Glycolysis regulates Hedgehog signalling via the plasma membrane potential.

Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator-the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady-state levels of ATP and stabilizes Smoothened (Smo), the 7-pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full-length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N-acylethanolamides-lipoprotein-borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo-a key step in pathway activation-in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential.

Tumor induction in Drosophila imaginal epithelia triggers modulation of fat body lipid droplets.

Our understanding of cancer-specific metabolic changes is currently unclear. In recent years, the fruit fly Drosophila melanogaster with its powerful genetic tools has become an attractive model for studying both tumor autonomous and the systemic processes resulting from the tumor growth. Here we investigated the effect of tumorigenesis on the modulation of lipid droplets (LDs) in the larval fat bodies (mammalian equivalent of adipose tissue). We have overexpressed Notch signaling alone or in combination with the developmental regulator Myocyte enhancer factor 2 (Mef2) using wing-specific and eye-specific drivers, quantified the size of LDs in the fat body of the different tumor bearing larvae, and estimated the expression of genes associated with lipolysis and lipogenesis. We have found that hyperplastic and neoplastic tumor induced by overexpression of Notch and co-expression of Notch and Mef2 respectively triggers impaired lipid metabolism marked by increased size of fat body LDs. The impaired lipid metabolism in tumor carrying larvae is linked to the altered expression of genes that participate in lipolysis and lipogenesis. These findings reveal modulation of LDs as one of the host's specific response upon tumor initiation. This information could potentially uncover mechanisms for designing innovative approaches to modulate cancer growth.

Frizzled-Dependent Planar Cell Polarity without Secreted Wnt Ligands.

Planar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP components. It has been suggested that Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. Here, we dissect the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for the study of PCP. We found that PCP was normal in quintuple mutant wings that rely solely on the membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. We then used a nanobody-based approach to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. We conclude that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization.

Context-Dependent Tumorigenic Effect of Testis-Specific Mitochondrial Protein Tiny Tim 2 in Drosophila Somatic Epithelia.

We have undertaken a study towards understanding the effect of ectopic expression of testis proteins in the soma in Drosophila. Here, we show that in the larval neuroepithelium, ectopic expression of the germline-specific component of the inner mitochondrial translocation complex tiny tim 2 (ttm2) brings about cell autonomous hyperplasia and extension of G2 phase. In the wing discs, cells expressing ectopic ttm2 upregulate Jun N-terminal kinase (JNK) signaling, present extended G2, become invasive, and elicit non-cell autonomous G2 extension and overgrowth of the wild-type neighboring tissue. Ectopic tomboy20, a germline-specific member of the outer mitochondrial translocation complex is also tumorigenic in wing discs. Our results demonstrate the tumorigenic potential of unscheduled expression of these two testis proteins in the soma. They also show that a unique tumorigenic event may trigger different tumor growth pathways depending on the tissular context.

Transcriptional host-pathogen responses of Pseudogymnoascus destructans and three species of bats with white-nose syndrome.

Understanding how context (e.g., host species, environmental conditions) drives disease susceptibility is an essential goal of disease ecology. We hypothesized that in bat white-nose syndrome (WNS), species-specific host-pathogen interactions may partly explain varying disease outcomes among host species. We characterized bat and pathogen transcriptomes in paired samples of lesion-positive and lesion-negative wing tissue from bats infected with Pseudogymnoascus destructans in three parallel experiments. The first two experiments analyzed samples collected from the susceptible Nearctic Myotis lucifugus and the less-susceptible Nearctic Eptesicus fuscus, following experimental infection and hibernation in captivity under controlled conditions. The third experiment applied the same analyses to paired samples from infected, free-ranging Myotis myotis, a less susceptible, Palearctic species, following natural infection and hibernation (n = 8 sample pairs/species). Gene expression by P. destructans was similar among the three host species despite varying environmental conditions among the three experiments and was similar within each host species between saprophytic contexts (superficial growth on wings) and pathogenic contexts (growth in lesions on the same wings). In contrast, we observed qualitative variation in host response: M. lucifugus and M. myotis exhibited systemic responses to infection, while E. fuscus up-regulated a remarkably localized response. Our results suggest potential phylogenetic determinants of response to WNS and can inform further studies of context-dependent host-pathogen interactions.

Knockdown of genes involved in axonal transport enhances the toxicity of human neuromuscular disease-linked MATR3 mutations in Drosophila.

Mutations in the nuclear matrix protein Matrin 3 (MATR3) have been identified in amyotrophic lateral sclerosis and myopathy. To investigate the mechanisms underlying MATR3 mutations in neuromuscular diseases and efficiently screen for modifiers of MATR3 toxicity, we generated transgenic MATR3 flies. Our findings indicate that expression of wild-type or mutant MATR3 in motor neurons reduces climbing ability and lifespan of flies, while their expression in indirect flight muscles (IFM) results in abnormal wing positioning and muscle degeneration. In both motor neurons and IFM, mutant MATR3 expression results in more severe phenotypes than wild-type MATR3, demonstrating that the disease-linked mutations confer pathogenicity. We conducted a targeted candidate screen for modifiers of the MATR3 abnormal wing phenotype and identified multiple enhancers involved in axonal transport. Knockdown of these genes enhanced protein levels and insolubility of mutant MATR3. These results suggest that accumulation of mutant MATR3 contributes to toxicity and implicate axonal transport dysfunction in disease pathogenesis.

CHIP modulates APP-induced autophagy-dependent pathological symptoms in Drosophila.

Dysregulation of autophagy is associated with the neurodegenerative processes in Alzheimer's disease (AD), yet it remains controversial whether autophagy is a cause or consequence of AD. We have previously expressed the full-length human APP in Drosophila and established a fly AD model that exhibits multiple AD-like symptoms. Here we report that depletion of CHIP effectively palliated APP-induced pathological symptoms, including morphological, behavioral, and cognitive defects. Mechanistically, CHIP is required for APP-induced autophagy dysfunction, which promotes Aß production via increased expression of BACE and Psn. Our findings suggest that aberrant autophagy is not only a consequence of abnormal APP activity, but also contributes to dysregulated APP metabolism and subsequent AD pathogenesis.

Flight muscles degenerate by programmed cell death after migration in the wheat aphid, Sitobion avenae.

OBJECTIVE: Previous studies showed that flight muscles degenerate after migration in some aphid species; however, the underlying molecular mechanism remains virtually unknown. In this study, using the wheat aphid, Sitobion avenae, we aim to investigate aphid flight muscle degeneration and the underlying molecular mechanism. RESULTS: Sitobion avenae started to differentiate winged or wingless morphs at the second instar, the winged aphids were fully determined at the third instar, and their wings were fully developed at the fourth instar. After migration, the aphid flight muscles degenerated via programmed cell death, which is evidenced by a Terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling assay. Then, we identified a list of differentially expressed genes before and after tethered flights using differential-display reverse transcription-PCR. One of the differentially expressed genes, ubiquitin-ribosomal S27a, was confirmed using qPCR. Ubiquitin-ribosomal S27a is drastically up regulated following the aphids' migration and before the flight muscle degeneration. Our data suggested that aphid flight muscles degenerate after migration. During flight muscle degeneration, endogenous proteins may be degraded to reallocate energy for reproduction.

Wing Abnormality in a Wild-Hatched Whooping Crane (Grus americana) Chick from the Nonmigratory Population in Louisiana, USA.

We describe a wing abnormality in a wild-hatched Whooping Crane (Grus americana) chick from the reintroduced Louisiana, US nonmigratory population. Despite its seemingly compromised flight ability, the chick fledged, reached independence, and lived until 13 mo of age. Necropsy revealed an axial malunion near the left carpus likely resulting from trauma.

Impact of Varroa destructor and deformed wing virus on emergence, cellular immunity, wing integrity and survivorship of Africanized honey bees in Mexico.

The ectoparasitic mite Varroa destructor is the primary health problem of honey bees (Apis mellifera) worldwide. Africanized honey bees in Brazil have demonstrated tolerance to the mite, but there is controversy about the degree of mite tolerance of Africanized bees in other countries. This study was conducted to quantify the effect of V. destructor parasitism on emergence, hemocyte concentration, wing integrity and longevity of Africanized honey bees in Mexico. Africanized bee brood were artificially infested with V. destructor mites and held in an incubator until emergence as adults and compared to non-infested controls. Deformed wing virus (DWV) presence was determined in the mites used to infest the bees. After emergence, the bees were maintained in an incubator to determine survivorship. The percentage of worker bees that emerged from parasitized cells (69%) was significantly lower than that of bees emerged from non-infested cells (96%). Newly-emerged parasitized bees had a significantly lower concentration of hemocytes in the hemolymph than non-parasitized bees. Additionally, the proportion of bees with deformed wings that emerged from V. destructor-parasitized cells was significantly higher (54%) than that of the control group (0%). The mean survival time of bees that emerged from infested and non-infested cells was 8.5 ±â€¯0.3 and 14.4 ±â€¯0.4 days, respectively, and the difference was significant. We conclude that V. destructor parasitism and DWV infections kill, cause deformities and inhibit cellular immunity in developing Africanized honey bees, and significantly reduce the lifespan of adult bees in Mexico. These results suggest that the tolerance of Africanized bees to V. destructor is related to adult bee mechanisms.

Deformed Wing Virus Infection in Honey Bees (Apis mellifera L.).

Deformed wing virus (DWV) is a single-stranded RNA virus of honey bees (Apis mellifera L.) transmitted by the parasitic mite Varroa destructor. Although DWV represents a major threat to honey bee health worldwide, the pathological basis of DWV infection is not well documented. The objective of this study was to investigate clinicopathological and histological aspects of natural DWV infection in honey bee workers. Emergence of worker honey bees was observed in 5 colonies that were clinically affected with DWV and the newly emerged bees were collected for histopathology. DWV-affected bees were 2 times slower to emerge and had 30% higher mortality compared to clinically normal bees. Hypopharyngeal glands in bees with DWV were hypoplastic, with fewer intracytoplasmic secretory vesicles; cells affected by apoptosis were observed more frequently. Mandibular glands were hypoplastic and were lined by cuboidal epithelium in severely affected bees compared to tall columnar epithelium in nonaffected bees. The DWV load was on average 1.7 × 106 times higher (P < .001) in the severely affected workers compared to aged-matched sister honey bee workers that were not affected by deformed wing disease based on gross examination. Thus, DWV infection is associated with prolonged emergence, increased mortality during emergence, and hypoplasia of hypopharyngeal and mandibular glands in newly emerged worker honey bees in addition to previously reported deformed wing abnormalities.

Drosophila jumu modulates apoptosis via a JNK-dependent pathway and is required for other processes in wing development.

Previous studies in several model organisms have revealed that members of the Forkhead (Fkh) transcription factor family have multiple functions. Drosophila Jumeau (Jumu), a member of this family, participates in cardiogenesis, hematopoiesis and immune system homeostasis. Here, we show that loss of jumu function positively regulates or triggers apoptosis via a JNK-dependent pathway in wing development. jumu mutants showed reduced wing size and increased apoptosis. Moreover, we observed a loss of the anterior cross vein (ACV) phenotype that was similar to that observed in wings in which JNK signaling has been ectopically activated. The JNK signaling markers puckered (puc) and p-JNK were also significantly increased in the wing discs of jumu mutants. In addition, apoptosis induced by the loss of jumu was rescued by knocking down JNK, indicating a role for JNK in reducing jumu-induced apoptosis. Jumu could also control wing margin development via the positive regulation of cut expression, and the observed wing margin defect did not result from a loss of jumu-induced apoptosis. Further, jumu deficiency in the pupal wing could induce multiple wing hairs via a Rho1-mediated planar cell polarity pathway, but abnormal Rho1 expression was not why jumu loss induced apoptosis via a JNK-dependent pathway in wing discs.

The Two Prevalent Genotypes of an Emerging Infectious Disease, Deformed Wing Virus, Cause Equally Low Pupal Mortality and Equally High Wing Deformities in Host Honey Bees.

Deformed wing virus (DWV) is an emerging infectious disease of the honey bee (Apis mellifera) that is considered a major cause of elevated losses of honey bee colonies. DWV comprises two widespread genotypes: the originally described genotype A, and genotype B. In adult honey bees, DWV-B has been shown to be more virulent than DWV-A. However, their comparative effects on earlier host developmental stages are unknown. Here, we experimentally inoculated honey bee pupae and tested for the relative impact of DWV-A versus DWV-B on mortality and wing deformities in eclosing adults. DWV-A and DWV-B caused similar, and only slightly elevated, pupal mortality (mean 18% greater mortality than control). Both genotypes caused similarly high wing deformities in eclosing adults (mean 60% greater wing deformities than control). Viral titer was high in all of the experimentally inoculated eclosing adults, and was independent of wing deformities, suggesting that the phenotype 'deformed wings' is not directly related to viral titer or viral genotype. These viral traits favor the emergence of both genotypes of DWV by not limiting the reproduction of its vector, the ectoparasitic Varroa destructor mite, in infected pupae, and thereby facilitating the spread of DWV in honey bees infested by the mite.

Inhibitory effects of viral infection on cancer development.

Immune responses evoked on viral infections prevent the dissemination of infection that otherwise leads to the development of diseases in host organisms. In the present study, we investigated whether viral infection influences tumorigenesis in cancer-bearing animals using a Drosophila model of cancer. Cancer was induced in the posterior part of wing imaginal discs through the simultaneous inhibition of apoptosis and cell-cycle checkpoints. The larvae and embryos of cancer-induced flies were infected with Drosophila C virus, a natural pathogen to Drosophila, and larval wing discs and adult wings were morphologically examined for cancer characteristics relative to uninfected controls. We found that viral infections brought about an approximately 30% reduction in the rate of cancer development in both wing discs and wings. These inhibitory effects were not observed when growth-defective virus was used to infect animals. These results indicate that productive viral infections repress tumorigenesis in Drosophila.

Successful Management of Open, Contaminated Metacarpal Fractures in an Adult Snowy Owl ( Bubo scandiacus) With a Minimal Type II External Skeletal Fixator.

An adult, male snowy owl ( Bubo scandiacus) was found down and unable to fly in western New York State. Physical examination and radiographs revealed a subacute, open wound and fractured major and minor metacarpals of the right wing. A minimal type II external skeletal fixator (ESF) device was placed on the right major metacarpal bone and the open wound was allowed to granulate and close. After evidence of bone union, the ESF device was removed. The owl performed auto-physiotherapy throughout the process and was released with sustained flight 2 months postoperatively. It was recaptured 7 weeks later and underwent further rehabilitation to allow successful release 11 months after surgical stabilization. To our knowledge, this is the first case report describing use of a type II ESF device on the metacarpus of a bird.

Use of a Compounded Poloxamer 407 Antibiotic Topical Therapy as Part of the Successful Management of Chronic Ulcerative Dermatitis in a Congo African Grey Parrot ( Psittacus erithacus).

A 23-year-old, 425-g male African grey parrot ( Psittacus erithacus) was evaluated for chronic ulcerative dermatitis of the axillary regions under both wings. Initial swab cultures of the sites had revealed a coagulase-positive methicillin-resistant Staphylococcus aureas (MRSA) with marked antibiotic resistance. A second swab culture obtained 8 weeks after the initial culture showed heavy growth of a coagulase-positive Staphylococcus species, which could not be speciated, but showed the same sensitivity as the previous culture. Previous treatment included systemic antibiotics and a topical antimicrobial cream, with variable response and only temporary resolution. On examination, full-thickness, ulcerative, necrotic dermatitis was present under both wings with intermittent bleeding and subdermal tissue exposure. Initial treatment included wound debridement, oral antibiotics, topical therapy, analgesics, and bandages. After a relapse, a poloxamer gel containing 2% doxycycline, 1% chloramphenicol, and 0.5% mupirocin was used in combination with oral antibiotics and analgesics. On follow-up examination, the skin lesions had completely resolved and the patient was doing well and remains normal 4 years later. This report emphasizes the importance of prompt, aggressive multi-modal therapy for MRSA and other dermal bacterial infections in pet birds that may represent zoonoses or have carrier-state zoonotic potential. Preparation by a compounding pharmacy of a transdermal poloxamer gel containing antibiotics shows promise for severe, infected, ulcerative skin lesions in birds when other therapies fail to achieve a cure.

Design and evaluation of a deformable wing configuration for economical hovering flight of an insect-like tailless flying robot.

Studies on wing kinematics indicate that flapping insect wings operate at higher angles of attack (AoAs) than conventional rotary wings. Thus, effectively flying an insect-like flapping-wing micro air vehicle (FW-MAV) requires appropriate wing design for achieving low power consumption and high force generation. Even though theoretical studies can be performed to identify appropriate geometric AoAs for a wing for achieving efficient hovering flight, designing an actual wing by implementing these angles into a real flying robot is challenging. In this work, we investigated the wing morphology of an insect-like tailless FW-MAV, which was named KUBeetle, for obtaining high vertical force/power ratio or power loading. Several deformable wing configurations with various vein structures were designed, and their characteristics of vertical force generation and power requirement were theoretically and experimentally investigated. The results of the theoretical study based on the unsteady blade element theory (UBET) were validated with reference data to prove the accuracy of power estimation. A good agreement between estimated and measured results indicated that the proposed UBET model can be used to effectively estimate the power requirement and force generation of an FW-MAV. Among the investigated wing configurations operating at flapping frequencies of 23 Hz to 29 Hz, estimated results showed that the wing with a suitable vein placed outboard exhibited an increase of approximately 23.7% ± 0.5% in vertical force and approximately 10.2% ± 1.0% in force/power ratio. The estimation was supported by experimental results, which showed that the suggested wing enhanced vertical force by approximately 21.8% ± 3.6% and force/power ratio by 6.8% ± 1.6%. In addition, wing kinematics during flapping motion was analyzed to determine the reason for the observed improvement.

Glycogen synthase kinase 3ß functions as a positive effector in the WNK signaling pathway.

The with no lysine (WNK) protein kinase family is conserved among many species. Some mutations in human WNK gene are associated with pseudohypoaldosteronism type II, a form of hypertension, and hereditary sensory and autonomic neuropathy type 2A. In kidney, WNK regulates the activity of STE20/SPS1-related, proline alanine-rich kinase and/or oxidative-stress responsive 1, which in turn regulate ion co-transporters. The misregulation of this pathway is involved in the pathogenesis of pseudohypoaldosteronism type II. In the neural system, WNK is involved in the specification of the cholinergic neuron, but the pathogenesis of hereditary sensory and autonomic neuropathy type 2A is still unknown. To better understand the WNK pathway, we isolated WNK-associated genes using Drosophila. We identified Glycogen synthase kinase 3ß (GSK3ß)/Shaggy (Sgg) as a candidate gene that was shown to interact with the WNK signaling pathway in both Drosophila and mammalian cells. Furthermore, GSK3ß was involved in neural specification downstream of WNK. These results suggest that GSK3ß/Sgg functions as a positive effector in the WNK signaling pathway.

Isolation of Aggressive Behavior Mutants in Drosophila Using a Screen for Wing Damage.

Aggression is a complex social behavior that is widespread in nature. To date, only a limited number of genes that affect aggression have been identified, in large part because the complexity of the phenotype makes screening difficult and time-consuming regardless of the species that is studied. We discovered that aggressive group-housed Drosophila melanogaster males inflict damage on each other's wings, and show that wing damage negatively affects their ability to fly and mate. Using this wing-damage phenotype, we screened males from ∼1400 chemically mutagenized strains and found ∼40 mutant strains with substantial wing damage. Five of these mutants also had increased aggressive behavior. To identify the causal mutation in one of our top aggressive strains, we used whole-genome sequencing and genomic duplication rescue strategies. We identified a novel mutation in the voltage-gated potassium channel Shaker (Sh) and show that a nearby previously identified Sh mutation also results in increased aggression. This simple screen can be used to dissect the molecular mechanisms underlying aggression.

Evidence for a novel function of Awd in maintenance of genomic stability.

The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/NME2 metastasis suppressor genes. Awd acts in multiple tissues where its function is critical in establishing and maintaining epithelial integrity. Here, we analysed awd gene function in Drosophila epithelial cells using transgene-mediated RNA interference and genetic mosaic analysis. We show that awd knockdown in larval wing disc epithelium leads to chromosomal instability (CIN) and induces apoptosis mediated by activation of c-Jun N-terminal kinase. Forced maintenance of Awd depleted cells, by expressing the cell death inhibitor p35, downregulates atypical protein kinase C and DE-Cadherin. Consistent with their loss of cell polarity and enhanced level of matrix metalloproteinase 1, cells delaminate from wing disc epithelium. Furthermore, the DNA content profile of these cells indicates that they are aneuploid. Overall, our data demonstrate a novel function for awd in maintenance of genomic stability. Our results are consistent with other studies reporting that NME1 down-regulation induces CIN in human cell lines and suggest that Drosophila model could be successfully used to study in vivo the impact of NME/Awd - induced genomic instability on tumour development and metastasis formation.