Reversal of MYB-dependent suppression of MAFB expression overrides leukaemia phenotype in MLL-rearranged AML.

The transcription factor MYB plays a pivotal role in haematopoietic homoeostasis and its aberrant expression is involved in the genesis and maintenance of acute myeloid leukaemia (AML). We have previously demonstrated that not all AML subtypes display the same dependency on MYB expression and that such variability is dictated by the nature of the driver mutation. However, whether this difference in MYB dependency is a general trend in AML remains to be further elucidated. Here, we investigate the role of MYB in human leukaemia by performing siRNA-mediated knock-down in cell line models of AML with different driver lesions. We show that the characteristic reduction in proliferation and the concomitant induction of myeloid differentiation that is observed in MLL-rearranged and t(8;21) leukaemias upon MYB suppression is not seen in AML cells with a complex karyotype. Transcriptome analyses revealed that MYB ablation produces consensual increase of MAFB expression in MYB-dependent cells and, interestingly, the ectopic expression of MAFB could phenocopy the effect of MYB suppression. Accordingly, in silico stratification analyses of molecular data from AML patients revealed a reciprocal relationship between MYB and MAFB expression, highlighting a novel biological interconnection between these two factors in AML and supporting new rationales of MAFB targeting in MLL-rearranged leukaemias.

Biopsychosocial sequelae and recovery trajectories from whiplash injury following a motor vehicle collision.

BACKGROUND CONTEXT: Five out of 10 injured in a motor vehicle collision (MVC) will develop persistent pain and disability. It is unclear if prolonged symptoms are related to peritraumatic pain/disability, psychological distress, muscle fat, lower extremity weakness. PURPOSE: To test if widespread muscle fat infiltration (MFI) was (1) unique to those with poor recovery, (2) present in the peritraumatic stage, (3) related to known risk factors. STUDY DESIGN/SETTING: A cohort study, single-center academic hospital. PATIENT SAMPLES: A total of 97 men and women (age 18-65) presenting to an urban academic emergency medicine department following MVC, but not requiring inpatient hospitalization. PRIMARY OUTCOME MEASURE: Neck disability at 12-months. METHODS: Participants underwent magnetic resonance imaging (MRI) to quantify neck and lower extremity MFI, completed questionnaires on pain/disability and psychological distress (< 1-week, 2-weeks, 3-, and 12-months) and underwent maximum volitional torque testing of their lower extremities (2-weeks, 3-, and 12-months). Percentage score on the Neck Disability Index at 12-months was used for a model of (1) Recovered (0%-8%), (2) Mild (10%-28%), and (3) Moderate/Severe (≥ 30%). This model was adjusted for BMI and age. RESULTS: Significant differences for neck MFI were revealed, with the Recovered group having significantly lower neck MFI than the Mild and Moderate/Severe groups at all time points. The Mild group had significantly more leg MFI at 12-months (p=.02) than the Recovered group. There were no other significant differences at any other time point. Lower extremity torques revealed no group differences. The Traumatic Injury Distress Scale (TIDS) and MFI of the neck at 1-week postinjury significantly predicted NDI score at 12-months. CONCLUSIONS: Higher neck MFI and distress may represent a risk factor though it is unclear whether this is a pre-existing phenotype or result of the trauma. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02157038.

Whiplash-Associated Dysphagia: Considerations of Potential Incidence and Mechanisms.

Non-specific self-reports of dysphagia have been described in people with whiplash-associated disorders (WAD) following motor vehicle collision (MVC); however, incidence and mechanistic drivers remain poorly understood. Alterations in oropharyngeal dimensions on magnetic resonance imaging (MRI), along with heightened levels of stress, pain, and changes in stress-dependent microRNA expression (e.g., miR-320a) have been also associated with WAD, suggesting multi-factorial issues may underpin any potential swallowing changes. In this exploratory paper, we examine key biopsychosocial parameters in three patients with persistent WAD reporting swallowing change and three nominating full recovery after whiplash with no reported swallowing change. Parameters included (1) oropharyngeal volume with 3D MRI, (2) peritraumatic miR-320a expression, and (3) psychological distress. These factors were explored to highlight the complexity of patient presentation and propose future considerations in relation to a potential deglutition disorder following WAD. The three participants reporting changes in swallowing all had smaller oropharyngeal volumes at < 1 week and at 3 months post injury and lower levels of peritraumatic miR-320a. At 3 months post MVC, oropharyngeal volumes between groups indicated a large effect size (Hedge's g = 0.96). Higher levels of distress were reported at both time points for those with persistent symptomatology, including self-reported dysphagia, however, this was not featured in those nominating recovery. This paper considers current evidence for dysphagia as a potentially under-recognized feature of WAD and highlights the need for future, larger-scaled, multidimensional investigation into the incidence and mechanisms of whiplash-associated dysphagia.

A cyclic nucleotide-gated channel in the brain regulates olfactory modulation through neuropeptide F in fruit fly Drosophila melanogaster.

Olfactory sensing and its modulation are important for the insects in recognizing diverse odors from the environment and in making correct decisions to survive. Identifying new genes involved in olfactory modulation and unveiling their mechanisms may lead us to understand decision making processes in the central nervous system. Here, we report a novel olfactory function of the cyclic nucleotide-gated (CNG) channel CG42260 in modulating ab3A olfactory sensory neurons, which specifically respond to food-derived odors in fruit fly Drosophila melanogaster. We found that two independent CG42260 mutants show reduced responses in the ab3A neurons. Unlike mammalian CNGs, CG42260 is not expressed in the odorant sensory neurons but broadly in the central nervous system including neuropeptide-producing cells. By using molecular genetic tools, we identified CG42260 expression in one pair of neuropeptide F (NPF) positive L1-l cells known to modulate food odor responsiveness. Knockdown of CG42260 in the NPF neurons reduced production of NPF in Ll-1 cells, which in turn, led to reduction of neuronal responses of the ab3A neurons. Our findings show the novel biological function of CG42260 in modulating olfactory responses to food odor through NPF.

A Commercially Available Capacitive Stretch-Sensitive Sensor for Measurement of Rotational Neck Movement in Healthy People: Proof of Concept.

Freedom of neck range of motion has been identified for decades as an important indicator of neck health. In the past, neck motion has been measured in clinical settings using straight-plane movements that do not represent real-world 'ecological' performance. The tools currently used are low-fidelity analog or digital tools that rely greatly on the orientation of the person with respect to gravity, or the evaluator's ability to accurately align protractor arms with key surface markers for angle measurement. A possible solution lies in the use of wearable sensors for tracking the motion of the neck without clinical instruction. For this purpose, the focus of this paper is on the assessment of a commercially available stretch sensitive sensor, C-Stretch® against a gold standard for motion tracking. The sensor's accuracy and agreement for measuring neck rotations were evaluated. The results show that the stretch sensitive sensor was accurate with an average RMSE of 5.86° (SD=$4.38^{\circ}, \mathrm{n}=2$) and highly correlated $r=0.88-0.99,(p\lt0.01)$ with Aurora, an electromagnetic tracking system. This work may lead to using wearable sensors as a cost-effective, lightweight, and safe alternative to assess real-world neck range of motion for clinical application.

Does a familial subtype of complex regional pain syndrome exist? Results of a systematic review.

Background and Objective: Complex regional pain syndrome (CRPS) is a chronic condition characterized by severe regional pain, allodynia, hyperalgesia, and functional impairment. The aim of this systematic review is to investigate whether a familial subtype of CRPS (fCRPS) exists and to determine whether people with fCRPS have specific characteristics. Methods: Databases CINAHL, Medline, PsycINFO, and PubMed were searched with no date limitation. Quality of reporting was assessed using the Scottish Intercollegiate Guidelines Network scale and the Joanna Briggs Institute's checklists. Results: Eight studies were included. Family relationships were defined as any immediate (i.e., parents or siblings) or blood relatives. A combination of participants with known or unknown causes for CRPS was recruited. The studies in this review support the potential for the existence of fCRPS, although this included less than 25% of those affected. People with potential fCRPS showed more severe symptoms, more sites involved, a higher percentage of spontaneous onset, and earlier age at onset. An elevated sibling recurrence risk ratio of 5.6 (95% confidence interval [CI], 3.0 to 9.8) was reported for people under 50. None of the studies established a pattern of heritability. Therefore, the most likely explanation for heritability would be a multifactorial model in which cumulative and interactive Gene × Environment effects may be involved. Conclusions: This systematic review supports the potential for the existence of fCRPS; however, all identified studies used uncontrolled case reports, case series, and case-control designs that cannot provide evidence of causation. Further studies are required to reveal the heritability and genetic structure of fCRPS.

Contexte et objectifs: Le syndrome de douleur régionale complexe (SDRC) est une maladie chronique qui se caractérise par de fortes douleurs régionales, une allodynie, une hyperalgésie et une déficience fonctionnelle. Le but de cette revue systématique était de vérifier s'il existe un sous-type familial de SDRC (SDRCf) et de déterminer si les personnes atteintes de SDRCf présentent des caractéristiques particulières.Méthodes: Des recherches ont été effectuées dans les bases de données CINAHL, Medline, PsychINFO et PubMed, sans limite de date. La qualité des rapports a été évaluée à l'aide de l'échelle du Scottish Intercollegiate Guidelines Network et des listes de vérification de l'Institut Joanna Briggs.Résultats: Huit études ont été incluses. Les relations familiales ont été définies comme toutes les relations immédiates (i.e. parents, frères ou soeurs), ou les parents consanguins. Une combinaison de participants pour lesquels les causes du SDRC étaient connues et inconnues ont été recrutés. Les études ayant fait partie de cette revue vont dans le sens de la possible existence d'un SDRCf, bien que cette constatation touchait moins de 25 % des personnes affectées. Les personnes souffrant d'un possible SDRCf présentaient des symptômes plus graves, un plus grand nombre de régions touchées, un pourcentage plus élevé de déclenchements spontanés et le déclenchement de la maladie à un plus jeune âge. Un rapport relatif de récurrence élevé, se situant à 5,6 (95 % IC, 3,0 à 9,8) chez les frères et soeurs, a été rapporté pour les personnes de moins de 50 ans. Aucune des études n'établissait de modèle d'héritabilité. L'explication la plus plausible pour l'héritabilité serait donc un modèle mutifactoriel dans lequel les effets cumulatifs des gènes et de l'environnement pourraient interagir.Conclusions: Les conclusions de cette revue systémaique vont dans le sens de l'existence possible du SDRCf; toutefois, toutes les études répertoriées ont eu recours à des devis non contrôlés comme des rapports de cas, des séries de cas et des études cas-témoins qui ne peuvent pas prouver le lien de causalité. D'autres études sont nécesaires pour révéler l'héritabilité et la structure génétique du SDRCf.

The exceptional X-ray evolution of SN 1996cr in high resolution.

We present X-ray spectra spanning 18 yr of evolution for SN 1996cr, one of the five nearest SNe detected in the modern era. Chandra HETG exposures in 2000, 2004, and 2009 allow us to resolve spectrally the velocity profiles of Ne, Mg, Si, S, and Fe emission lines and monitor their evolution as tracers of the ejecta-circumstellar medium interaction. To explain the diversity of X-ray line profiles, we explore several possible geometrical models. Based on the highest signal-to-noise 2009 epoch, we find that a polar geometry with two distinct opening angle configurations and internal obscuration can successfully reproduce all of the observed line profiles. The best-fitting model consists of two plasma components: (1) a mildly absorbed (2 × 1021 cm-2), cooler (≈2 keV) with high Ne, Mg, Si, and S abundances associated with a wide polar interaction region (half-opening angle ≈58°); (2) a moderately absorbed (2 × 1022 cm-2), hotter (≳20 keV) plasma with high Fe abundances and strong internal obscuration associated with a narrow polar interaction region (half-opening angle ≈20°). We extend this model to seven further epochs with lower signal-to-noise ratio and/or lower spectral-resolution between 2000 and 2018, yielding several interesting trends in absorption, flux, geometry, and expansion velocity. We argue that the hotter and colder components are associated with reflected and forward shocks, respectively, at least at later epochs. We discuss the physical implications of our results and plausible explosion scenarios to understand the X-ray data of SN 1996cr.

Correlation of Endoscopic and Histologic Severity Scores in Pediatric Ulcerative Colitis at First Presentation.

Detailed histologic scoring systems have been developed for the assessment of disease activity in ulcerative colitis. Literature from adult patients has shown some correlation between endoscopy and histology, and reproducibility of histologic scoring systems has also been supported. The effectiveness of endoscopic appearance at predicting histologic scores in pediatric patients has not been well studied, and none of the histologic scoring systems used in adults have had interobserver reproducibility assessed in pediatric patients. We reviewed endoscopic images and concurrent biopsies using Mayo and Geboes scores from the distal colon and rectum in untreated pediatric patients at the presentation of presumed ulcerative colitis based on clinical and endoscopic findings. Interobserver concordance was calculated by weighted-kappa statistic. The averaged histologic scores were compared to endoscopy scores using Spearman's coefficient. Correlation between endoscopic score and each histologic score was weakly to moderately positive, whereas interobserver agreement for histologic scores was fair to moderate, suggesting that the Geboes scoring system has value in pediatric patients. For each histologic parameter, the average score was lower than the average endoscopic score. Examination of larger pediatric cohorts, treated patients, correlations of clinical outcomes with individual histologic parameters, and alternate scoring systems may contextualize these findings.

The Multidimensional Symptom Index: A new patient-reported outcome for pain phenotyping, prognosis and treatment decisions.

BACKGROUND: There are few patient-reported outcomes routinely used that capture frequency and interference of different pain-related symptoms on a single scale. The purpose of this study was to describe the development and initial validation of the new Multidimensional Symptom Index (MSI). METHODS: Items were generated from patient interviews of the experience of chronic pain. Health valuations were created from rankings of 82 healthy subjects for each of 120 symptom (×10) × frequency (×3) × interference (×4) combinations using preference-based health valuations (0-100). Ranks for each symptom combination were then used in scale scoring. A sample of 300 patients with acute or chronic pain subsequently completed the MSI and a battery of other tools. Exploratory (EFA) and Confirmatory (CFA) factor analyses were triangulated with theory to arrive at the factor structure. Convergent validity was tested against established measures. RESULTS: Health rankings resulted in scores of 0-12 for each of the 10 symptom types. Factor analyses revealed two factors: MSI Somatic Symptoms and MSI Non-Somatic Symptoms. The MSI also quantified number of symptoms experienced (/10), mean frequency (/3) and mean interference (/4). The indices showed appropriate associations with the established PROs. CONCLUSIONS: The MSI is a new symptom-focused PRO that allows patient phenotyping and may have value for screening, prognosis and evaluating change. SIGNIFICANCE: This article presents the development and psychometric properties of a new measure of pain and related symptom frequency and interference. This measure could aid clinicians in establishing clinically relevant pain phenotypes for screening, prognosis and treatment decisions.

Mass Spectrometry-Based Screening Platform Reveals Orco Interactome in Drosophila melanogaster.

Animals use their odorant receptors to receive chemical information from the environment. Insect odorant receptors differ from the G protein-coupled odorant receptors in vertebrates and nematodes, and very little is known about their protein-protein interactions. Here, we introduce a mass spectrometric platform designed for the large-scale analysis of insect odorant receptor protein-protein interactions. Using this platform, we obtained the first Orco interactome from Drosophila melanogaster. From a total of 1,186 identified proteins, we narrowed the interaction candidates to 226, of which only two-thirds have been named. These candidates include the known olfactory proteins Or92a and Obp51a. Around 90% of the proteins having published names likely function inside the cell, and nearly half of these intracellular proteins are associated with the endomembrane system. In a basic loss-of-function electrophysiological screen, we found that the disruption of eight (i.e., Rab5, CG32795, Mpcp, Tom70, Vir-1, CG30427, Eaat1, and CG2781) of 28 randomly selected candidates affects olfactory responses in vivo. Thus, because this Orco interactome includes physiologically meaningful candidates, we anticipate that our platform will help guide further research on the molecular mechanisms of the insect odorant receptor family.

Cyanide Toxicity of Freshly Prepared Smoothies and Juices Frequently Consumed.

AIMS: This study was conducted to detect the presence of cyanide in popular fruit and vegetable smoothies and juices marketed as raw and natural. STUDY DESIGN: Eleven (11) popular varieties of drinks were analyzed for total cyanide (TCN). Drinks contained raw vegetables and fruits, flax seeds, whole apples with seeds, raw almond milk, and pasteurized almond milk as ingredients. PLACE AND STUDY DURATION: Samples were collected from health food eateries located within Las Vegas, Nevada (USA) during the summer of 2017. METHODOLOGY: Fifty milliliters (mL) of a homogenized smoothie and juice drink and 1 gram of flax seeds were subjected to the above-referenced methods for sample preparation per USEPA Methods 9012B (digestion) followed by USEPA method 9014 (colorimetry). RESULTS: The highest TCN was detected in drinks containing raw flax seed followed by unpasteurized raw almond milk, then fresh whole apple juice. No TCN was observed in drinks that contained none of the above mentioned items (e.g. flax seed, raw almond milk) or those utilizing pasteurized ingredients. CONCLUSION: This study observed that TCN is present in smoothies and juices containing raw flax seeds, fresh whole apples, and/or unpasteurized almond milk. Concentrations were detected as high as 341 µg L-1 in commercially available smoothies containing vegetables, raw flax seeds, almond milk and fruits. Smoothies with vegetables, fruits, unpasteurized almond milk, and no flax seeds contained 41 ug L-1 TCN, while similar smoothies with pasteurized almond milk contained negligible to 9.6 ug L-1 CN-. Unpasteurized almond milk and raw flax seeds were the major sources of TCN in drinks. With the increased demand for raw and natural foods, there is a potential sublethal exposure of TCN by consumers.

Central peptidergic modulation of peripheral olfactory responses.

BACKGROUND: Animal olfactory systems detect volatile environmental chemicals and integrate this information to direct the discovery of food and mates as well as danger avoidance. Rather than remaining constant, olfactory response thresholds are modulated by internal and external cues to adapt odor-guided behaviors to changing conditions. RESULTS: Here, we show in Drosophila melanogaster that neuropeptide F (NPF) modulates the responses of a specific population of antennal olfactory sensory neurons (OSNs) to food-derived odors. We show that knock-down of NPF in NPF neurons specifically reduces the responses of the ab3A neurons to ethyl butyrate, a volatile ester found in apples and other fruits. Knock-down of the NPF receptor (NPFR) in the ab3A neuron reduces their responses and disrupts the ability of the flies to locate food. We also identify a sexual dimorphism in ab3A responsiveness: ab3A neurons in females immediately post-eclosion are less responsive to ethyl butyrate than those of both age-matched males and older females. Not only does this change correlate with brain NPF levels, but also NPFR mutants show no such sexual dimorphism. Finally, by way of mechanism, we show that mutation of NPFR seems to cause intracellular clustering of OR22a, the odorant receptor expressed in the ab3A neurons. CONCLUSIONS: Interestingly, this modulation of the peripheral odorant responsiveness of the ab3A neurons by NPF is distinct from the modulation of presynaptic gain in the ab3A neurons previously observed with the similarly named but distinct neuropeptide sNPF. Rather than affecting the strength of the output at the level of the first synapse in the antennal lobe, NPF-NPFR signaling may affect the process of odorant detection itself by causing intracellular OR clustering.

A fat-derived metabolite regulates a peptidergic feeding circuit in Drosophila.

Here, we show that the enzymatic cofactor tetrahydrobiopterin (BH4) inhibits feeding in Drosophila. BH4 biosynthesis requires the sequential action of the conserved enzymes Punch, Purple, and Sepiapterin Reductase (Sptr). Although we observe increased feeding upon loss of Punch and Purple in the adult fat body, loss of Sptr must occur in the brain. We found Sptr expression is required in four adult neurons that express neuropeptide F (NPF), the fly homologue of the vertebrate appetite regulator neuropeptide Y (NPY). As expected, feeding flies BH4 rescues the loss of Punch and Purple in the fat body and the loss of Sptr in NPF neurons. Mechanistically, we found BH4 deficiency reduces NPF staining, likely by promoting its release, while excess BH4 increases NPF accumulation without altering its expression. We thus show that, because of its physically distributed biosynthesis, BH4 acts as a fat-derived signal that induces satiety by inhibiting the activity of the NPF neurons.

Transcriptional regulation of SPROUTY2 by MYB influences myeloid cell proliferation and stem cell properties by enhancing responsiveness to IL-3.

Myeloproliferative neoplasms (MPN), which overproduce blood cells in the bone marrow, have recently been linked with a genetically determined decrease in expression of the MYB transcription factor. Here, we use a mouse MYB knockdown model with an MPN-like phenotype to show how lower levels of MYB lead to stem cell characteristics in myeloid progenitors. The altered progenitor properties feature elevated cytokine responsiveness, especially to interleukin-3, which results from increased receptor expression and increased MAPK activity leading to enhanced phosphorylation of a key regulator of protein synthesis, ribosomal protein S6. MYB acts on MAPK signaling by directly regulating transcription of the gene encoding the negative modulator SPRY2. This mechanistic insight points to pathways that might be targeted therapeutically in MPN.

Insect odorant receptor trafficking requires calmodulin.

BACKGROUND: Like most animals, insects rely on their olfactory systems for finding food and mates and in avoiding noxious chemicals and predators. Most insect olfactory neurons express an odorant-specific odorant receptor (OR) along with Orco, the olfactory co-receptor. Orco binds ORs and permits their trafficking to the dendrites of antennal olfactory sensory neurons (OSNs), where together, they are suggested to form heteromeric ligand-gated non-selective cation channels. While most amino acid residues in Orco are well conserved across insect orders, one especially well-conserved region in Orco's second intracellular loop is a putative calmodulin (CaM) binding site (CBS). In this study, we explore the relationship between Orco and CaM in vivo in the olfactory neurons of Drosophila melanogaster. RESULTS: We first found OSN-specific knock-down of CaM at the onset of OSN development disrupts the spontaneous firing of OSNs and reduces Orco trafficking to the ciliated dendrites of OSNs without affecting their morphology. We then generated a series of Orco CBS mutant proteins and found that none of them rescue the Orco-null Orco 1 mutant phenotype, which is characterized by an OR protein trafficking defect that blocks spontaneous and odorant-evoked OSN activity. In contrast to an identically constructed wild-type form of Orco that does rescue the Orco 1 phenotype, all the Orco CBS mutants remain stuck in the OSN soma, preventing even the smallest odorant-evoked response. Last, we found CaM's modulation of OR trafficking is dependent on activity. Knock-down of CaM in all Orco-positive OSNs after OR expression is well established has little effect on olfactory responsiveness alone. When combined with an extended exposure to odorant, however, this late-onset CaM knock-down significantly reduces both olfactory sensitivity and the trafficking of Orco only to the ciliated dendrites of OSNs that respond to the exposed odorant. CONCLUSIONS: In this study, we show CaM regulates OR trafficking and olfactory responses in vivo in Drosophila olfactory neurons via a well-conserved binding site on the olfactory co-receptor Orco. As CaM's modulation of Orco seems to be dependent on activity, we propose a model in which the CaM/Orco interaction allows insect OSNs to maintain appropriate dendritic levels of OR regardless of environmental odorant concentrations.

HLA Matching at the Eplet Level Protects Against Chronic Lung Allograft Dysfunction.

Donor selection in lung transplantation (LTx) is historically based upon clinical urgency, ABO compatibility, and donor size. HLA matching is not routinely considered; however, the presence or later development of anti-HLA antibodies is associated with poorer outcomes, particularly chronic lung allograft dysfunction (CLAD). Using eplet mismatches, we aimed to determine whether donor/recipient HLA incompatibility was a significant predictor of CLAD. One hundred seventy-five LTx undertaken at the Alfred Hospital between 2008 and 2012 met criteria. Post-LTx monitoring was continued for at least 12 months, or until patient death. HLA typing was performed by sequence-based typing and Luminex sequence-specific oligonucleotide. Using HLAMatchmaker, eplet mismatches between each donor/recipient pairing were analyzed and correlated against incidences of CLAD. HLA-DRB1/3/4/5+DQA/B eplet mismatch was a significant predictor of CLAD (hazard ratio [HR] 3.77, 95% confidence interval [CI]: 1.71-8.29 p < 0.001). When bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS) were analyzed independently, HLA-DRB1/3/4/5 + DQA/B eplet mismatch was shown to significantly predict RAS (HR 8.3, 95% CI: 2.46-27.97 p < 0.001) but not BOS (HR 1.92, 95% CI: 0.64-5.72, p = 0.237). HLA-A/B eplet mismatch was shown not to be a significant predictor when analyzed independently but did provide additional stratification of results. This study illustrates the importance of epitope immunogenicity in defining donor-recipient immune compatibility in LTx.

Identification of a Peptidergic Pathway Critical to Satiety Responses in Drosophila.

Although several neural pathways have been implicated in feeding behaviors in mammals [1-7], it remains unclear how the brain coordinates feeding motivations to maintain a constant body weight (BW). Here, we identified a neuropeptide pathway important for the satiety and BW control in Drosophila. Silencing of myoinhibitory peptide (MIP) neurons significantly increased BW through augmented food intake and fat storage. Likewise, the loss-of-function mutation of mip also increased feeding and BW. Suppressing the MIP pathway induced satiated flies to behave like starved ones, with elevated sensitivity toward food. Conversely, activating MIP neurons greatly decreased food intake and BW and markedly blunted the sensitivity of starved flies toward food. Upon terminating the activation protocol of MIP neurons, the decreased BW reverts rapidly to the normal level through a strong feeding rebound, indicating the switch-like role of MIP pathway in feeding. Surprisingly, the MIP-mediated BW decrease occurred independently of sex peptide receptor (SPR), the only known receptor for MIP, suggesting the presence of a yet-unknown MIP receptor. Together, our results reveal a novel anorexigenic pathway that controls satiety in Drosophila and provide a new avenue to study how the brain actively maintains a constant BW.

An accelerated miRNA-based screen implicates Atf-3 in Drosophila odorant receptor expression.

The Drosophila olfactory system is highly stereotyped in form and function; olfactory sensory neurons (OSNs) expressing a specific odorant receptor (OR) always appear in the same antennal location and the axons of OSNs expressing the same OR converge on the same antennal lobe glomeruli. Although some transcription factors have been implicated in a combinatorial code specifying OR expression and OSN identity, it is clear other players remain unidentified. In hopes of mitigating the challenges of genome-wide screening, we examined the feasibility of a two-tiered approach comprising a primary "pooling" screen for miRNAs whose tissue-specific over-expression causes a phenotype of interest followed by a focused secondary screen using gene-specific RNAi. Since miRNAs down-regulate their targets, miRNA over-expression phenotypes should be attributable to target loss-of-function. It is the sequence-dependence of miRNA-target pairing that suggests candidates for the secondary screen. Since miRNAs are short, however, miRNA misexpression will likely uncover non-biological miRNA-target relationships. Rather than focusing on miRNA function itself where these non-biological relationships could be misleading, we propose using miRNAs as tools to focus a more traditional RNAi-based screen. Here we describe such a screen that uncovers a role for Atf3 in the expression of the odorant receptor Or47b.

Ca-α1T, a fly T-type Ca2+ channel, negatively modulates sleep.

Mammalian T-type Ca(2+) channels are encoded by three separate genes (Cav3.1, 3.2, 3.3). These channels are reported to be sleep stabilizers important in the generation of the delta rhythms of deep sleep, but controversy remains. The identification of precise physiological functions for the T-type channels has been hindered, at least in part, by the potential for compensation between the products of these three genes and a lack of specific pharmacological inhibitors. Invertebrates have only one T-type channel gene, but its functions are even less well-studied. We cloned Ca-α1T, the only Cav3 channel gene in Drosophila melanogaster, expressed it in Xenopus oocytes and HEK-293 cells, and confirmed it passes typical T-type currents. Voltage-clamp analysis revealed the biophysical properties of Ca-α1T show mixed similarity, sometimes falling closer to Cav3.1, sometimes to Cav3.2, and sometimes to Cav3.3. We found Ca-α1T is broadly expressed across the adult fly brain in a pattern vaguely reminiscent of mammalian T-type channels. In addition, flies lacking Ca-α1T show an abnormal increase in sleep duration most pronounced during subjective day under continuous dark conditions despite normal oscillations of the circadian clock. Thus, our study suggests invertebrate T-type Ca(2+) channels promote wakefulness rather than stabilizing sleep.

Genome-wide microRNA screening reveals that the evolutionary conserved miR-9a regulates body growth by targeting sNPFR1/NPYR.

MicroRNAs (miRNAs) regulate many physiological processes including body growth. Insulin/IGF signalling is the primary regulator of animal body growth, but the extent to which miRNAs act in insulin-producing cells (IPCs) is unclear. Here we generate a UAS-miRNA library of Drosophila stocks and perform a genetic screen to identify miRNAs whose overexpression in the IPCs inhibits body growth in Drosophila. Through this screen, we identify miR-9a as an evolutionarily conserved regulator of insulin signalling and body growth. IPC-specific miR-9a overexpression reduces insulin signalling and body size. Of the predicted targets of miR-9a, we find that loss of miR-9a enhances the level of sNPFR1. We show via an in vitro binding assay that miR-9a binds to sNPFR1 mRNA in insect cells and to the mammalian orthologue NPY2R in rat insulinoma cells. These findings indicate that the conserved miR-9a regulates body growth by controlling sNPFR1/NPYR-mediated modulation of insulin signalling.