and inhibit CLK/CYC-mediated transcription. The CLK/CYC heterodimer also rhythmically activates transcription of a large number of output genes by binding rhythmically to their E-boxes, thereby controlling circadian rhythms in cellular functions1. Along with these transcriptional mechanisms, multiple post-transcriptional and post-translational mechanisms, particularly the post-translational modifications of PER that control its timed nuclear localization and degradation, are critical for the generation of circa 24-hr period rhythms2.
The circadian pacemaker circuit is composed of approximately 150 clock-containing neurons in the brain and controls the circadian locomotor rhythms of adult flies. Clock neurons are classified into 7 major subgroups: two classes of ventral lateral neurons (small-ventral lateral neurons, s-LNvs; large-ventral lateral neurons, l-LNvs), dorsal lateral neurons (LNds), lateral posterior neurons (LPNs), and three classes of dorsal neurons (DN1s, DN2s, and DN3s). Four out of five s-LNvs and all the l-LNvs express the neuropeptide Pigment-dispersing factor (PDF). The larval circadian circuit is composed of three classes of clock neurons (LNvs, DN1s and DN2s) and controls circadian light response behavior3,4. Larval PDF-positive LNvs are identical to the adult PDF-positive s-LNvs, which are the master pacemakers that control circuit synchrony and coordinate behavioural output5.
Previous studies have demonstrated the contribution of neuronal excitability and network interactions to rhythm generation in Drosophila pacemaker clock neurons. Similar to what is observed in the mammalian circadian pacemaker neurons located in the suprachiasmatic nuclei (SCN)6,7, continuous neuronal silencing and blockage of synaptic transmission do not abolish the molecular clockwork in Drosophila clock neurons, although these manipulations impair behavioural output8,9. A number of studies have shown that PDF plays critical roles in controlling synchrony in clock neurons and behavioural rhythms, analogous to the roles of vasoactive intestinal peptide (VIP) in the SCN10,11,12. PDF acts on multiple clock neuron subtypes through possibly diverse and cell type-specific mechanisms of action, e.g., controlling the phase, amplitude or pace of molecular clocks13,14,15,16,17,18,19. A recent study also demonstrated that PDF is required to control the phase of clock neuron output20. However, despite these advances in characterizing the role of PDF signalling in the circadian circuit, a mechanistic understanding of the precise interplay between the molecular clock and PDF signalling requires further investigation. Other neuropeptides and neurotransmitters are also involved in circuit synchronization and the coordination of behavioural output21,22; however, their impact on the molecular clockwork remains largely uncharacterized.
Precise mechanistic analysis of the interplay between network interactions and the molecular clockwork requires a system that enables the manipulation of network communication while simultaneously monitoring the dynamics of the molecular clock. To this end, here we develop two novel fluorescent reporters that permit real-time recording of the transcriptional and post-transcriptional machineries of the Drosophila clock in cultured neurons and whole brains. Using these new tools combined with genetic and pharmacological manipulations, we demonstrate that PDF enhances CLK/CYC-mediated transcription cell-autonomously at night via an activity-dependent, cAMP-independent mechanism. Our results confirm and extend the role of PDF in the molecular clock and more broadly demonstrate the utility of the novel live-imaging tools for investigating the molecular-neural interactions important for the operation of the circadian pacemaker circuit.
Transcriptional activation from E-box-containing promoters and the feedback transcriptional inhibition by PER comprise the essential core loop of the molecular clock. Post-transcriptional regulatory features that control the stability, activity and subcellular localization of PER are also critical for the generation of circa 24-hr period rhythms23. Therefore, to monitor the functioning of the transcriptional and post-transcriptional machinery of the molecular clock at single-cell resolution, we generated two fluorescent reporters: one to monitor CLK/CYC transcriptional activity rhythms and the other to follow PER protein levels and localization (Fig. 1a).
Figure 1: Novel fluorescent reporters of CLK/CYC-mediated transcription and PER protein oscillations.
(a) Transcriptional (3 × 69-VNP) and PER protein (per-TdT) reporters. (b) 3 × 69-VNP and PER-TDT expression in third instar larval brains dissected every 4 hr in LD cycles. Representative confocal images of the LNvs are shown. Clock neurons in the 3 × 69-VNP line were labelled by 1982clk-gal4, UAS-mCD8::RFP. The LNvs in the per-TdT flies were labelled by gal1118-GAL4, UAS-mCD8::VENUS. Scale bars are 5 μm. (c) Quantification of the reporter fluorescence intensity in clock neurons. Mean ± SEM. A.U., arbitrary unit. A minimum of 30 LNvs, 17 DN1s and 11 DN2s were analysed at each timepoint. (d) Nuclear/cytoplasmic fluorescence ratio of PER-TDT in adult and larval LNvs.
The CLK/CYC transcriptional activity reporter 3 × 69-VNP expresses short-lived yellow fluorescent protein fused to a nuclear localization signal (VENUS-NLS-PEST, VNP)24 under the control of three tandem repeats of the clock regulatory sequence (CRS), which is the E-box-containing 69-bp enhancer of per. The CRS is necessary and sufficient for per-like spatial and temporal transcriptional expression25, and the CRS trimer has been shown to drive CLK/CYC-dependent, high-intensity and high-amplitude circadian reporter expression in vivo26. The PER protein reporter per-TdT was modified from the previously established BG-luc reporter27 and was designed to express the N-terminal two-thirds of PER fused with tandem TOMATO (TdT) red fluorescent protein, under the control of per regulatory sequences and the 3′UTR. The N-terminal PER moiety harbours most of the known functional domains, including two PER-ARNT-SIM (PAS) domains and an NLS sequence, but it lacks the CLK/CYC inhibition domain and thus does not replace endogenous PER function27.
As expected, both molecular clock reporters were expressed rhythmically in adult and larval brain clock neurons (Fig. 1b,c, and S1a–d). A detailed analysis of the simple circadian circuit of third-instar larvae revealed that 3 × 69-VNP peaked between ZT14 (2 hr after lights off under a 12 hr:12 hr light-dark (LD) cycle) and ZT18, which corresponds approximately to the peak phases of per mRNA accumulation, CLK/CYC binding to per E-boxes, and genome-wide CLK/CYC binding1,28,29. PER-TDT peaked approximately 8 hr after the 3 × 69-VNP peak in the LNvs and DN1s, recapitulating the characteristic lag between per mRNA and protein accumulation29. PER-TDT rhythms in the DN2s were anti-phasic to the LNvs and DN1s, which is consistent with endogenous PER cycling30 (Fig. 1c and S1a). Furthermore, the cycling profile of PER-TDT nuclear-cytoplasmic localization was identical to that of PER (Fig. 1d)31. The cycling expression of both reporters and the rhythms in PER-TDT nuclear/cytoplasmic ratio were largely maintained in DD (Fig. S1c and d). These results establish that the 3 × 69-VNP and per-TdT reporters provide reliable readouts of the molecular clockwork at a single-cell resolution; 3 × 69-VNP reports CLK/CYC-mediated transcriptional rhythms and also mimics per mRNA rhythms, whereas PER-TDT mimics PER protein oscillations.
Next, we tested whether our new reporters can be also used for monitoring rhythms in live preparations. Cultured whole brains of adult flies have been shown to generate rhythms in luciferase reporter activity in a tissue-autonomous manner32,33,34. We chose to use cultured third-instar larval brains for live imaging, as larval circadian circuits are numerically simpler yet composed of fully differentiated and functional clock neurons35. As expected, clock neurons in cultured larval brains displayed circadian rhythms in 3 × 69-VNP and PER-TDT reporter expression, although with a longer average period (~29 hr) (Fig. 2a–c, Movies S1 and S2). Each neuron expressed varying levels of fluorescence; thus, not all the neurons were detected. Among the detected neurons, 57% of the LNvs, 50% of DN1s and 76% of DN2s showed circadian rhythms in 3 × 69-VNP levels, and 75% of the LNvs showed rhythmic PER-TDT expression. Since the long periods were likely due to the combination of the longer endogenous rhythms of the reporter flies (free-running locomotor rhythms of 1982Clk > mCD8::RFP, 3 × 69-VNP flies: 25.1 ± 0.13 hr; gal1118 > mCD8::Venus, per-TdT flies: 25.3 ± 0.08 hr), the 3 hr temporal resolution and culture conditions, we limited the use of brain explants to relatively short (<8 hr) fluorescent live-imaging at 1 hr intervals in subsequent experiments.
Figure 2: Fluorescent reporter expression in clock neurons in cultured larval brains.
Whole-brain explants were prepared from LD-entrained larvae at ZT11 and then cultured and imaged in constant darkness (DD). Images were taken every 3 hr for at least 48 hr from ZT18 of the last LD cycle. In 3 × 69-VNP flies, all 3 larval clock neuron subtypes were labelled by driving expression of UAS-mCD8::RFP with 1982clk-gal4 and analysed. PER-TDT expression was monitored only in the LNvs labelled with gal1118, UAS-mCD8::Venus due to the technical difficulties establishing a stable transgenic line expressing both per-TdT and a pan-clock neuron driver. Relative fluorescence intensity measurements normalized to the value at t = 0 are shown separately for each clock neuron subtype. Representative single-cell traces of rhythmic cells are shown with 6th order polynomial trend lines (red dotted lines). τ indicates the period. (a) 3 × 69-VNP expression in the LNvs (n = 11), DN1s (n = 15) and DN2s (n = 8). (b) PER-TDT expression in the LNvs (n = 17). (c) Periods of the rhythmic neurons in brain explants. Mean period ± SEM of 3 × 69-VNP was 29.27 ± 0.91 hr, PER-TDT was 28.5 ± 1.38 hr.
The neuropeptide PDF mediates normal circadian locomotor rhythms by affecting the synchrony, amplitude and pace of the molecular clocks15,16,18,19 and by coordinating the phase of pacemaker neuron activity20. PDF activates adenylate cyclase and increases cAMP levels in PDF receptor (PDFR)-expressing neurons36,37,38. The rise in cAMP levels activates protein kinase A (PKA) and stabilizes TIM and PER14,17, which contributes to the phase resetting and speed control of the molecular clocks. PDF also enhances neuronal activity in a cell-autonomous manner36,39,40. Importantly, the stabilization of TIM via PDF signalling is activity-independent; therefore, PDF/PDFR signalling at least bifurcates into the pathway controlling neuronal excitability and the one controlling PER/TIM stability17. However, the possible effects of PDF signalling on clock gene transcription have not been directly examined.
To better dissect the effect of PDF signalling on the clockwork, we monitored 3 × 69-VNP and PER-TDT expression in larval brain explants following a bath application of PDF. LD-entrained larvae were dissected at ZT1 or ZT13 and immediately subjected to live imaging in DD. PDF was applied at ZT2 or ZT14 (i.e., 1 hr after the start of live imaging). PDF application at ZT2 did not alter 3 × 69-VNP or PER-TDT expression in any larval clock neuron subtype. In contrast, PDF application at ZT14 upregulated 3 × 69-VNP and PER-TDT levels in the LNvs and DN1s. PDF application did not detectably affect reporter expression in the DN2s (Fig. 3a and b). Reporter expression levels continued to rise in the LNvs and DN1s during the course of imaging following the application of PDF at ZT14. This is consistent with the previous finding by Klose et al. that signalling downstream of PDFR continues to be active as long as PDF is present in the medium in ex vivo preparations41. As the addition of PDF had no effect on the expression profiles of 3 × 69-VNP and PER-TDT in cultures prepared from the pdfr hypomorphic mutant larvae, even at ZT14, the effect of PDF is specific and occurs via PDFR (Fig. S2a). Furthermore, PDF upregulated 3 × 69-VNP and PER-TDT levels at ZT14 but not at ZT2 also in pdf01 mutants (Fig. S3). These results indicate that PDF upregulates CLK/CYC-mediated transcription and PER levels in the LNvs and DN1s at night but not during the day, and this nighttime-specific response of the molecular clock is controlled independently of the timing of PDF release.
Figure 3: Time-of-day-dependent modulation of the molecular clockwork by PDF.
LD-entrained larval brains were dissected and mounted at ZT1 or ZT13, and PDF (2 μM), DMSO or PDF (2 μM) + TTX (100 nM) was applied at ZT2 or ZT14. 3 × 69-VNP (a) and PER-TDT expression (b) in brain explants were monitored every hour by time-lapse microscopy. The mean fluorescence intensity ± SEM normalized to the value at the start of imaging (corresponding to ZT1 or ZT13) is shown. The red arrows indicate the time of drug application (ZT2 or ZT14). The colour of the asterisk indicates the group compared with the Vehicle-added group. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by two-way ANOVA with Sidak's correction for multiple comparisons. A minimum of 32 LNvs, 18 DN1s and 16 DN2s were analysed for each data point.
To test whether neuronal excitability is involved in the reporter upregulation induced by PDF at ZT14, we co-applied the voltage-gated sodium channel blocker tetrodotoxin (TTX) and PDF to the brain explants. PER-TDT upregulation by PDF was insensitive to TTX (Fig. 3b). This finding parallels the observation that PDF stabilizes TIM through a mechanism independent of neuronal activity17. In contrast, TTX blocked the PDF-mediated upregulation of 3 × 69-VNP (Fig. 3a), indicating that PDF affects CLK/CYC-mediated transcription via time-of-day- and activity-dependent mechanisms.
To further test if the upregulation of PER-TDT by PDF is mediated by PKA activation14, we applied the cAMP analogue Sp-adenosine-3′, 5′- cyclic monophosphorothioate triethyamine (Sp-cAMPS), which specifically activates PKA42, to the larval brain explants at ZT14. Sp-cAMPS did not affect 3 × 69-VNP levels but significantly increased PER-TDT levels (Fig. 4), consistent with the role of cAMP-PKA signalling in PER stabilization14. Taken together, these results demonstrate that PDF/PDFR-signalling modulates the molecular clockwork specifically at night via two pathways: one pathway involving an increase in CLK/CYC-mediated transcription in an activity-dependent mechanism and the other involving enhancement of PER stability through cAMP-PKA activation, independent of neuronal excitability.
Figure 4: PKA activity enhances PER stability without affecting CLK/CYC-mediated transcription.
LD-entrained larval brains were dissected and mounted at ZT13, and PKA activator Sp-cAMPS (150 μM) or H2O (vehicle) was applied at ZT14. Mean fluorescence intensities ± SEM of 3 × 69-VNP (a) and PER-TDT (b) normalized to the value at the start of imaging (ZT13) are shown. A minimum of 44 LNvs, 44 DN1s and 46 DN2s for PER-TDT and 64 LNvs, 28 DN1s and 38 DN2s for 3 × 69-VNP were analysed. The red arrows indicate the time of drug application. The colour of the asterisk indicates the group compared with the Vehicle-added group. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by two-way ANOVA with Sidak's correction for multiple comparisons.
TTX blocks spontaneous firing generated by cell-intrinsic mechanisms as well as the action potentials triggered by synaptic inputs43,44. To distinguish between these two possibilities, we monitored 3 × 69-VNP expression in dissociated cultured neurons in real time. Cultures were prepared from LD-entrained larvae expressing 3 × 69-VNP and the clock neuron marker (1982clk-gal4, UAS-mCD8::RFP) at approximately ZT11, then incubated for two days in DD and then subjected to time-lapse imaging starting at CT18. The cultured neurons were morphologically intact, and their neurites continued to grow and elaborate as previously shown45, even during the time-lapse imaging (Fig. S2b).
All of the 1982clk > mCD8::RFP clock neuron marker-positive cells co-expressed 3 × 69-VNP; however, 3 × 69-VNP levels showed steady increases over time without detectable rhythms. Remarkably, the addition of PDF caused a significant further increase in 3 × 69-VNP levels (Movie S3, Fig. 5a and d). The addition of PDF had no significant effect on 3 × 69-VNP expression in the culture prepared from the pdfr mutant larvae (Fig. 5b and e), confirming that the effect of PDF in vitro is specific and via PDFR, as in brain culture. Furthermore, TTX or the inhibitory neurotransmitter GABA completely inhibited the upregulation of 3 × 69-VNP levels in response to acute PDF treatment (Fig. 5c and d). These results indicate that PDF upregulates CLK/CYC-mediated transcription by a cell-autonomous, activity-dependent mechanism.
Figure 5: PDF cell-autonomously upregulates CLK/CYC-mediated transcription by an activity-dependent mechanism.
3 × 69-VNP expression in cultured clock neurons in response to various drug treatments. Graphs and heatmaps show the relative fluorescence intensity time course of single cells normalized to the value at t = 0. Dark red indicates values exceeding the uppermost colour scale of the heatmap. The red arrows indicate the time of drug application. (a) Wild-type clock neurons after the addition of PDF (20 μM) or vehicle at t = 0. (b) Clock neurons in pdfr mutants with PDF or vehicle treatment. (c) Wild-type clock neurons after the addition of TTX (100 nM) or GABA (10 μM), with or without PDF (20 μM) at t = 0. (d) Average 3 × 69-VNP fluorescence levels in wild-type clock neurons with the addition of different drugs. (e) Average 3 × 69-VNP levels in pdfr clock neurons treated with PDF or vehicle. Error bar, SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by two-way ANOVA with Sidak's correction for multiple comparisons.
Bioluminescence-based circadian reporters allow non-invasive, long-term recording with a high temporal resolution and have been successfully used to monitor rhythmicity in real time32,33,34. In contrast, fluorescent reporters can achieve the higher spatial resolution necessary to distinguish individual cells in a cluster and even to characterize intracellular protein dynamics. Using two new fluorescent reporters of the Drosophila molecular clock, we were able to separately interrogate two important aspects of the clockwork: CLK/CYC-mediated transcription from E-boxes and PER intracellular dynamics, both at a single-cell resolution and in real time.
Our study confirms and extends the mechanistic links between network interactions and molecular clockworks, particularly those mediated by PDF signalling. PDF has been shown to trigger multiple downstream effects even within the same neuron. We showed that PDF bath application upregulates PER-TDT in whole-brain cultures, consistent with previous findings indicating that PDF stabilizes PER and TIM via PKA activation independently of neuronal activity14,17. In addition, we demonstrated for the first time that PDF also enhances CLK/CYC-mediated transcription through a cell-autonomous, activity-dependent but cAMP-PKA signalling-independent mechanism. Furthermore, the effect of PDF on PER stability and clock gene transcription occurs at night but not during daytime (Figs 3,4 and 6).
Figure 6: Model of the interplay between the molecular clockwork and PDF signalling.
Nighttime-specific role of PDF in the LNv and DN1 clock neurons in the pacemaker circuit. At night, PDF depolarizes the membrane potential and leads to the upregulation of CLK/CYC-mediated transcription. PDF also promotes PER stability in a manner independent of neuronal activity via cAMP-PKA signalling. This nighttime-specific action of PDF on the molecular clock can amplify molecular oscillations and contribute to the synchrony of the pacemaker neurons. Dotted arrows indicate possible indirect controls.
PDF signalling occurs via both cAMP-dependent and cAMP-independent pathways in cockroach circadian pacemaker neurons to increase the intracellular Ca2+ concentration40. Similarly, we demonstrated here the presence of cAMP-independent pathway in Drosophila PDF signalling, which leads to the activation of clock gene transcription (Fig. 4a). Activity-dependent gene expression triggered by increases in Ca2+ concentration is a widespread phenomenon in the nervous system46. Although it is beyond the scope of this study to identify the mechanisms by which PDF leads to the activity-dependent (TTX-sensitive) upregulation of CLK/CYC-transcription, we speculate that Ca2+ influx downstream of PDF/PDFR signalling leads to an upregulation of clock gene transcription. As implied by Agrawal et al.47, PDFR-dependent increase in Ca2+ concentration may be mediated by IP3/Ca2+ signalling.
How does PDF confer its time-of-day-dependent effects on the molecular clock? PDF rhythmically accumulates at the s-LNv dorsal termini with a peak around ZT248, and is predicted to be released at a higher rate at ZT2 than at ZT1449. However, our observation of ZT14-specific reporter upregulation even in the absence of endogenous PDF (Fig. S3) indicates that intracellular timing mechanisms (but not the rhythms in PDF availability) gate the molecular clock transcriptional and post-transcriptional responses to PDF. Rhythms in PDFR expression might be a possible mechanism of time-of-day-dependent PDF response, although the precise daily patterns of PDFR expression are yet to be characterized. Klose et al. showed that the sensitivity of the s-LNvs to PDF, measured based on the cAMP concentration, exhibits daily rhythms and peaks at dawn41. This suggests that cAMP signalling is permissive for the PER stabilization effect of PDF even at its nadir and the mechanisms downstream of cAMP production gate the PER stabilization effect at night.
The electrical activity of clock neurons is rhythmic and highest from the late night to early morning49,50,51. As intracellular Ca2+ levels in the s-LNvs and DN1s peak at approximately the same time20, rhythms in neuronal excitability and Ca2+ may gate the transcriptional response to PDF. We showed that TTX inhibits the nighttime-specific upregulation of clock gene transcription via PDF in brain explant cultures (Fig. 3a). These results suggest the possibility that synaptic inputs to the LNvs and DN1s control the neuronal activity relevant for gating the transcriptional response to PDF. On the other hand, PDF addition to dissociated neurons uniformly triggered TTX-sensitive transcriptional upregulation despite the lack of rhythms (Fig. 5a,c and d). This result suggests that the transcriptional upregulation induced by PDF may be the default state, i.e., that the clock may negatively regulate clock gene transcription unresponsive to PDF in the morning. As TTX can also inhibit spontaneous firing occurring independently of synaptic inputs, these results also imply that cell-autonomous control of neuronal firing rhythms by the molecular clock may be involved in the mechanisms gating the response to PDF. Fluorescent circadian reporter imaging combined with neuronal silencing or excitation by genetic tools will facilitate further studies to investigate whether the neuronal activity relevant for the response to PDF is mediated by circuit properties or by cell-autonomous mechanisms.
The nighttime-specific effect of PDF on clock gene transcription may be related to the mechanism of phase shift. Light increases the firing rate of pacemaker neurons52 and likely induces PDF release from the LNvs. It has also been shown that acute induction of firing mimics the effect of light in phase shift12. Therefore, an increase in PDF release in the early night by a light pulse when PDF levels should normally be low may trigger phase delay via enhanced clock gene transcription. Our finding that PDF-mediated activation of clock gene transcription is activity-dependent (Figs 3a and 5) also supports this possibility.
The time-of-day-dependent modulation of the molecular clockwork by PDF may also enhance the synchronization of clock neurons. CLK/CYC-transcription is active during the late day-early night and reaches a maximum around ZT14. PER maximally accumulates somewhat later, in the middle or near the end of the night23. As reported previously14, the effect of PDF on PER stabilization likely occurs in the late night to slow down the pace of intrinsically fast-paced clock neurons, such as the s-LNvs and DN1a (same as larval LNvs and DN1s)19. Furthermore, by stimulating CLK/CYC-mediated transcription in the early night, PDF signalling can amplify molecular clock oscillations, which contributes to phase synchrony (Fig. 6). Interestingly, a computational study predicts that VIP, the functional ortholog of PDF, can mediate synchrony across SCN neurons by inducing per expression only if VIP signalling occurs in-phase with per transcription53. Our results strikingly parallel this model and suggest that neuropeptide signalling in-phase with core clock gene transcription is a conserved principle for achieving pacemaker neuron synchrony.
It is noteworthy that reporter upregulation upon PDF application to brain explants was observed in the LNvs and DN1s but not in the DN2s (Figs 3 and 4). Adult DN2s express PDFR and can be activated by PDF38,54. Although larval DN2s also express PDFR30, the signalling downstream of PDFR in these cells may differ from that in the LNvs and DN1s. This finding underscores that PDF has multiple cell-type-specific roles.
One of our most intriguing observations is the lack of discernible circadian rhythms in 3 × 69-VNP expression in dissociated neuron cultures (Fig. 5). This finding suggests a possibility that robust transcriptional rhythms may require intact network communication and parallels the notion that circuit properties strongly influence the robustness of the mammalian SCN clocks6,55.
A recent study by Mezan et al. from Kadener's group used another fluorescent circadian transcriptional reporters and showed that PDF signalling negatively regulates clock gene expression56. The differences in experimental setup and timescale of observations likely contributed to the differences in the conclusions between our and their studies. Whereas we focused on analysing acute response to PDF, Mezan et al. investigated the effect of pdfr mutation on tim transcription at steady-state or following the induction of CLK-GR transgene nuclear localization to increase CLK/CYC-mediated transcription. Before induction, CLK-GR acts dominant negatively to inhibit clock gene transcription, and it takes at least 24 hr to observe the clock gene tim transcriptional upregulation after CLK-GR induction. It will be interesting to compare short- and long-term effect of PDF on clock gene transcription using both experimental systems in future studies.
Cell type-specific differences in PDF signalling, downstream molecular components and relationships with neuronal activity are important issues to be addressed in future studies. There are likely other important neuropeptides and neurotransmitters that link neuronal communication with the molecular clockwork. Our new fluorescent reporters can be combined with optogenetic tools and genetically encoded calcium reporters to manipulate and analyse neuronal activity; however, the period estimated from the fluorescence time-lapse imaging could be imprecise due to its inherent lower temporal resolution compared with bioluminescence recordings. As is often the case in any Drosophila transgenic line, we also observed that genetic background affects the period length of the reporter lines independently of the copy number of the fluorescent reporters. Our results also suggest that culture conditions of brain explants impact the rhythmicity and period of the reporters. Given these advantages and caveats, the application of our fluorescent reporters to imaging in intact fly brains20,57 may offer powerful tools to further decipher the intricate interactions between neuronal signalling and the molecular clockwork.
Drosophila were reared at 25 °C on a corn-meal medium under 12 hr:12 hr light-dark (LD) cycles. The 1982clk-gal4 line was provided by N.R. Glossop58. The GAL4 enhancer trap line gal111859, and pdfr5304 60, pdf01 (Bloomington stock center, nb 26654) have been previously characterized.
UAS-mCD8::VENUS and UAS-mCD8::RFP constructs were generated by exchanging the GFP coding sequence of the pUAST/mCD8-GFP construct provided by L. Luo6161 to Venus cDNA or mRFP cDNA. The resulting constructs were introduced into the genome by P-element-mediated germline transformation.
To generate 3 × 69-VNP flies, the hs43 basal promoter sequence from pCaSpeR-hs43-lacz (GenBank accession number X81643), the Venus-NLS-PEST1 coding sequence24 and 1 kb of the per 3′ UTR were PCR-amplified and ligated into the vector containing three tandem copies of the 69-bp CRS of per (pCaSpeR-per69 × 3-luc), thus replacing the luciferase coding sequence and the SV40 3′ UTR26. The resulting pCaSpeR-3 × 69-Venus-UTR vector was used for P-element-mediated germline transformation.
To generate the per-TdT transgenic line, the pStinger vector was modified by removing the coding sequences of 5xUAS and EGFP-NLS and adding the attB sequence between the P-element 3′ end and a gypsy insulator. A 13.2 kb per genomic fragment (from the BamHI site at −4.2 kb to the EcoRI site located ca. 2 kb downstream of exon 8) was cloned into a modified pStinger vector. The tdTomato (TdT) coding sequence, amplified from the pRSET-B/tdTomato plasmid (generated by R. Tsien and provided by L. Luo), and a 1 kb per 3′UTR sequence were cloned into the pBS plasmid. A BamHI-PsiI fragment from this plasmid containing the TdT-3′UTR was then ligated to the BamHI-PsiI fragment of pStinger-per13.2, which resulted in the per (exon 1–5)-TdT-3′UTR fusion construct (per-TdT vector). The per-TdT strains were generated by integrating the per-TdT vector into the attP16 (2R) and attP40 (2L) landing sites by PhiC13-mediated site-specific integration. Injections were performed by BestGene Inc. The per-TdT (attP16) and per-TdT (attP40) lines were crossed to recombine both transgenes on the 2nd chromosome.
The brains of LD-entrained non-wandering L3 larvae were dissected in ice-cold saline solution62 at ZT11. The imaginal discs were left intact to prevent any tearing of the tissue. Dissected brains were kept on ice in modified Schneider's medium (SMactive)63 supplemented with 5 mM Bis-Tris (Sigma). For ex vivo brain culture, the brain explants were mounted on a glass-bottom dish (35 mm MatTek petri dish, 20 mm microwell with 0.16/0.19 mm coverglass) in a fibrinogen clot prepared by adding thrombin (bovine thrombin, Sigma) to fibrinogen (bovine fibrinogen, Calbiochem) as previously described64. The glass-bottom well was filled with the SMactive medium and covered by a Teflon membrane permeable to oxygen. The cultured brains were kept at 25 °C in 80% relative humidity and in the dark for a few hours prior to time-lapse imaging to allow the brains to set. Time-lapse imaging was performed in the same culture conditions, with images acquired every 3 hr.
For pharmacological experiments on brain explants, brains were dissected either at ZT1 or at ZT13 and cultured in dissecting saline solution (equivalent to hemolymph-like saline) without a Teflon membrane. The brain explants were immediately imaged to establish a baseline. Approximately 1 hr after the start of the time-lapse imaging, the neuropeptide PDF (custom-made by Chi Scientific, H-NSELINSLLSLPKNMNDA-OH; 2 μM) was added alone or in combination with 100 nM TTX (Cayman Chemical). For the control condition, the vehicle (DMSO) was added. Sp-cAMPS (sc-201571 from Santa Cruz Biotechnology, 150 μM) was added alone and the vehicle (H2O) was used as a control. Time-lapse imaging was performed in the same conditions as described above with images acquired every hour for 8 hr.
Dissociated neuron culture was performed as previously described45,64 with the following modifications. In brief, the dissected brains were enzymatically treated with 50 units/mL papain (Worthington) and mechanically dissociated. The cell suspension was then plated on glass-bottom dishes (35 mm MatTek petri dish, 10 mm microwell with 0.16/0.19 mm coverglass) coated with concanavalin A (Sigma). Once the cells were attached to the glass bottom, the dish was flooded with SMactive. The dissociated neuron culture was incubated at 25 °C in 80% relative humidity and constant darkness for 2 days prior to time-lapse imaging. For pharmacological experiments, 2 or 20 μM PDF, 100 nM TTX, 10 μM GABA (Sigma), or the vehicle (DMSO or ddH2O) was added to the cell culture medium just before the start of the time-lapse imaging. Time-lapse experiments were conducted in the same culture conditions, and the images were captured every 3 hr.
A Leica TCS SP5 tandem scanner confocal microscope was used for fluorescence imaging. The same parameter settings were used to image all samples of the same type (dissected brains, cultured brains or cultured neurons). Freshly dissected larval and adult brains were scanned using a 40x water-immersion objective with the galvo scanner at 400 Hz. For time-lapse imaging, 20x, 40x or 63x objectives were used, and the images were acquired using the resonant scanner at 8,000 Hz with high-sensitivity HyD detectors. A bi-directional scan was used together with an 8x line average. Z-section steps of 1.7 μm × 8 and 2 μm × ~40 were used for imaging the dissociated cultured neurons and ex vivo brain culture, respectively. A 514-nm laser was used to excite the VENUS fluorophore (0.68 μW/cm2 to image VNP, 0.40 μW/cm2 for mCD8::VENUS), and a 561-nm laser was used for the TdTomato and mRFP fluorophores (14.8 μW/cm2 for PER-TDT, 5.71 μW/cm2 for mCD8::RFP). The laser intensity was measured at the level of the sample with a microscope slide power meter (Thorlabs, S170C).
The fluorescence intensities in the time-lapse movies of the cultured neurons were measured using Imaris software (Bitplane). A 3D mask (region of interest in 3D) was built for each cell by thresholding the 3 × 69-VNP or PER-TDT fluorescence levels after background subtraction. The intensity SUM in each 3D mask was then extracted from the statistical data that were automatically generated by the program. For presentation purposes only, some of the images and time-lapse movies were processed with a 10x iterative deconvolution using AutoQuant (MediaCybernetics) and Imaris. When necessary, time-lapse images were also treated for 3D correction drift using the ImageJ plugin (NIH).
Fluorescence-intensity time-series data were normalized to the value at t = 0. Heatmaps representing the fluorescence-intensity time course were generated with an in-house R script. Each row represents the data of a single cell. Rows were ordered by the highest intensity over 24 hr.
For rhythm analysis, a combination of manual inspection and Maximum Entropy Spectral Analysis (MESA) was used to detect rhythmicity and estimate the period. MESA was chosen because, unlike other methods, it is adapted for the detection of rhythms in short or noisy time series66. First, time course plots of the intensity were generated using Excel or Prism (GraphPad Prism version 6.0c for Mac, GraphPad Software, San Diego, California, USA, www.graphpad.com). Because we found that 6th-order polynomial regression models best fit the data, we superimposed the 6th-order polynomial trend lines on the graphs to facilitate the detection of rhythmicity. The time-series data with circadian rhythms, defined by a peak-to-peak interval from 18 to 36 hr, were then identified by manual inspection. However, the polynomial trend lines were not used to determine the period. In parallel, the intensity time-series data without normalization were analysed with MESA without filtering. The time-series data were scored as circadian only when both the manual and MESA analyses detected the rhythms with a period between 18 and 36 hr.
Statistical analyses were performed with GraphPad Prism software. To compare the effects of the drug treatments in the pharmacological experiments (Figs 3–5), a two-way ANOVA with a Sidak test to correct for multiple comparisons was used.
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We thank N.R. Glossop and the Bloomington Drosophila Stock Center for fly stocks and L. Luo and R. Tsien for plasmids. We thank C. Helfrich-Förster, S. Kadener and U. Schibler for helpful comments on the manuscript. This work was supported by the JST PRESTO program, the Swiss National Science Foundation (31003A_149893), the European Research Council (ERC-StG-311194), the Novartis Foundation for Medical-Biomedical Research (13A39) and the University of Geneva.
Howard Hughes Medical Institute, National Center for Behavioral Genomics, Department of Biology, Brandeis University, 415 South Street, Waltham, Massachusetts 02451, USA.
V.S., L.V. and E.N. performed research; V.S., L.V., J.M.N. and E.N. analysed data; M.R. and E.N. designed research; and V.S., M.R. and E.N. wrote the paper.


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Marriott opens new hotel with European flair in downtown Portland (photos)
Updated: Mar. 22, 2017, 8:35 p.m. |
Published: Mar. 22, 2017, 7:35 p.m.
Stephanie Yao Long | The Oregonian/OregonLive
One of the newest hotels in town has its beginnings almost 20 years ago in Spain with founder Antonio Catalan. He's still there, in the name. The AC Hotel Portland Downtown opened last month with 204 rooms rising 13 floors high at the corner of SW 3rd Avenue and Taylor Street.
Marriott brought this European chain of hotels under their banner in 2011, opening the first AC Hotel in the United States three years later in New Orleans.
A visitor to the Portland version can expect the comforts found in any Portland home: from Water Avenue coffee in the lobby coffee shop to cookbooks authored by local chefs to artwork from local artists.
Then there are the unique luxuries, such as the Alpha Dominche Steampunk espresso maker in the lobby coffee shop that works off a computer pad and looks like something onboard a starship.
Get into one of the rooms and the differences between an AC Hotel and other mainstream hotels really start to affect your away-from-home routine. There are no closets or drawers to forget stuff in, but one long ledge that acts as a luggage stand and bench. A platform bed continues the open feel since it reduces the need for a bed skirt. There isn't an alarm clock anywhere. And forget about going home with the hotel's travel-size toiletries. In the glass shower you'll only find three bulk-sized bottles of body wash, shampoo and conditioner. This contributed to the building's Silver LEED certification.
Relax at the end of the day in the AC Lounge downstairs. It opens at 4 p.m. with an "evening ritual" around 5 p.m. that consists of a complimentary tasting of a craft cocktail that will change seasonally. Right now it's the Rose City, inspired by a Manhattan, utilizing Bull Run Bourbon in its mix.
Portland is experiencing a flurry of hotel construction. There are 15 hotels under construction or recently completed in Portland's core, according to Travel Portland, the regional tourism bureau. Together, they comprise nearly 3,000 new hotel rooms.

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In Conversation with Liza Perrat..
I am delighted to welcome back to Jaffareadstoo
Liza Perrat
Perrat Publishing
*~Welcome Liza~*
What can you tell us about Wolfsangel that won't give too much away?
Wolfsangel is the second book in L'Auberge des Anges series, and follows the descendants of the Charpentier family a hundred and fifty years after the French Revolution, when the village comes under the heel of the German occupation. Even before I had finished writing the first book in the series –– Spirit of Lost Angels –– I knew the characters had more tales to tell. However, to answer your question, Jo, Wolfsangel was directly inspired by a true-life WWII war crime, but telling you about this particular event would spoil the story! I'll just say that this tragic site has haunted me since I visited it many years ago, and I have included some information about it in the Author's Note section at the back of Wolfsangel.
Where did you get the inspiration for the title of the book?
The women of Spirit of Lost Angels, Wolfsangel and the third in the series –– Midwife Héloïse – Blood Rose Angel –– all share the same profession: healer, midwife and angel-maker. But they also have a stronger link: an angel pendant carved from bone, which is handed down through the generations of women of L'Auberge des Anges. So, I wanted to keep the word "angel" in all the titles. It is also alludes to the symbol for Das Reich's 2nd SS Panzer Division, was notorious for the particular war crime on which my story is based: the wolf's hook, or Wolfsangel.
And lastly, the Wolfs' angel is a reference to the Jewish family who feature in this story –– Max, Sabine, Talia and Jacob Wolf.
Why did you choose to set this book during WWII?
After Spirit of Lost Angels, I wanted to continue the story of the Charpentier family, their farmhouse and their village, and what might have been their lives during different historical eras and upheavals. During WWII, the Germans did occupy the region of France in which Wolfsangel is set, and there was a large French Resistance presence. Consequently, the local historical association has a lot of documentation on this subject. I was also fortunate enough to meet "Agent du Roc", an ex-Resistance member who gave me first-hand accounts of what it was really like. Two of the Resistance members in the novel –– Pierre and Antoine –– are based on him. I also stumbled upon an excellent Occupation and Resistance exhibition in a village of the Monts du Lyonnais –– St-Martin-en-Haut –– which features in Wolfsangel as a "safe hideout" for various Resistance members. Photos of the exhibition can be seen here:
http://www.pinterest.com/triskelebooks/wolfsangel/
Wolfsangel is your second novel in the L'Auberge des Anges series – did you feel more of an obligation to make this book even better than the first?
I strive to improve my writing with each book, and to make every one better than the last. Before Spirit of Lost Angels, I wrote two books, both of which will stay asleep, forever, on my hard drive! I regard them as part of the learning curve. That said though, the story for Wolfsangel seemed to come more easily than the others; it was the kind of story that wrote itself. And those, I've found, from the days I wrote short stories, always seem to turn out the best.
The cover of Wolfsangel is very evocative - who designed the cover and why did you go for that particular design?
Oh I'm glad you like the cover, Jo; I'm so thrilled with it too. I worked closely with my very talented Triskele Books colleague and designer, JD Smith. She does the covers for all our Triskele books, as well as our promotional material –– posters, bookmarks, etc. I wanted to evoke the tragic church scene, with the dark clouds. I wanted there illustrate hope, in the bright part of the sky, with the bird. I also tried to go for the strong, rebellious heroine look.
What can you tell us about the next story in the series?
As I said, a bone-carved angel pendant is handed down from mother to daughter in the first two books, and the stories and legends that surround this angel talisman are many and varied. The characters are not certain from where it hails, and from what type of bone it is sculpted. Some say it was probably carved from seal or ox, or walrus tusk. Others believe it was mammoth bone and a few whisper that it was carved from human bone.
I haven't quite decided where the angel pendant comes from, though I have several ideas, which I'm currently exploring in the third and final novel in the series: Midwife Héloïse – Blood Rose Angel, set in the same French village, during the 14th century Black Plague years.
Here is the working blurb, to give you some idea:
1348. A bone-carved angel talisman –– family heirloom, evil curse, holy relic. And Héloïse Dumortier, the midwife-healer woman who wears it –– heretic, Devil's servant, saint.
When the Black Plague arrives, sweeping across France, the superstitious villagers begin to ask who or what is to blame for this pestilence. Fire and foul winds from the East, or the stinking miasmas of stagnant lakes? Is it some malign conjunction of the planets, or God's wrath against sinners? Perhaps it is the curse of a mere woman.
Amidst the terror, grief and contagion, Héloïse must find the courage and compassion to care for birthing women and plague-stricken victims. She must also fight against those who accuse her.
Liza grew up in Wollongong, Australia, where she worked as a nurse and midwife for fifteen years. When she met her French husband on a Bangkok bus, she moved to France, where she has been living with her husband and three children for twenty years. She works part-time as a French-English medical translator, and a novelist.
Several of her short stories have won awards, notably the Writers Bureau annual competition of 2004 and her stories have been published widely in anthologies and small press magazines. Her articles on French culture and tradition have been published in international magazines such as France Magazine and France Today.
She has completed four novels and one short-story collection, and is represented by Judith Murdoch of the Judith Murdoch Literary Agency.
Spirit of Lost Angels, the first in the historical L'Auberge des Anges series was published under the Triskele Books label in May, 2012. The second in the series –– Wolfsangel –– was published in October, 2013, and Liza is working on the third novel in the series –– Midwife Héloïse – Blood Rose Angel –– set during the 14th century Black Plague years.
Liza is a co-founder and member of Triskele Books, an independent writers' collective with a commitment to quality and a strong sense of place.
Liza reviews books for the Historical Novel Society and Words with Jam magazine.
Contact and Other Information
E-mail: liza.perrat@gmail.com
Website: www.lizaperrat.com
Blog: http://lizaperrat.blogspot.com
Twitter: @LizaPerrat
Facebook: https://www.facebook.com/liza.perrat.5
Author Collective: www.triskelebooks.co.uk Blog: http://triskelebooks.blogspot.com/
Twitter: @TriskeleBooks
Liza has very kindly offered an amazing giveaway opportunity
Liza ~ Jaffa and I are delighted to chat with you about Wolfsangel.
It's always a real pleasure to have you visit with us.
Do come back and see us again soon.
My thoughts on Wolfsangel
Wolfsangel is the second book in the L'Auberge des Anges trilogy and opens as elderly Céleste Roussel commemorates a tragedy which happened in the French village of Lucie-sur-Vionne some 68 years earlier. This emotional opening chapter sets the scene for a powerful and dramatic story, which takes the reader back to 1943, and to a dark time in France's history, when the German occupation of this tiny French village had a profound effect on those who lived and worked under the shadow of cruelty and repression.
Céleste Roussel, and the women of her family, have been connected to the village of Lucie-sur-Vionne, and their home at L'Auberge des Anges for centuries. Strengthened by their ancestry, and running like a silken thread throughout the narrative, is the continuing connection the women of L'Auberge des Anges have with an angel talisman, which exudes strength and positivity to those who wear it. In 1943, Céleste Roussel is the latest keeper of the talisman, she is a spirited young woman, quick witted, courageous and as brave as a lion, but she is also impetuous, capricious and entirely unpredictable. However, it is her burgeoning relationship with a Reich officer which threatens not just her sanity, but also the safety of those who are precious to her.
With superb skill, the author has manipulated the narrative into a powerful depiction of a dark and dangerous time. Lucie-sur-Vionne is so vividly described that I stood in the market square, and felt that first frisson of excitement as Céleste met the violet eyes of the German officer, Martin Diehl. I rode with her, on the same rickety bicycle, through dank, dark woodland, and cheered with relief when the stealthy movements of the local résistance succeeded in one dangerous mission after another. The squalor, the danger, and the sheer unpredictability of living life constantly looking over your shoulder is so realistic, that you feel as if you are seeing the story unfold in real time.
Beautifully researched, and based on historical factual evidence, the story has an emotional depth which pervades and which reveals a story of courage, bravery and unforgettable heroism.
I loved it and can't wait for the third and final novel in the L'Auberge des Anges series.
Labels: Author Interview, Conversation, Giveaway, Historical Fiction
Anne Williams 18 November 2013 at 11:33
To my eternal shame, I still haven't got round to reading the first in this series - but I'd love to win an e-copy of this one, it sounds wonderful. Thanks for such an interesting feature Josie, and for hosting the giveaway...
Cheers, Anne
http://beingannereading.blogspot.co.uk/
Great to see you here Anne - I am sure you would really enjoy Wolfsangel.
Liza Perrat 27 November 2013 at 10:23
Thanks for your interest, Anne, and good luck with the giveaway. Best, Liza
Thanks for a wonderful review and interview, Jo! And also for hosting the giveaway. Best, Liza
Hi Liza ~ it's always a delight to feature one of your books on our blog. Come back and see us again soon with Book 3 !!
I certainly will be back with Book 3, Jo!
Emma and Silvia 24 November 2013 at 22:46
I have just ordered the first book in the series, hoping to have a chance to win the second one. Not the e-book, hopefully, as I am an old-fashioned reader!
Hi Emma and Silvia - thanks for visiting ~ good luck in the giveaway :)
Thanks for your interest, Emma and Silvia, and best of luck in the giveaway!
Thanks to everyone who entered this giveaway - the lucky winners will be announced soon !!
I am delighted to say that the winners are Silvia, Anne and Nancy - I hope you enjoy Wolfsangel as much as I did :)
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Bird Box-style videos banned by YouTube
The video platform updated its guidelines to prevent people endangering themselves
By Martyn Landi, Press Association Technology Correspondent
The latest stunt involved one prankster walking blindfolded on a live railway (Image: Ben Phillips Youtube)
YouTube has updated its guidelines to ban prank and dangerous challenge videos.
The Google-owned video platform said that while it is a home for light-hearted prank videos, it wanted to reinforce that any stunts that place people in danger are against site policy.
The platform has been home to a variety of challenges - such as the ice bucket challenge to raise money for charity - but one inspired by Netflix show Bird Box - where users are encouraged to carry out a range of activities, including driving, while blindfolded - has caused concerns.
In a new post to the FAQ section on its website, the company said: "YouTube is home to many beloved viral challenges and pranks, like Jimmy Kimmel's Terrible Christmas Presents prank or the water bottle flip challenge.
Netflix told Bird Box fans not to "end up in the hospital due to memes" (Image: Netflix)
"That said, we've always had policies to make sure what's funny doesn't cross the line into also being harmful or dangerous. Our Community Guidelines prohibit content that encourages dangerous activities that are likely to result in serious harm, and today (we are) clarifying what this means for dangerous challenges and pranks."
Last year a public awareness campaign was launched in the US after an increase in poisoning reports linked to a video challenge to eat detergent pods went viral on the site.
"We've updated our external guidelines to make it clear that challenges like the Tide pod challenge or the Fire challenge, that can cause death and/or have caused death in some instances, have no place on YouTube," the site said.
"We've made it clear that our policies prohibiting harmful and dangerous content also extend to pranks with a perceived danger of serious physical injury.
The challenge involves taking in part in tasks while wearing a blindfold, emulating characters in Netflix hit film Bird Box (Image: Publicity Picture)
"We don't allow pranks that make victims believe they're in serious physical danger - for example, a home invasion prank or a drive-by shooting prank.
"We also don't allow pranks that cause children to experience severe emotional distress, meaning something so bad that it could leave the child traumatised for life."
YouTube is one of several social media and internet services that has been accused of failing to properly police its platform, with legislators in the UK and the US warning they may introduce regulation if firms do not become more proactive.
YouTube said it had worked with child psychologists to help develop its guidelines around the types of pranks which are acceptable for the site.
Videos that showed "the fake death of a parent or severe abandonment or shaming for mistakes" were cited as those that "crossed the line".


text:
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redpajama_set_name: RedPajamaGithub


text:
The Real Impact of Mission Support Grants
Learn from those who have partnered with AHF Since 2010, Mission Support Grants have provided core operating support to nonprofits advancing health and wellness for the most vulnerable in San Diego and Imperial Counties—those under 250% of the federal poverty level,...
2017 Mission Support Grants Overview
AHF provides core operating support to organizations in San Diego and Imperial Counties. In 2017, 26 health and wellness focused organizations were selected for funding. Below are the key findings from the Interim Report. Source: 2017 Self Reported Data from Mission...
AHF provides core operating support to organizations in San Diego and Imperial Counties. In 2016, 19 health and wellness focused organizations were selected for funding. Below are the key findings from the Interim Report.
And the Mission Support Grants Go To….
Drum roll please… Award season is upon us. Musicians recently rocked through the Grammys on February 15 while Hollywood recently embraced the glitz and glam of the Academy Awards. Keeping with the spirit of the awards, we are here to announce the recipients of...
Take Your Heart Out on a Date
It's time to show your heart some TLC. February is underway and love is in the air. As you go about your day, I'm sure you have recently noticed the transition to pink decorations and sugary treats as we quickly approach every romantic's favorite holiday, Valentine's...
January News
2022 i2 Challenge Grant Awards Announced
Final Pitches Made for i2 $1 Million Challenge Grant-Announcement Coming Soon!
New homes in time for the holidays!
David Palmer on Five Finalists Vying for $1 Million Innovation Initiative (i2) Award from Alliance Healthcare Foundation
Jonathan Dale on Majority of Californians Are Prediabetic or Diabetic: What Do We Do?
DR. Ofelia Dirige on Majority of Californians Are Prediabetic or Diabetic: What Do We Do?
Barbara Mandel on Majority of Californians Are Prediabetic or Diabetic: What Do We Do?
Susan on iEngageU Recap: Tackling the ADA 30-Day Wellness Challenge
CalAIM
Impact Investments
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Mission Support – Imperial County
Mission Support – San Diego
Past Grantees
Support New Grants


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Second 59Racing B12Hr McLaren entry confirmed > View
Home » IndyCar » IndyCar crown 'definitely achievable' for Dixon now
IndyCar crown 'definitely achievable' for Dixon now
By Daniel Herrero
Tuesday 20th August, 2019 - 1:00pm
Scott Dixon
Scott Dixon has declared winning the 2019 IndyCar Series to be 'definitely achievable' having gained more ground on points leader Josef Newgarden at Pocono.
Dixon finished second to Will Power at the 'Tricky Triangle' as Newgarden came home in fifth position, and the New Zealander is now 52 points off the top of the table, in fourth in the standings.
There are three races remaining this season, with 50 points on offer for a win in the first two, 100 for the finale, and bonuses for pole, leading at least one lap, and leading the greatest number of laps.
While the five-time and current champion is therefore just over a standard race behind, and would also have to usurp Alexander Rossi and Simon Pagenaud, Dixon pointed to the circumstances in which he won his fourth title as reason to remain hopeful.
In 2015, he came from 47 points back heading into the final round at Sonoma to take the big trophy on a countback when he won the race and previous series leader Juan Pablo Montoya finished sixth.
Dixon also pointed to the return to Laguna Seca, which has not hosted a top level American open-wheeler race since the 2004 CART event, for the finale as a wildcard.
"I think, honestly, until you're out of it, you're never really out of it," said the Chip Ganassi Racing (CGR) stalwart.
"I don't know what the points gap was going in '15 for us, behind Montoya. (But) It's definitely achievable from the point deficit right now.
"There's still a long ways to go. I think the next two will be pretty interesting. St.Louis (Gateway) is going to be a lot of fun. Then Portland will be interesting.
"For a lot of us, too, it was interesting to get back to Laguna for a while," he added, referring to a private test there in February.
"Some teams struggled more than they thought they would. Laguna will be a pretty interesting race with double points."
While Dixon is clearly a hunter at present, a contrast to 2018 when he took over the series lead midway through the campaign and continued to tighten his grip, there are similarities to last year.
Then, he finished top three in six of the last nine races of the season and fifth in another two of those.
From the most recent four races, Dixon has a win and three second places, a run which has seen him gain 42 points on Newgarden, who has been on top of the table after all but one race so far this year.
"Last year, we took the lead of the championship at Texas and carried it through, which was very different, felt a lot different. It's a lot more stressful that way," said Dixon.
"But, no, I think the team, even Dario's (Franchitti, former team-mate, now CGR adviser) championships were similar to that scenario. The start of the year, midway you're kind of there, thereabouts, but really came on in the latter parts of the year.
"It's (champion) the best over the full year. Sometimes that down part creates determination not just in yourself but also in the team.
"It also allows you a little bit of difference, maybe in strategy can you take more risks, you can go for race wins. If you get on a roll, it just starts to feel good for everybody and it kind of continues a little bit longer.
"I don't know, there have been many championships won many different ways.
"Josef took the lead for the championship the first race, so we'll see."
The Gateway event takes place on August 23-24 (local time).
Next Story McLaren IndyCar return designed to boost F1 Previous Story Rossi at odds with 'disgraceful' Sato over Pocono pile-up
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McDonald is an extinct town in Barry County, in the U.S. state of Missouri. The community once held the county seat of Barry County.

McDonald was laid out in 1841, and named in honor of a family of settlers. A post office called McDonald was established in 1840, and remained in operation until 1854.

References

Ghost towns in Missouri
Former populated places in Barry County, Missouri

redpajama_set_name: RedPajamaWikipedia


Setting Up An Office - to Assist Aliyah in Australia!
2016: A Jubilee Year of Restoration for Israel and the Church?
Basic Hebrew Words: "Truth" and "Lies"
Can Christians support the Third Temple?
Hanukkah and the Battle for the Temple Mount 4/4 - The days of Hanukkah are back!
Was the Site of the Temple lost?
When is the rapture / the Lord's return? 2017/2018?
Copyright © 2019 Enoch Lavender Ministries - Bible Prophecy blog articles and video clips. All Rights Reserved. Designed by JoomlArt.com. Joomla! is Free Software released under the GNU General Public License.


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Katie Meili est une nageuse américaine née le  à Carrollton. Elle remporte la médaille de bronze du 100 mètres brasse aux Jeux olympiques d'été de 2016 à Rio de Janeiro.

Liens externes

Nageuse américaine
Nageuse aux Jeux olympiques d'été de 2016
Championne olympique de natation
Championne olympique américaine
Médaillée de bronze olympique américaine
Naissance au Texas
Naissance en avril 1991
Championne du monde de natation en petit bassin


District Office: Neighborhood Center of Utica, Inc.
Brief Description: Evacuation drills must be conducted at least monthly during various hours of operation of the school age child care program.
Brief Description: When a school-age child care program is in operation, an adequate number of qualified staff must be on duty to protect the health and safety of the children in care.
Brief Description: Each program must hold two shelter-in-place drills annually during which procedures and supplies are reviewed. Parents must be made aware of this drill in advance.
Brief Description: before such person has any involvement in child care work.
Brief Description: the information necessary to determine whether the applicant is the subject of an indicated report of child abuse and maltreatment as required by section 414.10(b)(i) of this Part.
Be the first to review this childcare provider. Write a review about YMCA of the Greater Tri-Valley. Let other families know what's great, or what could be improved. Please read our brief review guidelines to make your review as helpful as possible.


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Gurja Himal () is a mountain in Gandaki Province, Nepal. Gurja Himal is part of the Dhaulagiri massif and it has an elevation of .

In 2018, an avalanche from the mountain killed nine people which was labelled as the  worst climbing disaster in Nepal after the 2015 Mount Everest avalanches. One of the victims was South Korean mountain climber Kim Chang-ho.

Gurja Himal was first climbed on 1 November 1969 by a Japanese expedition.

References

External links

 Gurja Himal at Nepal Himal Peak Profile

Seven-thousanders of the Himalayas
Mountains of the Gandaki Province


Malta does not recognise the right to conscientious objection for professional soldiers.
Conscription has never existed in Malta1. According to the Armed Forces Act, the Armed Forces of Malta can only be raised "by voluntary enlistment" (Article 3)2.
The legal basis for enlistment into the Armed Forces of Malta is the 1970 Malta Armed Forces Act3.
The minimum age for joining the Armed Forces of Malta is 18 years, and the maximum age is presently set at 25 years, and 23 years for Officer Cadets4.
There is also the option to join the Emergency Voluntary Reserve Force, in which case the maximum age for joining is 35 years5. This is a part-time force.
Recruitment is by public announcement. There is at least one call for applications annually. This is published in the Malta Government Gazzette and advertised in all the local newspapers6.
Volunteers must sign up for at least 3 years.
conscientious objection to serving in the military7. However, this does not guarantee a right to conscientious objection, it only excludes a substitute service for conscientious objectors from the definition of forced labour.
As Malta does not have conscription, and does not have laws giving the power to conscript, the issue of conscientious objection for conscripts does presently not arise.
According to Article 9 of the Armed Forces Act 1970, the service of voluntary soldiers can be extended in an emergency or war for up to twelve months. In fact, in this case any discharge is automatically postponed8. Article 10 makes similar provisions in case a war is imminent.
Article 14 allows for the purchase of discharge before the end of a contract, but this is not possible in case service has been extended according to Article 10 (see above).
is more than two years.
Similar punishments are possible for being absent without leave (article 51).


text:
Inlays Onlays
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Our dentists and the Farmington Family and Implant Dentistry team can take care of your tooth bonding in Farmington, NY. The dental bonding process is very similar to the process of filling in tooth decay. Whereas the term "filling" is generally reserved for tooth decay, bonding refers to reshaping the tooth in a more aesthetic or cosmetic sense. Typically the tooth is prepped and smoothed before applying a resin material to reshape or reform the tooth. Once applied and dried, the tooth is then reworked to appear just like a natural tooth. Cosmetic bonding can be used to fill gaps, change the shape, color or other aesthetics of a tooth. Using this method of tooth restoration is usually more economical than other forms of cosmetic dentistry such as porcelain veneers.
Do you have a small chip in your front tooth? Slight overlap? Uneven length? If these are minimal yet bothersome to you, you could likely benefit from contouring or reshaping the tooth. Even an occlusion (bite) that is slightly imbalanced can sometimes be fixed with contouring. However, to fix a tooth by contouring, you cannot jeopardize your bite. Another method will need to be taken if a bite imbalance is at risk.
Contouring/reshaping is performed by taking a handpiece and removing a small amount of enamel from the tooth to reach the desired appearance. Anesthesia is not necessary for pain during this since it is only the enamel being removed and no nerve endings are present in your enamel. If you haven't yet gone through orthodontic treatment but are planning on it, we suggest looking into contouring after as the aesthetic of the teeth will change.
Farmington Family & Implant Dentistry
Farmington, NY 14425
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Bo benefited from my sister's occasional absentmindedness. Away, I called one night, chatted for a while to my sister and was surprised to have her call me straight back. Bo, she wailed, ate my dinner. I waited. I just made creamed corn and soy cheese toast when you called, and put it down on the foot stool. I'm a horrible sister; all I could do was laugh. Bo, not one to miss a food opportunity, was apparently approving of that vegan delicacy if not the vege rice.
If the furry-ones adored her, my sister loved them equally. The three of them were getting on in years, and she nursed them all through ill health at various times when we weren't around. But we were all together when we had to let first Jack, then a few months later Baddy, go. My sister and I were up together, and in turns, all night the night before Baddy left us.
It wasn't long after my sister moved out to the city to share a house with friends that Bo made her not totally unexpected but sudden exit. It was awful news to break to my sister, and she was heartbroken that she hadn't been there.
For years after she moved out, forgetting she had paid weekly board which we spent on hiring a cleaner to save all our sanities, my sister petitioned to pay every time we had breakfast, lunch, dinner or a coffee. I have to pay you back, were words that drove me crazy.
In time, our younger sister decided to spend a year living & working in the city, employed at the same firm as me, living with the other sister. It was then the elder of my younger two sisters was able to embrace the concept of pay it forward. She's also the sister whose apartment the G.O. and I live in.
One of the great things about families is if you need somewhere to stay, they will find a bed for you… even if it is a fold-out couch in the living room, which is what we now currently have on offer in our two room city apartment. It's surprising how many people take it up.
I never had a sister, but I had a cousin who, through unfortunate circumstances, needed a place to live until she was 'legal'. She lived with our family and shared my room for a year. She was 17 and I was 13. It was so fun. There was never a cross word between us and we bonded closer than many sisters. We are sisters of choice, I guess one could say. Nice post Elladee!
Thank you 🙂 After being an only child for a few days shy of 17 years, it was great to be a sister, and to have sisters and a brother. Even though we are halves, we still share much nature & nurture commonality, as I imagine you and your cousin-big sister do also.
Ohhh, sisters, they are very special.
They are 🙂 I had to wait a while to take delivery of mine but it was worth it.
True, and I'd do it again if circumstances warranted. Some days I miss being able to leave the domestic goddess stuff for someone else.
Thank you 🙂 I'm glad you thought so.
How lovely that your family is there for each other.
Thank you 🙂 I think the whole concept of pay it forward… onto a future person-need is far more sensible than paying back to someone who already had the means to assist.
I actually think slotting into re-living with a brother or sister is much easier than doing so with parents! Don't know why, but it's something I clarified on my last trip 'home'. Lovely blog, Ella. I came here to find the 2nd of the short stories I missed and was delighted to read this first.
Or re-opened old wounds perhaps … I know where you're coming from on that!
Being an only one I've never experienced that sibling affection, but looking at my two daughters I can see it is something special.
I was an only until 3 days before my 17th birthday. Now I have 3 half siblings. I missed it growing up and although I lived in a small town with lots of friends, had close family and went to boarding school, it's not the same. They are adults now as well, so it's really nice. It's a lottery. Some people cannot get on with their siblings at all.


text:
package Paws::ElasticTranscoder::UpdatePipelineResponse;
  use Moose;
  has Pipeline => (is => 'ro', isa => 'Paws::ElasticTranscoder::Pipeline');
  has Warnings => (is => 'ro', isa => 'ArrayRef[Paws::ElasticTranscoder::Warning]');

  has _request_id => (is => 'ro', isa => 'Str');
1;

### main pod documentation begin ###

=head1 NAME

Paws::ElasticTranscoder::UpdatePipelineResponse

=head1 ATTRIBUTES

=head2 Pipeline => L<Paws::ElasticTranscoder::Pipeline>

The pipeline updated by this C<UpdatePipelineResponse> call.

=head2 Warnings => ArrayRef[L<Paws::ElasticTranscoder::Warning>]

Elastic Transcoder returns a warning if the resources used by your
pipeline are not in the same region as the pipeline.

Using resources in the same region, such as your Amazon S3 buckets,
Amazon SNS notification topics, and AWS KMS key, reduces processing
time and prevents cross-regional charges.

=head2 _request_id => Str

=cut