The Trissin/TrissinR signaling pathway in the circadian network regulates evening activity in Drosophila melanogaster under constant dark conditions.

The circadian clock in Drosophila is governed by a neural network comprising approximately 150 neurons, known as clock neurons, which are intricately interconnected by various neurotransmitters. The neuropeptides that play functional roles in these clock neurons have been identified; however, the roles of some neuropeptides, such as Trissin, remain unclear. Trissin is expressed in lateral dorsal clock neurons (LNds), while its receptor, TrissinR, is expressed in dorsal neuron 1 (DN1) and LNds. In this study, we investigated the role of the Trissin/TrissinR signaling pathway within the circadian network in Drosophila melanogaster. Analysis involving our newly generated antibody against the Trissin precursor revealed that Trissin expression in the LNds cycles in a circadian manner. Behavioral analysis further demonstrated that flies with Trissin or TrissinR knockout or knockdown showed delayed evening activity offset under constant darkness conditions. Notably, this observed delay in evening activity offset in TrissinRNAi flies was restored via the additional knockdown of Ion transport peptide (ITP), indicating that the Trissin/TrissinR signaling pathway transmits information via ITP. Therefore, this pathway may be a key regulator of the timing of evening activity offset termination, orchestrating its effects in collaboration with the neuropeptide, ITP.

Evaluating Emotional Outcomes of Medical Students in Pediatric Emergency Medicine Telesimulation.

The coronavirus disease 2019 (COVID-19) pandemic has challenged the feasibility of traditional in-person simulation-based clinical training due to the public health recommendation on social distancing. During the pandemic, telesimulation training was implemented to avoid multiple students and faculties gathering in confined spaces. While medical trainees' perceived emotions have been acknowledged as a critical outcome of the in-person simulation-based training, the impact of telesimulation on trainees' emotions has been unexamined. We conducted an educational team-based simulation study with a pediatric case of septic shock. Seventeen and twenty-four medical students participated in the telesimulation training and in-person simulation training, respectively. The institutional pandemic social restrictions at the time of each training session determined the participant assignment to either the telesimulation training or in-person simulation training. All participants responded to the Japanese version of the Medical Emotion Scale, which includes 20 items rated on a five-point Likert-type scale before, during, and after the simulation sessions. The measured emotions were categized into four emotion groups according to two dimensions: positive or negative and activating or deactivating emotions. The one-way analysis of variance between the telesimulation and in-person simulation training revealed no significant differences in the emotions perceived by the participants before, during, and after the simulation training sessions. The perceived emotions of medical students were comparable between the telesimulation and in-person simulation training. Further longitudinal studies with larger samples and multiple variables are needed to generalize the effectiveness of telesimulation.

Symbiont specificity differs among green hydra strains.

The symbiotic hydra Hydra viridissima has a stable symbiotic relationship with the green alga Chlorella. This hydra appears to cospeciate with the symbiotic alga, and some strains are known to have strain-specific host/symbiont combinations. To investigate the mechanism of the specificity between host and symbiont, we explored the effect of the removal or exchange of symbionts in two distantly related H. viridissima strains (K10 and M9). In the K10 strain, severe morphological and behavioural changes were found in symbiont-removed and symbiont-exchanged polyps. Interestingly, both polyps showed a similar gene expression pattern. The gene ontology (GO) enrichment analysis revealed that the removal or exchange of symbionts caused the downregulation of genes involved in the electron transport chain and the upregulation of genes involved in translation in the K10 strain. On the other hand, symbiont-removed and symbiont-exchanged M9 polyps showed modest changes in their morphology and behaviour compared with the K10 strain. Furthermore, the patterns of the gene expression changes in the M9 strain were quite different between the symbiont-removed and symbiont-exchanged polyps. Our results suggested that the regulation of energy balance is one of the crucial mechanisms for maintaining symbiotic relationships in green hydra, and this mechanism differs between the strains.

Glial immune-related pathways mediate effects of closed head traumatic brain injury on behavior and lethality in Drosophila.

In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects.

Creating Clinical Reasoning Assessment Tools in Different Languages: Adaptation of the Pediatric Emergency Medicine Script Concordance Test to Japanese.

Introduction: Clinical reasoning is a crucial skill in the practice of pediatric emergency medicine and a vital element of the various competencies achieved during the clinical training of resident doctors. Pediatric emergency physicians are often required to stabilize patients and make correct diagnoses with limited clinical information, time and resources. The Pediatric Emergency Medicine Script Concordance Test (PEM-SCT) has been developed specifically for assessing physician's reasoning skills in the context of the uncertainties in pediatric emergency practice. In this study, we developed the Japanese version of the PEM-SCT (Jpem-SCT) and confirmed its validity by collecting relevant evidence. Methods: The Jpem-SCT was developed by translating the PEM-SCT into Japanese using the Translation, Review, Adjudication, Pretest, Documentation team translation model, which follows cross-cultural survey guidelines for proper translation and cross-cultural and linguistic equivalences between the English and Japanese version of the survey. First, 15 experienced pediatricians participated in the pre-test session, serving as a reference panel for modifying the test descriptions, incorporating Japanese context, and establishing the basis for the scoring process. Then, a 1-h test containing 60 questions was administered to 75 trainees from three academic institutions. Following data collection, we calculated the item-total correlations of the scores to optimize selection of the best items in the final version of the Jpem-SCT. The reliability of the finalized Jpem-SCT was calculated using Cronbach's α coefficient for ensuring generalizability of the evidence. We also conducted multiple regression analysis of the test score to collect evidence on validity of the extrapolation. Results: The final version of the test, based on item-total correlation data analysis, contained 45 questions. The participant's specialties were as follows: Transitional interns 12.0%, pediatric residents 56.0%, emergency medicine residents 25.3%, and PEM fellows 6.7%. The mean score of the final version of the Jpem-SCT was 68.6 (SD 9.8). The reliability of the optimized test (Cronbach's α) was 0.70. Multiple regression analysis showed that being a transitional intern was a negative predictor of test scores, indicating that clinical experience relates to performance on the Jpem-SCT. Conclusion: This pediatric emergency medicine Script Concordance Test was reliable and valid for assessing the development of clinical reasoning by trainee doctors during residency training.

Immature symbiotic system between horizontally transmitted green algae and brown hydra.

Some strains of brown hydra (Hydra vulgaris) are able to harbor the green algae Chlorococcum in their endodermal epithelial cells as symbionts. However, the relationship between brown hydra and chlorococcum is considered to be incipient symbiosis because most artificially introduced symbionts are not stable and because symbiotic H. vulgaris strains are rare in the wild. In this study, we compared the gene expression levels of the newly established symbiotic hydra (strain 105G), the native symbiotic strain (J7), and their non-symbiotic polyps to determine what changes would occur at the early stage of the evolution of symbiosis. We found that both the 105G and J7 strains showed comparable expression patterns, exhibiting upregulation of lysosomal enzymes and downregulation of genes related to nematocyte development and function. Meanwhile, genes involved in translation and the respiratory chain were upregulated only in strain 105G. Furthermore, treatment with rapamycin, which inhibits translation activity, induced the degeneration of the symbiotic strains (105G and J7). This effect was severe in strain 105G. Our results suggested that evolving the ability to balance the cellular metabolism between the host and the symbiont is a key requirement for adapting to endosymbiosis with chlorococcum.

Point-of-Care Ultrasound for Pediatric Endotracheal Tube Placement Confirmation by Advanced Practice Transport Nurses.

OBJECTIVE: This study aimed to identify which point-of-care ultrasound (POCUS) techniques and sonographic signs were reliably acquired and interpreted by transport nurses for the confirmation of endotracheal tube placement in children. METHODS: We developed and assessed a multimodal POCUS training curriculum for transport nurses that evaluated 5 sonographic signs using 3 scanning techniques. RESULTS: Twenty-one transport nurses were enrolled in the curriculum. The mean scores (95% confidence interval [CI]) of the knowledge test were 82% (95% CI, 63%-93%), 88% (95% CI, 68%-95%), and 74% (95% CI, 53%-87%) for tracheal, lung, and hemidiaphragmatic scans, respectively. For the simulation image evaluation, 94% (95% CI, 77%-99%) of tracheal scan images, 97% (95% CI, 81%-99%) of lung scan images, and 88% (95% CI, 69%-96%) of hemidiaphragmatic scan images were determined clinically useful. For the pediatric intensive care unit image evaluation, 100% (95% CI, 86%-100%) of tracheal scan images, 100% (95% CI, 86%-100%) of lung scan images, and 79% (95% CI, 59%-91%) of hemidiaphragmatic scan images were determined clinically useful. A tracheal dilation sign was rarely captured by POCUS. CONCLUSION: Transport nurses can acquire POCUS skills to confirm endotracheal tube placement in children. A combination of a double trachea sign, a lung sliding sign, and a lung pulse sign could be a feasible POCUS approach.

A sleep-like state in Hydra unravels conserved sleep mechanisms during the evolutionary development of the central nervous system.

Sleep behaviors are observed even in nematodes and arthropods, yet little is known about how sleep-regulatory mechanisms have emerged during evolution. Here, we report a sleep-like state in the cnidarian Hydra vulgaris with a primitive nervous organization. Hydra sleep was shaped by homeostasis and necessary for cell proliferation, but it lacked free-running circadian rhythms. Instead, we detected 4-hour rhythms that might be generated by ultradian oscillators underlying Hydra sleep. Microarray analysis in sleep-deprived Hydra revealed sleep-dependent expression of 212 genes, including cGMP-dependent protein kinase 1 (PRKG1) and ornithine aminotransferase. Sleep-promoting effects of melatonin, GABA, and PRKG1 were conserved in Hydra However, arousing dopamine unexpectedly induced Hydra sleep. Opposing effects of ornithine metabolism on sleep were also evident between Hydra and Drosophila, suggesting the evolutionary switch of their sleep-regulatory functions. Thus, sleep-relevant physiology and sleep-regulatory components may have already been acquired at molecular levels in a brain-less metazoan phylum and reprogrammed accordingly.

Hydra vulgaris exhibits day-night variation in behavior and gene expression levels.

BACKGROUND: Day-night behavioral variation is observed in most organisms, and is generally controlled by circadian clocks and/or synchronization to environmental cues. Hydra species, which are freshwater cnidarians, are thought to lack the core clock genes that form transcription-translation feedback loops in clock systems. In this study, we examined whether hydras exhibit diel rhythms in terms of behavior and gene expression levels without typical clock genes. RESULTS: We found that the total behavior of hydras was elevated during the day and decreased at night under a 12-h light-dark cycle. Polyp contraction frequency, one component of behavior, exhibited a clear diel rhythm. However, neither total behavior nor polyp contraction frequency showed rhythmic changes under constant light and constant dark conditions. To identify the genes underlying diel behavior, we performed genome-wide transcriptome analysis of hydras under light-dark cycles. Using three different analytic algorithms, we found that 380 genes showed robust diel oscillations in expression. Some of these genes shared common features with diel cycle genes of other cnidarian species with endogenous clock systems. CONCLUSION: Hydras show diel behavioral rhythms under light-dark cycles despite the absence of canonical core clock genes. Given the functions of the genes showing diel oscillations in hydras and the similarities of those genes with the diel cycle genes of other cnidarian species with circadian clocks, it is possible that diel cycle genes play an important role across cnidarian species regardless of the presence or absence of core clock genes under light-dark cycles.

Just-in-Time Training for Intraosseous Needle Placement and Defibrillator Use in a Pediatric Emergency Department.

OBJECTIVES: Just-in-time training (JITT) is a method of simulation-based training where the training occurs within the clinical environment in a concise manner. Just-in-time training has shown effects at the learner, patient, and system-wide levels. We evaluated a JITT curriculum for the procedures of intraosseous (IO) needle placement and defibrillator use in a pediatric emergency department (ED) by comparing the trainees' comfort level in performing those procedures independently (Kirkpatrick level 2a) and trainees' knowledge of the procedures/equipment (Kirkpatrick level 2b) before and after the JITT. METHODS: The study enrolled all fourth year medical students and residents (family medicine and pediatrics) who rotated through a children's hospital ED. The JITT curriculum included group discussion on storage locations of procedure equipment in the ED and clinical indications/contraindications followed by hands-on procedure training. One of 2 attending physicians facilitated the 10- to 20-minute JITT in the ED during their shifts. Trainees completed an anonymous survey to delineate medical training level, previous procedure experiences, procedure-related knowledge, and comfort level to perform the procedures independently. Identical surveys were completed before and after the JITT. The data were analyzed using percentage for categorical variables. For comparisons between pre-JITT and post-JITT survey data, χ tests or Fisher exact tests were used. RESULTS: There were 65 surveys included (34 pre-JITT and 31 post-JITT surveys). The comfort level to perform procedures independently increased from pre-JITT 0% to post-JITT 48% (P < 0.001) for IO needle placement and from pre-JITT 3% to post-JITT 32% (P = 0.0016) for defibrillator use. The procedure-related knowledge also increased by ##greater than or equal to 50% post-JITT (P < 0.0001). CONCLUSIONS: Our JITT curriculum significantly increased the comfort level of the trainees to perform IO needle insertion and defibrillator use independently. Procedure-related knowledge also increased. By increasing their comfort to perform these procedures independently, we aim to increase the likelihood that trainees can be competent contributing members of an acute medical response team in these respective roles.

Ataxin-2: A versatile posttranscriptional regulator and its implication in neural function.

Ataxin-2 (ATXN2) is a eukaryotic RNA-binding protein that is conserved from yeast to human. Genetic expansion of a poly-glutamine tract in human ATXN2 has been implicated in several neurodegenerative diseases, likely acting through gain-of-function effects. Emerging evidence, however, suggests that ATXN2 plays more direct roles in neural function via specific molecular and cellular pathways. ATXN2 and its associated protein complex control distinct steps in posttranscriptional gene expression, including poly-A tailing, RNA stabilization, microRNA-dependent gene silencing, and translational activation. Specific RNA substrates have been identified for the functions of ATXN2 in aspects of neural physiology, such as circadian rhythms and olfactory habituation. Genetic models of ATXN2 loss-of-function have further revealed its significance in stress-induced cytoplasmic granules, mechanistic target of rapamycin signaling, and cellular metabolism, all of which are crucial for neural homeostasis. Accordingly, we propose that molecular evolution has been selecting the ATXN2 protein complex as an important trans-acting module for the posttranscriptional control of diverse neural functions. This explains how ATXN2 intimately interacts with various neurodegenerative disease genes, and suggests that loss-of-function effects of ATXN2 could be therapeutic targets for ATXN2-related neurological disorders. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Deciphering the Genes for Taste Receptors for Fructose in Drosophila.

Taste sensitivity to sugars plays an essential role in the initiation of feeding behavior. In Drosophila melanogaster, recent studies have identified several gustatory receptor (Gr) genes required for sensing sweet compounds. However, it is as yet undetermined how these GRs function as taste receptors tuned to a wide range of sugars. Among sugars, fructose has been suggested to be detected by a distinct receptor from other sugars. While GR43A has been reported to sense fructose in the brain, it is not expressed in labellar gustatory receptor neurons that show taste response to fructose. In contrast, the Gr64a-Gr64f gene cluster was recently shown to be associated with fructose sensitivity. Here we sought to decipher the genes required for fructose response among Gr64a-Gr64f genes. Unexpectedly, the qPCR analyses for these genes show that labellar expression levels of Gr64d and Gr64e are higher in fructose low-sensitivity flies than in high-sensitivity flies. Moreover, gustatory nerve responses to fructose in labellar sensilla are higher in Gr64d and Gr64f mutant lines than in mutant flies of the other Gr64a-Gr64f genes. These data suggest the possibility that deletion of GR64D or GR64F may indirectly induce enhanced fructose sensitivity in the labellum. Finally, we conclude that response to fructose cannot be explained by a single one of the Gr64a-Gr64f genes.

cryptochrome genes form an oscillatory loop independent of the per/tim loop in the circadian clockwork of the cricket Gryllus bimaculatus.

BACKGROUND: Animals exhibit circadian rhythms with a period of approximately 24 h in various physiological functions, including locomotor activity. This rhythm is controlled by an endogenous oscillatory mechanism, or circadian clock, which consists of cyclically expressed clock genes and their product proteins. cryptochrome (cry) genes are thought to be involved in the clock mechanism, and their functions have been examined extensively in holometabolous insects, but in hemimetabolous insects their role is less well understood. RESULTS: In the present study, the role of cry genes was investigated using RNAi technology in a hemimetabolous insect, the cricket Gryllus bimaculatus. Using a molecular cloning approach, we obtained cDNAs for two cry genes: Drosophila-type cry1 (Gb'cry1) and mammalian-type cry2 (Gb'cry2). Gb'cry2 has six splicing variants, most of which showed rhythmic mRNA expression. Gb'cry1RNAi treatment had only a limited effect at the behavioral and molecular levels, while Gb'cry2RNAi had a significant effect on behavioral rhythms and molecular oscillatory machinery, alone or in combination with Gb'cry1RNAi. In Gb'cry1/Gb'cry2 double-RNAi crickets, most clock genes showed arrhythmic expression, except for timeless, which retained clear rhythmic expression. Molecular analysis revealed that some combination of Gb'cry1 and Gb'cry2 variants suppressed CLK/CYC transcriptional activity in cultured cells. CONCLUSION: Based on these results, we propose a new model of the cricket's circadian clock, including a molecular oscillatory loop for Gb'cry2, which can operate independent of the Gb'per/Gb'tim loop.

Mated Drosophila melanogaster females consume more amino acids during the dark phase.

To maintain homeostasis, animals must ingest appropriate quantities, determined by their internal nutritional state, of suitable nutrients. In the fruit fly Drosophila melanogaster, an amino acid deficit induces a specific appetite for amino acids and thus results in their increased consumption. Although multiple processes of physiology, metabolism, and behavior are under circadian control in many organisms, it is unclear whether the circadian clock also modulates such motivated behavior driven by an internal need. Differences in levels of amino acid consumption by flies between the light and dark phases of the day:night cycle were examined using a capillary feeder assay following amino acid deprivation. Female flies exhibited increased consumption of amino acids during the dark phase compared with the light phase. Investigation of mutants lacking a functional period gene (per0), a well-characterized clock gene in Drosophila, found no difference between the light and dark phases in amino acid consumption by per0 flies. Furthermore, increased consumption of amino acids during the dark phase was observed in mated but not in virgin females, which strongly suggested that mating is involved in the rhythmic modulation of amino acid intake. Egg production, which is induced by mating, did not affect the rhythmic change in amino acid consumption, although egg-laying behavior showed a per0-dependent change in rhythm. Elevated consumption of amino acids during the dark phase was partly induced by the action of a seminal protein, sex peptide (SP), on the sex peptide receptor (SPR) in females. Moreover, we showed that the increased consumption of amino acids during the dark phase is induced in mated females independently of their internal level of amino acids. These results suggest that a post-mating SP/SPR signal elevates amino acid consumption during the dark phase via the circadian clock.

Genetics of Circadian Rhythms.

Nearly all organisms exhibit time-dependent behavior and physiology across a 24-hour day known as circadian rhythms. These outputs are manifestations of endogenous cyclic gene expression patterns driven by the activity of a core transcription/translation feedback loop. Cyclic gene expression determines highly tissue-specific functional activity regulating such processes as metabolic state, endocrine activity, and neural excitability. Entrainment of these cellular clocks is achieved through exogenous daily inputs, such as light and food. Dysregulation of the transcription/translation feedback loop has been shown to result in a wide range of disorders and diseases driving increased interest in circadian therapies.

C-terminal binding protein (CtBP) activates the expression of E-box clock genes with CLOCK/CYCLE in Drosophila.

In Drosophila, CLOCK/CYCLE heterodimer (CLK/CYC) is the primary activator of circadian clock genes that contain the E-box sequence in their promoter regions (hereafter referred to as "E-box clock genes"). Although extensive studies have investigated the feedback regulation of clock genes, little is known regarding other factors acting with CLK/CYC. Here we show that Drosophila C-terminal binding protein (dCtBP), a transcriptional co-factor, is involved in the regulation of the E-box clock genes. In vivo overexpression of dCtBP in clock cells lengthened or abolished circadian locomotor rhythm with up-regulation of a subset of the E-box clock genes, period (per), vrille (vri), and PAR domain protein 1ε (Pdp1ε). Co-expression of dCtBP with CLK in vitro also increased the promoter activity of per, vri, Pdp1ε and cwo depending on the amount of dCtBP expression, whereas no effect was observed without CLK. The activation of these clock genes in vitro was not observed when we used mutated dCtBP which carries amino acid substitutions in NAD+ domain. These results suggest that dCtBP generally acts as a putative co-activator of CLK/CYC through the E-box sequence.

Membrane-bound transporter controls the circadian transcription of clock genes in Drosophila.

Little is known about molecular mechanisms that control the Drosophila circadian clock beyond the transcriptional-translational feedback regulation of clock genes as an intracellular process. In this study, Early gene at 23 (E23) was identified as a novel clock gene that encodes the membrane-bound ABC transporter that is induced by the molting hormone ecdysone. E23 expresses in pacemaker neurons in fly head, and its knockdown flies lengthened circadian period with an increased expression of the clock gene vrille. E23 and vrille responded to both ecdysone and clock signals, whereas E23 protein specifically suppressed the ecdysone response and is necessary for rhythmicity. Thus, E23 forms its own feedback loop in the ecdysone response to control circadian oscillation through ecdysone-mediated vrille expression. The ecdysone signaling pathway with E23 is essential not only in developmental stage but also for the circadian behavior in adult fly.

Ets family members induce lymphangiogenesis through physical and functional interaction with Prox1.

Prox1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.

Self-assembly cloning: a rapid construction method for recombinant molecules from multiple fragments.

Enzyme-free cloning (EFC) can rapidly produce an in-frame fusion gene with multiple fragments. To practically apply EFC, we investigated the extent and sequence of complementary staggered overhangs necessary to direct self-assembly of multiple fragments as well as a size limitation of the constructed DNA molecule. Six-base pair overhangs with 50% GC content were sufficient to direct self-assembly. A functional plasmid that exceeded 10 kb, which includes an in-frame fusion domain, was efficiently constructed from four PCR fragments in one step by our improved method.

A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock.

The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tiling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation.