Science.gov

Sample records for drosophila assay system

  1. Methods to assay Drosophila behavior.

    PubMed

    Nichols, Charles D; Becnel, Jaime; Pandey, Udai B

    2012-01-01

    Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases(1). We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials(2-4). The rapid iterative negative geotaxis (RING) assay(5) has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously

  2. The Drosophila visual system

    PubMed Central

    Zhu, Yan

    2013-01-01

    A compact genome and a tiny brain make Drosophila the prime model to understand the neural substrate of behavior. The neurogenetic efforts to reveal neural circuits underlying Drosophila vision started about half a century ago, and now the field is booming with sophisticated genetic tools, rich behavioral assays, and importantly, a greater number of scientists joining from different backgrounds. This review will briefly cover the structural anatomy of the Drosophila visual system, the animal’s visual behaviors, the genes involved in assembling these circuits, the new and powerful techniques, and the challenges ahead for ultimately identifying the general principles of biological computation in the brain.   A typical brain utilizes a great many compact neural circuits to collect and process information from the internal biological and external environmental worlds and generates motor commands for observable behaviors. The fruit fly Drosophila melanogaster, despite of its miniature body and tiny brain, can survive in almost any corner of the world.1 It can find food, court mate, fight rival conspecific, avoid predators, and amazingly fly without crashing into trees. Drosophila vision and its underlying neuronal machinery has been a key research model for at least half century for neurogeneticists.2 Given the efforts invested on the visual system, this animal model is likely to offer the first full understanding of how visual information is computed by a multi-cellular organism. Furthermore, research in Drosophila has revealed many genes that play crucial roles in the formation of functional brains across species. The architectural similarities between the visual systems of Drosophila and vertebrate at the molecular, cellular, and network levels suggest new principles discovered at the circuit level on the relationship between neurons and behavior in Drosophila shall also contribute greatly to our understanding of the general principles for how bigger brains work.3

  3. Paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the Drosophila melanogaster giant fiber system.

    PubMed

    Mejia, Monica; Heghinian, Mari D; Busch, Alexandra; Marí, Frank; Godenschwege, Tanja A

    2012-01-01

    Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on Dα7 nicotinic acetylcholine receptors (nAChRs). Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles. Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster" from Augustin et al, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in

  4. Developmental Toxicity Assays Using the Drosophila Model

    PubMed Central

    Rand, Matthew D.; Montgomery, Sara L.; Prince, Lisa; Vorojeikina, Daria

    2014-01-01

    The fruit fly (Drosophila melanogaster) has long been a premier model for developmental biologists and geneticists. The utility of Drosophila for toxicology studies has only recently gained broader recognition as a tool to elaborate molecular genetic mechanisms of toxic substances. In this article two practical applications of Drosophila for developmental toxicity assays are described. The first assay takes advantage of newly developed methods to render the fly embryo accessible to small molecules, toxicants and drugs. The second assay engages straightforward exposures to developing larvae and easy to score outcomes of adult development. With the extensive collections of flies that are publicly available and the ease with which to create transgenic flies, these two assays have a unique power for identifying and characterizing molecular mechanisms and cellular pathways specific to the mode of action of a number of toxicants and drugs. PMID:24789363

  5. Drosophila tools and assays for the study of human diseases.

    PubMed

    Ugur, Berrak; Chen, Kuchuan; Bellen, Hugo J

    2016-03-01

    Many of the internal organ systems of Drosophila melanogaster are functionally analogous to those in vertebrates, including humans. Although humans and flies differ greatly in terms of their gross morphological and cellular features, many of the molecular mechanisms that govern development and drive cellular and physiological processes are conserved between both organisms. The morphological differences are deceiving and have led researchers to undervalue the study of invertebrate organs in unraveling pathogenic mechanisms of diseases. In this review and accompanying poster, we highlight the physiological and molecular parallels between fly and human organs that validate the use of Drosophila to study the molecular pathogenesis underlying human diseases. We discuss assays that have been developed in flies to study the function of specific genes in the central nervous system, heart, liver and kidney, and provide examples of the use of these assays to address questions related to human diseases. These assays provide us with simple yet powerful tools to study the pathogenic mechanisms associated with human disease-causing genes. PMID:26935102

  6. Drosophila tools and assays for the study of human diseases

    PubMed Central

    Ugur, Berrak; Chen, Kuchuan; Bellen, Hugo J.

    2016-01-01

    ABSTRACT Many of the internal organ systems of Drosophila melanogaster are functionally analogous to those in vertebrates, including humans. Although humans and flies differ greatly in terms of their gross morphological and cellular features, many of the molecular mechanisms that govern development and drive cellular and physiological processes are conserved between both organisms. The morphological differences are deceiving and have led researchers to undervalue the study of invertebrate organs in unraveling pathogenic mechanisms of diseases. In this review and accompanying poster, we highlight the physiological and molecular parallels between fly and human organs that validate the use of Drosophila to study the molecular pathogenesis underlying human diseases. We discuss assays that have been developed in flies to study the function of specific genes in the central nervous system, heart, liver and kidney, and provide examples of the use of these assays to address questions related to human diseases. These assays provide us with simple yet powerful tools to study the pathogenic mechanisms associated with human disease-causing genes. PMID:26935102

  7. A Single-fly Assay for Foraging Behavior in Drosophila

    PubMed Central

    Zaninovich, Orel A.; Kim, Susy M.; Root, Cory R.; Green, David S.; Ko, Kang I.; Wang, Jing W.

    2013-01-01

    For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis. PMID:24299900

  8. The Drosophila Auditory System

    PubMed Central

    Boekhoff-Falk, Grace; Eberl, Daniel F.

    2013-01-01

    Development of a functional auditory system in Drosophila requires specification and differentiation of the chordotonal sensilla of Johnston’s organ (JO) in the antenna, correct axonal targeting to the antennal mechanosensory and motor center (AMMC) in the brain, and synaptic connections to neurons in the downstream circuit. Chordotonal development in JO is functionally complicated by structural, molecular and functional diversity that is not yet fully understood, and construction of the auditory neural circuitry is only beginning to unfold. Here we describe our current understanding of developmental and molecular mechanisms that generate the exquisite functions of the Drosophila auditory system, emphasizing recent progress and highlighting important new questions arising from research on this remarkable sensory system. PMID:24719289

  9. An assay for social interaction in Drosophila fragile X mutants

    PubMed Central

    Bolduc, Francois V.; Valente, Dan; Nguyen, Antoinette T.; Mitra, Partha P.; Tully, Tim

    2010-01-01

    We developed a novel assay to examine social interactions in Drosophila and, as a first attempt, apply it here at examining the behavior of Drosophila Fragile X Mental Retardation gene (dfmr1) mutants. Fragile X syndrome is the most common cause of single gene intellectual disability (ID) and is frequently associated with autism. Our results suggest that dfmr1 mutants are less active than wild-type flies and interact with each other less often. In addition, mutants for one allele of dfmr1, dfmr1B55, are more likely to come in close contact with a wild-type fly than another dfmr1B55 mutant. Our results raise the possibility of defective social expression with preserved receptive abilities. We further suggest that the assay may be applied in a general strategy of examining endophenoypes of complex human neurological disorders in Drosophila, and specifically in order to understand the genetic basis of social interaction defects linked with ID. PMID:20519966

  10. Induction of mutations by tritiated water and 3H-thymidine in Drosophila melanogaster assayed by the somatic zeste-white eye mutation system.

    PubMed

    Rasmuson, A; Xamena, N; Creus, A; Marcos, R

    1988-01-01

    In order to study the mutagenic effect of exposure to tritium, Drosophila melanogaster larvae were treated with tritiated water (3H2O) or tritiated thymidine (3H-TdR) during development. Dose rates ranged from 0.0058 to 0.058 rad/h per nucleus for 3H-TdR and from 0.049 to 0.122 rad/h for 3H2O. Induction of mutations was measured by the appearance of somatic mutations in the eyes of an unstable strain of Drosophila melanogaster. Both substances caused a significant increase in mutation frequency. With the assumption that each mutation observed in this assay is caused by one DNA break, the effectiveness of tritium to create DNA breaks is estimated to be 0.20 breaks per decay for 3H-TdR and 0.27 breaks per decay for 3H2O. PMID:3128734

  11. Statistical Analysis of Nondisjunction Assays in Drosophila

    PubMed Central

    Zeng, Yong; Li, Hua; Schweppe, Nicole M.; Hawley, R. Scott; Gilliland, William D.

    2010-01-01

    Many advances in the understanding of meiosis have been made by measuring how often errors in chromosome segregation occur. This process of nondisjunction can be studied by counting experimental progeny, but direct measurement of nondisjunction rates is complicated by not all classes of nondisjunctional progeny being viable. For X chromosome nondisjunction in Drosophila female meiosis, all of the normal progeny survive, while nondisjunctional eggs produce viable progeny only if fertilized by sperm that carry the appropriate sex chromosome. The rate of nondisjunction has traditionally been estimated by assuming a binomial process and doubling the number of observed nondisjunctional progeny, to account for the inviable classes. However, the correct way to derive statistics (such as confidence intervals or hypothesis testing) by this approach is far from clear. Instead, we use the multinomial-Poisson hierarchy model and demonstrate that the old estimator is in fact the maximum-likelihood estimator (MLE). Under more general assumptions, we derive asymptotic normality of this estimator and construct confidence interval and hypothesis testing formulae. Confidence intervals under this framework are always larger than under the binomial framework, and application to published data shows that use of the multinomial approach can avoid an apparent type 1 error made by use of the binomial assumption. The current study provides guidance for researchers designing genetic experiments on nondisjunction and improves several methods for the analysis of genetic data. PMID:20660647

  12. Microinjection wound assay and in vivo localization of epidermal wound response reporters in Drosophila embryos.

    PubMed

    Juarez, Michelle T; Patterson, Rachel A; Li, Wilson; McGinnis, William

    2013-01-01

    The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding. PMID:24300796

  13. Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer.

    PubMed

    Lin, Chun-Chieh; Riabinina, Olena; Potter, Christopher J

    2016-01-01

    A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)(1-3). With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors(4-11). Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions. PMID:27585032

  14. An assay of behavioral plasticity in Drosophila larvae

    PubMed Central

    Min, Virginia A.; Condron, Barry G.

    2010-01-01

    Stress, or threats to homeostasis, is a universal part of life. Organisms face changing and challenging situations everyday, and the ability to respond to such stress is essential for survival. When subjected to acute stress, the body responds molecularly and behaviorally in order to recover a steady state. We developed a simple and robust assay of behavioral plasticity for Drosophila larvae in which well-defined behavioral responses and recovery can be observed and quantified. After experiencing different control and bright light treatments, populations of photophobic fly larvae were placed a defined distance from a food source to which they crawled. Half-times (t½), or times at which half the total number of larvae reached the food, were used to compare different treatments and larval populations. Repeated control treatments with a main experimental strain gave tight, reproducible t½ ranges. Control treatments with the wild type strains Oregon R and Canton S, the “rover” and “sitter” alleles of the forager locus, and eyeless mutants gave comparable results to those of the experimental strain. Exposure to bright light for a defined time period resulted in a reproducible slowing of locomotion. However, given a defined recovery period, the larvae recover full, normal locomotion. In addition, bright light treatments with Canton S gave comparable results to those of the experimental strain. Eyeless mutants, which are partially blind, do not show a response to bright light treatment. Thus, our assay measures the behavioral responses to bright light in Drosophila larvae and therefore might be useful as a general assay for studying behavioral plasticity and, potentially, adaptation to a stressful stimulus. PMID:15922026

  15. Assessing neurodegenerative phenotypes in Drosophila dopaminergic neurons by climbing assays and whole brain immunostaining.

    PubMed

    Barone, Maria Cecilia; Bohmann, Dirk

    2013-01-01

    Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's (5). Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human α-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function (2). Accordingly, this model has been successfully used to identify potential therapeutic targets in PD (8). Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line (3, 10). PMID:23644755

  16. Assaying Blood Cell Populations of the Drosophila melanogaster Larva

    PubMed Central

    Petraki, Sophia; Alexander, Brandy; Brückner, Katja

    2015-01-01

    In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage. HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms. PMID:26650404

  17. Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

    PubMed Central

    Ajjuri, Rami R.; O'Donnell, Janis M.

    2013-01-01

    Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular

  18. An Assay to Detect In Vivo Y Chromosome Loss in Drosophila Wing Disc Cells

    PubMed Central

    Szabad, Janos; Bellen, Hugo J.; Venken, Koen J. T.

    2012-01-01

    Loss of the Y chromosome in Drosophila has no impact on cell viability and therefore allows us to assay the impact of environmental agents and genetic alterations on chromosomal loss. To detect in vivo chromosome loss in cells of the developing Drosophila wing primordia, we first engineered a Y chromosome with an attP docking site. By making use of the ΦC31 integrase system, we site-specifically integrated a genomic transgene encompassing the multiple wing hair (mwh) locus into this attP site, leading to a mwh+Y chromosome. This chromosome fully rescues the mwh mutant phenotype, an excellent recessive wing cell marker mutation. Loss of this mwh+Y chromosome in wing primordial cells then leads to manifestation of the mwh mutant phenotype in mwh-homozygous cells. The forming mwh clones permit us to quantify the effect of agents and genetic alterations by assaying frequency and size of the mwh mosaic spots. To illustrate the use of the mwh+Y loss system, the effects of four known mutagens (X-rays, colchicine, ethyl methanesulfonate, and formaldehyde) and two genetic conditions (loss- and gain-of-function lodestar mutant alleles) are documented. The procedure is simple, sensitive, and inexpensive. PMID:22973547

  19. Rover waste assay system

    SciTech Connect

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  20. An improved method for nematode infection assays in Drosophila larvae

    PubMed Central

    Dobes, Pavel; Wang, Zhi; Markus, Robert; Theopold, Ulrich; Hyrsl, Pavel

    2012-01-01

    The infective juveniles (IJs) of entomopathogenic nematodes (EPNs) seek out host insects and release their symbiotic bacteria into their body cavity causing septicaemia, which eventually leads to host death. The interaction between EPNs and their hosts are only partially understood, in particular the host immune responses appears to involve pathways other than phagocytosis and the canonical transcriptional induction pathways. These pathways are genetically tractable and include for example clotting factors and lipid mediators. The aim of this study was to optimize the nematode infections in Drosophila melanogaster larvae, a well-studied and genetically tractable model organism. Here we show that two nematode species namely Steinernema feltiae and Heterorhabditis bacteriophora display different infectivity toward Drosophila larvae with the latter being less pathogenic. The effects of supporting media and IJ dosage on the mortality of the hosts were assessed and optimized. Using optimum conditions, a faster and efficient setup for nematode infections was developed. This newly established infection model in Drosophila larvae will be applicable in large scale screens aimed at identifying novel genes/pathways involved in innate immune responses. PMID:22614785

  1. An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

    PubMed Central

    Sandhu, Simran; Kollah, Arnavaz; Chan, Robin; Lewellyn, Lara; Grotewiel, Mike

    2015-01-01

    Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUDs, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila. PMID:25939022

  2. An inexpensive, scalable behavioral assay for measuring ethanol sedation sensitivity and rapid tolerance in Drosophila.

    PubMed

    Sandhu, Simran; Kollah, Arnavaz P; Lewellyn, Lara; Chan, Robin F; Grotewiel, Mike

    2015-01-01

    Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila. PMID:25939022

  3. Small flies to tackle big questions: assaying complex bacterial virulence mechanisms using Drosophila melanogaster.

    PubMed

    Fauvarque, Marie-Odile

    2014-06-01

    A successful raid on a fortress requires ingenious strategies in addition to a large number of soldiers. When a microorganism faces a potential host many factors are important, including not only the capacity to proliferate but also the ability to hide, escape or subvert the defence arsenal of the infected organism. This ability confers microbial pathogenicity and relies on complex virulence mechanisms, which are tightly regulated during the course of the infection. The amazing versatility of some microbes that can infect a wide broad of hosts undoubtedly relies on virulence factors intent on fighting evolutionarily conserved innate immune mechanisms. This makes the use of alternative invertebrate models, which are of outstanding interest because they demand less ethical consideration and lower experimental costs, extremely relevant. These simpler organisms are used to analyse genes and mechanisms involved in resistance or tolerance to microorganisms. They can also be used to study bacterial virulence factors that allow proliferation or persistence in the host. In particular, the Drosophila fruit fly has a complex immune response (similar to the mammalian innate immune response) and is particularly appropriate for deciphering many events underlying bacterial pathogenicity from acute virulence to biofilm formation. As highlighted in this review, Drosophila has been notably extensively used to study virulence traits of the opportunistic bacteria Pseudomonas aeruginosa, such as proliferation or persistence, translocation through an epithelial barrier, subversion of the phagocytic machinery, in vivo biofilm formation and enhanced virulence provided by commensal flora or a polymicrobial community. Moreover, these small flies now appear to be a useful system for assaying chemicals with therapeutic potential. PMID:24628939

  4. Mechanisms of systemic wound response in Drosophila.

    PubMed

    Lee, Won-Jae; Miura, Masayuki

    2014-01-01

    In response to cellular and tissue losses caused by physical or chemical injuries, organisms must activate multiple wound repair systems at the cellular, tissue, and organismal levels. The systemic wound response (SWR) that occurs via interorgan communication between local wound sites and remote organs ensures that the host is protected efficiently in response to a local wound. The local wound response around the wound site is fairly well documented, but the molecular mechanisms that allow the host to launch SWR are poorly understood. Recent studies on the Drosophila adult model system have shown that the local wound response is not restricted to the wound site because it plays an essential role in generating signals transmitted to remote organs that subsequently achieve SWR. By exploiting the genetic methods available for investigating Drosophila, we are just beginning to understand the complex interorgan networks that operate during SWRs. This review discusses the basic processes involved with classical integumental wound responses and tissue regeneration, such as epithelial cell movement, hemocyte recruitment, apoptosis, melanization, and generation of reactive oxygen species, as well as the recently described intestinal epithelial cell renewal program that occurs in response to gut cell damages. Furthermore, we discuss how these local wound responses integrate with organ-to-organ communication to launch SWR. Genetic analysis of SWRs using the Drosophila model system will provide a unique opportunity to dissect the molecular mechanisms that control wound-induced organ-to-organ communication. PMID:24512709

  5. Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

    PubMed Central

    Chiu, Joanna C.; Low, Kwang Huei; Pike, Douglas H.; Yildirim, Evrim; Edery, Isaac

    2010-01-01

    Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (≡24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties. PMID:20972399

  6. Proposal of an in vivo comet assay using haemocytes of Drosophila melanogaster.

    PubMed

    Carmona, Erico R; Guecheva, Temenouga N; Creus, Amadeu; Marcos, Ricardo

    2011-03-01

    This study presents the first application of an in vivo alkaline comet assay using haemocytes of Drosophila melanogaster larvae. These cells, which play a role similar to that of mammalian blood, can be easily obtained and represent an overall exposure of the treated larvae. To validate the assay, we evaluated the response of these cells to three well-known mutagenic agents: ethyl methanesulfonate (EMS), potassium dichromate (PD), and gamma radiation (γ-irradiation). Third-instar Drosophila larvae were exposed to different concentrations of EMS (1, 2, and 4 mM) and PD (0.5, 1, and 2.5 mM) and to different doses of γ-irradiation (2, 4, and 8 Gγ). Subsequently, haemolymph was extracted from the larvae, and haemocytes were isolated by centrifugation and used in the comet assay. Haemocytes exhibited a significant dose-related increase in DNA damage, indicating that these cells are clearly sensitive to the treatments. These results suggest that the proposed in vivo comet test, using larvae haemocytes of D. melanogaster, may be a useful in vivo assay for genotoxicity assessment. PMID:20740640

  7. Performance of 181 chemicals in a Drosophila assay predominantly monitoring interchromosomal mitotic recombination.

    PubMed

    Vogel, E W; Nivard, M J

    1993-01-01

    An evaluation is presented of the effects of 181 chemicals in the (white/white+) (w/w+) eye mosaic assay, an in vivo short-term test measuring genetic damage in somatic cells of Drosophila after treatment of larvae. The genetic principle of this system is loss of heterozygosity for the wild-type reporter gene w+, an event predominantly resulting from homologous interchromosomal mitotic recombination between the two X chromosomes of female genotypes. The w/w+ eye mosaic test detects a broad spectrum of DNA modifications, since all distinct classes of genotoxins are monitored. Non-DNA-reactive chemicals are in principle not detected by this system. Occasional positive responses obtained for chemicals such as amitrole, ethionine and hexachloeroethane are probably not related to the mechanism responsible for their tumorigenicity. The principle outcome of this analysis is the necessity for classification of responses into three categories. (i) Positive, '++'. The 92 chemicals (Tables II and III) falling into this category were clearly recombinagenic in the assay, meaning that dose-response relations were obtained (or could have been established as was evident from the strong responses obtained at one or two exposure doses). Among the 92 chemicals were 49 promutagens including volatile chemicals such as vinyl bromide and vinyl chloride. (ii) Marginally positive, '+w'. The definition of a weakly positive response is the absence of a dose-response relationship due to the fact that a weak but reproducible effect, in most cases no more than a doubling of the spontaneous clone frequency, is inherently related to toxicity. The 40 chemicals (Tables IV and V) belonging to this category mainly represented four distinct types. (a) Procarcinogens, such as 2-acetylaminofluorene, dibenz[a,h]anthracene, p-dimethylaminoazobenzene, 2-naphthylamine and safrole, for which metabolic conversion was the apparent problem in the assay. (b) Electrophilic chemicals of high nucleophilic

  8. A HRM Real-Time PCR Assay for Rapid and Specific Identification of the Emerging Pest Spotted-Wing Drosophila (Drosophila suzukii)

    PubMed Central

    Dhami, Manpreet K.; Kumarasinghe, Lalith

    2014-01-01

    Spotted wing drosophila (Drosophila suzukii) is an emerging pest that began spreading in 2008 and its distribution now includes 13 countries across two continents. Countries where it is established have reported significant economic losses of fresh produce, such as cherries due to this species of fly. At larval stages, it is impossible to identify due to its striking similarities with other cosmopolitan and harmless drosophilids. Molecular methods allow identification but the current technique of DNA barcoding is time consuming. We developed and validated a rapid, highly sensitive and specific assay based on real-time PCR and high resolution melt (HRM) analysis using EvaGreen DNA intercalating dye chemistry. Performance characteristics of this qualitative assay, validation and applicability in a New Zealand quarantine framework are discussed. Application of this robust and independently validated assay across the spectrum of key food production and border protection industries will allow us to reduce the further spread of this damaging species worldwide. PMID:24927410

  9. Methodological aspects of the white-ivory assay of Drosophila melanogaster.

    PubMed

    Ferreiro, J A; Sierra, L M; Comendador, M A

    1995-10-01

    The white-ivory somatic assay of Drosophila melanogaster was developed to detect genotoxic agents which induce loss of a tandem duplication. Although the mechanism of this loss is not known, some suggestions point to intrachromosomal recombination as the main reversion mechanism. Since the few papers published to date on this assay present controversial methodologies, prior to a larger study of chemicals with different mechanisms of action, we have carried out an analysis to optimize some conditions of this assay. For this purpose, we have used three different strains and four well characterized mutagenic chemicals: N-ethyl-N-nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and hexamethyl phosphoramide (HMPA). The results obtained allow us to conclude that: (i) the best strain for this assay is C(1)DX,y,f/Dp(1:1:1:1)wi,y2, although the use of strain FM6,l(1)66a/Dp(1:1:1:1)wi,y2;st/st could be considered for some mechanistical studies; (ii) developmental reasons make it necessary to use as estimate of reversion frequency the proportion of eyes showing at least one spot; (iii) reversion frequency cannot be used as estimate of mutation efficiency, neither can spot size evaluate time of spot induction; (iv) the four chemicals clearly induce loss of the wi duplication; according to their activities they rank ENU > HMPA > MMS approximately EMS. PMID:7477046

  10. A Follicle Rupture Assay Reveals an Essential Role for Follicular Adrenergic Signaling in Drosophila Ovulation

    PubMed Central

    Deady, Lylah D.; Sun, Jianjun

    2015-01-01

    Ovulation is essential for the propagation of the species and involves a proteolytic degradation of the follicle wall for the release of the fertilizable oocyte. However, the precise mechanisms for regulating these proteolytic events are largely unknown. Work from our lab and others have shown that there are several parallels between Drosophila and mammalian ovulation at both the cellular and molecular levels. During ovulation in Drosophila, posterior follicle cells surrounding a mature oocyte are selectively degraded and the residual follicle cells remain in the ovary to form a corpus luteum after follicle rupture. Like in mammals, this rupturing process also depends on matrix metalloproteinase 2 (Mmp2) activity localized at the posterior end of mature follicles, where oocytes exit. In the present study, we show that Mmp2 activity is regulated by the octopaminergic signaling in mature follicle cells. Exogenous octopamine (OA; equivalent to norepinephrine, NE) is sufficient to induce follicle rupture when isolated mature follicles are cultured ex vivo, in the absence of the oviduct or ovarian muscle sheath. Knocking down the alpha-like adrenergic receptor Oamb (Octoampine receptor in mushroom bodies) in mature follicle cells prevents OA-induced follicle rupture ex vivo and ovulation in vivo. We also show that follicular OA-Oamb signaling induces Mmp2 enzymatic activation but not Mmp2 protein expression, likely via intracellular Ca2+ as the second messenger. Our work develops a novel ex vivo follicle rupture assay and demonstrates the role for follicular adrenergic signaling in Mmp2 activation and ovulation in Drosophila, which is likely conserved in other species. PMID:26473732

  11. Characterization of a method for quantitating food consumption for mutation assays in Drosophila

    SciTech Connect

    Thompson, E.D.; Reeder, B.A.; Bruce, R.D. )

    1991-01-01

    Quantitation of food consumption is necessary when determining mutation responses to multiple chemical exposures in the sex-linked recessive lethal assay in Drosophila. One method proposed for quantitating food consumption by Drosophila is to measure the incorporation of 14C-leucine into the flies during the feeding period. Three sources of variation in the technique of Thompson and Reeder have been identified and characterized. First, the amount of food consumed by individual flies differed by almost 30% in a 24 hr feeding period. Second, the variability from vial to vial (each containing multiple flies) was around 15%. Finally, the amount of food consumed in identical feeding experiments performed over the course of 1 year varied nearly 2-fold. The use of chemical consumption values in place of exposure levels provided a better means of expressing the combined mutagenic response. In addition, the kinetics of food consumption over a 3 day feeding period for exposures to cyclophosphamide which produce lethality were compared to non-lethal exposures. Extensive characterization of lethality induced by exposures to cyclophosphamide demonstrate that the lethality is most likely due to starvation, not chemical toxicity.

  12. Context-specific comparison of sleep acquisition systems in Drosophila

    PubMed Central

    Garbe, David S.; Bollinger, Wesley L.; Vigderman, Abigail; Masek, Pavel; Gertowski, Jill; Sehgal, Amita; Keene, Alex C.

    2015-01-01

    ABSTRACT Sleep is conserved across phyla and can be measured through electrophysiological or behavioral characteristics. The fruit fly, Drosophila melanogaster, provides an excellent model for investigating the genetic and neural mechanisms that regulate sleep. Multiple systems exist for measuring fly activity, including video analysis and single-beam (SB) or multi-beam (MB) infrared (IR)-based monitoring. In this study, we compare multiple sleep parameters of individual flies using a custom-built video-based acquisition system, and commercially available SB- or MB-IR acquisition systems. We report that all three monitoring systems appear sufficiently sensitive to detect changes in sleep duration associated with diet, age, and mating status. Our data also demonstrate that MB-IR detection appeared more sensitive than the SB-IR for detecting baseline nuances in sleep architecture, while architectural changes associated with varying life-history and environment were generally detected across all acquisition types. Finally, video recording of flies in an arena allowed us to measure the effect of ambient environment on sleep. These experiments demonstrate a robust effect of arena shape and size as well as light levels on sleep duration and architecture, and highlighting the versatility of tracking-based sleep acquisition. These findings provide insight into the context-specific basis for choosing between Drosophila sleep acquisition systems, describe a novel cost-effective system for video tracking, and characterize sleep analysis using the MB-IR sleep analysis. Further, we describe a modified dark-place preference sleep assay using video tracking, confirming that flies prefer to sleep in dark locations. PMID:26519516

  13. Drosophila melanogaster as a Model System to Study Mitochondrial Biology

    PubMed Central

    Fernández-Moreno, Miguel Angel; Farr, Carol L.; Kaguni, Laurie S.; Garesse, Rafael

    2016-01-01

    Summary Mitochondria play an essential role in cellular homeostasis. Although in the last few decades our knowledge of mitochondria has increased substantially, the mechanisms involved in the control of mitochondrial biogenesis remain largely unknown. The powerful genetics of Drosophila combined with a wealth of available cell and molecular biology techniques, make this organism an excellent system to study mitochondria. In this chapter we will review briefly the opportunities that Drosophila offers as a model system and describe in detail how to purify mitochondria from Drosophila and to perform the analysis of developmental gene expression using in situ hybridization. PMID:18314716

  14. Drosophila blood as a model system for stress sensing mechanisms.

    PubMed

    Shim, Jiwon

    2015-04-01

    The Drosophila lymph gland is the hematopoietic organ in which stem-like progenitors proliferate and give rise to myeloid-type blood cells. Mechanisms involved in Drosophila hematopoiesis are well established and known to be conserved in the vertebrate system. Recent studies in Drosophila lymph gland have provided novel insights into how external and internal stresses integrate into blood progenitor maintenance mechanisms and the control of blood cell fate decision. In this review, I will introduce a developmental overview of the Drosophila hematopoietic system, and recent understandings of how the system uses developmental signals not only for hematopoiesis but also as sensors for stress and environmental changes to elicit necessary blood responses. PMID:25560697

  15. Aging Studies in Drosophila melanogaster

    PubMed Central

    Sun, Yaning; Yolitz, Jason; Wang, Cecilia; Spangler, Edward; Zhan, Ming; Zou, Sige

    2015-01-01

    Summary Drosophila is a genetically tractable system ideal for investigating the mechanisms of aging and developing interventions for promoting healthy aging. Here we describe methods commonly used in Drosophila aging research. These include basic approaches for preparation of diets and measurements of lifespan, food intake and reproductive output. We also describe some commonly used assays to measure changes in physiological and behavioral functions of Drosophila in aging, such as stress resistance and locomotor activity. PMID:23929099

  16. Differential effects of arsenite and arsenate to Drosophila melanogaster in a combined adult/developmental toxicity assay

    SciTech Connect

    Goldstein, S.H.; Babich, H.

    1989-02-01

    Current concern of the environmental consequences of chemical wastes in soils has led to the development of microbial, plant, and, to a lesser extent, animal bioassays for terrestrial ecosystems. This paper evaluated a Drosophila assay that yields data both on acute toxicity to adults and on developmental toxicity to offspring and which is applicable for screening extracts from soils contaminated with chemical wastes. Acute toxicity assays with Drosophila have been used to evaluate the relative potencies of chemicals. The acute toxicity to adults and the developmental exposure bioassays were designed to be performed as separate tests. This paper combined these two tests into a single bioassay, using arsenic compounds as the test agents. Arsenite and arsenate were selected to evaluate the sensitivity of this combined assay in distinguishing between the toxicities of closely related chemicals.

  17. Genotoxic testing of titanium dioxide anatase nanoparticles using the wing-spot test and the comet assay in Drosophila.

    PubMed

    Carmona, Erico R; Escobar, Bibi; Vales, Gerard; Marcos, Ricard

    2015-01-15

    Titanium dioxide nanoparticles (TiO2 NPs) are widely used for preparations of sunscreens, cosmetics, food and personal care products. However, the possible genotoxic risk associated with this nano-scale material exposure is not clear, especially in whole organisms. In the present study, we explored the in vivo genotoxic activity of TiO2 NPs as well as their TiO2 bulk form using two well-established genotoxic assays, the wing spot test and the comet assay in Drosophila melanogaster. To determine the extent of tissue damage induced by TiO2 NPs in Drosophila larvae, the trypan blue dye exclusion test was also applied. Both compounds were supplied to third instar larvae by ingestion at concentration ranging from 0.08 to 1.60 mg/mL. The results obtained in the present study indicate that TiO2 NPs can reach and induce cytotoxic effects on midgut and imaginal disc tissues of larvae, but they do not promote genotoxicity in the wing-spot test of Drosophila. However, when both nano- and large-size forms of TiO2 were evaluated with the comet assay in Drosophila hemocytes, a significant increase in DNA damage, with a direct dose-response pattern, was observed for TiO2 NPs. The results obtained with the comet assay suggest that the primary DNA damage associated with TiO2 NPs exposure in Drosophila could be associated with specific physico-chemical properties of nano-TiO2, since no effects were observed with the bulk form. This study remarks the usefulness of using more than one genetic end-point in the evaluation of the genotoxic potential of nanomaterials. PMID:25726144

  18. Method for selecting exposure levels for the Drosophila sex-linked recessive lethal assay

    SciTech Connect

    Thompson, E.D.; Reeder, B.A.

    1987-01-01

    The use of the Drosophila sex-linked recessive lethal assay for detecting mutagenicity of chemicals is well established. When compounds are tested by feeding adult flies, the National Toxicology Program protocol specifies a 3-day feeding regimen at an exposure level that produces about 30% mortality. Uptake of the test compound is monitored by feeding behavior, amount of excretion, or abdomen size. An alternate method for determining uptake is to add radiolabeled sucrose to the feeding solution and then to determine the amount of radioactivity in the flies. We have found that the addition of radiolabeled sucrose underestimates consumption for feeding exposures longer than 24 hr because sucrose is metabolized and as much as 30% of the label is excreted, presumably as /sup 14/CO/sub 2/ or /sup 3/H/sub 2/O. Here we describe a method for determining uptake of chemicals by adding /sup 14/C-leucine to the feeding solution. The incorporation of /sup 14/c-leucine is essentially linear over the 3-day feeding period, which permits accurate estimates of food consumption. Use of this method demonstrates that lower exposure levels of a chemical that do not produce mortality actually results in higher consumption by the flies. The method is proposed as a prescreen to select the appropriate exposure level for the sex-linked recessive lethal assay.

  19. A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

    PubMed Central

    Rand, Matthew D.

    2014-01-01

    The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 °C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules. PMID:25046169

  20. Drosophila GRAIL: An intelligent system for gene recognition in Drosophila DNA sequences

    SciTech Connect

    Xu, Ying; Einstein, J.R.; Uberbacher, E.C.; Helt, G.; Rubin, G.

    1995-06-01

    An AI-based system for gene recognition in Drosophila DNA sequences was designed and implemented. The system consists of two main modules, one for coding exon recognition and one for single gene model construction. The exon recognition module finds a coding exon by recognition of its splice junctions (or translation start) and coding potential. The core of this module is a set of neural networks which evaluate an exon candidate for the possibility of being a true coding exon using the ``recognized`` splice junction (or translation start) and coding signals. The recognition process consists of four steps: generation of an exon candidate pool, elimination of improbable candidates using heuristic rules, candidate evaluation by trained neural networks, and candidate cluster resolution and final exon prediction. The gene model construction module takes as input the clustered exon candidates and builds a ``best`` possible single gene model using an efficient dynamic programming algorithm. 129 Drosophila sequences consisting of 441 coding exons including 216358 coding bases were extructed from GenBank and used to build statistical matrices and to train the neural networks. On this training set the system recognized 97% of the coding messages and predicted only 5% false messages. Among the ``correctly`` predicted exons, 68% match the actual exon exactly and 96% have at least one edge predicted correctly. On an independent test set consisting of 30 Drosophila sequences, the system recognized 96% of the coding messages and predicted 7% false messages.

  1. The Dopaminergic System in the Aging Brain of Drosophila

    PubMed Central

    White, Katherine E.; Humphrey, Dickon M.; Hirth, Frank

    2010-01-01

    Drosophila models of Parkinson's disease are characterized by two principal phenotypes: the specific loss of dopaminergic (DA) neurons in the aging brain and defects in motor behavior. However, an age-related analysis of these baseline parameters in wildtype Drosophila is lacking. Here we analyzed the DA system and motor behavior in aging Drosophila. DA neurons in the adult brain can be grouped into bilateral symmetric clusters, each comprising a stereotypical number of cells. Analysis of TH > mCD8::GFP and cell type-specific MARCM clones revealed that DA neurons show cluster-specific, stereotypical projection patterns with terminal arborization in target regions that represent distinct functional areas of the adult brain. Target areas include the mushroom bodies, involved in memory formation and motivation, and the central complex, involved in the control of motor behavior, indicating that similar to the mammalian brain, DA neurons in the fly brain are involved in the regulation of specific behaviors. Behavioral analysis revealed that Drosophila show an age-related decline in startle-induced locomotion and negative geotaxis. Motion tracking however, revealed that walking activity, and exploration behavior, but not centrophobism increase at late stages of life. Analysis of TH > Dcr2, mCD8::GFP revealed a specific effect of Dcr2 expression on walking activity but not on exploratory or centrophobic behavior, indicating that the siRNA pathway may modulate distinct DA behaviors in Drosophila. Moreover, DA neurons were maintained between early- and late life, as quantified by TH > mCD8::GFP and anti-TH labeling, indicating that adult onset, age-related degeneration of DA neurons does not occur in the aging brain of Drosophila. Taken together, our data establish baseline parameters in Drosophila for the study of Parkinson's disease as well as other disorders affecting DA neurons and movement control. PMID:21165178

  2. Drosophila as a model system to study autophagy

    PubMed Central

    Zirin, Jonathan; Perrimon, Norbert

    2013-01-01

    Originally identified as a response to starvation in yeast, autophagy is now understood to fulfill a variety of roles in higher eukaryotes, from the maintenance of cellular homeostasis to the cellular response to stress, starvation, and infection. Although genetics and biochemical studies in yeast have identified many components involved in autophagy, the findings that some of the essential components of the yeast pathway are missing in higher organisms underscore the need to study autophagy in more complex systems. This review focuses on the use of the fruitfly, Drosophila melanogaster as a model system for analysis of autophagy. Drosophila is an organism well-suited for genetic analysis and represents an intermediate between yeast and mammals with respect to conservation of the autophagy machinery. Furthermore, the complex biology and physiology of Drosophila presents an opportunity to model human diseases in a tissue specific and analogous context. PMID:20798940

  3. Whole-central nervous system functional imaging in larval Drosophila

    PubMed Central

    Lemon, William C.; Pulver, Stefan R.; Höckendorf, Burkhard; McDole, Katie; Branson, Kristin; Freeman, Jeremy; Keller, Philipp J.

    2015-01-01

    Understanding how the brain works in tight concert with the rest of the central nervous system (CNS) hinges upon knowledge of coordinated activity patterns across the whole CNS. We present a method for measuring activity in an entire, non-transparent CNS with high spatiotemporal resolution. We combine a light-sheet microscope capable of simultaneous multi-view imaging at volumetric speeds 25-fold faster than the state-of-the-art, a whole-CNS imaging assay for the isolated Drosophila larval CNS and a computational framework for analysing multi-view, whole-CNS calcium imaging data. We image both brain and ventral nerve cord, covering the entire CNS at 2 or 5 Hz with two- or one-photon excitation, respectively. By mapping network activity during fictive behaviours and quantitatively comparing high-resolution whole-CNS activity maps across individuals, we predict functional connections between CNS regions and reveal neurons in the brain that identify type and temporal state of motor programs executed in the ventral nerve cord. PMID:26263051

  4. Rapid enzyme kinetic assays of individual Drosophila and comparisons of field-caught D. melanogaster and D. simulans.

    PubMed

    Clark, A G; Keith, L E

    1989-06-01

    Techniques for performing numerous enzyme kinetic assays with minimum time and effort would be valuable to studies of the evolutionary genetics of metabolic control and the quantitative genetics of determinants of kinetic parameters. Microtiter plate readers have been used for a variety of repetitious analytical techniques, and instruments are available that can take repetitive readings with sufficient speed to perform kinetic assays. The ability of these instruments to assay rapidly the kinetic properties of small samples makes them potentially useful for a number of problems in population genetics. While the ability to handle large numbers of samples is very attractive, the small sample volumes and optical imprecision of microtiter plates result in some sacrifice in accuracy. This paper presents methods for performing kinetic assays on individual field-caught Drosophila, quantifies the precision of these methods, and characterizes differences among Drosophila melanogaster and D. simulans from samples caught in California and Pennsylvania. Comparisons between field-caught and laboratory reared D. melanogaster show that most of the characters are very similar, with the exception of alpha GPDH, which has a threefold higher mean activity among field-caught flies. The phenotypic correlations are presented with a brief discussion of their relevance to assessing the evolution of metabolic control of these enzymes. PMID:2508620

  5. Functional characterization of dopamine transporter in vivo using Drosophila melanogaster behavioral assays.

    PubMed

    Ueno, Taro; Kume, Kazuhiko

    2014-01-01

    Dopamine mediates diverse functions such as motivation, reward, attention, learning/memory and sleep/arousal. Recent studies using model organisms including the fruit fly, have elucidated various physiological functions of dopamine, and identified specific neural circuits for these functions. Flies with mutations in the Drosophila dopamine transporter (dDAT) gene show enhanced dopamine signaling, and short sleep and memory impairment phenotypes. However, understanding the mechanism by which dopamine signaling causes these phenotypes requires an understanding of the dynamics of dopamine release. Here we report the effects of dDAT expression on behavioral traits. We show that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. dDAT expression in other cell types such as Kenyon cells and glial cells can also rescue the short sleep phenotype of dDAT mutants. dDAT mutants also show a down-regulation of the D1-like dopamine receptor dDA1, and this phenotype is rescued when dDAT is expressed in the same cell types in which it rescues sleep. On the other hand, dDAT overexpression in mushroom bodies, which are the target of memory forming dopamine neurons, abolishes olfactory aversive memory. Our data demonstrate that expression of extrasynaptic dopamine transporters can rescue some aspects of dopamine signaling in dopamine transporter mutants. These results provide novel insights into regulatory systems that modulate dopamine signaling. PMID:25232310

  6. Genotoxic potency in Drosophila melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as assayed in the DNA repair test.

    PubMed

    Fujikawa, K; Fort, F L; Samejima, K; Sakamoto, Y

    1993-12-01

    Drosophila melanogaster stock consisting of meiotic recombination deficient (Rec-) double mutant mei-9a mei-41D5 males and Rec+ females was exposed at the larval stage to an aromatic amine or a polycyclic aromatic hydrocarbon. After emergence as adult flies, the males and the females were scored separately. When the treatment caused a dose-dependent reduction in the male to female ratio from the control level; the experiment was repeated with a larval stock consisting of Rec+ males and Rec+ females under comparable conditions. A preferential killing effect upon Rec- larvae was taken as evidence of DNA damaging effect of the test compound. Among 16 compounds tested, 1-AP, B(a)P, 2-AF, DAF, 4-AAF, 2-AAF, 1-AA, 2-AA, DMA, B(a)A and DMBA were registered as positive; Py and 3-MC were weakly positive; and B(e)P, Fluo and Ant were negative. The selective killing effects of the compounds in each of the pyrene, fluorene and anthracene series varied drastically as a function of structure in a way similar to that reported for the genotoxicity in Drosophila and the carcinogenicity in rodents. The Drosophila DNA repair assay will serve as a simple adjunct to the already available means for studying the genotoxic potency of aromatic amines and polycyclic aromatic hydrocarbons. PMID:7694108

  7. An infrared system for monitoring Drosophila motility during microgravity.

    PubMed

    Miller, Mark S; Fortney, Michael D; Keller, Tony S

    2002-12-01

    Presently, the precise mechanisms of the aging process are unknown. Examination and comprehension of the aging process in other species could lead to significant advances in the understanding of human aging. Drosophila melanogaster (fruit fly), commonly used for aging studies, is a widely studied organism in terms of behavior, development, and genetics. Previous microgravity experiments have shown a significant decrease in the life span of young male Drosophila after microgravity exposure. This decrease in lifespan may be related to locomotor activity, a convenient measure of overall physiological performance. This study describes the design and performance of a Drosophila Infrared Motility Monitoring System (DIMMS). The DIMMS uses a unique design of two infrared (IR) beams per fly to measure the locomotor activity of 240 flies. Locomotor activity is measured in terms of number of IR crossings per unit time, instantaneous velocity, and continuous velocity. Ground-based results using the DIMMS equipment agree well with previous values for Drosophila locomotor velocity. DIMMS is an improvement over equipment previously used due to its ability to continuously monitor locomotor activity throughout short-duration microgravity exposure. DIMMS is also lightweight, compact, and power efficient. DIMMS has been flight tested onboard NASA's KC-135 reduced gravity research aircraft and a Nike-Orion sounding rocket. PMID:14638462

  8. An infrared system for monitoring Drosophila motility during microgravity

    NASA Technical Reports Server (NTRS)

    Miller, Mark S.; Fortney, Michael D.; Keller, Tony S.

    2002-01-01

    Presently, the precise mechanisms of the aging process are unknown. Examination and comprehension of the aging process in other species could lead to significant advances in the understanding of human aging. Drosophila melanogaster (fruit fly), commonly used for aging studies, is a widely studied organism in terms of behavior, development, and genetics. Previous microgravity experiments have shown a significant decrease in the life span of young male Drosophila after microgravity exposure. This decrease in lifespan may be related to locomotor activity, a convenient measure of overall physiological performance. This study describes the design and performance of a Drosophila Infrared Motility Monitoring System (DIMMS). The DIMMS uses a unique design of two infrared (IR) beams per fly to measure the locomotor activity of 240 flies. Locomotor activity is measured in terms of number of IR crossings per unit time, instantaneous velocity, and continuous velocity. Ground-based results using the DIMMS equipment agree well with previous values for Drosophila locomotor velocity. DIMMS is an improvement over equipment previously used due to its ability to continuously monitor locomotor activity throughout short-duration microgravity exposure. DIMMS is also lightweight, compact, and power efficient. DIMMS has been flight tested onboard NASA's KC-135 reduced gravity research aircraft and a Nike-Orion sounding rocket.

  9. Model systems, taxonomic bias, and sexual selection: beyond Drosophila.

    PubMed

    Zuk, Marlene; Garcia-Gonzalez, Francisco; Herberstein, Marie Elisabeth; Simmons, Leigh W

    2014-01-01

    Although model systems are useful in entomology, allowing generalizations based on a few well-known species, they also have drawbacks. It can be difficult to know how far to generalize from information in a few species: Are all flies like Drosophila? The use of model systems is particularly problematic in studying sexual selection, where variability among taxa is key to the evolution of different behaviors. A bias toward the use of a few insect species, particularly from the genus Drosophila, is evident in the sexual selection and sexual conflict literature over the past several decades, although the diversity of study organisms has increased more recently. As the number of model systems used to study sexual conflict increased, support for the idea that sexual interactions resulted in harm to females decreased. Future work should choose model systems thoughtfully, combining well-known species with those that can add to the variation that allows us to make more meaningful generalizations. PMID:24160422

  10. Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

    PubMed Central

    Khalil, Sarah; Jacobson, Eliana; Chambers, Moria C.; Lazzaro, Brian P.

    2015-01-01

    The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology. PMID:25992475

  11. Expert system for transuranic waste assay

    SciTech Connect

    Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

    1989-01-01

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs.

  12. The iFly Tracking System for an Automated Locomotor and Behavioural Analysis of Drosophila melanogaster

    PubMed Central

    Kohlhoff, Kai J.; Jahn, Thomas R.; Lomas, David A.; Dobson, Christopher M.; Crowther, Damian C.; Vendruscolo, Michele

    2016-01-01

    The use of animal models in medical research provides insights into molecular and cellular mechanisms of human disease, and helps identify and test novel therapeutic strategies. Drosophila melanogaster – the common fruit fly – is one of the most established model organisms, as its study can be performed more readily and with far less expense than for other model animal systems, such as mice, fish, or indeed primates. In the case of fruit flies, standard assays are based on the analysis of longevity and basic locomotor functions. Here we present the iFly tracking system, which enables to increase the amount of quantitative information that can be extracted from these studies, and to reduce significantly the duration and costs associated with them. The iFly system uses a single camera to simultaneously track the trajectories of up to 20 individual flies with about 100μm spatial and 33ms temporal resolution. The statistical analysis of fly movements recorded with such accuracy makes it possible to perform a rapid and fully automated quantitative analysis of locomotor changes in response to a range of different stimuli. We anticipate that the iFly method will reduce very considerably the costs and the duration of the testing of genetic and pharmacological interventions in Drosophila models, including an earlier detection of behavioural changes and a large increase in throughput compared to current longevity and locomotor assays. PMID:21698336

  13. Tissue communication in a systemic immune response of Drosophila.

    PubMed

    Yang, Hairu; Hultmark, Dan

    2016-07-01

    Several signaling pathways, including the JAK/STAT and Toll pathways, are known to activate blood cells (hemocytes) in Drosophila melanogaster larvae. They are believed to regulate the immune response against infections by parasitoid wasps, such as Leptopilina boulardi, but how these pathways control the hemocytes is not well understood. Here, we discuss the recent discovery that both muscles and fat body take an active part in this response. Parasitoid wasp infection induces Upd2 and Upd3 secretion from hemocytes, leading to JAK/STAT activation mainly in hemocytes and in skeletal muscles. JAK/STAT activation in muscles, but not in hemocytes, is required for an efficient encapsulation of wasp eggs. This suggests that Upd2 and Upd3 are important cytokines, coordinating different tissues for the cellular immune response in Drosophila. In the fat body, Toll signaling initiates a systemic response in which hemocytes are mobilized and activated hemocytes (lamellocytes) are generated. However, the contribution of Toll signaling to the defense against wasps is limited, probably because the wasps inject inhibitors that prevent the activation of the Toll pathway. In conclusion, parasite infection induces a systemic response in Drosophila larvae involving major organ systems and probably the physiology of the entire organism. PMID:27116253

  14. Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

    PubMed Central

    McQuilton, Peter; Forero, Manuel G; Mizuguchi, Kenji; Sutcliffe, Ben; Gu, Chun-Jing; Fenton, Janine C; Hidalgo, Alicia

    2008-01-01

    Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects. By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. We show that Spätzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases. PMID:19018662

  15. Drosophila C Virus Systemic Infection Leads to Intestinal Obstruction

    PubMed Central

    Chtarbanova, Stanislava; Lamiable, Olivier; Lee, Kwang-Zin; Galiana, Delphine; Troxler, Laurent; Meignin, Carine; Hetru, Charles; Hoffmann, Jules A.; Daeffler, Laurent

    2014-01-01

    ABSTRACT Drosophila C virus (DCV) is a positive-sense RNA virus belonging to the Dicistroviridae family. This natural pathogen of the model organism Drosophila melanogaster is commonly used to investigate antiviral host defense in flies, which involves both RNA interference and inducible responses. Although lethality is used routinely as a readout for the efficiency of the antiviral immune response in these studies, virus-induced pathologies in flies still are poorly understood. Here, we characterize the pathogenesis associated with systemic DCV infection. Comparison of the transcriptome of flies infected with DCV or two other positive-sense RNA viruses, Flock House virus and Sindbis virus, reveals that DCV infection, unlike those of the other two viruses, represses the expression of a large number of genes. Several of these genes are expressed specifically in the midgut and also are repressed by starvation. We show that systemic DCV infection triggers a nutritional stress in Drosophila which results from intestinal obstruction with the accumulation of peritrophic matrix at the entry of the midgut and the accumulation of the food ingested in the crop, a blind muscular food storage organ. The related virus cricket paralysis virus (CrPV), which efficiently grows in Drosophila, does not trigger this pathology. We show that DCV, but not CrPV, infects the smooth muscles surrounding the crop, causing extensive cytopathology and strongly reducing the rate of contractions. We conclude that the pathogenesis associated with systemic DCV infection results from the tropism of the virus for an important organ within the foregut of dipteran insects, the crop. IMPORTANCE DCV is one of the few identified natural viral pathogens affecting the model organism Drosophila melanogaster. As such, it is an important virus for the deciphering of host-virus interactions in insects. We characterize here the pathogenesis associated with DCV infection in flies and show that it results from the

  16. Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay

    PubMed Central

    2011-01-01

    Background Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels. Results Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners. Conclusion Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development. PMID:21276241

  17. Automated optical sensing system for biochemical assays

    NASA Astrophysics Data System (ADS)

    Oroszlan, Peter; Duveneck, Gert L.; Ehrat, Markus; Widmer, H. M.

    1994-03-01

    In this paper, we present a new system called FOBIA that was developed and optimized with respect to automated operation of repetitive assay cycles with regenerable bioaffinity sensors. The reliability and precision of the new system is demonstrated by an application in a competitive assay for the detection of the triazine herbicide Atrazine. Using one sensor in more than 300 repetitive cycles, a signal precision better than 5% was achieved.

  18. Enhancing Undergraduate Teaching and Research with a "Drosophila" Virginizing System

    ERIC Educational Resources Information Center

    Venema, Dennis R.

    2006-01-01

    Laboratory exercises using "Drosophila" crosses are an effective pedagogical method to complement traditional lecture and textbook presentations of genetics. Undergraduate thesis research is another common setting for using "Drosophila." A significant barrier to using "Drosophila" for undergraduate teaching or research is the time and skill…

  19. Antiviral immunity in Drosophila requires systemic RNAi spread

    PubMed Central

    Saleh, Maria-Carla; Tassetto, Michel; van Rij, Ronald P.; Goic, Bertsy; Gausson, Valérie; Berry, Bassam; Jacquier, Caroline; Antoniewski, Christophe; Andino, Raul

    2014-01-01

    Multicellular organisms evolved sophisticated defense systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations1-4. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity5-7. However, the antiviral RNAi defense in flies is thought to be a local, cell-autonomous process, since flies are considered unable to generate a systemic RNAi response8. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway9 is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus (DCV) and Sindbis virus. Mortality in dsRNA-uptake defective flies was accompanied by 100-to 105-fold increases in viral titers and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Strikingly, infection with Sindbis-GFP suppressed expression of host-encoded GFP at a distal site. Thus, similar to protein-based immunity in vertebrates, the antiviral RNAi-response in flies also relies on the systemic spread of a virus-specific immunity signal. PMID:19204732

  20. A Miniaturized Video System for Monitoring Drosophila Behavior

    NASA Technical Reports Server (NTRS)

    Bhattacharya, Sharmila; Inan, Omer; Kovacs, Gregory; Etemadi, Mozziyar; Sanchez, Max; Marcu, Oana

    2011-01-01

    Long-term spaceflight may induce a variety of harmful effects in astronauts, resulting in altered motor and cognitive behavior. The stresses experienced by humans in space - most significantly weightlessness (microgravity) and cosmic radiation - are difficult to accurately simulate on Earth. In fact, prolonged and concomitant exposure to microgravity and cosmic radiation can only be studied in space. Behavioral studies in space have focused on model organisms, including Drosophila melanogaster. Drosophila is often used due to its short life span and generational cycle, small size, and ease of maintenance. Additionally, the well-characterized genetics of Drosophila behavior on Earth can be applied to the analysis of results from spaceflights, provided that the behavior in space is accurately recorded. In 2001, the BioExplorer project introduced a low-cost option for researchers: the small satellite. While this approach enabled multiple inexpensive launches of biological experiments, it also imposed stringent restrictions on the monitoring systems in terms of size, mass, data bandwidth, and power consumption. Suggested parameters for size are on the order of 100 mm3 and 1 kg mass for the entire payload. For Drosophila behavioral studies, these engineering requirements are not met by commercially available systems. One system that does meet many requirements for behavioral studies in space is the actimeter. Actimeters use infrared light gates to track the number of times a fly crosses a boundary within a small container (3x3x40 mm). Unfortunately, the apparatus needed to monitor several flies at once would be larger than the capacity of the small satellite. A system is presented, which expands on the actimeter approach to achieve a highly compact, low-power, ultra-low bandwidth solution for simultaneous monitoring of the behavior of multiple flies in space. This also provides a simple, inexpensive alternative to the current systems for monitoring Drosophila

  1. Phylogeography, Interaction Patterns and the Evolution of Host Choice in Drosophila-Parasitoid Systems in Ryukyu Archipelago and Taiwan.

    PubMed

    Novković, Biljana; Kimura, Masahito T

    2015-01-01

    Island biotas provide a great opportunity to study not only the phylogeographic patterns of a group of species, but also to explore the differentiation in their coevolutionary interactions. Drosophila and their parasitoids are exemplary systems for studying complex interaction patterns. However, there is a lack of studies combining interaction-based and molecular marker-based methods. We applied an integrated approach combining phylogeography, interaction, and host-choice behavior studies, with the aim to understand how coevolutionary interactions evolve in Drosophila-parasitoid island populations. The study focused on the three most abundant Drosophila species in Ryukyu archipelago and Taiwan: D. albomicans, D. bipectinata, and D. takahashii, and the Drosophila-parasitoid Leptopilina ryukyuensis. We determined mitochondrial COI haplotypes for samples representing five island populations of Drosophila and four island populations of L. ryukyuensis. We additionally sequenced parts of the autosomal Gpdh for Drosophila samples, and the ITS2 for parasitoid samples. Phylogenetic and coalescent analyses were used to test for demographic events and to place them in a temporal framework. Geographical differences in Drosophila-parasitoid interactions were studied in host-acceptance, host-suitability, and host-choice experiments. All four species showed species-specific phylogeographic patterns. A general trend of the haplotype diversity increasing towards the south was observed. D. albomicans showed very high COI haplotype diversity, and had the most phylogeographically structured populations, with differentiation into the northern and the southern population-group, divided by the Kerama gap. Differentiation in host suitability was observed only between highly structured populations of D. albomicans, possibly facilitated by restricted gene flow. Differentiation in host-acceptance in D. takahashii, and host-acceptance and host-choice in L. ryukyuensis was found, despite there

  2. Phylogeography, Interaction Patterns and the Evolution of Host Choice in Drosophila-Parasitoid Systems in Ryukyu Archipelago and Taiwan

    PubMed Central

    2015-01-01

    Island biotas provide a great opportunity to study not only the phylogeographic patterns of a group of species, but also to explore the differentiation in their coevolutionary interactions. Drosophila and their parasitoids are exemplary systems for studying complex interaction patterns. However, there is a lack of studies combining interaction-based and molecular marker-based methods. We applied an integrated approach combining phylogeography, interaction, and host-choice behavior studies, with the aim to understand how coevolutionary interactions evolve in Drosophila-parasitoid island populations. The study focused on the three most abundant Drosophila species in Ryukyu archipelago and Taiwan: D. albomicans, D. bipectinata, and D. takahashii, and the Drosophila-parasitoid Leptopilina ryukyuensis. We determined mitochondrial COI haplotypes for samples representing five island populations of Drosophila and four island populations of L. ryukyuensis. We additionally sequenced parts of the autosomal Gpdh for Drosophila samples, and the ITS2 for parasitoid samples. Phylogenetic and coalescent analyses were used to test for demographic events and to place them in a temporal framework. Geographical differences in Drosophila-parasitoid interactions were studied in host-acceptance, host-suitability, and host-choice experiments. All four species showed species-specific phylogeographic patterns. A general trend of the haplotype diversity increasing towards the south was observed. D. albomicans showed very high COI haplotype diversity, and had the most phylogeographically structured populations, with differentiation into the northern and the southern population-group, divided by the Kerama gap. Differentiation in host suitability was observed only between highly structured populations of D. albomicans, possibly facilitated by restricted gene flow. Differentiation in host-acceptance in D. takahashii, and host-acceptance and host-choice in L. ryukyuensis was found, despite there

  3. Mutational Analysis of Drosophila Basigin Function in the Visual System

    PubMed Central

    Munro, Michelle; Akkam, Yazan; Curtin, Kathryn D.

    2009-01-01

    Drosophila basigin is a cell-surface glycoprotein of the Ig superfamily and a member of a protein family that includes mammalian EMMPRIN/CD147/basigin, neuroplastin, and embigin. Our previous work on Drosophila basigin has shown that it is required for normal photoreceptor cell structure and normal neuron-glia interaction in the fly visual system. Specifically, the photoreceptor neurons of mosaic animals that are mutant in the eye for basigin show altered cell structure with nuclei, mitochondria and rER misplaced and variable axon diameter compared to wild-type. In addition, glia cells in the optic lamina that contact photoreceptor axons are misplaced and show altered structure. All these defects are rescued by expression of either transgenic fly basigin or transgenic mouse basigin in the photoreceptors demonstrating that mouse basigin can functionally replace fly basigin. To determine what regions of the basigin protein are required for each of these functions, we have created mutant basigin transgenes coding for proteins that are altered in conserved residues, introduced these into the fly genome, and tested them for their ability to rescue both photoreceptor cell structure defects and neuron-glia interaction defects of basigin. The results suggest that the highly conserved transmembrane domain and the extracellular domains are crucial for basigin function in the visual system while the short intracellular tail may not play a role in these functions. PMID:19782733

  4. Mutational analysis of Drosophila basigin function in the visual system.

    PubMed

    Munro, Michelle; Akkam, Yazan; Curtin, Kathryn D

    2010-01-01

    Drosophila basigin is a cell-surface glycoprotein of the Ig superfamily and a member of a protein family that includes mammalian EMMPRIN/CD147/basigin, neuroplastin, and embigin. Our previous work on Drosophila basigin has shown that it is required for normal photoreceptor cell structure and normal neuron-glia interaction in the fly visual system. Specifically, the photoreceptor neurons of mosaic animals that are mutant in the eye for basigin show altered cell structure with nuclei, mitochondria and rER misplaced and variable axon diameter compared to wild-type. In addition, glia cells in the optic lamina that contact photoreceptor axons are misplaced and show altered structure. All these defects are rescued by expression of either transgenic fly basigin or transgenic mouse basigin in the photoreceptors demonstrating that mouse basigin can functionally replace fly basigin. To determine what regions of the basigin protein are required for each of these functions, we have created mutant basigin transgenes coding for proteins that are altered in conserved residues, introduced these into the fly genome, and tested them for their ability to rescue both photoreceptor cell structure defects and neuron-glia interaction defects of basigin. The results suggest that the highly conserved transmembrane domain and the extracellular domains are crucial for basigin function in the visual system while the short intracellular tail may not play a role in these functions. PMID:19782733

  5. Ontogeny of Drosophila melanogaster in a system of dysgenic crosses

    SciTech Connect

    Grishaeva, T.M.; Ivashchenko, N.I.

    1995-09-01

    Three families of mobile elements that induce P-M, H-E, and I-R hybrid dysgenesis in Drosophila melanogaster were activated by crossing flies of different cytotypes. Manifestation of gonadal sterility in F{sub 1} hybrid progeny was dependent on the temperature of development. The systems differed significantly in lethality of F{sub 2} hybrids at various stages of ontogeny (embyros, larvae, pupae, and adult flies). The highest embryo lethality was found in the P-M system at the cleavage stage. In the I-R and H-E systems, the peak of embryonic death corresponded to the stages of blastoderm and organogenesis, respectively. Experimental results are discussed in view of molecular and cytological characteristics of interacting strains and existing hypotheses for regulation of transposition of P, hobo, and I mobile elements. 44 refs., 4 figs., 4 tabs.

  6. Expert system technology for nondestructive waste assay

    SciTech Connect

    Becker, G.K.; Determan, J.C.

    1998-07-01

    Nondestructive assay waste characterization data generated for use in the National TRU Program must be of known and demonstrable quality. Each measurement is required to receive an independent technical review by a qualified expert. An expert system prototype has been developed to automate waste NDA data review of a passive/active neutron drum counter system. The expert system is designed to yield a confidence rating regarding measurement validity. Expert system rules are derived from data in a process involving data clustering, fuzzy logic, and genetic algorithms. Expert system performance is assessed against confidence assignments elicited from waste NDA domain experts. Performance levels varied for the active, passive shielded, and passive system assay modes of the drum counter system, ranging from 78% to 94% correct classifications.

  7. Effect of sterol metabolism in the yeast-Drosophila system on the frequency of radiation-induced aneuploidy in the Drosophila melanogaster oocytes

    SciTech Connect

    Savitskii, V.V.; Luchnikova, E.M.; Inge-Vechtomov, S.G.

    1986-01-01

    The effect of sterol metabolism on induced mutagenesis of Drosophila melanogaster was studied in the ecogenetic system of yeast-Drosophila. Sterol deficiency was created in Drosophila by using the biomass of live cells of Saccharomyces cerevisiae strain 9-2-P712 till mutation in locus nys/sup r1/ blocking the synthesis of ergosterol as the food. It was found that rearing of Drosophila females on the mutant yeast increases the frequency of loss and nondisjunction of X chromosomes induced in mature oocytes by X rays (1000 R). Addition of 0.1% of cholesterol solution in 10% ethanol to the yeast biomass restores the resistance of oocyte to X irradiation to the control level. The possible hormonal effect on membrane leading to increased radiation-induced aneuploidy in Drosophila and the role of sterol metabolism in determining the resistance to various damaging factors are discussed.

  8. Neutron Assay System for Confinement Vessel Disposition

    SciTech Connect

    Frame, Katherine C; Bourne, Mark M; Crooks, William J; Evans, Louise; Mayo, Douglas R; Miko, David K; Salazar, William R; Stange, Sy; Valdez, Jose I; Vigil, Georgiana M

    2012-07-13

    Waste will be removed from confinement vessels remaining from 1970s-era experiments. Los Alamos has 9+ spherical confinement vessels remaining from experiments. Each vessel contains {approx} 500 lbs of radioactive debris such as actinide metals and oxides, metals, powdered silica, graphite, and wires and hardware. In order to dispose of the vessels, debris and contamination must be removed. Neutron assay system was designed to assay vessels before and after cleanout. System requirements are: (1) Modular and moveable; (2) Capable of detecting {approx}100g {sup 239}Pu equivalent in a 2-inch thick steel sphere with 6 foot diameter; and (3) Capable of safeguards-quality assays. Initial design parameters arethe use of 4-atm {sup 3}He tubes with length of 6 feet, and {sup 3}He tubes embedded in polyethelene for moderation. This paper describes the calibration of the Confinement Vessel Assay System (CVAS) and quantification of its uncertainties. Assay uncertainty depends on five factors: (1) Statistical uncertainty in the assay measurement; (2) Statistical uncertainty in the background measurement; (3) Statistical uncertainty in the isotopics determination - This should be much smaller than the other uncertainties; (4) Systematic uncertainty due to position bias; and (5) Systematic uncertainty due to fluctuations in cosmic ray spallation. This one can be virtually eliminated by performing the background measurement with an empty vessel - but that may not be possible. We used modeling and experiments to quantify the systematic uncertainties. The calibration assumes a uniform distribution of material, but reality will be different. MCNPX modeling was used to quantify the positional bias. The model was benchmarked to build confidence in its results. Material at top of vessel is 44% greater than amount assayed, according to singles. Material near 19-tube detector is 38% less than amount assayed, according to singles. Cosmic ray spallation contributes significantly to the

  9. Neutron Assay System for Confinement Vessel Disposition

    SciTech Connect

    Frame, Katherine C.; Bourne, Mark M.; Crooks, William J.; Evans, Louise; Mayo, Douglas R.; Miko, David K.; Salazar, William R.; Stange, Sy; Valdez, Jose I.; Vigil, Georgiana M.

    2012-07-13

    Los Alamos National Laboratory has a number of spherical confinement vessels (CVs) remaining from tests involving nuclear materials. These vessels have an inner diameter of 6 feet with 1-inch thick steel walls. The goal of the Confinement Vessel Disposition (CVD) project is to remove debris and reduce contamination inside the CVs. The Confinement Vessel Assay System (CVAS) was developed to measure the amount of special nuclear material (SNM) in CVs before and after cleanout. Prior to cleanout, the system will be used to perform a verification measurement of each vessel. After cleanout, the system will be used to perform safeguards-quality assays of {le}100-g {sup 239}Pu equivalent in a vessel for safeguards termination. The CVAS has been tested and calibrated in preparation for verification and safeguards measurements.

  10. Test procedure for boxed waste assay system

    SciTech Connect

    Wachter, J.

    1994-12-07

    This document, prepared by Los Alamos National Laboratory`s NMT-4 group, details the test methodology and requirements for Acceptance/Qualification testing of a Boxed Waste Assay System (BWAS) designed and constructed by Pajarito Scientific Corporation. Testing of the BWAS at the Plutonium Facility (TA55) at Los Alamos National Laboratory will be performed to ascertain system adherence to procurement specification requirements. The test program shall include demonstration of conveyor handling capabilities, gamma ray energy analysis, and imaging passive/active neutron accuracy and sensitivity. Integral to these functions is the system`s embedded operating and data reduction software.

  11. Matrix effects of TRU (transuranic) assays using the SWEPP PAN assay system

    SciTech Connect

    Smith, J.R.

    1990-08-01

    The Drum Assay System (DAS) at the Stored Waste Experimental Pilot Plant (SWEPP) is a second-generation active-passive neutron assay system. It has been used to assay over 5000 208-liter drums of transuranic waste from the Rocky Flats Plant (RFP). Data from these assays have been examined and compared with the assays performed at Rocky Flats, mainly utilize counting of {sup 239}Pu gamma rays. For the most part the passive assays are in very good agreement with the Rocky Flats assays. The active assays are strongly correlated with the results of the other two methods, but require matrix-dependent correction factors beyond those provided by the system itself. A set of matrix-dependent correction factors has been developed from the study of the assay results. 3 refs., 4 figs., 3 tabs.

  12. Nondestructive boxed transuranic (TRU) waste assay systems

    NASA Astrophysics Data System (ADS)

    Caldwell, John T.; Jones, Stephanie A.; Lucero, Randy F.

    1999-01-01

    A brief history of boxed waste assay systems (primarily those developed at Los Alamos National Laboratory) is presented. The characteristics and design process involved with current generation systems--as practiced by BII--are also discussed in some detail. Finally, a specific boxed waste assay system and acceptance test results are presented. This system was developed by BII and installed at the Waste Receiving and Packaging (WRAP) facility in Hanford, Washington in early 1997. The WRAP system combines imaging passive/active neutron (IPAN) techniques with gamma- ray energy analysis (GEA) to assay crates up to 2.5 m X 2.5 m X 6.5 m in size. (Systems that incorporate both these methodologies are usually denoted IPAN/GEA types.) Two separate gamma-ray measurements are accomplished utilizing 16 arrayed NaI detectors and a moveable HPGe detector, while 3He detectors acquire both active and passive neutron data. These neutron measurements use BII's proprietary imaging methodology. Acceptance testing of the system was conducted at Hanford in January 1998. The system's operating performance was evaluated based on accuracy and sensitivity requirements for three different matrix types. Test results indicate an average 13% active mode accuracy for 10 nCi/g loadings of Pu waste and 5% passive mode accuracy for 10 g loadings of Pu waste. Sensitivity testing demonstrated an active mode lower limit of detection of less than 5 nCi/g of 239Pu for the medium matrix and less than 20 pCi/g of fission and activation products at 3(sigma) above background.

  13. Tumor promotion: models and assay systems.

    PubMed

    Fitzgerald, D J; Yamasaki, H

    1990-01-01

    Tumor promotion is defined operationally from two-stage models of experimental carcinogenesis. It is, therefore, in a strict sense, possible to identify tumor promoters only from such models. The development and use of in vitro two-stage cell transformation assays was a logical extension toward in vitro short-term testing for tumor promoters. Another approach is to apply mechanistic knowledge of the tumor promotion process in developing end points for such assays. In this context, we have been examining the role of blocked gap-junctional intercellular communication (GJIC) in tumor promotion, using in vitro and in vivo systems. Many promoters have been shown to block GJIC in vitro; our studies support the idea that inhibition of GJIC does play an important role in the promotion stage of BALB/c 3T3 cell transformation. In animal studies, we have shown that the rat liver tumor promoter phenobarbital can decrease the level of expression of the 32 Kd gap junction protein gene specifically in liver upon systemic exposure in rats. Further examination of the role of GJIC in tumor promotion is indeed warranted. Also, deployment of in vitro GJIC and transformation assay systems should provide useful short-term tests for detecting tumor promoting activity of environmental chemicals. PMID:1973858

  14. System and method for assaying a radionuclide

    DOEpatents

    Cadieux, James R; King, III, George S; Fugate, Glenn A

    2014-12-23

    A system for assaying a radionuclide includes a liquid scintillation detector, an analyzer connected to the liquid scintillation detector, and a delay circuit connected to the analyzer. A gamma detector and a multi-channel analyzer are connected to the delay circuit and the gamma detector. The multi-channel analyzer produces a signal reflective of the radionuclide in the sample. A method for assaying a radionuclide includes selecting a sample, detecting alpha or beta emissions from the sample with a liquid scintillation detector, producing a first signal reflective of the alpha or beta emissions, and delaying the first signal a predetermined time. The method further includes detecting gamma emissions from the sample, producing a second signal reflective of the gamma emissions, and combining the delayed first signal with the second signal to produce a third signal reflective of the radionuclide.

  15. System and method for assaying radiation

    DOEpatents

    DiPrete, David P; Whiteside, Tad; Pak, Donald J; DiPrete, Cecilia C

    2013-11-12

    A system for assaying radiation includes a sample holder configured to hold a liquid scintillation solution. A photomultiplier receives light from the liquid scintillation solution and generates a signal reflective of the light. A control circuit biases the photomultiplier and receives the signal from the photomultiplier reflective of the light. A light impermeable casing surrounds the sample holder, photomultiplier, and control circuit. A method for assaying radiation includes placing a sample in a liquid scintillation solution, placing the liquid scintillation solution in a sample holder, and placing the sample holder inside a light impermeable casing. The method further includes positioning a photomultiplier inside the light impermeable casing and supplying power to a control circuit inside the light impermeable casing.

  16. Drosophila melanogaster as a genetic model system to study neurotransmitter transporters

    PubMed Central

    Martin, Ciara A.; Krantz, David E.

    2014-01-01

    The model genetic organism Drosophila melanogaster, commonly known as the fruit fly, uses many of the same neurotransmitters as mammals and very similar mechanisms of neurotransmitter storage, release and recycling. This system offers a variety of powerful molecular-genetic methods for the study of transporters, many of which would be difficult in mammalian models. We review here progress made using Drosophila to understand the function and regulation of neurotransmitter transporters and discuss future directions for its use. PMID:24704795

  17. A microtiter-based assay for protein kinase activity suitable for the analysis of large numbers of samples, and its application to the study of Drosophila learning mutants.

    PubMed

    Buxbaum, J D; Dudai, Y

    1988-02-15

    We have developed a microtiter-based assay for protein kinase activity which depends on the immobilization of substrate proteins to nitrocellulose. The technique makes use of a filtration manifold, allowing as much as a 10-fold increase in efficiency as compared to other protein kinase assays. We have used this assay to measure cAMP-dependent protein kinase (PKA) in Drosophila learning and memory mutants, with exogenous and endogenous substrates. An alteration was found in the affinity of PKA in the mutant turnip. The procedure should be useful for rapid screening of mutants and drugs and could be adapted to additional types of protein kinases as well as protein phosphatases. PMID:3369684

  18. A novel sulfonylurea receptor family member expressed in the embryonic Drosophila dorsal vessel and tracheal system.

    PubMed

    Nasonkin, I; Alikasifoglu, A; Ambrose, C; Cahill, P; Cheng, M; Sarniak, A; Egan, M; Thomas, P M

    1999-10-01

    Sulfonylurea receptors (SURx) are required subunits of the ATP-sensitive potassium channel. SURx alone is electrophysiologically inert. However, when SURx is combined with an inward rectifier Kir6.2 subunit, ATP-sensitive potassium channel activity is generated. We report the identification, characterization, and localization of Dsur, a novel Drosophila gene that is highly related to the vertebrate SUR family. The Dsur coding sequence contains structural features characteristic of the ABC transporter family and, in addition, harbors 1.7 kilobases of a distinctive sequence that does not share homology with any known gene. When Dsur alone is expressed in Xenopus oocytes glibenclamide-sensitive potassium channel activity occurs. During Drosophila embryogenesis, the Dsur gene is specifically expressed in the developing tracheal system and dorsal vessel. Studies of the Drosophila genome support that only a single Dsur gene is present. Our data reveal conservation of glibenclamide-sensitive potassium channels in Drosophila and suggest that Dsur may play an important role during Drosophila embryogenesis. The lack of gene duplication in the Drosophila system provides a unique opportunity for functional studies of SUR using a genetic approach. PMID:10506204

  19. Evaluation of titanium dioxide nanocrystal-induced genotoxicity by the cytokinesis-block micronucleus assay and the Drosophila wing spot test.

    PubMed

    Reis, Érica de Melo; Rezende, Alexandre Azenha Alves de; Oliveira, Pollyanna Francielli de; Nicolella, Heloiza Diniz; Tavares, Denise Crispim; Silva, Anielle Christine Almeida; Dantas, Noelio Oliveira; Spanó, Mário Antônio

    2016-10-01

    Titanium dioxide nanocrystals (TiO2 NCs) crystalline structures include anatase, rutile and brookite. This study evaluated the genotoxic effects of 3.4 and 6.2 nm anatase TiO2 NCs and 78.0 nm predominantly rutile TiO2 NCs through an in vitro micronucleus (MN) assay using V79 cells and an in vivo somatic mutation and recombination test in Drosophila wings. The MN assay was performed with nontoxic concentrations of TiO2 NCs. Only anatase (3.4 nm) at the highest concentration (120 μM) induced genotoxicity in V79 cells. In the in vivo test, Drosophila melanogaster larvae obtained from standard (ST) or high bioactivation (HB) crosses were treated with TiO2 NCs. In the ST cross, no mutagenic effects were observed. However, in the HB cross, TiO2 NCs (3.4 nm) were mutagenic at 1.5625 and 3.125 mM, while 78.0 nm NCs increased mutant spots at all concentrations tested except 3.125 mM. Only the smallest anatase TiO2 NCs induced mutagenic effects in vitro and in vivo. For rutile TiO2 NCs, no clastogenic/aneugenic effects were observed in the MN assay. However, they were mutagenic in Drosophila. Therefore, both anatase and rutile TiO2 NCs induced mutagenicity. Further research is necessary to clarify the TiO2 NCs genotoxic/mutagenic action mechanisms. PMID:27562929

  20. Trithorax regulates systemic signaling during Drosophila imaginal disc regeneration.

    PubMed

    Skinner, Andrea; Khan, Sumbul Jawed; Smith-Bolton, Rachel K

    2015-10-15

    Although tissue regeneration has been studied in a variety of organisms, from Hydra to humans, many of the genes that regulate the ability of each animal to regenerate remain unknown. The larval imaginal discs of the genetically tractable model organism Drosophila melanogaster have complex patterning, well-characterized development and a high regenerative capacity, and are thus an excellent model system for studying mechanisms that regulate regeneration. To identify genes that are important for wound healing and tissue repair, we have carried out a genetic screen for mutations that impair regeneration in the wing imaginal disc. Through this screen we identified the chromatin-modification gene trithorax as a key regeneration gene. Here we show that animals heterozygous for trithorax are unable to maintain activation of a developmental checkpoint that allows regeneration to occur. This defect is likely to be caused by abnormally high expression of puckered, a negative regulator of Jun N-terminal kinase (JNK) signaling, at the wound site. Insufficient JNK signaling leads to insufficient expression of an insulin-like peptide, dILP8, which is required for the developmental checkpoint. Thus, trithorax regulates regeneration signaling and capacity. PMID:26487779

  1. Missense variants in the middle domain of DNM1L in cases of infantile encephalopathy alter peroxisomes and mitochondria when assayed in Drosophila.

    PubMed

    Chao, Yu-Hsin; Robak, Laurie A; Xia, Fan; Koenig, Mary K; Adesina, Adekunle; Bacino, Carlos A; Scaglia, Fernando; Bellen, Hugo J; Wangler, Michael F

    2016-05-01

    Defects in organelle dynamics underlie a number of human degenerative disorders, and whole exome sequencing (WES) is a powerful tool for studying genetic changes that affect the cellular machinery. WES may uncover variants of unknown significance (VUS) that require functional validation. Previously, a pathogenic de novo variant in the middle domain of DNM1L (p.A395D) was identified in a single patient with a lethal defect of mitochondrial and peroxisomal fission. We identified two additional patients with infantile encephalopathy and partially overlapping clinical features, each with a novel VUS in the middle domain of DNM1L (p.G350R and p.E379K). To evaluate pathogenicity, we generated transgenic Drosophila expressing wild-type or variant DNM1L. We find that human wild-type DNM1L rescues the lethality as well as specific phenotypes associated with the loss of Drp1 in Drosophila. Neither the p.A395D variant nor the novel variant p.G350R rescue lethality or other phenotypes. Moreover, overexpression of p.A395D and p.G350R in Drosophila neurons, salivary gland and muscle strikingly altered peroxisomal and mitochondrial morphology. In contrast, the other novel variant (p.E379K) rescued lethality and did not affect organelle morphology, although it was associated with a subtle mitochondrial trafficking defect in an in vivo assay. Interestingly, the patient with the p.E379K variant also has a de novo VUS in pyruvate dehydrogenase 1 (PDHA1) affecting the same amino acid (G150) as another case of PDHA1 deficiency suggesting the PDHA1 variant may be pathogenic. In summary, detailed clinical evaluation and WES with functional studies in Drosophila can distinguish different functional consequences of newly-described DNM1L alleles. PMID:26931468

  2. The Drosophila neural lineages: a model system to study brain development and circuitry.

    PubMed

    Spindler, Shana R; Hartenstein, Volker

    2010-06-01

    In Drosophila, neurons of the central nervous system are grouped into units called lineages. Each lineage contains cells derived from a single neuroblast. Due to its clonal nature, the Drosophila brain is a valuable model system to study neuron development and circuit formation. To better understand the mechanisms underlying brain development, genetic manipulation tools can be utilized within lineages to visualize, knock down, or over-express proteins. Here, we will introduce the formation and development of lineages, discuss how one can utilize this model system, offer a comprehensive list of known lineages and their respective markers, and then briefly review studies that have utilized Drosophila neural lineages with a look at how this model system can benefit future endeavors. PMID:20306203

  3. The Drosophila neural lineages: a model system to study brain development and circuitry

    PubMed Central

    Spindler, Shana R.

    2010-01-01

    In Drosophila, neurons of the central nervous system are grouped into units called lineages. Each lineage contains cells derived from a single neuroblast. Due to its clonal nature, the Drosophila brain is a valuable model system to study neuron development and circuit formation. To better understand the mechanisms underlying brain development, genetic manipulation tools can be utilized within lineages to visualize, knock down, or over-express proteins. Here, we will introduce the formation and development of lineages, discuss how one can utilize this model system, offer a comprehensive list of known lineages and their respective markers, and then briefly review studies that have utilized Drosophila neural lineages with a look at how this model system can benefit future endeavors. PMID:20306203

  4. Enhancing Undergraduate Teaching and Research with a Drosophila Virginizing System

    PubMed Central

    2006-01-01

    Laboratory exercises using Drosophila crosses are an effective pedagogical method to complement traditional lecture and textbook presentations of genetics. Undergraduate thesis research is another common setting for using Drosophila. A significant barrier to using Drosophila for undergraduate teaching or research is the time and skill required to accurately collect virgins for use in controlled crosses. Erroneously collecting males or nonvirgin females contaminates crosses with unintended genotypes and confounds the results. Collecting adequate numbers of virgins requires large amounts of time, even for those skilled in virgin collection. I have adapted an effective method for virgin collection that eliminates these concerns and is straightforward to use in undergraduate settings. Using a heat-shock–induced, conditional lethal transgene specifically in males, male larvae can be eliminated from a culture before adults eclose. Females thus eclose in the absence of males and remain virgin, eliminating the need to laboriously score and segregate freshly eclosed females. This method is reliable, easily adaptable to any desired phenotypic marker, and readily scaleable to provide sufficient virgins for large laboratory classes or undergraduate research projects. In addition, it allows instructors lacking Drosophila expertise to use this organism as a pedagogical tool. PMID:17146043

  5. Molecular Sieve Regeneration System for assaying HTO from detritiation systems

    SciTech Connect

    Nasise, J.E.; Anderson, J.L.; Naruse, Y.

    1992-07-01

    A Molecular Sieve Regeneration System (MSRS) is being added to the existing Tritium Waste Treatment system (TWT) within the Tritium Systems Test Assembly (TSTA) at the Los Alamos National Laboratory. This system is an upgrade to the TWT to provide accurate measurements of the liquid waste generated from this system. Within the TWT, hydrogen isotopes are removed from the effluent gas stream by the catalytic conversion to water and the subsequent removal of water by molecular sieve trapping prior to the release to the environment. Within the TWT and similar systems, molecular sieve regeneration is required to rejuvenate the beds. The major difference of the MSRS and other regeneration systems is the capability of direct assay of long-term storage waste containers. This is accomplished with loop-flow regeneration, water collection, and tritiated water assay by scintillation and calorimetric techniques. This paper describes the MSRS in detail and how it is interfaced with the Tritium Waste Treatment system.

  6. Molecular Sieve Regeneration System for assaying HTO from detritiation systems

    SciTech Connect

    Nasise, J.E.; Anderson, J.L. ); Naruse, Y. )

    1992-01-01

    A Molecular Sieve Regeneration System (MSRS) is being added to the existing Tritium Waste Treatment system (TWT) within the Tritium Systems Test Assembly (TSTA) at the Los Alamos National Laboratory. This system is an upgrade to the TWT to provide accurate measurements of the liquid waste generated from this system. Within the TWT, hydrogen isotopes are removed from the effluent gas stream by the catalytic conversion to water and the subsequent removal of water by molecular sieve trapping prior to the release to the environment. Within the TWT and similar systems, molecular sieve regeneration is required to rejuvenate the beds. The major difference of the MSRS and other regeneration systems is the capability of direct assay of long-term storage waste containers. This is accomplished with loop-flow regeneration, water collection, and tritiated water assay by scintillation and calorimetric techniques. This paper describes the MSRS in detail and how it is interfaced with the Tritium Waste Treatment system.

  7. 233U Assay A Neutron NDA System

    SciTech Connect

    Hensley, D.C.; Lucero, A.J.; Pierce, L.

    1998-11-17

    The assay of highly enriched {sup 233}U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched {sup 235}U do not convert easily over to the assay of {sup 233}U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with {gamma} ray isotopics information should give a good overall determination of {sup 233}U material now stored in bldg. 3019 at the Oak Ridge National Laboratory.

  8. Drosophila melanogaster, a model system for comparative studies on the responses to real and simulated microgravity.

    PubMed

    Marco, R; Laván, D A; van Loon, J J W A; Leandro, L J; Larkin, O J; Dijkstra, C; Anthony, P; Villa, A; Davey, M R; Lowe, K C; Power, J B; Medina, F J

    2007-07-01

    A key requirement to enhance our understanding of the response of biological organisms to different levels of gravity is the availability of experimental systems that can simulate microgravity and hypergravity in ground-based laboratories. This paper compares the results obtained from analysing gene expression profiles of Drosophila in space versus those obtained in a random position machine (RPM) and by centrifugation. The correlation found validates the use of the RPM simulation technique to establish the effects of real microgravity on biological systems. This work is being extended to investigate Drosophila development in another gravity modifying instrument, the levitation magnet. PMID:18372731

  9. The synchronous active neutron detection assay system

    SciTech Connect

    Pickrell, M.M.; Kendall, P.K.

    1994-08-01

    We have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit a 14-MeV neutron generator developed by Schlumberger. The technique, termed synchronous active neutron detection (SAND), follows a method used routinely in other branches of physics to detect very small signals in presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed ``lock-in`` amplifiers. We have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. The Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. Results are preliminary but promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly; it also appears resilient to background neutron interference. The interrogating neutrons appear to be non-thermal and penetrating. Work remains to fully explore relevant physics and optimize instrument design.

  10. Systems, devices, and methods for agglutination assays using sedimentation

    DOEpatents

    Schaff, Ulrich Y.; Sommer, Gregory J.; Singh, Anup K.

    2016-01-26

    Embodiments of the present invention include methods for conducting agglutination assays using sedimentation. Aggregates may be exposed to sedimentation forces and travel through a density medium to a detection area. Microfluidic devices, such as microfluidic disks, are described for conducting the agglutination assays, as are systems for conducting the assays.

  11. The synchronous active neutron detection assay system

    SciTech Connect

    Pickrell, M.M.; Kendall, P.K.

    1994-09-01

    The authors have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. They are using a Schlumberger neutron generator for the direct measurement of the fissile material content in spent fuel, in place of the indirect measures used at present. The technique they are investigating is termed synchronous active neutron detection (SAND). It closely follows a method that has been used routinely in other branches of physics for the detection of very small signals in the presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed ``lock-in`` amplifiers. They have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. The results to data are preliminary but quite promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly. It also appears to be quite resilient to background neutron interference.

  12. Automated rapid iterative negative geotaxis assay and its use in a genetic screen for modifiers of Aβ(42)-induced locomotor decline in Drosophila.

    PubMed

    Liu, Haiyan; Han, Meng; Li, Qingyi; Zhang, Xiao; Wang, Wen-An; Huang, Fu-De

    2015-10-01

    The negative-geotaxis climbing assay is used to efficiently study aging and neurodegeneration in Drosophila. To make it suitable for large-scale study, a method called the rapid iterative negative geotaxis (RING) assay has been established by simultaneously photographing the climbing of multiple groups of flies when they are manually tapped down in test tubes. Here, we automated the assay by using a well-controlled electric motor to drive the tapping, and a homemade program to analyze the climbing height of flies. Using the automated RING (aRING) assay, we found that the climbing ability of a strain of wild-type flies, males in particular, declined rapidly before day 21 after eclosion, but slowly from day 21 to 35. We also found that the expression of arctic mutant Aβ42 accelerated the age-dependent decline in the climbing ability of flies. Moreover, using aRING, we examined the effect of third chromosome deficiencies on the accelerated locomotor decline in Aβ42-expressing flies, and isolated 7 suppressors and 15 enhancers. PMID:26077703

  13. Variables Affecting Two Electron Transport System Assays

    PubMed Central

    Burton, G. Allen; Lanza, Guy R.

    1986-01-01

    Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4°C. Extraction efficiencies of formazan from sediment varied with alcohol type; methanol was unsatisfactory. Phosphate buffer (0.06 M) produced higher activity than did either U.S. Environmental Protection Agency reconstituted hard water or Tris buffer sediment diluents. Intracellular formazan crystals were dissolved within minutes when in contact with immersion oil. Greater crystal production (respiration) detected by a tetrazolium salt assay occurred at increased substrate concentrations. Test diluents containing macrophyte exudates produced greater activity than did phosphate buffer, U.S. Environmental Protection Agency water, or ultrapure water diluents. Both assays showed decreases in sediment or bacterial activity through time. PMID:16347067

  14. Drosophila as a model for the two myeloid blood cell systems in vertebrates

    PubMed Central

    Gold, Katrina S.; Brückner, Katja

    2016-01-01

    Fish, mice and men rely on two coexisting myeloid blood cell systems. One is sustained by hematopoietic progenitor cells, which reside in specialized microenvironments in hematopoietic organs and give rise to cells of the monocyte lineage. The other system corresponds to the independent lineage of self-renewing tissue macrophages, which colonize organs during embryonic development and are maintained during later life by proliferation in local tissue microenvironments. However, little is known about the nature of these microenvironments and their regulation. Moreover, many vertebrate tissues contain a mix of both tissue-resident and monocyte-derived macrophages, posing a challenge to the study of lineage-specific regulatory mechanisms and function. This review highlights how research in the simple model organism Drosophila melanogaster can address many of these outstanding questions in the field. Drawing parallels between hematopoiesis in Drosophila and vertebrates, we illustrate the evolutionary conservation of the two myeloid systems across animal phyla. Much like vertebrates, Drosophila possesses a lineage of self-renewing tissue-resident macrophages, as well as a ‘definitive’ lineage of macrophages that derive from hematopoiesis in the progenitor-based lymph gland. We summarize key findings from Drosophila hematopoiesis that illustrate how local microenvironments, systemic signals, immune challenges and nervous inputs regulate adaptive responses of tissue-resident macrophages and progenitor-based hematopoiesis to achieve optimal fitness of the animal. PMID:24946019

  15. Drosophila Stathmin: A Microtubule-destabilizing Factor Involved in Nervous System Formation

    PubMed Central

    Ozon, Sylvie; Guichet, Antoine; Gavet, Olivier; Roth, Siegfried; Sobel, André

    2002-01-01

    Stathmin is a ubiquitous regulatory phosphoprotein, the generic element of a family of neural phosphoproteins in vertebrates that possess the capacity to bind tubulin and interfere with microtubule dynamics. Although stathmin and the other proteins of the family have been associated with numerous cell regulations, their biological roles remain elusive, as in particular inactivation of the stathmin gene in the mouse resulted in no clear deleterious phenotype. We identified stathmin phosphoproteins in Drosophila, encoded by a unique gene sharing the intron/exon structure of the vertebrate stathmin and stathmin family genes. They interfere with microtubule assembly in vitro, and in vivo when expressed in HeLa cells. Drosophila stathmin expression is regulated during embryogenesis: it is high in the migrating germ cells and in the central and peripheral nervous systems, a pattern resembling that of mammalian stathmin. Furthermore, RNA interference inactivation of Drosophila stathmin expression resulted in germ cell migration arrest at stage 14. It also induced important anomalies in nervous system development, such as loss of commissures and longitudinal connectives in the ventral cord, or abnormal chordotonal neuron organization. In conclusion, a single Drosophila gene encodes phosphoproteins homologous to the entire vertebrate stathmin family. We demonstrate for the first time their direct involvement in major biological processes such as development of the reproductive and nervous systems. PMID:11854423

  16. Drosophila stathmin: a microtubule-destabilizing factor involved in nervous system formation.

    PubMed

    Ozon, Sylvie; Guichet, Antoine; Gavet, Olivier; Roth, Siegfried; Sobel, André

    2002-02-01

    Stathmin is a ubiquitous regulatory phosphoprotein, the generic element of a family of neural phosphoproteins in vertebrates that possess the capacity to bind tubulin and interfere with microtubule dynamics. Although stathmin and the other proteins of the family have been associated with numerous cell regulations, their biological roles remain elusive, as in particular inactivation of the stathmin gene in the mouse resulted in no clear deleterious phenotype. We identified stathmin phosphoproteins in Drosophila, encoded by a unique gene sharing the intron/exon structure of the vertebrate stathmin and stathmin family genes. They interfere with microtubule assembly in vitro, and in vivo when expressed in HeLa cells. Drosophila stathmin expression is regulated during embryogenesis: it is high in the migrating germ cells and in the central and peripheral nervous systems, a pattern resembling that of mammalian stathmin. Furthermore, RNA interference inactivation of Drosophila stathmin expression resulted in germ cell migration arrest at stage 14. It also induced important anomalies in nervous system development, such as loss of commissures and longitudinal connectives in the ventral cord, or abnormal chordotonal neuron organization. In conclusion, a single Drosophila gene encodes phosphoproteins homologous to the entire vertebrate stathmin family. We demonstrate for the first time their direct involvement in major biological processes such as development of the reproductive and nervous systems. PMID:11854423

  17. Conservation of neural nicotinic acetylcholine receptors from Drosophila to vertebrate central nervous systems.

    PubMed Central

    Bossy, B; Ballivet, M; Spierer, P

    1988-01-01

    Nicotinic acetylcholine receptors (nAChR) are found both in vertebrate and insect central nervous systems. We have isolated a Drosophila gene by crosshybridization with a vertebrate probe. Structural conservation of domains of the deduced protein and of intron/exon boundaries indicate that the Drosophila gene encodes an nAChR alpha-like subunit (ALS). That the Drosophila gene product most resembles the neuronal set of vertebrate nAChRs alpha-subunits is also indicated by the failure of an ALS-beta-galactosidase fusion protein to bind alpha-bungarotoxin on blots in contrast to vertebrate endplate alpha-subunit constructions. The ALS encoding gene exceeds 54 kb in length and the transcript has a very long and unusual 5' leader. As we found previously for a gene whose product is also involved in cholinergic synapses, acetylcholinesterase, the leader encodes short open reading frames, which might be involved in translation control. We also note the presence of opa repeats in the gene, as has been found for various Drosophila genes expressed in the nervous system. Images PMID:2840281

  18. Two Enhancers Control Transcription of Drosophila muscleblind in the Embryonic Somatic Musculature and in the Central Nervous System

    PubMed Central

    Cerro-Herreros, Estefanía; Artero, Ruben

    2014-01-01

    The phylogenetically conserved family of Muscleblind proteins are RNA-binding factors involved in a variety of gene expression processes including alternative splicing regulation, RNA stability and subcellular localization, and miRNA biogenesis, which typically contribute to cell-type specific differentiation. In humans, sequestration of Muscleblind-like proteins MBNL1 and MBNL2 has been implicated in degenerative disorders, particularly expansion diseases such as myotonic dystrophy type 1 and 2. Drosophila muscleblind was previously shown to be expressed in embryonic somatic and visceral muscle subtypes, and in the central nervous system, and to depend on Mef2 for transcriptional activation. Genomic approaches have pointed out candidate gene promoters and tissue-specific enhancers, but experimental confirmation of their regulatory roles was lacking. In our study, luciferase reporter assays in S2 cells confirmed that regions P1 (515 bp) and P2 (573 bp), involving the beginning of exon 1 and exon 2, respectively, were able to initiate RNA transcription. Similarly, transgenic Drosophila embryos carrying enhancer reporter constructs supported the existence of two regulatory regions which control embryonic expression of muscleblind in the central nerve cord (NE, neural enhancer; 830 bp) and somatic (skeletal) musculature (ME, muscle enhancer; 3.3 kb). Both NE and ME were able to boost expression from the Hsp70 heterologous promoter. In S2 cell assays most of the ME enhancer activation could be further narrowed down to a 1200 bp subregion (ME.3), which contains predicted binding sites for the Mef2 transcription factor. The present study constitutes the first characterization of muscleblind enhancers and will contribute to a deeper understanding of the transcriptional regulation of the gene. PMID:24667536

  19. Programmed cell death acts at different stages of Drosophila neurodevelopment to shape the central nervous system.

    PubMed

    Pinto-Teixeira, Filipe; Konstantinides, Nikolaos; Desplan, Claude

    2016-08-01

    Nervous system development is a process that integrates cell proliferation, differentiation, and programmed cell death (PCD). PCD is an evolutionary conserved mechanism and a fundamental developmental process by which the final cell number in a nervous system is established. In vertebrates and invertebrates, PCD can be determined intrinsically by cell lineage and age, as well as extrinsically by nutritional, metabolic, and hormonal states. Drosophila has been an instrumental model for understanding how this mechanism is regulated. We review the role of PCD in Drosophila central nervous system development from neural progenitors to neurons, its molecular mechanism and function, how it is regulated and implemented, and how it ultimately shapes the fly central nervous system from the embryo to the adult. Finally, we discuss ideas that emerged while integrating this information. PMID:27404003

  20. Mapping chromatic pathways in the Drosophila visual system.

    PubMed

    Lin, Tzu-Yang; Luo, Jiangnan; Shinomiya, Kazunori; Ting, Chun-Yuan; Lu, Zhiyuan; Meinertzhagen, Ian A; Lee, Chi-Hon

    2016-02-01

    In Drosophila, color vision and wavelength-selective behaviors are mediated by the compound eye's narrow-spectrum photoreceptors R7 and R8 and their downstream medulla projection (Tm) neurons Tm5a, Tm5b, Tm5c, and Tm20 in the second optic neuropil or medulla. These chromatic Tm neurons project axons to a deeper optic neuropil, the lobula, which in insects has been implicated in processing and relaying color information to the central brain. The synaptic targets of the chromatic Tm neurons in the lobula are not known, however. Using a modified GFP reconstitution across synaptic partners (GRASP) method to probe connections between the chromatic Tm neurons and 28 known and novel types of lobula neurons, we identify anatomically the visual projection neurons LT11 and LC14 and the lobula intrinsic neurons Li3 and Li4 as synaptic targets of the chromatic Tm neurons. Single-cell GRASP analyses reveal that Li4 receives synaptic contacts from over 90% of all four types of chromatic Tm neurons, whereas LT11 is postsynaptic to the chromatic Tm neurons, with only modest selectivity and at a lower frequency and density. To visualize synaptic contacts at the ultrastructural level, we develop and apply a "two-tag" double-labeling method to label LT11's dendrites and the mitochondria in Tm5c's presynaptic terminals. Serial electron microscopic reconstruction confirms that LT11 receives direct contacts from Tm5c. This method would be generally applicable to map the connections of large complex neurons in Drosophila and other animals. PMID:26179639

  1. Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila.

    PubMed

    McGuire, Sean E; Mao, Zhengmei; Davis, Ronald L

    2004-02-17

    Targeted gene expression has become a standard technique for the study of biological questions in Drosophila. Until recently, transgene expression could be targeted in the dimension of either time or space, but not both. Several new systems have recently been developed to direct transgene expression simultaneously in both time and space. We describe here two such systems that we developed in our laboratory. The first system provides a general method for temporal and regional gene expression targeting (TARGET) with the conventional GAL4-upstream activator sequence (UAS) system and a temperature-sensitive GAL80 molecule, which represses GAL4 transcriptional activity at permissive temperatures. The second system, termed Gene-Switch, is based on a GAL4-progesterone receptor chimera that is hormone-inducible. We have used both systems for simultaneous spatial and temporal rescue of memory dysfunction in the rutabaga (rut) memory mutant of Drosophila. In this protocol, we provide guidelines for the use of these two novel systems, which should have general utility in studying Drosophila biology and in using the fly as a model for human disease. PMID:14970377

  2. A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila.

    PubMed

    Xu, Jiang; Ren, Xingjie; Sun, Jin; Wang, Xia; Qiao, Huan-Huan; Xu, Bo-Wen; Liu, Lu-Ping; Ni, Jian-Quan

    2015-04-20

    The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions. PMID:25953352

  3. FGF /FGFR Signal Induces Trachea Extension in the Drosophila Visual System

    PubMed Central

    Chu, Wei-Chen; Lee, Yuan-Ming; Henry Sun, Yi

    2013-01-01

    The Drosophila compound eye is a large sensory organ that places a high demand on oxygen supplied by the tracheal system. Although the development and function of the Drosophila visual system has been extensively studied, the development and contribution of its tracheal system has not been systematically examined. To address this issue, we studied the tracheal patterns and developmental process in the Drosophila visual system. We found that the retinal tracheae are derived from air sacs in the head, and the ingrowth of retinal trachea begin at mid-pupal stage. The tracheal development has three stages. First, the air sacs form near the optic lobe in 42-47% of pupal development (pd). Second, in 47-52% pd, air sacs extend branches along the base of the retina following a posterior-to-anterior direction and further form the tracheal network under the fenestrated membrane (TNUFM). Third, the TNUFM extend fine branches into the retina following a proximal-to-distal direction after 60% pd. Furthermore, we found that the trachea extension in both retina and TNUFM are dependent on the FGF(Bnl)/FGFR(Btl) signaling. Our results also provided strong evidence that the photoreceptors are the source of the Bnl ligand to guide the trachea ingrowth. Our work is the first systematic study of the tracheal development in the visual system, and also the first study demonstrating the interactions of two well-studied systems: the eye and trachea. PMID:23991208

  4. Regulation of Sleep by Insulin-like Peptide System in Drosophila melanogaster

    PubMed Central

    Cong, Xiaona; Wang, Haili; Liu, Zhenxing; He, Chunxia; An, Chunju; Zhao, Zhangwu

    2015-01-01

    Study Objectives: Most organisms have behavioral and physiological circadian rhythms, which are controlled by an endogenous clock. Although genetic analysis has revealed the intracellular mechanism of the circadian clock, the manner in which this clock communicates its temporal information to produce systemic regulation is still largely unknown. Design: Sleep behavior was measured using the Drosophila Activity Monitoring System (DAMS) monitor under a 12 h light:12 h dark cycle and constant darkness (DD), and 5 min without recorded activity were defined as a bout of sleep. Results: Here we show that Drosophila insulin-like peptides (DILPs) and their receptor (DInR) regulate sleep behavior. All mutants of the seven dilps and the mutant of their receptor exhibit decreases of total sleep except dilp4 mutants, whereas upregulation of DILP and DInR in the nervous system led to increased sleep. Histological analysis identified four previously unidentified neurons expressing DILP: D1, P1, L1, and L2, of which L1 and L2 belong to the LNd and LNv clock neurons that separately regulate different times of sleep. In addition, dilp2 levels significantly decrease when flies were fasted, which is consistent with a previous report that starvation inhibits sleep, further indicating that the dilp system is involved in sleep regulation. Conclusion: Taken together, the results indicate that the Drosophila insulin-like peptide system is a crucial regulator of sleep. Citation: Cong X, Wang H, Liu Z, He C, An C, Zhao Z. Regulation of sleep by insulin-like peptide system in Drosophila melanogaster. SLEEP 2015;38(7):1075–1083. PMID:25581915

  5. Toll-6 and Toll-7 function as neurotrophin receptors in the Drosophila central nervous system

    PubMed Central

    McIlroy, Graham; Foldi, Istvan; Aurikko, Jukka; Wentzell, Jill S.; Lim, Mei Ann; Fenton, Janine C.; Gay, Nicholas J.; Hidalgo, Alicia

    2015-01-01

    Neurotrophin receptors corresponding to vertebrate Trk, p75NTR or Sortilin have not been identified in Drosophila, thus it is unknown how neurotrophism may be implemented in insects. Two Drosophila neurotrophins, DNT1 and DNT2, have nervous system functions, but their receptors are unknown. The Toll receptor superfamily has ancient evolutionary origins and a universal function in innate immunity. Here we show that Toll paralogues unrelated to the mammalian neurotrophin receptors function as neurotrophin receptors in fruit-flies. Toll-6 and Toll-7 are expressed in the central nervous system throughout development, and regulate locomotion, motoraxon targeting and neuronal survival. DNT1 and 2 interact genetically with Toll-6 and 7, bind to Toll-7 and 6 promiscuously, and are distributed in vivo in complementary or overlapping domains. We conclude that in fruit-flies, Tolls are not only involved in development and immunity but also in neurotrophism, revealing an unforeseen relationship between the neurotrophin and Toll protein families. PMID:23892553

  6. Fine-scale topography in sensory systems: insights from Drosophila and vertebrates

    PubMed Central

    Kaneko, Takuya; Ye, Bing

    2015-01-01

    To encode the positions of sensory stimuli, sensory circuits form topographic maps in the central nervous system through specific point-to-point connections between pre- and post-synaptic neurons. In vertebrate visual systems, the establishment of topographic maps involves the formation of a coarse topography followed by that of fine-scale topography that distinguishes the axon terminals of neighboring neurons. It is known that intrinsic differences in the form of broad gradients of guidance molecules instruct coarse topography while neuronal activity is required for fine-scale topography. On the other hand, studies in the Drosophila visual system have shown that intrinsic differences in cell adhesion among the axon terminals of neighboring neurons instruct the fine-scale topography. Recent studies on activity-dependent topography in the Drosophila somatosensory system have revealed a role of neuronal activity in creating molecular differences among sensory neurons for establishing fine-scale topography, implicating a conserved principle. Here we review the findings in both Drosophila and vertebrates and propose an integrated model for fine-scale topography. PMID:26091779

  7. Fine-scale topography in sensory systems: insights from Drosophila and vertebrates.

    PubMed

    Kaneko, Takuya; Ye, Bing

    2015-09-01

    To encode the positions of sensory stimuli, sensory circuits form topographic maps in the central nervous system through specific point-to-point connections between pre- and postsynaptic neurons. In vertebrate visual systems, the establishment of topographic maps involves the formation of a coarse topography followed by that of fine-scale topography that distinguishes the axon terminals of neighboring neurons. It is known that intrinsic differences in the form of broad gradients of guidance molecules instruct coarse topography while neuronal activity is required for fine-scale topography. On the other hand, studies in the Drosophila visual system have shown that intrinsic differences in cell adhesion among the axon terminals of neighboring neurons instruct the fine-scale topography. Recent studies on activity-dependent topography in the Drosophila somatosensory system have revealed a role of neuronal activity in creating molecular differences among sensory neurons for establishing fine-scale topography, implicating a conserved principle. Here we review the findings in both Drosophila and vertebrates and propose an integrated model for fine-scale topography. PMID:26091779

  8. Drosophila melanogaster as a model system for assessing development under conditions of microgravity

    NASA Technical Reports Server (NTRS)

    Abbott, M. K.; Hilgenfeld, R. B.; Denell, R. E.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    More is known about the regulation of early developmental events in Drosophila than any other animal. In addition, its size and short life cycle make it a facile experimental system. Since developmental perturbations have been demonstrated when both oogenesis and embryogenesis occur in the space environment, there is a strong rationale for using this organism for the elucidation of specific gravity-sensitive developmental events.

  9. Bacteria sensing mechanisms in Drosophila gut: Local and systemic consequences.

    PubMed

    Capo, Florence; Charroux, Bernard; Royet, Julien

    2016-11-01

    All insects are colonized by microorganisms on their exoskeleton, their gut and even in some cases within their own somatic and germ line cells. This microbiota that can represent up to a few percent of the insect biomass may have a pervasive impact on many aspects of insect biology including physiology, nutrient acquisition, ageing, behaviour and resistance to infection. Mainly through ingestion of contaminated food, the mouth-gut axis represents the first and principal access of external bacteria to the host. Soon after ingestion, the feeding insect needs to rapidly and accurately identify the ingested microbes and decide whether to preserve them if beneficial or neutral, or to eliminate them if potentially harmful. We will review here the recent data acquired in Drosophila on the mechanisms that invertebrate enterocytes rely on to detect the presence of bacteria in the gut. We will compare these modes of bacteria sensing to those in other immune competent tissues and try to rationalize differences that may exist. We will also analyse the physiological consequences of bacteria detection not only locally for the gut itself but also for remote tissues. Finally, we will describe the physiological disorders that can occur due to inaccurate bacteria identification by the gut epithelium. PMID:26778296

  10. The calcium-dependent proteolytic system calpain-calpastatin in Drosophila melanogaster.

    PubMed Central

    Pintér, M; Friedrich, P

    1988-01-01

    Ca2+-dependent proteolytic activity was detected at pH 7.5 in head extracts of the fruit fly Drosophila melanogaster. This activity was abolished by iodoacetate, but was unaffected by phenylmethanesulphonyl fluoride. These properties resemble those of the Ca2+-dependent thiol-proteinase calpain. The activity appeared at Mr 280,000 on Sepharose CL-6B gel chromatography. DEAE-cellulose chromatography revealed two activity peaks, with elution positions corresponding to vertebrate calpains I and II. The fly head enzymes were inhibited by a heat-stable and trypsin-sensitive component of the fly head extract, which also inhibited calpains from rat kidney. The inhibitor emerged from Sepharose CL-6B columns at Mr 310,000 and from DEAE-cellulose at a position corresponding to the protein inhibitor calpastatin from other sources. It is concluded that Drosophila heads comprise the Ca2+-dependent calpain-calpastatin proteolytic system. PMID:2845920

  11. An Integrated Drosophila Model System Reveals Unique Properties for F14512, a Novel Polyamine-Containing Anticancer Drug That Targets Topoisomerase II

    PubMed Central

    Chelouah, Sonia; Monod-Wissler, Caroline; Bailly, Christian; Barret, Jean-Marc; Guilbaud, Nicolas; Vispé, Stéphane; Käs, Emmanuel

    2011-01-01

    F14512 is a novel anti-tumor molecule based on an epipodophyllotoxin core coupled to a cancer-cell vectoring spermine moiety. This polyamine linkage is assumed to ensure the preferential uptake of F14512 by cancer cells, strong interaction with DNA and potent inhibition of topoisomerase II (Topo II). The antitumor activity of F14512 in human tumor models is significantly higher than that of other epipodophyllotoxins in spite of a lower induction of DNA breakage. Hence, the demonstrated superiority of F14512 over other Topo II poisons might not result solely from its preferential uptake by cancer cells, but could also be due to unique effects on Topo II interactions with DNA. To further dissect the mechanism of action of F14512, we used Drosophila melanogaster mutants whose genetic background leads to an easily scored phenotype that is sensitive to changes in Topo II activity and/or localization. F14512 has antiproliferative properties in Drosophila cells and stabilizes ternary Topo II/DNA cleavable complexes at unique sites located in moderately repeated sequences, suggesting that the drug specifically targets a select and limited subset of genomic sequences. Feeding F14512 to developing mutant Drosophila larvae led to the recovery of flies expressing a striking phenotype, "Eye wide shut," where one eye is replaced by a first thoracic segment. Other recovered F14512-induced gain- and loss-of-function phenotypes similarly correspond to precise genetic dysfunctions. These complex in vivo results obtained in a whole developing organism can be reconciled with known genetic anomalies and constitute a remarkable instance of specific alterations of gene expression by ingestion of a drug. "Drosophila-based anticancer pharmacology" hence reveals unique properties for F14512, demonstrating the usefulness of an assay system that provides a low-cost, rapid and effective complement to mammalian models and permits the elucidation of fundamental mechanisms of action of candidate

  12. Electroretinograms in Drosophila: A Robust and Genetically Accessible Electrophysiological System for the Undergraduate Laboratory

    PubMed Central

    Vilinsky, Ilya; Johnson, Karl G.

    2012-01-01

    Laboratory courses in neurophysiology fulfill a critical need for inquiry-based training in undergraduate programs in neuroscience and biology. These courses typically use classical electrophysiological preparations to explore the basic features of neuronal function. However, current neuroscience research also focuses on elucidating the molecular and genetic mechanisms of neuronal function, using model systems that include mutant and transgenic animals. To bridge laboratory training in neurophysiology with modern molecular genetics, we describe a teaching model based on electroretinography of the fruit fly Drosophila melanogaster, a long-established model system for basic neuroscience research. Drosophila are easily maintained, economical, and have hundreds of neurophysiologically relevant mutant strains and genetic tools readily available. The Drosophila electroretinogram (ERG) is a simple and accessible extracellular recording of a neural signal in the fly eye in response to flashes of light. The signal is multifaceted and the response is sensitive to stimulation parameters such as intensity, duration and wavelength, thus forming a rich source of analysis for students. Most importantly, different mutations affecting key components of intracellular signaling, synaptic transmission or neuronal function can affect the ERG waveform in characteristic ways. Recording wild type and mutant ERGs allows students to examine firsthand the connection between genetics, biochemical pathways, and electrophysiology. This neurophysiology laboratory course can facilitate and enhance an understanding of the cellular and molecular contributions to neurophysiological recordings. PMID:23494679

  13. Intracellular Trafficking in Drosophila Visual System Development: A Basis for Pattern Formation Through Simple Mechanisms

    PubMed Central

    Chan, Chih-Chiang; Epstein, Daniel; Hiesinger, P. Robin

    2012-01-01

    Intracellular trafficking underlies cellular functions ranging from membrane remodeling to receptor activation. During multicellular organ development, these basic cell biological functions are required as both passive machinery and active signaling regulators. Exocytosis, endocytosis, and recycling of several key signaling receptors have long been known to actively regulate morphogenesis and pattern formation during Drosophila eye development. Hence, intracellular membrane trafficking not only sets the cell biological stage for receptor-mediated signaling but also actively controls signaling through spatiotemporally regulated receptor localization. In contrast to eye development, the role of intracellular trafficking for the establishment of the eye-to-brain connectivity map has only recently received more attention. It is still poorly understood how guidance receptors are spatiotemporally regulated to serve as meaningful synapse formation signals. Yet, the Drosophila visual system provides some of the most striking examples for the regulatory role of intracellular trafficking during multicellular organ development. In this review we will first highlight the experimental and conceptual advances that motivate the study of intracellular trafficking during Drosophila visual system development. We will then illuminate the development of the eye, the eye-to-brain connectivity map and the optic lobe from the perspective of cell biological dynamics. Finally, we provide a conceptual framework that seeks to explain how the interplay of simple genetically encoded intracellular trafficking events governs the seemingly complex cellular behaviors, which in turn determine the developmental product. PMID:21714102

  14. The peripheral nervous system supports blood cell homing and survival in the Drosophila larva

    PubMed Central

    Makhijani, Kalpana; Alexander, Brandy; Tanaka, Tsubasa; Rulifson, Eric; Brückner, Katja

    2011-01-01

    Interactions of hematopoietic cells with their microenvironment control blood cell colonization, homing and hematopoiesis. Here, we introduce larval hematopoiesis as the first Drosophila model for hematopoietic colonization and the role of the peripheral nervous system (PNS) as a microenvironment in hematopoiesis. The Drosophila larval hematopoietic system is founded by differentiated hemocytes of the embryo, which colonize segmentally repeated epidermal-muscular pockets and proliferate in these locations. Importantly, we show that these resident hemocytes tightly colocalize with peripheral neurons and we demonstrate that larval hemocytes depend on the PNS as an attractive and trophic microenvironment. atonal (ato) mutant or genetically ablated larvae, which are deficient for subsets of peripheral neurons, show a progressive apoptotic decline in hemocytes and an incomplete resident hemocyte pattern, whereas supernumerary peripheral neurons induced by ectopic expression of the proneural gene scute (sc) misdirect hemocytes to these ectopic locations. This PNS-hematopoietic connection in Drosophila parallels the emerging role of the PNS in hematopoiesis and immune functions in vertebrates, and provides the basis for the systematic genetic dissection of the PNS-hematopoietic axis in the future. PMID:22071105

  15. Assaying benzene, a parquet varnish, and a synthetic thinner with respect to induction of in vivo chromosome loss in wing primordial cells of Drosophila.

    PubMed

    Soós, István; Szabad, János

    2014-03-15

    An assay detecting the in vivo loss of mwh(+)Y, a genetically engineered Y chromosome, in cells of the Drosophila wing primordia was published recently. Loss of the mwh(+)Y chromosome in any of the wing-disk cells - in a multiple wing hairs homozygous background - leads to the formation of an mwh mosaic spot (clone) in the emerging wing. The frequency and the size of the mwh clones allow detection and quantitative evaluation of environmental and/or genetic agents inducing chromosome loss. Using this novel technique, we analyzed the potential of vapors of benzene, a parquet varnish, and a synthetic thinner to induce chromosome loss. Exposure to 0.047μg/ml benzene vapor for one day or to 0.175μg/ml for four hours resulted in a significantly elevated mwh clone-frequency confirming the ability of benzene to induce chromosome loss. A one-day exposure to vapors of a parquet varnish or a 6-h exposure to vapors of a synthetic thinner slightly, yet significantly elevated the frequency of chromosome loss. Results of the present paper show the potential of vapors of the analyzed parquet varnish and synthetic thinner to induce chromosome loss, and illustrate the usefulness of the new technique. PMID:24530351

  16. Genotoxic profile of inhibitors of topoisomerases I (camptothecin) and II (etoposide) in a mitotic recombination and sex-chromosome loss somatic eye assay of Drosophila melanogaster.

    PubMed

    Sortibrán, América Nitxin Castañeda; Téllez, María Guadalupe Ordaz; Rodríguez-Arnaiz, Rosario

    2006-04-30

    Genotoxic carcinogens which interact with DNA may produce double-strand breaks as normal intermediates of homologous mitotic recombination, and may give rise to structural chromosome aberrations and inter-chromosomal deletion-recombination. The genotoxic profile of two inhibitors of DNA topoisomerases were evaluated using an in vivo somatic w/w+ eye assay of Drosophila melanogaster for the detection of loss of heterozygosity (LOH) by homologous mitotic recombination, intra-chromosomal recombination and structural chromosomal aberrations. We studied camptothecin (CPT) as a topoisomerase-I-interactive agent and etoposide (ETOP) as a topoisomerase II inhibitor. These drugs act by stabilizing a ternary complex consisting of topoisomerases covalently linked to DNA at single-strand or at double-strand breaks, thereby preventing the relegation step of the breakage/rejoining reaction mediated by the enzyme. The genotoxic profiles were determined from the appearance of eye tissue in adult flies, in which LOH and expression of the reporter gene white produced light clones. The results demonstrated that both compounds were significantly genotoxic, with CPT being more effective than ETOP. Inter-chromosomal mitotic recombination was the major mechanism responsible for the induction of light spots by both compounds in XX females. Loss of the ring X chromosome (rX), was significantly enhanced by CPT, and this topoisomerase blocker also produced intra-chromosomal recombination (XY males). PMID:16529987

  17. Expression of a Drosophila melanogaster acetylcholine receptor-related gene in the central nervous system

    SciTech Connect

    Wadsworth, S.C.; Rosenthal, L.S.; Kammermeyer, K.L.; Potter, M.B.; Nelson, D.J.

    1988-02-01

    The authors isolated Drosophila melanogaster genomic sequences with nucleotide and amino acid sequence homology to subunits of vertebrate acetylcholine receptor by hybridization with a Torpedo acetylcholine receptor subunit cDNA probe. Five introns are present in the portion of the Drosophila gene encoding the unprocessed protein and are positionally conserved relative to the human acetylcholine receptor alpha-subunit gene. The Drosophila genomic clone hybridized to salivary gland polytene chromosome 3L within region 64B and was termed AChR64B. A 3-kilobasae poly(A)-containing transcript complementary to the AChR64B clone was readily detectable by RNA blot hybridizations during midembryogenesis, during metamorphosis, and in newly enclosed adults. AChR64B transcripts were localized to the cellular regions of the central nervous system during embryonic, larval, pupal, and adult stages of development. During metamorphosis, a temporal relationship between the morphogenesis of the optic lobe and expression of AChR64B transcripts was observed.

  18. Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster

    SciTech Connect

    Rodriguez, A.; Zhou, Zhijian; Tang, My Lien

    1996-06-01

    We are using Drosophila as a model system for analysis of immunity and tumor formation and have conducted two types of screens using enhancer detector strains to find genes related to these processes: genes expressed in the immune system (type A; hemocytes, lymph glands and fat body) and genes increased in expression by bacterial infection (type B). For type A, tissue-specific reporter gene activity was determined. For type B, a variation of enhancer detection was devised in which {beta}-galactosidase is assayed spectrophotometrically with and without bacterial infection. Because of immune system involvement in melanotic tumor formation, a third type was hypothesized to be found among types A and B: genes that, when mutated, have a melanotic tumor phenotype. Enhancer detector strains (2800) were screened for type A, 900 for B, and 11 retained for further analysis. Complementation tests, cytological mapping, P-element mobilization, and determination of lethal phase and mutant phenotype have identified six novel genes, Dorothy, wizard, toto, viking, Thor and dappled, and one previously identified gene, Collagen IV. All are associated with reporter gene expression in at least one immune system tissue. Thor has increased expression upon infection. Mutations of wizard and dappled have a melanotic tumor phenotype. 72 refs., 6 figs., 3 tabs.

  19. Microarray assays for solar system exploration

    NASA Astrophysics Data System (ADS)

    Steele, Andrew; Toporski, Jan; McKay, David S.; Schweitzer, Mary; Pincus, Seth; Pérez-Mercader, Juan; Parro García, Victor

    2001-08-01

    The detection of evidence of extinct and extant life is a key issue in astrobiological research, particularly with respect to future exploration of the solar system. Simple life forms may have evolved and developed on planetary bodies such as Mars or Europa. At this point in time, tests whether life once was or still is present can only be carried out by means of in situ experiments. Here, we discuss the potential and advantages of immunological concepts for life detection and the development of a miniaturized automated immunoassay flight device.

  20. Multiple spectral inputs improve motion discrimination in the Drosophila visual system.

    PubMed

    Wardill, Trevor J; List, Olivier; Li, Xiaofeng; Dongre, Sidhartha; McCulloch, Marie; Ting, Chun-Yuan; O'Kane, Cahir J; Tang, Shiming; Lee, Chi-Hon; Hardie, Roger C; Juusola, Mikko

    2012-05-18

    Color and motion information are thought to be channeled through separate neural pathways, but it remains unclear whether and how these pathways interact to improve motion perception. In insects, such as Drosophila, it has long been believed that motion information is fed exclusively by one spectral class of photoreceptor, so-called R1 to R6 cells; whereas R7 and R8 photoreceptors, which exist in multiple spectral classes, subserve color vision. Here, we report that R7 and R8 also contribute to the motion pathway. By using electrophysiological, optical, and behavioral assays, we found that R7/R8 information converge with and shape the motion pathway output, explaining flies' broadly tuned optomotor behavior by its composite responses. Our results demonstrate that inputs from photoreceptors of different spectral sensitivities improve motion discrimination, increasing robustness of perception. PMID:22605779

  1. Additional data in support of the quadruplicated white-ivory reversion system to test for somatic genotoxicity in Drosophila melanogaster.

    PubMed

    Xamena, N; Egido, A; Velázquez, A; Creus, A; Marcos, R

    1991-06-01

    Cyclophosphamide, ethyl methanesulfonate, propyleneimine and tritiated water were tested in a new short-term somatic mutation bioassay, previously described by Green and coworkers (1986), to evaluate the suitability of the quadruplicated white-ivory system of Drosophila melanogaster for genotoxicity testing of chemicals. A 2.9-kb tandemly duplicated sequence of w+ within a w+ gene is responsible for the white-ivory phenotype. Reversion of wi to w+ is, in general, associated with the loss of the appended 2.9 kb or other alterations affecting this duplicated region. The appearance of light (white or nearly white) phenotypes could be due to the loss of some wi copies. Thus, in the eyes of adult males resulting from treated larvae, we can detect 2 types of sectors (red and light) on the orange-yellow background. Our results indicate that the genetic system used in this somatic assay is sensitive to the genotoxic effects of the 4 compounds tested. All 4 compounds tested were positive. Tritiated water had weak effects, cyclophosphamide was characterized by small revertant clones and ethyl methanesulfonate and propyleneimine by large clone size. From the available data, we feel that the wi system can be considered of potential value for genotoxicity testing. PMID:1646957

  2. Field experience with a mobile tomographic nondestructive assay system

    SciTech Connect

    Prettyman, T.H.; Betts, S.E.; Taggart, D.P.; Estep, R.J.; Nicholas, N.J.; Lucas, M.C.; Harlan, R.A.

    1995-12-01

    A mobile tomographic gamma-ray scanner (TGS) developed by Los Alamos National Laboratory was recently demonstrated at the Rocky Flats Environmental Technology Site and is currently in use at Los Alamos waste storage areas. The scanner was developed to assay radionuclides in low-level, transuranic, and mixed waste in containers ranging in size from 2 ft{sup 3} boxes to 83-gallon overpacks. The tomographic imaging capability provides a complete correction for source distribution and matrix attenuation effects, enabling accurate assays of Pu-239 and other gamma-ray emitting isotopes. In addition, the system can reliably detect self-absorbing material such as plutonium metal shot, and can correct for bias caused by self-absorption. The system can be quickly configured to execute far-field scans, segmented gamma-ray scans, and a host of intermediate scanning protocols, enabling higher throughput (up to 20 drums per 8-hour shift). In this paper, we will report on the results of field trials of the mobile system at Rocky Flats and Los Alamos. Assay accuracy is confirmed for cases in which TGS assays can be compared with assays (e.g. with calorimetry) of individual packages within the drums. The mobile tomographic technology is expected to considerably reduce characterization costs at DOE production and environmental technology sites.

  3. Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila

    PubMed Central

    2012-01-01

    In Drosophila, most neurogenetic research is carried out in vivo. Mammalian research demonstrates that primary cell culture techniques provide a powerful model to address cell autonomous and non-autonomous processes outside their endogenous environment. We developed a cell culture system in Drosophila using wildtype and genetically manipulated primary neural tissue for long-term observations. We assessed the molecular identity of distinct neural cell types by immunolabeling and genetically expressed fluorescent cell markers. We monitored mitotic activity of cell cultures derived from wildtype and tumorous larval brains. Our system provides a powerful approach to unveil developmental processes in the nervous system and to complement studies in vivo. PMID:22554060

  4. Lack of Dietary Polyunsaturated Fatty Acids Causes Synapse Dysfunction in the Drosophila Visual System

    PubMed Central

    Ziegler, Anna B.; Ménagé, Cindy; Grégoire, Stéphane; Garcia, Thibault; Ferveur, Jean-François; Bretillon, Lionel; Grosjean, Yael

    2015-01-01

    Polyunsaturated fatty acids (PUFAs) are essential nutrients for animals and necessary for the normal functioning of the nervous system. A lack of PUFAs can result from the consumption of a deficient diet or genetic factors, which impact PUFA uptake and metabolism. Both can cause synaptic dysfunction, which is associated with numerous disorders. However, there is a knowledge gap linking these neuronal dysfunctions and their underlying molecular mechanisms. Because of its genetic manipulability and its easy, fast, and cheap breeding, Drosophila melanogaster has emerged as an excellent model organism for genetic screens, helping to identify the genetic bases of such events. As a first step towards the understanding of PUFA implications in Drosophila synaptic physiology we designed a breeding medium containing only very low amounts of PUFAs. We then used the fly’s visual system, a well-established model for studying signal transmission and neurological disorders, to measure the effects of a PUFA deficiency on synaptic function. Using both visual performance and eye electrophysiology, we found that PUFA deficiency strongly affected synaptic transmission in the fly’s visual system. These defects were rescued by diets containing omega-3 or omega-6 PUFAs alone or in combination. In summary, manipulating PUFA contents in the fly’s diet was powerful to investigate the role of these nutrients on the fly´s visual synaptic function. This study aims at showing how the first visual synapse of Drosophila can serve as a simple model to study the effects of PUFAs on synapse function. A similar approach could be further used to screen for genetic factors underlying the molecular mechanisms of synaptic dysfunctions associated with altered PUFA levels. PMID:26308084

  5. Lack of Dietary Polyunsaturated Fatty Acids Causes Synapse Dysfunction in the Drosophila Visual System.

    PubMed

    Ziegler, Anna B; Ménagé, Cindy; Grégoire, Stéphane; Garcia, Thibault; Ferveur, Jean-François; Bretillon, Lionel; Grosjean, Yael

    2015-01-01

    Polyunsaturated fatty acids (PUFAs) are essential nutrients for animals and necessary for the normal functioning of the nervous system. A lack of PUFAs can result from the consumption of a deficient diet or genetic factors, which impact PUFA uptake and metabolism. Both can cause synaptic dysfunction, which is associated with numerous disorders. However, there is a knowledge gap linking these neuronal dysfunctions and their underlying molecular mechanisms. Because of its genetic manipulability and its easy, fast, and cheap breeding, Drosophila melanogaster has emerged as an excellent model organism for genetic screens, helping to identify the genetic bases of such events. As a first step towards the understanding of PUFA implications in Drosophila synaptic physiology we designed a breeding medium containing only very low amounts of PUFAs. We then used the fly's visual system, a well-established model for studying signal transmission and neurological disorders, to measure the effects of a PUFA deficiency on synaptic function. Using both visual performance and eye electrophysiology, we found that PUFA deficiency strongly affected synaptic transmission in the fly's visual system. These defects were rescued by diets containing omega-3 or omega-6 PUFAs alone or in combination. In summary, manipulating PUFA contents in the fly's diet was powerful to investigate the role of these nutrients on the fly´s visual synaptic function. This study aims at showing how the first visual synapse of Drosophila can serve as a simple model to study the effects of PUFAs on synapse function. A similar approach could be further used to screen for genetic factors underlying the molecular mechanisms of synaptic dysfunctions associated with altered PUFA levels. PMID:26308084

  6. Nerve-racking - apoptotic and non-apoptotic roles of caspases in the nervous system of Drosophila.

    PubMed

    Melzer, Juliane; Broemer, Meike

    2016-07-01

    Studies using Drosophila as a model system have contributed enormously to our knowledge of caspase function and regulation. Caspases are best known as central executioners of apoptosis but also control essential physiological processes in a non-apoptotic manner. The Drosophila genome codes for seven caspases and in this review we provide an overview of current knowledge about caspase function in the nervous system. Caspases regulate neuronal death at all developmental stages and in various neuronal populations. In contrast, non-apoptotic roles are less well understood. The development of new genetically encoded sensors for caspase activity provides unprecedented opportunities to study caspase function in the nervous system in more detail. In light of these new tools we discuss the potential of Drosophila as a model to discover new apoptotic and non-apoptotic neuronal roles of caspases. PMID:26900934

  7. Heterogeneity of the Peripheral Circadian Systems in Drosophila melanogaster: A Review

    PubMed Central

    Ito, Chihiro; Tomioka, Kenji

    2016-01-01

    Circadian rhythms in organisms are involved in many aspects of metabolism, physiology, and behavior. In many animals, these rhythms are produced by the circadian system consisting of a central clock located in the brain and peripheral clocks in various peripheral tissues. The oscillatory machinery and entrainment mechanism of peripheral clocks vary between different tissues and organs. The relationship between the central and peripheral clocks is also tissue-dependent. Here we review the heterogeneous nature of peripheral circadian clocks in the fruit fly Drosophila melanogaster and their dependence on the central clock, and discuss their significance in the temporal organization of physiology in peripheral tissues/organs. PMID:26858652

  8. Heterogeneity of the Peripheral Circadian Systems in Drosophila melanogaster: A Review.

    PubMed

    Ito, Chihiro; Tomioka, Kenji

    2016-01-01

    Circadian rhythms in organisms are involved in many aspects of metabolism, physiology, and behavior. In many animals, these rhythms are produced by the circadian system consisting of a central clock located in the brain and peripheral clocks in various peripheral tissues. The oscillatory machinery and entrainment mechanism of peripheral clocks vary between different tissues and organs. The relationship between the central and peripheral clocks is also tissue-dependent. Here we review the heterogeneous nature of peripheral circadian clocks in the fruit fly Drosophila melanogaster and their dependence on the central clock, and discuss their significance in the temporal organization of physiology in peripheral tissues/organs. PMID:26858652

  9. An integrated optical coherence microscopy imaging and optical stimulation system for optogenetic pacing in Drosophila melanogaster (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Alex, Aneesh; Li, Airong; Men, Jing; Jerwick, Jason; Tanzi, Rudolph E.; Zhou, Chao

    2016-03-01

    Electrical stimulation is the clinical standard for cardiac pacing. Although highly effective in controlling cardiac rhythm, the invasive nature, non-specificity to cardiac tissues and possible tissue damage limits its applications. Optogenetic pacing of the heart is a promising alternative, which is non-invasive and more specific, has high spatial and temporal precision, and avoids the shortcomings in electrical stimulation. Drosophila melanogaster, which is a powerful model organism with orthologs of nearly 75% of human disease genes, has not been studied for optogenetic pacing in the heart. Here, we developed a non-invasive integrated optical pacing and optical coherence microscopy (OCM) imaging system to control the heart rhythm of Drosophila at different developmental stages using light. The OCM system is capable of providing high imaging speed (130 frames/s) and ultrahigh imaging resolutions (1.5 μm and 3.9 μm for axial and transverse resolutions, respectively). A light-sensitive pacemaker was developed in Drosophila by specifically expressing the light-gated cation channel, channelrhodopsin-2 (ChR2) in transgenic Drosophila heart. We achieved non-invasive and specific optical control of the Drosophila heart rhythm throughout the fly's life cycle (larva, pupa, and adult) by stimulating the heart with 475 nm pulsed laser light. Heart response to stimulation pulses was monitored non-invasively with OCM. This integrated non-invasive optogenetic control and in vivo imaging technique provides a novel platform for performing research studies in developmental cardiology.

  10. Regulation of Sleep by Neuropeptide Y-Like System in Drosophila melanogaster

    PubMed Central

    He, Chunxia; Yang, Yunyan; Zhang, Mingming; Price, Jeffrey L.; Zhao, Zhangwu

    2013-01-01

    Sleep is important for maintenance of normal physiology in animals. In mammals, neuropeptide Y (NPY), a homolog of Drosophila neuropeptide F (NPF), is involved in sleep regulation, with different effects in human and rat. However, the function of NPF on sleep in Drosophila melanogaster has not yet been described. In this study, we investigated the effects of NPF and its receptor-neuropeptide F receptor (NPFR1) on Drosophila sleep. Male flies over-expressing NPF or NPFR1 exhibited increased sleep during the nighttime. Further analysis demonstrated that sleep episode duration during nighttime was greatly increased and sleep latency was significantly reduced, indicating that NPF and NPFR1 promote sleep quality, and their action on sleep is not because of an impact of the NPF signal system on development. Moreover, the homeostatic regulation of flies after sleep deprivation was disrupted by altered NPF signaling, since sleep deprivation decreased transcription of NPF in control flies, and there were less sleep loss during sleep deprivation and less sleep gain after sleep deprivation in flies overexpressing NPF and NPFR1 than in control flies, suggesting that NPF system auto-regulation plays an important role in sleep homeostasis. However, these effects did not occur in females, suggesting a sex-dependent regulatory function in sleep for NPF and NPFR1. NPF in D1 brain neurons showed male-specific expression, providing the cellular locus for male-specific regulation of sleep by NPF and NPFR1. This study brings a new understanding into sleep studies of a sexually dimorphic regulatory mode in female and male flies. PMID:24040211

  11. Results of field tests of a transportable calorimeter assay system

    SciTech Connect

    Rakel, D.A.; Lemming, J.F.; Rodenburg, W.W.; Duff, M.F.; Jarvis, J.Y.

    1981-01-01

    A transportable calorimetric assay system, developed for use by US Department of Energy inspectors, is described. The results of field tests at three DOE sites are presented. The samples measured in these tests represent a variety of forms (ash, oxide, metal buttons), isotopic composition, and total plutonium content.

  12. DEVELOPMENT OF A CARCINOGEN ASSAY SYSTEM UTILIZING ESTUARINE FISHES

    EPA Science Inventory

    The objective of this project was the development of systems to assay the effects of chemical carcinogens on marine teleosts. It was determined that the LC-50 for benzidine with respect to Cyprinodon variegatus was ca. 64 ppm. Weekly contaminations of 1 ppm benzidine caused some ...

  13. Drosophila myogenesis.

    PubMed

    Bothe, Ingo; Baylies, Mary K

    2016-09-12

    The skeletal muscle system is the largest organ in motile animals, constituting between 35 and 55% of the human body mass, and up to 75% of the body mass in flying organisms like Drosophila. The flight muscles alone in flying insects comprise up to 65% of total body mass. Not only is the musculature the largest organ system, it is also exquisitely complex, with single muscles existing in different shapes and sizes. These different morphologies allow for such different functions as the high-frequency beating of a wing in a hummingbird, the dilation of the pupil in a human eye, or the maintenance of posture in a giraffe's neck. PMID:27623256

  14. Probing the Fractal Pattern of Heartbeats in Drosophila Pupae by Visible Optical Recording System

    PubMed Central

    Lin, Chen; Chang, Yi-Chung; Cheng, Ya-Chen; Lai, Po-Jung; Yeh, Chien-Hung; Hsieh, Wan-Hsin; Hu, Kun; Wu, June-Tai; Lee, Hsiu-Hsiang; Lo, Men-Tzung; Ho, Yi-Lwun

    2016-01-01

    Judiciously tuning heart rates is critical for regular cardiovascular function. The fractal pattern of heartbeats — a multiscale regulation in instantaneous fluctuations — is well known for vertebrates. The most primitive heart system of the Drosophila provides a useful model to understand the evolutional origin of such a fractal pattern as well as the alterations of fractal pattern during diseased statuses. We developed a non-invasive visible optical heart rate recording system especially suitable for long-term recording by using principal component analysis (PCA) instead of fluorescence recording system to avoid the confounding effect from intense light irradiation. To deplete intracellular Ca2+ levels, the expression of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) was tissue-specifically knocked down. The SERCA group shows longer heart beat intervals (Mean ± SD: 1009.7 ± 151.6 ms) as compared to the control group (545.5 ± 45.4 ms, p < 0.001). The multiscale correlation of SERCA group (scaling exponent: 0.77 ± 0.07), on the other hand, is weaker than that of the control Drosophila (scaling exponent: 0.85 ± 0.03) (p = 0.016). PMID:27535299

  15. Probing the Fractal Pattern of Heartbeats in Drosophila Pupae by Visible Optical Recording System.

    PubMed

    Lin, Chen; Chang, Yi-Chung; Cheng, Ya-Chen; Lai, Po-Jung; Yeh, Chien-Hung; Hsieh, Wan-Hsin; Hu, Kun; Wu, June-Tai; Lee, Hsiu-Hsiang; Lo, Men-Tzung; Ho, Yi-Lwun

    2016-01-01

    Judiciously tuning heart rates is critical for regular cardiovascular function. The fractal pattern of heartbeats - a multiscale regulation in instantaneous fluctuations - is well known for vertebrates. The most primitive heart system of the Drosophila provides a useful model to understand the evolutional origin of such a fractal pattern as well as the alterations of fractal pattern during diseased statuses. We developed a non-invasive visible optical heart rate recording system especially suitable for long-term recording by using principal component analysis (PCA) instead of fluorescence recording system to avoid the confounding effect from intense light irradiation. To deplete intracellular Ca(2+) levels, the expression of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) was tissue-specifically knocked down. The SERCA group shows longer heart beat intervals (Mean ± SD: 1009.7 ± 151.6 ms) as compared to the control group (545.5 ± 45.4 ms, p < 0.001). The multiscale correlation of SERCA group (scaling exponent: 0.77 ± 0.07), on the other hand, is weaker than that of the control Drosophila (scaling exponent: 0.85 ± 0.03) (p = 0.016). PMID:27535299

  16. Hybrid Lethal Systems in the Drosophila Melanogaster Species Complex. I. the Maternal Hybrid Rescue (Mhr) Gene of Drosophila Simulans

    PubMed Central

    Sawamura, K.; Taira, T.; Watanabe, T. K.

    1993-01-01

    Hybrid females from Drosophila simulans females X Drosophila melanogaster males die as embryos while hybrid males from the reciprocal cross die as late larvae. The other two classes are sterile adults. Letting C, X, and Y designate egg cytoplasm, X, and Y chromosomes, respectively, and subscripts m and s stand for melanogaster and simulans, C(m)X(m)Y(s) males are lethal in the larval stage and are rescued by the previously reported genes, Lhr (Lethal hybrid rescue) in simulans or Hmr (Hybrid male rescue) in melanogaster. We report here another rescue gene located on the second chromosome of simulans, mhr (maternal hybrid rescue) that, when present in the mother, rescues C(s)X(m)X(s) females from embryonic lethality. It has been postulated that the hybrids not carrying the X(s) like C(m)X(m)Y(s) males are larval lethal and that the hybrids carrying both the C(s) and the X(m) like C(s)X(m)X(s) females are embryonic lethal. According to these postulates C(s)X(m)Y(s) males (obtained by mating attached-X simulans females to melanogaster males) should be doubly lethal, at both embryo and larval stages. When both rescuing genes are present, Hmr in the father and mhr in the mother, males of this genotype are fully viable, as predicted. PMID:8436276

  17. The Serotonergic Central Nervous System of the Drosophila Larva: Anatomy and Behavioral Function

    PubMed Central

    Apostolopoulou, Anthi A.; Widmann, Annekathrin; Pfitzenmaier, Johanna E.; Maiolo, Elena M.; Selcho, Mareike; Pauls, Dennis; von Essen, Alina; Gupta, Tripti; Sprecher, Simon G.; Birman, Serge; Riemensperger, Thomas; Stocker, Reinhard F.; Thum, Andreas S.

    2012-01-01

    The Drosophila larva has turned into a particularly simple model system for studying the neuronal basis of innate behaviors and higher brain functions. Neuronal networks involved in olfaction, gustation, vision and learning and memory have been described during the last decade, often up to the single-cell level. Thus, most of these sensory networks are substantially defined, from the sensory level up to third-order neurons. This is especially true for the olfactory system of the larva. Given the wealth of genetic tools in Drosophila it is now possible to address the question how modulatory systems interfere with sensory systems and affect learning and memory. Here we focus on the serotonergic system that was shown to be involved in mammalian and insect sensory perception as well as learning and memory. Larval studies suggested that the serotonergic system is involved in the modulation of olfaction, feeding, vision and heart rate regulation. In a dual anatomical and behavioral approach we describe the basic anatomy of the larval serotonergic system, down to the single-cell level. In parallel, by expressing apoptosis-inducing genes during embryonic and larval development, we ablate most of the serotonergic neurons within the larval central nervous system. When testing these animals for naïve odor, sugar, salt and light perception, no profound phenotype was detectable; even appetitive and aversive learning was normal. Our results provide the first comprehensive description of the neuronal network of the larval serotonergic system. Moreover, they suggest that serotonin per se is not necessary for any of the behaviors tested. However, our data do not exclude that this system may modulate or fine-tune a wide set of behaviors, similar to its reported function in other insect species or in mammals. Based on our observations and the availability of a wide variety of genetic tools, this issue can now be addressed. PMID:23082175

  18. Regulated protein depletion by the auxin-inducible degradation system in Drosophila melanogaster.

    PubMed

    Trost, Martina; Blattner, Ariane C; Lehner, Christian F

    2016-01-01

    The analysis of consequences resulting after experimental elimination of gene function has been and will continue to be an extremely successful strategy in biological research. Mutational elimination of gene function has been widely used in the fly Drosophila melanogaster. RNA interference is used extensively as well. In the fly, exceptionally precise temporal and spatial control over elimination of gene function can be achieved in combination with sophisticated transgenic approaches and clonal analyses. However, the methods that act at the gene and transcript level cannot eliminate protein products which are already present at the time when mutant cells are generated or RNA interference is started. Targeted inducible protein degradation is therefore of considerable interest for controlled rapid elimination of gene function. To this end, a degradation system was developed in yeast exploiting TIR1, a plant F box protein, which can recruit proteins with an auxin-inducible degron to an E3 ubiquitin ligase complex, but only in the presence of the phytohormone auxin. Here we demonstrate that the auxin-inducible degradation system functions efficiently also in Drosophila melanogaster. Neither auxin nor TIR1 expression have obvious toxic effects in this organism, and in combination they result in rapid degradation of a target protein fused to the auxin-inducible degron. PMID:27010248

  19. Differentiated muscles are mandatory for gas-filling of the Drosophila airway system

    PubMed Central

    Wang, Yiwen; Cruz, Tina; Irion, Uwe; Moussian, Bernard

    2015-01-01

    ABSTRACT At the end of development, organs acquire functionality, thereby ensuring autonomy of an organism when it separates from its mother or a protective egg. In insects, respiratory competence starts when the tracheal system fills with gas just before hatching of the juvenile animal. Cellular and molecular mechanisms of this process are not fully understood. Analyses of the phenotype of Drosophila embryos with malformed muscles revealed that they fail to gas-fill their tracheal system. Indeed, we show that major regulators of muscle formation like Lame duck and Blown fuse are important, while factors involved in the development of subsets of muscles including cardiac and visceral muscles are dispensable for this process, suggesting that somatic muscles (or parts of them) are essential to enable tracheal terminal differentiation. Based on our phenotypic data, we assume that somatic muscle defect severity correlates with the penetrance of the gas-filling phenotype. This argues that a limiting molecular or mechanical muscle-borne signal tunes tracheal differentiation. We think that in analogy to the function of smooth muscles in vertebrate lungs, a balance of physical forces between muscles and the elasticity of tracheal walls may be decisive for tracheal terminal differentiation in Drosophila. PMID:26621831

  20. GAL4/UAS-WGA system as a powerful tool for tracing Drosophila transsynaptic neural pathways.

    PubMed

    Tabuchi, K; Sawamoto, K; Suzuki, E; Ozaki, K; Sone, M; Hama, C; Tanifuji-Morimoto, T; Yuasa, Y; Yoshihara, Y; Nose, A; Okano, H

    2000-01-01

    Visualization of specific transsynaptic neural pathways is an indispensable technique for understanding the relationship between structure and function in the nervous system. Here, we demonstrate the application of the wheat germ agglutinin (WGA) transgene technique for tracing transsynaptic neural pathways in Drosophila. The intracellular localization of WGA was examined by immunoelectron microscopy. WGA signals were detected in granule-like structures in both the outer photoreceptor cells expressing WGA and the second-order laminar neurons. Misexpression of tetanus toxin (TNT), which inactivates N-synaptobrevin, in the outer photoreceptor cells resulted in the elimination of on/off transients in electroretinogram (ERG) recordings and in a great reduction in WGA transfer into laminar neurons, suggesting that anterograde WGA transsynaptic transfer is dependent mainly on synaptic transmission. Retrograde WGA transfer was also detected upon its forced expression in muscle cells. WGA primarily expressed in muscle cells was taken up by motoneuron axons and transported to their cell bodies in the ventral nerve cord, suggesting that WGA can trace motoneuronal pathways in combination with the muscle-specific GAL4 driver. Thus, the GAL4/UAS-WGA system should facilitate the dissection of the Drosophila neural circuit formation and/or synaptic activity in various regions and at various developmental stages. PMID:10658189

  1. Confinement Vessel Assay System: Calibration and Certification Report

    SciTech Connect

    Frame, Katherine C.; Bourne, Mark M.; Crooks, William J.; Evans, Louise; Gomez, Cipriano; Mayo, Douglas R.; Miko, David K.; Salazar, William R.; Stange, Sy; Vigil, Georgiana M.

    2012-07-17

    Los Alamos National Laboratory has a number of spherical confinement vessels (CVs) remaining from tests involving nuclear materials. These vessels have an inner diameter of 6 feet with 1 to 2 inch thick steel walls. The goal of the Confinement Vessel Disposition (CVD) project is to remove debris and reduce contamination inside the vessels. The Confinement Vessel Assay System (CVAS) was developed to measure the amount of SNM in CVs before and after cleanout. Prior to cleanout, the system will be used to perform a verification measurement of each vessel. After cleanout, the system will be used to perform safeguards-quality assays of {le} 100-g {sup 239}Pu equivalent in a vessel for safeguards termination. The system was calibrated in three different mass regions (low, medium, and high) to cover the entire plutonium mass range that will be assayed. The low mass calibration and medium mass calibration were verified for material positioned in the center of an empty vessel. The systematic uncertainty due to position bias was estimated using an MCNPX model to simulate the response of the system to material localized at various points along the inner surface of the vessel. The background component due to cosmic ray spallation was determined by performing measurements of an empty vessel and comparing to measurements in the same location with no vessel present. The CVAS has been tested and calibrated in preparation for verification and safeguards measurements of CVs before and after cleanout.

  2. Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system

    PubMed Central

    Caldwell, James T.; Melkani, Girish C.; Huxford, Tom; Bernstein, Sanford I.

    2011-01-01

    Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization. PMID:22178692

  3. Birth order dependent growth cone segregation determines synaptic layer identity in the Drosophila visual system

    PubMed Central

    Kulkarni, Abhishek; Ertekin, Deniz; Lee, Chi-Hon; Hummel, Thomas

    2016-01-01

    The precise recognition of appropriate synaptic partner neurons is a critical step during neural circuit assembly. However, little is known about the developmental context in which recognition specificity is important to establish synaptic contacts. We show that in the Drosophila visual system, sequential segregation of photoreceptor afferents, reflecting their birth order, lead to differential positioning of their growth cones in the early target region. By combining loss- and gain-of-function analyses we demonstrate that relative differences in the expression of the transcription factor Sequoia regulate R cell growth cone segregation. This initial growth cone positioning is consolidated via cell-adhesion molecule Capricious in R8 axons. Further, we show that the initial growth cone positioning determines synaptic layer selection through proximity-based axon-target interactions. Taken together, we demonstrate that birth order dependent pre-patterning of afferent growth cones is an essential pre-requisite for the identification of synaptic partner neurons during visual map formation in Drosophila. DOI: http://dx.doi.org/10.7554/eLife.13715.001 PMID:26987017

  4. Birth order dependent growth cone segregation determines synaptic layer identity in the Drosophila visual system.

    PubMed

    Kulkarni, Abhishek; Ertekin, Deniz; Lee, Chi-Hon; Hummel, Thomas

    2016-01-01

    The precise recognition of appropriate synaptic partner neurons is a critical step during neural circuit assembly. However, little is known about the developmental context in which recognition specificity is important to establish synaptic contacts. We show that in the Drosophila visual system, sequential segregation of photoreceptor afferents, reflecting their birth order, lead to differential positioning of their growth cones in the early target region. By combining loss- and gain-of-function analyses we demonstrate that relative differences in the expression of the transcription factor Sequoia regulate R cell growth cone segregation. This initial growth cone positioning is consolidated via cell-adhesion molecule Capricious in R8 axons. Further, we show that the initial growth cone positioning determines synaptic layer selection through proximity-based axon-target interactions. Taken together, we demonstrate that birth order dependent pre-patterning of afferent growth cones is an essential pre-requisite for the identification of synaptic partner neurons during visual map formation in Drosophila. PMID:26987017

  5. Drosophila RhoGEF4 encodes a novel RhoA-specific guanine exchange factor that is highly expressed in the embryonic central nervous system.

    PubMed

    Nahm, Minyeop; Lee, Mihye; Baek, Seung-Hak; Yoon, Jin-Ho; Kim, Hong-Hee; Lee, Zang Hee; Lee, Seungbok

    2006-12-15

    Rho family small GTPases act as molecular switches that regulate neuronal morphogenesis, including axon growth and guidance, dendritic spine formation, and synapse formation. These proteins are positively regulated by guanine nucleotide exchange factors (GEFs) of the Dbl family. This study describes the identification and characterization of Drosophila RhoGEF4 (DRhoGEF4), a novel Dbl family protein that is specifically expressed in the central nervous system during Drosophila embryogenesis. The predicted amino acid sequence of DRhoGEF4 contains a Dbl homology (DH) domain and an adjacent C-terminal pleckstrin homology (PH) domain, which are most closely related to those of mammalian frabins. In this study, the DH-PH motif is shown to enhance the dissociation of GDP from either RhoA or Rac1 but not from Cdc42 in vitro. In addition, p21-binding domain pull-down assays demonstrate that DRhoGEF4 activates RhoA, but neither Rac1 nor Cdc42 in HEK293 cells. Finally, overexpression of DRhoGEF4 is able to induce assembly of stress fibers in cultured NIH3T3 cells. Taken together, these findings suggest that DRhoGEF4 may participate in cytoskeleton-related cellular events by specifically activating RhoA in neuronal morphogenesis. PMID:17011730

  6. Field Deployable Tritium Assay System Host Graphical User Interface Software

    Energy Science and Technology Software Center (ESTSC)

    1998-05-12

    The FDTASHOST software is a Graphical User Interface for the Field Deployable Tritium Assay System (FDTAS - Invention Disclosure SRS-96-09-091 has been submitted). The program runs on the Host computer which is located in the Laboratory and connected to the FDTAS remote field system via a modem over a phone line. The operator receives status information and messages from the Remote system. The operator can enter in commands to be executed by the remote systemmore » using the mouse and a pull down menu.« less

  7. A Cell Marker System and Mosaic Patterns during Early Embryonic Development in Drosophila melanogaster

    PubMed Central

    Zusman, Susan B.; Wieschaus, Eric

    1987-01-01

    An embryonic cell marker system has been developed in Drosophila melanogaster that has enabled us to identify the genotype of cells as early as the cellular blastoderm stage of development. This system allows unambiguous detection of embryos homozygous for most X-linked lethal mutations at stages prior to when their first defects become obvious. By examining gynandromorphs at this stage, we have observed that the number of nuclei per unit area in male regions is about half that in female regions. An examination of early cleavage stage embryos whose DNA has been stained with Hoechst 33258 and whose actin has been stained with phalloidin suggests that this difference is due to a cell cycle delay in cells losing the ring-X. These experiments also demonstrate the existence of a mechanism which controls the timing of nuclear divisions in cycle 10–14 embryos. PMID:3108074

  8. Extensive local adaptation within the chemosensory system following Drosophila melanogaster's global expansion

    PubMed Central

    Arguello, J. Roman; Cardoso-Moreira, Margarida; Grenier, Jennifer K.; Gottipati, Srikanth; Clark, Andrew G.; Benton, Richard

    2016-01-01

    How organisms adapt to new environments is of fundamental biological interest, but poorly understood at the genetic level. Chemosensory systems provide attractive models to address this problem, because they lie between external environmental signals and internal physiological responses. To investigate how selection has shaped the well-characterized chemosensory system of Drosophila melanogaster, we have analysed genome-wide data from five diverse populations. By couching population genomic analyses of chemosensory protein families within parallel analyses of other large families, we demonstrate that chemosensory proteins are not outliers for adaptive divergence between species. However, chemosensory families often display the strongest genome-wide signals of recent selection within D. melanogaster. We show that recent adaptation has operated almost exclusively on standing variation, and that patterns of adaptive mutations predict diverse effects on protein function. Finally, we provide evidence that chemosensory proteins have experienced relaxed constraint, and argue that this has been important for their rapid adaptation over short timescales. PMID:27292132

  9. Toxin activity assays, devices, methods and systems therefor

    DOEpatents

    Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory Jon

    2016-04-05

    Embodiments of the present invention are directed toward devices, system and method for conducting toxin activity assay using sedimentation. The toxin activity assay may include generating complexes which bind to a plurality of beads in a fluid sample. The complexes may include a target toxin and a labeling agent, or may be generated due to presence of active target toxin and/or labeling agent designed to be incorporated into complexes responsive to the presence of target active toxin. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a lower density than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

  10. Can Drosophila melanogaster represent a model system for the detection of reproductive adverse drug reactions?

    PubMed

    Avanesian, Agnesa; Semnani, Sahar; Jafari, Mahtab

    2009-08-01

    Once a molecule is identified as a potential drug, the detection of adverse drug reactions is one of the key components of its development and the FDA approval process. We propose using Drosophila melanogaster to screen for reproductive adverse drug reactions in the early stages of drug development. Compared with other non-mammalian models, D. melanogaster has many similarities to the mammalian reproductive system, including putative sex hormones and conserved proteins involved in genitourinary development. Furthermore, the D. melanogaster model would present significant advantages in time efficiency and cost-effectiveness compared with mammalian models. We present data on methotrexate (MTX) reproductive adverse events in multiple animal models, including fruit flies, as proof-of-concept for the use of the D. melanogaster model. PMID:19482095

  11. A Systems-Level Interrogation Identifies Regulators of Drosophila Blood Cell Number and Survival

    PubMed Central

    Makhijani, Kalpana; Alexander, Brandy; Perrimon, Norbert; Brückner, Katja

    2015-01-01

    In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems. PMID:25749252

  12. Assessing the role of cell-surface molecules in central synaptogenesis in the Drosophila visual system.

    PubMed

    Berger-Müller, Sandra; Sugie, Atsushi; Takahashi, Fumio; Tavosanis, Gaia; Hakeda-Suzuki, Satoko; Suzuki, Takashi

    2013-01-01

    A hallmark of the central nervous system is its spatial and functional organization in synaptic layers. During neuronal development, axons form transient contacts with potential post-synaptic elements and establish synapses with appropriate partners at specific layers. These processes are regulated by synaptic cell-adhesion molecules. In the Drosophila visual system, R7 and R8 photoreceptor subtypes target distinct layers and form en passant pre-synaptic terminals at stereotypic loci of the axonal shaft. A leucine-rich repeat transmembrane protein, Capricious (Caps), is known to be selectively expressed in R8 axons and their recipient layer, which led to the attractive hypothesis that Caps mediates R8 synaptic specificity by homophilic adhesion. Contradicting this assumption, our results indicate that Caps does not have a prominent role in synaptic-layer targeting and synapse formation in Drosophila photoreceptors, and that the specific recognition of the R8 target layer does not involve Caps homophilic axon-target interactions. We generated flies that express a tagged synaptic marker to evaluate the presence and localization of synapses in R7 and R8 photoreceptors. These genetic tools were used to assess how the synaptic profile is affected when axons are forced to target abnormal layers by expressing axon guidance molecules. When R7 axons were mistargeted to the R8-recipient layer, R7s either maintained an R7-like synaptic profile or acquired a similar profile to r8s depending on the overexpressed protein. When R7 axons were redirected to a more superficial medulla layer, the number of presynaptic terminals was reduced. These results indicate that cell-surface molecules are able to dictate synapse loci by changing the axon terminal identity in a partially cell-autonomous manner, but that presynapse formation at specific sites also requires complex interactions between pre- and post-synaptic elements. PMID:24386266

  13. Local and systemic effects of targeted zinc redistribution in Drosophila neuronal and gastrointestinal tissues.

    PubMed

    Richards, Christopher D; Burke, Richard

    2015-12-01

    While the effects of systemic zinc ion deficiency and toxicity on animal health are well documented, the impacts of localized, tissue-specific disturbances in zinc homeostasis are less well understood. Previously we have identified zinc dyshomeostasis scenarios caused by the targeted manipulation of zinc transport genes in the Drosophila eye. Over expression of the uptake transporter dZIP42C.1 (dZIP1) combined with knockdown of the efflux transporter dZNT63C (dZNT1) causes a zinc toxicity phenotype, as does over expression of dZIP71B or dZNT86D. However, all three genotypes result in different morphologies, responses to dietary zinc, and genetic interactions with the remaining zinc transport genes, indicating that each causes a different redistribution of zinc within affected cells. dZNT86D (eGFP) over expression generates a completely different phenotype, interpreted as a Golgi zinc deficiency. Here we assess the effect of each of these transgenes when targeted to a range of Drosophila tissues. We find that dZIP71B is a particularly potent zinc uptake gene, causing early developmental lethality when targeted to multiple different tissue types. dZNT86D over expression (Golgi-only zinc toxicity) is less deleterious, but causes highly penetrant adult cuticle, sensory bristle and wing expansion defects. The dZIP42C.1 over expression, dZNT63C knockdown combination causes only moderate adult cuticle defects and sensitivity to dietary zinc when expressed in the midgut. The Golgi-only zinc deficiency caused by dZNT86D (eGFP) expression results in mild cuticle defects, highly penetrant wing expansion defects and developmental lethality when targeted to the central nervous system and, uniquely, the fat bodies. PMID:26411574

  14. Pu-238 assay performance with the Canberra IQ3 system

    SciTech Connect

    Booth, L.; Gillespie, B.; Seaman, G.

    1997-11-01

    Canberra Industries has recently completed a demonstration project at the Westinghouse Savannah River Site (WSRC) to characterize 55-gallon drums containing Pu-238 contaminated waste. The goal of this project was to detect and quantify Pu-238 contaminated waste. The goal of this project was to detect and quantify Pu-238 waste to detection limits of less than 50 nCi/g using gamma assay techniques. This would permit reclassification of these drums from transuranic (TRU) waste to low-level waste (LLW). The instrument used for this assay was a Canberra IQ3 high sensitivity gamma assay system, mounted in a trailer. The results of the measurements demonstrate achievement of detection levels as low as 1 nCi/g for low density waste drums, and good correlation with known concentrations in several test drums. In addition, the data demonstrates significant advantages for using large area low-energy germanium detectors for achieving the lowest possible MDAs for gamma rays in the 80-250 keV range. 1 fig., 2 tabs.

  15. Devices, systems, and methods for conducting sandwich assays using sedimentation

    SciTech Connect

    Schaff, Ulrich Y; Sommer, Gregory J; Singh, Anup K; Hatch, Anson V

    2015-02-03

    Embodiments of the present invention are directed toward devices, systems, and method for conducting sandwich assays using sedimentation. In one example, a method includes generating complexes on a plurality of beads in a fluid sample, individual ones of the complexes comprising a capture agent, a target analyte, and a labeling agent. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a density lower than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

  16. Development of a Calibration Strip for Immunochromatographic Assay Detection Systems

    PubMed Central

    Gao, Yue-Ming; Wei, Jian-Chong; Mak, Peng-Un; Vai, Mang-I.; Du, Min; Pun, Sio-Hang

    2016-01-01

    With many benefits and applications, immunochromatographic (ICG) assay detection systems have been reported on a great deal. However, the existing research mainly focuses on increasing the dynamic detection range or application fields. Calibration of the detection system, which has a great influence on the detection accuracy, has not been addressed properly. In this context, this work develops a calibration strip for ICG assay photoelectric detection systems. An image of the test strip is captured by an image acquisition device, followed by performing a fuzzy c-means (FCM) clustering algorithm and maximin-distance algorithm for image segmentation. Additionally, experiments are conducted to find the best characteristic quantity. By analyzing the linear coefficient, an average value of hue (H) at 14 min is chosen as the characteristic quantity and the empirical formula between H and optical density (OD) value is established. Therefore, H, saturation (S), and value (V) are calculated by a number of selected OD values. Then, H, S, and V values are transferred to the RGB color space and a high-resolution printer is used to print the strip images on cellulose nitrate membranes. Finally, verification of the printed calibration strips is conducted by analyzing the linear correlation between OD and the spectral reflectance, which shows a good linear correlation (R2 = 98.78%). PMID:27367694

  17. Development of a Calibration Strip for Immunochromatographic Assay Detection Systems.

    PubMed

    Gao, Yue-Ming; Wei, Jian-Chong; Mak, Peng-Un; Vai, Mang-I; Du, Min; Pun, Sio-Hang

    2016-01-01

    With many benefits and applications, immunochromatographic (ICG) assay detection systems have been reported on a great deal. However, the existing research mainly focuses on increasing the dynamic detection range or application fields. Calibration of the detection system, which has a great influence on the detection accuracy, has not been addressed properly. In this context, this work develops a calibration strip for ICG assay photoelectric detection systems. An image of the test strip is captured by an image acquisition device, followed by performing a fuzzy c-means (FCM) clustering algorithm and maximin-distance algorithm for image segmentation. Additionally, experiments are conducted to find the best characteristic quantity. By analyzing the linear coefficient, an average value of hue (H) at 14 min is chosen as the characteristic quantity and the empirical formula between H and optical density (OD) value is established. Therefore, H, saturation (S), and value (V) are calculated by a number of selected OD values. Then, H, S, and V values are transferred to the RGB color space and a high-resolution printer is used to print the strip images on cellulose nitrate membranes. Finally, verification of the printed calibration strips is conducted by analyzing the linear correlation between OD and the spectral reflectance, which shows a good linear correlation (R² = 98.78%). PMID:27367694

  18. Navigation-specific neural coding in the visual system of Drosophila.

    PubMed

    Dewar, Alex D M; Wystrach, Antoine; Graham, Paul; Philippides, Andrew

    2015-10-01

    Drosophila melanogaster are a good system in which to understand the minimal requirements for widespread visually guided behaviours such as navigation, due to their small brains (adults possess only 100,000 neurons) and the availability of neurogenetic techniques which allow the identification of task-specific cell types. Recently published data describe the receptive fields for two classes of visually responsive neurons (R2 and R3/R4d ring neurons in the central complex) that are essential for visual tasks such as orientation memory for salient objects and simple pattern discriminations. What is interesting is that these cells have very large receptive fields and are very small in number, suggesting that each sub-population of cells might be a bottleneck in the processing of visual information for a specific behaviour, as each subset of cells effectively condenses information from approximately 3000 visual receptors in the eye, to fewer than 50 neurons in total. It has recently been shown how R1 ring neurons, which receive input from the same areas as the R2 and R3/R4d cells, are necessary for place learning in Drosophila. However, how R1 neurons enable place learning is unknown. By examining the information provided by different populations of hypothetical visual neurons in simulations of experimental arenas, we show that neurons with ring neuron-like receptive fields are sufficient for defining a location visually. In this way we provide a link between the type of information conveyed by ring neurons and the behaviour they support. PMID:26310914

  19. Drosophila Melanogaster as a Model System for Studies of Islet Amyloid Polypeptide Aggregation

    PubMed Central

    Schultz, Sebastian Wolfgang; Nilsson, K. Peter R.; Westermark, Gunilla Torstensdotter

    2011-01-01

    Background Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death. Methodology/Principal Findings We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elavC155,Gal4 showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures. Conclusions/Significance These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology. PMID:21695120

  20. Field Deployable Tritium Assay System Remote Control Software

    Energy Science and Technology Software Center (ESTSC)

    1998-05-12

    The FDTASREM software is a command control based application for the Field Deployable Tritium Assay System (FDTAS-Invention Disclosure SRS-96-091 has been submitted). The program runs on the Remote computer which is located at the field site with the FDTAS sampling and analysis components. The application executes commands received over the connected phone line from the operator via the FDTAS Host GUI running in the laboratory some distance away. The FDTASREM controls interface with the FDTASmore » auto sampler and the analysis systems. It tells the sampler to take a sample from a specified location and send it to the analyzer. Once the sample is sent to the analyzer, FDTASREM sequences the internal valves and pumps to deliver the sample and cocktail to the counting chamber. Once the analysis is complete, the program can execute the clean command and prepare the system for the next sample.« less

  1. Generation of a sensitive TNFR2-specific murine assays system.

    PubMed

    Ando, D; Kamada, H; Inoue, M; Taki, S; Furuya, T; Abe, Y; Nagano, K; Tsutsumi, Y; Tsunoda, S

    2016-05-01

    Tumor necrosis factor (TNF)/TNF receptors (TNFR1/TNFR2) are considered to be potential drug targets to treat refractory diseases, including autoimmune diseases and malignant tumors. However, their specific functions, especially in the case of TNFR2, are poorly understood. In this study, we constructed a mouse TNFR2 (mTNFR2)-mediated biological assay system that shows no effects of mouse TNFR1 (mTNFR1) in order to screen mTNFR2-selective stimulating agents. Mouse TNFR1(-/-)R2(-/-) preadipocytes were transfected with the gene encoding the mTNFR2/mouse Fas (mFas) chimeric receptor in which the extracellular and transmembrane domains of mTNFR2 were fused to the intracellular domain of mFas. Our results demonstrated that this cell line exhibits highly sensitive mTNFR2-mediated cytotoxic effects. We propose that this mTNFR2-mediated biological assay system would be a useful tool to screen for mTNFR2-selective stimulating agents. PMID:27348964

  2. Embryonic Origins of a Motor System:Motor Dendrites Form a Myotopic Mapin Drosophila

    PubMed Central

    2003-01-01

    The organisational principles of locomotor networks are less well understood than those of many sensory systems, where in-growing axon terminals form a central map of peripheral characteristics. Using the neuromuscular system of the Drosophila embryo as a model and retrograde tracing and genetic methods, we have uncovered principles underlying the organisation of the motor system. We find that dendritic arbors of motor neurons, rather than their cell bodies, are partitioned into domains to form a myotopic map, which represents centrally the distribution of body wall muscles peripherally. While muscles are segmental, the myotopic map is parasegmental in organisation. It forms by an active process of dendritic growth independent of the presence of target muscles, proper differentiation of glial cells, or (in its initial partitioning) competitive interactions between adjacent dendritic domains. The arrangement of motor neuron dendrites into a myotopic map represents a first layer of organisation in the motor system. This is likely to be mirrored, at least in part, by endings of higher-order neurons from central pattern-generating circuits, which converge onto the motor neuron dendrites. These findings will greatly simplify the task of understanding how a locomotor system is assembled. Our results suggest that the cues that organise the myotopic map may be laid down early in development as the embryo subdivides into parasegmental units. PMID:14624243

  3. Structure of the voltage-dependent potassium channel is highly conserved from Drosophila to vertebrate central nervous systems.

    PubMed Central

    Baumann, A; Grupe, A; Ackermann, A; Pongs, O

    1988-01-01

    Voltage-sensitive potassium channels are found in vertebrate and invertebrate central nervous systems. We have isolated a rat brain cDNA by cross-hybridization with a probe of the Drosophila Shaker gene complex. Structural conservation of domains of the deduced protein indicate that the rat brain cDNA encodes a voltage-sensitive potassium channel. Of the deduced amino acid sequence, 82% is homologous to the Drosophila Shaker protein indicating that voltage-sensitive potassium channels have been highly conserved during evolution. Selective pressure was highest on sequences facing the intracellular side and on proposed transmembrane segments S4-S6, suggesting that these domains are crucial for voltage-dependent potassium channel function. The corresponding rat mRNA apparently belongs to a family of mRNA molecules which are preferentially expressed in the central nervous system. Images PMID:3191911

  4. SWEPP Assay System Version 2.0 software design description

    SciTech Connect

    East, L.V.; Marwil, E.S.

    1996-08-01

    The Idaho National Engineering Laboratory (INEL) Stored Waste Examination Pilot Plant (SWEPP) operations staff use nondestructive analysis methods to characterize the radiological contents of contact-handled radioactive waste containers. Containers of waste from Rocky Flats Environmental Technology Site and other Department of Energy (DOE) sites are currently stored at SWEPP. Before these containers can be shipped to the Waste Isolation Pilot Plant (WIPP), SWEPP must verify compliance with storage, shipping, and disposal requirements. This program has been in operation since 1985 at the INEL Radioactive Waste Management Complex (RWMC). One part of the SWEPP program measures neutron emissions from the containers and estimates the mass of plutonium and other transuranic (TRU) isotopes present. A Passive/Active Neutron (PAN) assay system developed at the Los Alamos National Laboratory is used to perform these measurements. A computer program named NEUT2 was originally used to perform the data acquisition and reduction functions for the neutron measurements. This program was originally developed at Los Alamos and extensively modified by a commercial vendor of PAN systems and by personnel at the INEL. NEUT2 uses the analysis methodology outlined, but no formal documentation exists on the program itself. The SWEPP Assay System (SAS) computer program replaced the NEUT2 program in early 1994. The SAS software was developed using an `object model` approach and is documented in accordance with American National Standards Institute (ANSI) and Institute of Electrical and Electronic Engineers (IEEE) standards. The new program incorporates the basic analysis algorithms found in NEUT2. Additional functionality and improvements include a graphical user interface, the ability to change analysis parameters without program code modification, an `object model` design approach and other features for improved flexibility and maintainability.

  5. Behavioural system identification of visual flight speed control in Drosophila melanogaster

    PubMed Central

    Rohrseitz, Nicola; Fry, Steven N.

    2011-01-01

    Behavioural control in many animals involves complex mechanisms with intricate sensory-motor feedback loops. Modelling allows functional aspects to be captured without relying on a description of the underlying complex, and often unknown, mechanisms. A wide range of engineering techniques are available for modelling, but their ability to describe time-continuous processes is rarely exploited to describe sensory-motor control mechanisms in biological systems. We performed a system identification of visual flight speed control in the fruitfly Drosophila, based on an extensive dataset of open-loop responses previously measured under free flight conditions. We identified a second-order under-damped control model with just six free parameters that well describes both the transient and steady-state characteristics of the open-loop data. We then used the identified control model to predict flight speed responses after a visual perturbation under closed-loop conditions and validated the model with behavioural measurements performed in free-flying flies under the same closed-loop conditions. Our system identification of the fruitfly's flight speed response uncovers the high-level control strategy of a fundamental flight control reflex without depending on assumptions about the underlying physiological mechanisms. The results are relevant for future investigations of the underlying neuromotor processing mechanisms, as well as for the design of biomimetic robots, such as micro-air vehicles. PMID:20525744

  6. Confinement Vessel Assay System: Design and Implementation Report

    SciTech Connect

    Frame, Katherine C.; Bourne, Mark M.; Crooks, William J.; Evans, Louise; Mayo, Douglas R.; Gomez, Cipriano D.; Miko, David K.; Salazar, William R.; Stange, Sy; Vigil, Georgiana M.

    2012-07-18

    Los Alamos National Laboratory has a number of spherical confinement vessels remaining from tests involving nuclear materials. These vessels have an inner diameter of 6 feet with 1- to 2-inch thick steel walls. The goal of the Confinement Vessel Disposition (CVD) project is to remove debris and reduce contamination inside the vessels. We have developed a neutron assay system for the purposes of Materials Control and Accountability (MC&A) measurements of the vessel prior to and after cleanout. We present our approach to confronting the challenges in designing, building, and testing such a system. The system was designed to meet a set of functional and operational requirements. A Monte Carlo model was developed to aid in optimizing the detector design as well as to predict the systematic uncertainty associated with confinement vessel measurements. Initial testing was performed to optimize and determine various measurement parameters, and then the system was characterized using {sup 252}Cf placed a various locations throughout the measurement system. Measurements were also performed with a {sup 252}Cf source placed inside of small steel and HDPE shells to study the effect of moderation. These measurements compare favorably with their MCNPX model equivalent, making us confident that we can rely on the Monte Carlo simulation to predict the systematic uncertainty due to variations in response to material that may be localized at different points within a vessel.

  7. The uranium cylinder assay system for enrichment plant safeguards

    SciTech Connect

    Miller, Karen A; Swinhoe, Martyn T; Marlow, Johnna B; Menlove, Howard O; Rael, Carlos D; Iwamoto, Tomonori; Tamura, Takayuki; Aiuchi, Syun

    2010-01-01

    Safeguarding sensitive fuel cycle technology such as uranium enrichment is a critical component in preventing the spread of nuclear weapons. A useful tool for the nuclear materials accountancy of such a plant would be an instrument that measured the uranium content of UF{sub 6} cylinders. The Uranium Cylinder Assay System (UCAS) was designed for Japan Nuclear Fuel Limited (JNFL) for use in the Rokkasho Enrichment Plant in Japan for this purpose. It uses total neutron counting to determine uranium mass in UF{sub 6} cylinders given a known enrichment. This paper describes the design of UCAS, which includes features to allow for unattended operation. It can be used on 30B and 48Y cylinders to measure depleted, natural, and enriched uranium. It can also be used to assess the amount of uranium in decommissioned equipment and waste containers. Experimental measurements have been carried out in the laboratory and these are in good agreement with the Monte Carlo modeling results.

  8. Genetic and molecular studies of apterous: a gene implicated in the juvenile hormone system of Drosophila.

    PubMed

    Shtorch, A; Werczberger, R; Segal, D

    1995-01-01

    The apterous (ap) gene in Drosophila melanogaster encodes a homeodomain transcription factor. It is required for the development of the wings and of a subset of embryonic muscles. The gene has been implicated in the juvenile hormone (JH) system because mutations in ap lead to JH deficiency, and are associated with defective histolysis of the larval fat body, arrested vitellogenesis, sterility, and aberrant sexual behavior, all of which are dependent on JH. We describe here the use of hemizygotes and germ-line clones, of X-ray- and hybrid dysgenesis-induced lethal ap alleles to determine the primary role of the gene during development. We find that ap lethality is polyphasic, but occurs primarily at the larval and pupal stages. The lethal phenotype is not associated with any overt morphological abnormality, suggesting that death occurs from a systemic malfunction. Strong interallelic complementation for the wing phenotype was found between some ap mutations induced by X-rays or by hybrid-dysgenesis. By Northern blot analysis, we demonstrate an increase in ap expression in pupae and adults as compared to embryos and larvae, suggesting that it is developmentally regulated. Finally, primer extension is used to determine the transcription start site of the gene. PMID:7579572

  9. Asymmetric Rab 11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system.

    PubMed

    Emery, Gregory; Hutterer, Andrea; Berdnik, Daniela; Mayer, Bernd; Wirtz-Peitz, Frederik; Gaitan, Marcos Gonzalez; Knoblich, Juergen A

    2005-09-01

    Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. Here, we describe a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. We show that trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab 11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab 11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, we demonstrate that recycling endosomes are essential for Delta activity. Our results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments. PMID:16137758

  10. CoinFLP: a system for efficient mosaic screening and for visualizing clonal boundaries in Drosophila.

    PubMed

    Bosch, Justin A; Tran, Ngoc Han; Hariharan, Iswar K

    2015-02-01

    Screens in mosaic Drosophila tissues that use chemical mutagenesis have identified many regulators of growth and patterning. Many of the mutant phenotypes observed were contingent upon the presence of both wild-type and mutant cells in the same tissue. More recently, large collections of RNAi lines or cDNAs expressed under Gal4/UAS control have been used to alter gene expression uniformly in specific tissues. However, these newer approaches are not easily combined with the efficient generation of genetic mosaics. The CoinFLP system described here enables mosaic screens in the context of gene knockdown or overexpression by automatically generating a reliable ratio of mutant to wild-type tissue in a developmentally controlled manner. CoinFLP-Gal4 generates mosaic tissues composed of clones of which only a subset expresses Gal4. CoinFLP-LexGAD/Gal4 generates tissues composed of clones that express either Gal4 or LexGAD, thus allowing the study of interactions between different types of genetically manipulated cells. By combining CoinFLP-LexGAD/Gal4 with the split-GFP system GRASP, boundaries between genetically distinct cell populations can be visualized at high resolution. PMID:25605786

  11. Lineage mapping identifies molecular and architectural similarities between the larval and adult Drosophila central nervous system

    PubMed Central

    Lacin, Haluk; Truman, James W

    2016-01-01

    Neurogenesis in Drosophila occurs in two phases, embryonic and post-embryonic, in which the same set of neuroblasts give rise to the distinct larval and adult nervous systems, respectively. Here, we identified the embryonic neuroblast origin of the adult neuronal lineages in the ventral nervous system via lineage-specific GAL4 lines and molecular markers. Our lineage mapping revealed that neurons born late in the embryonic phase show axonal morphology and transcription factor profiles that are similar to the neurons born post-embryonically from the same neuroblast. Moreover, we identified three thorax-specific neuroblasts not previously characterized and show that HOX genes confine them to the thoracic segments. Two of these, NB2-3 and NB3-4, generate leg motor neurons. The other neuroblast is novel and appears to have arisen recently during insect evolution. Our findings provide a comprehensive view of neurogenesis and show how proliferation of individual neuroblasts is dictated by temporal and spatial cues. DOI: http://dx.doi.org/10.7554/eLife.13399.001 PMID:26975248

  12. Mating system variation drives rapid evolution of the female transcriptome in Drosophila pseudoobscura

    PubMed Central

    Immonen, Elina; Snook, Rhonda R; Ritchie, Michael G

    2014-01-01

    Interactions between the sexes are believed to be a potent source of selection on sex-specific evolution. The way in which sexual interactions influence male investment is much studied, but effects on females are more poorly understood. To address this deficiency, we examined gene expression in virgin female Drosophila pseudoobscura following 100 generations of mating system manipulations in which we either elevated polyandry or enforced monandry. Gene expression evolution following mating system manipulation resulted in 14% of the transcriptome of virgin females being altered. Polyandrous females elevated expression of a greater number of genes normally enriched in ovaries and associated with mitosis and meiosis, which might reflect female investment into reproductive functions. Monandrous females showed a greater number of genes normally enriched for expression in somatic tissues, including the head and gut and associated with visual perception and metabolism, respectively. By comparing our data with a previous study of sex differences in gene expression in this species, we found that the majority of the genes that are differentially expressed between females of the selection treatments show female-biased expression in the wild-type population. A striking exception is genes associated with male-specific reproductive tissues (in D. melanogaster), which are upregulated in polyandrous females. Our results provide experimental evidence for a role of sex-specific selection arising from differing sexual interactions with males in promoting rapid evolution of the female transcriptome. PMID:25360260

  13. Mental Retardation Genes in Drosophila: New Approaches to Understanding and Treating Developmental Brain Disorders

    ERIC Educational Resources Information Center

    Restifo, Linda L.

    2005-01-01

    "Drosophila melanogaster" is emerging as a valuable genetic model system for the study of mental retardation (MR). MR genes are remarkably similar between humans and fruit flies. Cognitive behavioral assays can detect reductions in learning and memory in flies with mutations in MR genes. Neuroanatomical methods, including some at single-neuron…

  14. The synchronous active neutron detection system for spent fuel assay

    SciTech Connect

    Pickrell, M.M.; Kendall, P.K.

    1994-10-01

    The authors have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit the unique operating features of a 14-MeV neutron generator developed by Schlumberger. This generator and a novel detection system will be applied to the direct measurement of the fissile material content in spent fuel in place of the indirect measures used at present. The technique they are investigating is termed synchronous active neutron detection (SAND). It closely follows a method that has been used routinely in other branches of physics to detect very small signals in the presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed {open_quotes}lock-in{close_quotes} amplifiers. The authors have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. This approach is possible because the Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. The results to date are preliminary but quite promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly. It also appears to be quite resilient to background neutron interference. The interrogating neutrons appear to be nonthermal and penetrating. Although a significant amount of work remains to fully explore the relevant physics and optimize the instrument design, the underlying concept appears sound.

  15. Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host–Microbe Model System

    PubMed Central

    Bhatnagar, Srijak; Eisen, Jonathan A.; Kopp, Artyom

    2011-01-01

    Drosophila melanogaster is emerging as an important model of non-pathogenic host–microbe interactions. The genetic and experimental tractability of Drosophila has led to significant gains in our understanding of animal–microbial symbiosis. However, the full implications of these results cannot be appreciated without the knowledge of the microbial communities associated with natural Drosophila populations. In particular, it is not clear whether laboratory cultures can serve as an accurate model of host–microbe interactions that occur in the wild, or those that have occurred over evolutionary time. To fill this gap, we characterized natural bacterial communities associated with 14 species of Drosophila and related genera collected from distant geographic locations. To represent the ecological diversity of Drosophilids, examined species included fruit-, flower-, mushroom-, and cactus-feeders. In parallel, wild host populations were compared to laboratory strains, and controlled experiments were performed to assess the importance of host species and diet in shaping bacterial microbiome composition. We find that Drosophilid flies have taxonomically restricted bacterial communities, with 85% of the natural bacterial microbiome composed of only four bacterial families. The dominant bacterial taxa are widespread and found in many different host species despite the taxonomic, ecological, and geographic diversity of their hosts. Both natural surveys and laboratory experiments indicate that host diet plays a major role in shaping the Drosophila bacterial microbiome. Despite this, the internal bacterial microbiome represents only a highly reduced subset of the external bacterial communities, suggesting that the host exercises some level of control over the bacteria that inhabit its digestive tract. Finally, we show that laboratory strains provide only a limited model of natural host–microbe interactions. Bacterial taxa used in experimental studies are rare or absent in

  16. Drosophila calmodulin mutants with specific defects in the musculature or in the nervous system.

    PubMed Central

    Wang, Bo; Sullivan, Kathleen M C; Beckingham, Kathy

    2003-01-01

    We have studied lethal mutations in the single calmodulin gene (Cam) of Drosophila to gain insight into the in vivo functions of this important calcium sensor. As a result of maternal calmodulin (CaM) in the mature egg, lethality is delayed until the postembryonic stages. Prior to death in the first larval instar, Cam nulls show a striking behavioral abnormality (spontaneous backward movement) whereas a mutation, Cam7, that results in a single amino acid change (V91G) produces a very different phenotype: short indented pupal cases and pupal death with head eversion defects. We show here that the null behavioral phenotype originates in the nervous system and involves a CaM function that requires calcium binding to all four sites of the protein. Further, backward movement can be induced in hypomorphic mutants by exposure to high light levels. In contrast, the V91G mutation specifically affects the musculature and causes abnormal calcium release in response to depolarization of the muscles. Genetic interaction studies suggest that failed regulation of the muscle calcium release channel, the ryanodine receptor, is the major defect underlying the Cam7 phenotype. PMID:14668380

  17. Developmental origin of wiring specificity in the olfactory system of Drosophila.

    PubMed

    Jefferis, Gregory S X E; Vyas, Raj M; Berdnik, Daniela; Ramaekers, Ariane; Stocker, Reinhard F; Tanaka, Nobuaki K; Ito, Kei; Luo, Liqun

    2004-01-01

    In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites. PMID:14645123

  18. Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila

    PubMed Central

    Ameku, Tomotsune

    2016-01-01

    Mating and gametogenesis are two essential components of animal reproduction. Gametogenesis must be modulated by the need for gametes, yet little is known of how mating, a process that utilizes gametes, may modulate the process of gametogenesis. Here, we report that mating stimulates female germline stem cell (GSC) proliferation in Drosophila melanogaster. Mating-induced increase in GSC number is not simply owing to the indirect effect of emission of stored eggs, but rather is stimulated by a male-derived Sex Peptide (SP) and its receptor SPR, the components of a canonical neuronal pathway that induces a post-mating behavioral switch in females. We show that ecdysteroid, the major insect steroid hormone, regulates mating-induced GSC proliferation independently of insulin signaling. Ovarian ecdysteroid level increases after mating and transmits its signal directly through the ecdysone receptor expressed in the ovarian niche to increase the number of GSCs. Impairment of ovarian ecdysteroid biosynthesis disrupts mating-induced increase in GSCs as well as egg production. Importantly, feeding of ecdysteroid rescues the decrease in GSC number caused by impairment of neuronal SP signaling. Our study illustrates how female GSC activity is coordinately regulated by the neuroendocrine system to sustain reproductive success in response to mating. PMID:27310920

  19. Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila.

    PubMed

    Ameku, Tomotsune; Niwa, Ryusuke

    2016-06-01

    Mating and gametogenesis are two essential components of animal reproduction. Gametogenesis must be modulated by the need for gametes, yet little is known of how mating, a process that utilizes gametes, may modulate the process of gametogenesis. Here, we report that mating stimulates female germline stem cell (GSC) proliferation in Drosophila melanogaster. Mating-induced increase in GSC number is not simply owing to the indirect effect of emission of stored eggs, but rather is stimulated by a male-derived Sex Peptide (SP) and its receptor SPR, the components of a canonical neuronal pathway that induces a post-mating behavioral switch in females. We show that ecdysteroid, the major insect steroid hormone, regulates mating-induced GSC proliferation independently of insulin signaling. Ovarian ecdysteroid level increases after mating and transmits its signal directly through the ecdysone receptor expressed in the ovarian niche to increase the number of GSCs. Impairment of ovarian ecdysteroid biosynthesis disrupts mating-induced increase in GSCs as well as egg production. Importantly, feeding of ecdysteroid rescues the decrease in GSC number caused by impairment of neuronal SP signaling. Our study illustrates how female GSC activity is coordinately regulated by the neuroendocrine system to sustain reproductive success in response to mating. PMID:27310920

  20. Modulatory Action by the Serotonergic System: Behavior and Neurophysiology in Drosophila melanogaster

    PubMed Central

    Majeed, Zana R.; Abdeljaber, Esraa; Soveland, Robin; Cornwell, Kristin; Bankemper, Aubrey; Koch, Felicitas; Cooper, Robin L.

    2016-01-01

    Serotonin modulates various physiological processes and behaviors. This study investigates the role of 5-HT in locomotion and feeding behaviors as well as in modulation of sensory-motor circuits. The 5-HT biosynthesis was dysregulated by feeding Drosophila larvae 5-HT, a 5-HT precursor, or an inhibitor of tryptophan hydroxylase during early stages of development. The effects of feeding fluoxetine, a selective serotonin reuptake inhibitor, during early second instars were also examined. 5-HT receptor subtypes were manipulated using RNA interference mediated knockdown and 5-HT receptor insertional mutations. Moreover, synaptic transmission at 5-HT neurons was blocked or enhanced in both larvae and adult flies. The results demonstrate that disruption of components within the 5-HT system significantly impairs locomotion and feeding behaviors in larvae. Acute activation of 5-HT neurons disrupts normal locomotion activity in adult flies. To determine which 5-HT receptor subtype modulates the evoked sensory-motor activity, pharmacological agents were used. In addition, the activity of 5-HT neurons was enhanced by expressing and activating TrpA1 channels or channelrhodopsin-2 while recording the evoked excitatory postsynaptic potentials (EPSPs) in muscle fibers. 5-HT2 receptor activation mediates a modulatory role in a sensory-motor circuit, and the activation of 5-HT neurons can suppress the neural circuit activity, while fluoxetine can significantly decrease the sensory-motor activity. PMID:26989517

  1. Genetic Tools for the Analysis of Drosophila Stomatogastric Nervous System Development

    PubMed Central

    Bowser, Micah; Kidd, Thomas

    2015-01-01

    The Drosophila stomatogastric nervous system (SNS) is a compact collection of neurons that arises from the migration of neural precursors. Here we describe genetic tools allowing functional analysis of the SNS during the migratory phase of development. We constructed GAL4 lines driven by fragments of the Ret promoter, which yielded expression in a subset of migrating neural SNS precursors and also included a distinct set of midgut associated cells. Screening of additional GAL4 lines driven by fragments of the Gfrl/Munin, forkhead, twist and goosecoid (Gsc) promoters identified a Gsc fragment with expression from initial selection of SNS precursors until the end of embryogenesis. Inhibition of EGFR signaling using three identified lines disrupted the correct patterning of the frontal and recurrent nerves. To manipulate the environment traveled by SNS precursors, a FasII-GAL4 line with strong expression throughout the entire intestinal tract was identified. The transgenic lines described offer the ability to specifically manipulate the migration of SNS precursors and will allow the modeling and in-depth analysis of neuronal migration in ENS disorders such as Hirschsprung’s disease. PMID:26053861

  2. Controlled expression of Drosophila homeobox loci using the Hostile takeover system

    PubMed Central

    Javeed, Naureen; Tardi, Nicholas J.; Maher, Maggie; Singari, Swetha; Edwards, Kevin A.

    2015-01-01

    Background Hostile takeover (Hto) is a Drosophila protein trapping system that allows the investigator to both induce a gene and tag its product. The Hto transposon carries a GAL4-regulated promoter expressing an exon encoding a FLAG-mCherry tag. Upon expression, the Hto exon can splice to a downstream genomic exon, generating a fusion transcript and tagged protein. Results Using rough-eye phenotypic screens, Hto inserts were recovered at eight homeobox or Pax loci: cut, Drgx/CG34340, Pox neuro, araucan, shaven/D-Pax2, Zn finger homeodomain 2, Sex combs reduced (Scr), and the abdominal-A region. The collection yields diverse misexpression phenotypes. Ectopic Drgx was found to alter the cytoskeleton and cell adhesion in ovary follicle cells. Hto expression of cut, araucan or shaven gives phenotypes similar to those of the corresponding UAS-cDNA constructs. The cut and Pox neuro phenotypes are suppressed by the corresponding RNAi constructs. The Scr and abdominal-A inserts do not make fusion proteins, but may act by chromatin- or RNA-based mechanisms. Conclusions Hto can effectively express tagged homeodomain proteins from their endogenous loci; the Minos vector allows inserts to be obtained even in transposon cold-spots. Hto screens may recover homeobox genes at high rates because they are particularly sensitive to misexpression. PMID:25820349

  3. Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila

    PubMed Central

    Pindyurin, Alexey V.; Pagie, Ludo; Kozhevnikova, Elena N.; van Arensbergen, Joris; van Steensel, Bas

    2016-01-01

    Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages. PMID:27001518

  4. Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila.

    PubMed

    Pindyurin, Alexey V; Pagie, Ludo; Kozhevnikova, Elena N; van Arensbergen, Joris; van Steensel, Bas

    2016-07-01

    Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages. PMID:27001518

  5. Genetics on the Fly: A Primer on the Drosophila Model System.

    PubMed

    Hales, Karen G; Korey, Christopher A; Larracuente, Amanda M; Roberts, David M

    2015-11-01

    Fruit flies of the genus Drosophila have been an attractive and effective genetic model organism since Thomas Hunt Morgan and colleagues made seminal discoveries with them a century ago. Work with Drosophila has enabled dramatic advances in cell and developmental biology, neurobiology and behavior, molecular biology, evolutionary and population genetics, and other fields. With more tissue types and observable behaviors than in other short-generation model organisms, and with vast genome data available for many species within the genus, the fly's tractable complexity will continue to enable exciting opportunities to explore mechanisms of complex developmental programs, behaviors, and broader evolutionary questions. This primer describes the organism's natural history, the features of sequenced genomes within the genus, the wide range of available genetic tools and online resources, the types of biological questions Drosophila can help address, and historical milestones. PMID:26564900

  6. Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays

    NASA Astrophysics Data System (ADS)

    Hassibi, Arjang; Lee, Thomas H.; Davis, Ronald W.; Pourmand, Nader

    2003-07-01

    A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

  7. Drosophila DEG/ENaC pickpocket genes are expressed in the tracheal system, where they may be involved in liquid clearance

    PubMed Central

    Liu, Lei; Johnson, Wayne A.; Welsh, Michael J.

    2003-01-01

    The Drosophila tracheal system and mammalian airways are branching networks of tubular epithelia that deliver oxygen to the organism. In mammals, the epithelial Na+ channel (ENaC) helps clear liquid from airways at the time of birth and removes liquid from the airspaces in adults. We tested the hypothesis that related Drosophila degenerin (DEG)/ENaC family members might play a similar role in the fly. Among 16 Drosophila DEG/ENaC genes, called pickpocket (PPK) genes, we found 9 expressed in the tracheal system. By in situ hybridization, expression appeared in late-stage embryos after tracheal tube formation, with individual PPK genes showing distinct temporal and spatial expression patterns as development progressed. Promoters for several PPK genes drove reporter gene expression in the larval and adult tracheal systems. Adding the DEG/ENaC channel blocker amiloride to the medium inhibited liquid clearance from the trachea of first instar larvae. Moreover, when RNA interference was used to silence PPK4 and PPK11, larvae failed to clear tracheal liquid. These data suggest substantial molecular diversity of DEG/ENaC channel expression in the Drosophila tracheal system where the PPK proteins likely play a role in Na+ absorption. Extensive similarities between Drosophila and mammalian airways offer opportunities for genetic studies that may decipher further the structure and function of DEG/ENaC proteins and development of the airways. PMID:12571352

  8. Tetrahydropterin as a possible natural cofactor in the drosophila phenylalanine hydroxylation system

    SciTech Connect

    Bel, Y.; Jacobson, K.B.; Ferre, J. . Dept. of Genetics; Oak Ridge National Lab., TN; Valencia Univ. . Dept. of Genetics)

    1989-01-01

    The aim of the present work is the study of phenylalanine hydroxylase (PH) activity of Drosophila melanogaster wild type with different cofactors: the two natural occurring tetrahydropteridines (BH{sub 4} and PH{sub 4}) and the synthetic 6,7-dimethyltetrahydropterin (DMPH{sub 4}), as well as the determination of this activity at different developmental stages. 7 refs., 2 figs.

  9. Predetermined embryonic glial cells form the distinct glial sheaths of the Drosophila peripheral nervous system

    PubMed Central

    von Hilchen, Christian M.; Bustos, Álvaro E.; Giangrande, Angela; Technau, Gerhard M.; Altenhein, Benjamin

    2013-01-01

    One of the numerous functions of glial cells in Drosophila is the ensheathment of neurons to isolate them from the potassium-rich haemolymph, thereby establishing the blood-brain barrier. Peripheral nerves of flies are surrounded by three distinct glial cell types. Although all embryonic peripheral glia (ePG) have been identified on a single-cell level, their contribution to the three glial sheaths is not known. We used the Flybow system to label and identify each individual ePG in the living embryo and followed them into third instar larva. We demonstrate that all ePG persist until the end of larval development and some even to adulthood. We uncover the origin of all three glial sheaths and describe the larval differentiation of each peripheral glial cell in detail. Interestingly, just one ePG (ePG2) exhibits mitotic activity during larval stages, giving rise to up to 30 glial cells along a single peripheral nerve tract forming the outermost perineurial layer. The unique mitotic ability of ePG2 and the layer affiliation of additional cells were confirmed by in vivo ablation experiments and layer-specific block of cell cycle progression. The number of cells generated by this glial progenitor and hence the control of perineurial hyperplasia correlate with the length of the abdominal nerves. By contrast, the wrapping and subperineurial glia layers show enormous hypertrophy in response to larval growth. This characterisation of the embryonic origin and development of each glial sheath will facilitate functional studies, as they can now be addressed distinctively and genetically manipulated in the embryo. PMID:23903191

  10. Neuroblast lineage-specific origin of the neurons of the Drosophila larval olfactory system

    PubMed Central

    Das, Abhijit; Gupta, Tripti; Davla, Sejal; Godino, Laura Lucia Prieto; Diegelmann, Sören; Reddy, O. Venkateswara; VijayRaghavan, K.; Reichert, Heinrich; Lovick, Jennifer; Hartenstein, Volker

    2014-01-01

    The complete neuronal repertoire of the central brain of Drosophila originates from only approximately 100 pairs of neural stem cells, or neuroblasts. Each neuroblast produces a highly stereotyped lineage of neurons which innervate specific compartments of the brain. Neuroblasts undergo two rounds of mitotic activity: embryonic divisions produce lineages of primary neurons that build the larval nervous system; after a brief quiescence, the neuroblasts go through a second round of divisions in larval stage to produce secondary neurons which are integrated into the adult nervous system. Here we investigate the lineages that are associated with the larval antennal lobe, one of the most widely studied neuronal systems in fly. We find that the same five neuroblasts responsible for the adult antennal lobe also produce the antennal lobe of the larval brain. However, there are notable differences in the composition of larval (primary) lineages and their adult (secondary) counterparts. Significantly, in the adult, two lineages (lNB/BAlc and adNB/BAmv3) produce uniglomerular projection neurons connecting the antennal lobe with the mushroom body and lateral horn; another lineage, vNB/BAla1, generates multiglomerular neurons reaching the lateral horn directly. lNB/BAlc, as well as a fourth lineage, vlNB/BAla2, generate a diversity of local interneurons. We describe a fifth, previously unknown lineage, BAlp4, which connects the posterior part of the antennal lobe and the neighboring tritocerebrum (gustatory center) with a higher brain center located adjacent to the mushroom body. In the larva, only one of these lineages, adNB/BAmv3, generates all uniglomerular projection neurons. Also as in the adult, lNB/BAlc and vlNB/BAla2 produce local interneurons which, in terms of diversity in architecture and transmitter expression, resemble their adult counterparts. In addition, lineages lNB/BAlc and vNB/BAla1, as well as the newly described BAlp4, form numerous types of projection

  11. Neuroblast lineage-specific origin of the neurons of the Drosophila larval olfactory system.

    PubMed

    Das, Abhijit; Gupta, Tripti; Davla, Sejal; Prieto-Godino, Lucia L; Diegelmann, Sören; Reddy, O Venkateswara; Raghavan, K Vijay; Reichert, Heinrich; Lovick, Jennifer; Hartenstein, Volker

    2013-01-15

    The complete neuronal repertoire of the central brain of Drosophila originates from only approximately 100 pairs of neural stem cells, or neuroblasts. Each neuroblast produces a highly stereotyped lineage of neurons which innervate specific compartments of the brain. Neuroblasts undergo two rounds of mitotic activity: embryonic divisions produce lineages of primary neurons that build the larval nervous system; after a brief quiescence, the neuroblasts go through a second round of divisions in larval stage to produce secondary neurons which are integrated into the adult nervous system. Here we investigate the lineages that are associated with the larval antennal lobe, one of the most widely studied neuronal systems in fly. We find that the same five neuroblasts responsible for the adult antennal lobe also produce the antennal lobe of the larval brain. However, there are notable differences in the composition of larval (primary) lineages and their adult (secondary) counterparts. Significantly, in the adult, two lineages (lNB/BAlc and adNB/BAmv3) produce uniglomerular projection neurons connecting the antennal lobe with the mushroom body and lateral horn; another lineage, vNB/BAla1, generates multiglomerular neurons reaching the lateral horn directly. lNB/BAlc, as well as a fourth lineage, vlNB/BAla2, generate a diversity of local interneurons. We describe a fifth, previously unknown lineage, BAlp4, which connects the posterior part of the antennal lobe and the neighboring tritocerebrum (gustatory center) with a higher brain center located adjacent to the mushroom body. In the larva, only one of these lineages, adNB/BAmv3, generates all uniglomerular projection neurons. Also as in the adult, lNB/BAlc and vlNB/BAla2 produce local interneurons which, in terms of diversity in architecture and transmitter expression, resemble their adult counterparts. In addition, lineages lNB/BAlc and vNB/BAla1, as well as the newly described BAlp4, form numerous types of projection

  12. Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood Screening in India

    PubMed Central

    2016-01-01

    This review is confined to triplex nucleic acid testing (NAT) assays to be used on fully automated platform. Around the world, these assays are being used at various transfusion medicine centres or blood banks to screen blood units for HBV, HCV and HIV. These assay systems can screen up to 1000 blood units for HBV, HCV and HIV simultaneously in a day. This area has been dominated by mainly two manufacturers: M/s Gen-Probe-Novartis and M/s Roche Molecular Systems. The triplex NAT assay systems of both manufacturers are licensed by United States Food and Drug Administration. There is not much awareness about the technology and procedures used in these assays. The main objective of this review is to create awareness about the technology and procedure of these assays. PMID:27042485

  13. Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood Screening in India.

    PubMed

    Rajput, Manoj Kumar

    2016-02-01

    This review is confined to triplex nucleic acid testing (NAT) assays to be used on fully automated platform. Around the world, these assays are being used at various transfusion medicine centres or blood banks to screen blood units for HBV, HCV and HIV. These assay systems can screen up to 1000 blood units for HBV, HCV and HIV simultaneously in a day. This area has been dominated by mainly two manufacturers: M/s Gen-Probe-Novartis and M/s Roche Molecular Systems. The triplex NAT assay systems of both manufacturers are licensed by United States Food and Drug Administration. There is not much awareness about the technology and procedures used in these assays. The main objective of this review is to create awareness about the technology and procedure of these assays. PMID:27042485

  14. Why Drosophila to Study Phototransduction?

    PubMed Central

    Pak, William L.

    2010-01-01

    This review recounts the early history of Drosophila phototransduction genetics, covering the period between approximately 1966 to 1979. Early in this period, the author felt that there was an urgent need for a new approach in phototransduction research. Through inputs from a number of colleagues, he was led to consider isolating Drosophila mutants that are defective in the electroretinogram. Thanks to the efforts of dedicated associates and technical staff, by the end of this period, he was able to accumulate a large number of such mutants. Particularly important in this effort was the use of the mutant assay protocol based on the “prolonged depolarizing afterpotential.” This collection of mutants formed the basis of the subsequent intensive investigations of the Drosophila phototransduction cascade by many investigators. PMID:20536286

  15. SoxNeuro orchestrates central nervous system specification and differentiation in Drosophila and is only partially redundant with Dichaete

    PubMed Central

    2014-01-01

    Background Sox proteins encompass an evolutionarily conserved family of transcription factors with critical roles in animal development and stem cell biology. In common with vertebrates, the Drosophila group B proteins SoxNeuro and Dichaete are involved in central nervous system development, where they play both similar and unique roles in gene regulation. Sox genes show extensive functional redundancy across metazoans, but the molecular basis underpinning functional compensation mechanisms at the genomic level are currently unknown. Results Using a combination of genome-wide binding analysis and gene expression profiling, we show that SoxNeuro directs embryonic neural development from the early specification of neuroblasts through to the terminal differentiation of neurons and glia. To address the issue of functional redundancy and compensation at a genomic level, we compare SoxNeuro and Dichaete binding, identifying common and independent binding events in wild-type conditions, as well as instances of compensation and loss of binding in mutant backgrounds. Conclusions We find that early aspects of group B Sox functions in the central nervous system, such as stem cell maintenance and dorsoventral patterning, are highly conserved. However, in contrast to vertebrates, we find that Drosophila group B1 proteins also play prominent roles during later aspects of neural morphogenesis. Our analysis of the functional relationship between SoxNeuro and Dichaete uncovers evidence for redundant and independent functions for each protein, along with unexpected examples of compensation and interdependency, thus providing new insights into the general issue of transcription factor functional redundancy. PMID:24886562

  16. The gene search system. A method for efficient detection and rapid molecular identification of genes in Drosophila melanogaster.

    PubMed Central

    Toba, G; Ohsako, T; Miyata, N; Ohtsuka, T; Seong, K H; Aigaki, T

    1999-01-01

    We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of cloned genes, and 18% matched reported expressed sequence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of the protein-coding region. We discovered two new genes that showed sequence similarity to human genes, Ras-related protein 2 and microsomal glutathione S-transferase. The system can be useful as a tool for the functional mapping of the Drosophila genome. PMID:9927464

  17. The Drosophila F-box protein Archipelago controls levels of the Trachealess transcription factor in the embryonic tracheal system

    PubMed Central

    Mortimer, Nathan T.; Moberg, Kenneth H.

    2007-01-01

    The archipelago gene (ago) encodes the F-box specificity subunit of an SCF(skp-cullin-f box) ubiquitin ligase that inhibits cell proliferation in Drosophila melanogaster and suppresses tumorigenesis in mammals. ago limits mitotic activity by targeting cell cycle and cell growth proteins for ubiquitin-dependent degradation, but the diverse developmental roles of other F-box proteins suggests that it is likely to have additional protein targets. Here we show that ago is required for the post-mitotic shaping of the Drosophila embryonic tracheal system, and that it acts in this tissue by targeting the Trachealess (Trh) protein, a conserved bHLH-PAS transcription factor. ago restricts Trh levels in vivo and antagonizes transcription of the breathless FGF receptor, a known target of Trh in the tracheal system. At a molecular level, the Ago protein binds Trh and is required for proteasome-dependent elimination of Trh in response to expression of the Dysfusion protein. ago mutations that elevate Trh levels in vivo are defective in binding forms of Trh found in Dysfusion-positive cells. These data identify a novel function for the ago ubiquitin-ligase in tracheal morphogenesis via Trh and its target breathless, and suggest that ago has distinct functions in mitotic and post-mitotic cells that influence its role in development and disease. PMID:17976568

  18. Homeostatic epithelial renewal in the gut is required for dampening a fatal systemic wound response in Drosophila.

    PubMed

    Takeishi, Asuka; Kuranaga, Erina; Tonoki, Ayako; Misaki, Kazuyo; Yonemura, Shigenobu; Kanuka, Hirotaka; Miura, Masayuki

    2013-03-28

    Effective defense responses involve the entire organism. To maintain body homeostasis after tissue damage, a systemic wound response is induced in which the response of each tissue is tightly orchestrated to avoid incomplete recovery or an excessive, damaging response. Here, we provide evidence that in the systemic response to wounding, an apoptotic caspase pathway is activated downstream of reactive oxygen species in the midgut enterocytes (ECs), cells distant from the wound site, in Drosophila. We show that a caspase-pathway mutant has defects in homeostatic gut cell renewal and that inhibiting caspase activity in fly ECs results in the production of systemic lethal factors after wounding. Our results indicate that wounding remotely controls caspase activity in ECs, which activates the tissue stem cell regeneration pathway in the gut to dampen the dangerous systemic wound reaction. PMID:23523355

  19. Performance validation of commercially available mobile waste-assay systems: Preliminary report

    SciTech Connect

    Schanfein, M.; Bonner, C.; Maez, R.

    1997-11-01

    Prior to disposal, nuclear waste must be accurately characterized to identify and quantify the radioactive content to reduce the radioactive hazard to the public. Validation of the waste-assay systems` performance is critical for establishing the credibility of the assay results for storage and disposal purposes. Canberra Nuclear has evaluated regulations worldwide and identified standard, modular, neutron- and gamma-waste-assay systems that can be used to characterize a large portion of existing and newly generated transuranic (TRU) and low-level waste. Before making claims of guaranteeing any system`s performance for specific waste types, the standardized systems` performance be evaluated. 7 figs., 11 tabs.

  20. A Bioluminescence Assay System for Imaging Metal Cationic Activities in Urban Aerosols.

    PubMed

    Kim, Sung-Bae; Naganawa, Ryuichi; Murata, Shingo; Nakayama, Takayoshi; Miller, Simon; Senda, Toshiya

    2016-01-01

    A bioluminescence-based assay system was fabricated for an efficient determination of the activities of air pollutants. The following four components were integrated into this assay system: (1) an 8-channel assay platform uniquely designed for simultaneously sensing multiple optical samples, (2) single-chain probes illuminating toxic chemicals or heavy metal cations from air pollutants, (3) a microfluidic system for circulating medium mimicking the human body, and (4) the software manimulating the above system. In the protocol, we briefly introduce how to integrate the components into the system and the application to the illumination of the metal cationic activities in air pollutants. PMID:27424913

  1. The Physcomitrella patens System for Transient Gene Expression Assays.

    PubMed

    Thévenin, Johanne; Xu, Wenjia; Vaisman, Louise; Lepiniec, Loïc; Dubreucq, Bertrand; Dubos, Christian

    2016-01-01

    Transient expression assays are valuable techniques to study in vivo the transcriptional regulation of gene expression. These methods allow to assess the transcriptional properties of a given transcription factor (TF) or a complex of regulatory proteins against specific DNA motifs, called cis-regulatory elements. Here, we describe a fast, efficient, and reliable method based on the use of Physcomitrella patens protoplasts that allows the study of gene expression in a qualitative and quantitative manner by combining the advantage of GFP (green fluorescent protein) as a marker of promoter activity with flow cytometry for accurate measurement of fluorescence in individual cells. PMID:27557766

  2. Drosophila, a genetic model system to study cocaine-related behaviors: A review with focus on LIM-only proteins

    PubMed Central

    Heberlein, Ulrike; Tsai, Linus T.–Y.; Kapfhamer, David; Lasek, Amy W.

    2010-01-01

    Summary In the last decade, the fruit fly Drosophila melanogaster, highly accessible to genetic, behavioral and molecular analyses, has been introduced as a novel model organism to help decipher the complex genetic, neurochemical, and neuroanatomical underpinnings of behaviors induced by drugs of abuse. Here we review these data, focusing specifically on cocaine-related behaviors. Several of cocaine's most characteristic properties have been recapitulated in Drosophila. First, cocaine induces motor behaviors in flies that are remarkably similar to those observed in mammals. Second, repeated cocaine administration induces behavioral sensitization a form of behavioral plasticity believed to underlie certain aspects of addiction. Third, a key role for dopaminergic systems in mediating cocaine's effects has been demonstrated through both pharmacological and genetic methods. Finally, and most importantly, unbiased genetic screens, feasible because of the simplicity and scale with which flies can be manipulated in the laboratory, have identified several novel genes and pathways whose role in cocaine behaviors had not been anticipated. Many of these genes and pathways have been validated in mammalian models of drug addiction. We focus in this review on the role of LIM-only proteins in cocaine-induced behaviors. PMID:18694769

  3. Gustatory processing and taste memory in Drosophila.

    PubMed

    Masek, Pavel; Keene, Alex C

    2016-06-01

    Taste allows animals to discriminate the value and potential toxicity of food prior to ingestion. Many tastants elicit an innate attractive or avoidance response that is modifiable with nutritional state and prior experience. A powerful genetic tool kit, well-characterized gustatory system, and standardized behavioral assays make the fruit fly, Drosophila melanogaster, an excellent system for investigating taste processing and memory. Recent studies have used this system to identify the neural basis for acquired taste preference. These studies have revealed a role for dopamine-mediated plasticity of the mushroom bodies that modulate the threshold of response to appetitive tastants. The identification of neural circuitry regulating taste memory provides a system to study the genetic and physiological processes that govern plasticity within a defined memory circuit. PMID:27328844

  4. An Entry/Gateway® cloning system for general expression of genes with molecular tags in Drosophila melanogaster

    PubMed Central

    Akbari, Omar S; Oliver, Daniel; Eyer, Katie; Pai, Chi-Yun

    2009-01-01

    Background Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway® vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells. Results The Entry/Gateway® system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway® based system, which includes six P-element destination vectors (P-DEST) for expressing tagged proteins (eGFP, mRFP, or myc) in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei. Conclusion We have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster. This system

  5. Turbidimetric assay for tetracyclines in feeds using a microtiter plate system.

    PubMed

    Brady, M S; Katz, S E

    1991-01-01

    The microtiter plate system for turbidimetric assay of chlortetracycline (CTC) and oxytetracycline (OTC) levels in feeds uses a 96 well microtiter plate, a multichannel pipette, and an ELISA reader to measure turbidity. Feeds are extracted for both tetracyclines using AOAC extraction systems. For CTC, the range of the standard curve is 0.001-0.005 microgram CTC/mL; for OTC, the range is 0.004-0.016 microgram OTC/mL. Repeatability of CTC assays, as shown by the coefficient of variation (CV), ranged from 0.54 to 5.65% for same-day assays and from 2.01 to 9.39% for assays on different days. For OTC, CVs ranged from 2.69 to 10.01% for same-day assays and 3.24 to 9.08% for different-day assays. Average recoveries for CTC were 108.7% for same-day assays and 106.8% for different-day assays; for OTC, average recoveries were 112.4% and 106.5% for same-day and different-day assays, respectively. PMID:1874689

  6. Fruit flies on the front line: the translational impact of Drosophila

    PubMed Central

    Perrimon, Norbert; Bonini, Nancy M.; Dhillon, Paraminder

    2016-01-01

    ABSTRACT Drosophila melanogaster has been adopted as one of the most-used model systems since it was first introduced by Thomas Morgan for the study of heredity in the early 20th century. Its experimental tractability and similarity of its biological pathways to those of humans have placed the model at the forefront of research into human development and disease. With the ongoing accumulation of genetic tools and assays, the fly community has at its fingertips the resources to generate diverse Drosophila disease models for the study of genes and pathways involved in a wide range of disorders. In recent years, the fly has also been used successfully for drug screening. In this Editorial, we introduce a Special Collection of reviews, interviews and original research articles that highlight some of the many ways that Drosophila has made, and continues to make, an impact on basic biological insights and translational science. PMID:26935101

  7. Drosophila spermiogenesis

    PubMed Central

    Fabian, Lacramioara; Brill, Julie A.

    2012-01-01

    Drosophila melanogaster spermatids undergo dramatic morphological changes as they differentiate from small round cells approximately 12 μm in diameter into highly polarized, 1.8 mm long, motile sperm capable of participating in fertilization. During spermiogenesis, syncytial cysts of 64 haploid spermatids undergo synchronous differentiation. Numerous changes occur at a subcellular level, including remodeling of existing organelles (mitochondria, nuclei), formation of new organelles (flagellar axonemes, acrosomes), polarization of elongating cysts and plasma membrane addition. At the end of spermatid morphogenesis, organelles, mitochondrial DNA and cytoplasmic components not needed in mature sperm are stripped away in a caspase-dependent process called individualization that results in formation of individual sperm. Here, we review the stages of Drosophila spermiogenesis and examine our current understanding of the cellular and molecular mechanisms involved in shaping male germ cell-specific organelles and forming mature, fertile sperm. PMID:23087837

  8. Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila

    PubMed Central

    ROIGNANT, JEAN-YVES; CARRÉ, CLÉMENT; MUGAT, BRUNO; SZYMCZAK, DIMITRI; LEPESANT, JEAN-ANTOINE; ANTONIEWSKI, CHRISTOPHE

    2003-01-01

    RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21–23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in Drosophila. DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of one gene without transitive effect directed to sequences downstream from or upstream of the initial trigger region. Moreover, specific expression of a dsRNA, using either cell-specific GAL4 drivers or random clonal activation of a GAL4 driver, mediated a cell-autonomous RNAi. Together, our results provide evidence that transitive and systemic aspects of RNAi are not conserved in Drosophila and demonstrate that dsRNA-producing transgenes allow powerful reverse genetic approaches to be conducted in this model organism, by knocking down gene functions at the resolution of a single-cell type and of a single isoform. PMID:12592004

  9. Microfluidic System for Automated Cell-based Assays.

    PubMed

    Lee, Philip J; Ghorashian, Navid; Gaige, Terry A; Hung, Paul J

    2007-12-01

    Microfluidic cell culture is a promising technology for applications in the drug screening industry. Key benefits include improved biological function, higher quality cell-based data, reduced reagent consumption, and lower cost. In this work, we demonstrate how a microfluidic cell culture design was adapted to be compatible with the standard 96-well plate format. Key design features include the elimination of tubing and connectors, the ability to maintain long term continuous perfusion cell culture using a passive gravity driven pump, and direct analysis on the outlet wells of the microfluidic plate. A single microfluidic culture plate contained 8 independent flow units, each with 10(4) cells at a flow rate of 50 μl/day (6 minute residence time). The cytotoxicity of the anti-cancer drug etoposide was measured on HeLa cells cultured in this format, using a commercial lactate dehydrogenase (LDH) plate reader assay. The integration of microfluidic cell culture methods with commercial automation capabilities offers an exciting opportunity for improved cell-based screening. PMID:18172509

  10. Advanced Assay Systems for Radionuclide Contamination in Soils

    SciTech Connect

    J. R. Giles; L. G. Roybal; M. V. Carpenter; C. P. Oertel; J. A. Roach

    2008-02-01

    Through the support of the Department of Energy (DOE) Office of Environmental Management (EM) Technical Assistance Program, the Idaho National Laboratory (INL) has developed and deployed a suite of systems that rapidly scan, characterize, and analyze surface soil contamination. The INL systems integrate detector systems with data acquisition and synthesis software and with global positioning technology to provide a real-time, user-friendly field deployable turn-key system. INL real-time systems are designed to characterize surface soil contamination using methodologies set forth in the Multi-Agency Radiation Surveys and Site Investigation Manual (MARSSIM). MARSSIM provides guidance for planning, implementing, and evaluating environmental and facility radiological surveys conducted to demonstrate compliance with a dose or risk-based regulation and provides real-time information that is immediately available to field technicians and project management personnel. This paper discusses the history of the development of these systems and describes some of the more recent examples and their applications.

  11. Isolation and characterization of an insulin-degrading enzyme from Drosophila melanogaster

    SciTech Connect

    Garcia, J.V.; Fenton, B.W.; Rosner, M.R.

    1988-06-14

    An insulin-degrading enzyme (IDE) from the cytoplasm of Drosophila Kc cells has been purified and characterized. The purified enzyme is a monomer with an s value of 7.2 S, an apparent K/sub m/ for porcine insulin of 3 ..mu..M, and a specific activity of 3.3 nmol of porcine insulin degraded/(min x mg). N-Terminal sequence analysis of the gel-purified enzyme gave a single, serine-rich sequence. The Drosophila IDE shares a number of properties in common with its mammalian counterpart. The enzyme could be specifically affinity-labeled with (/sup 125/I)insulin, has a molecular weight of 110K, and has a pI of 5.3. Although Drosophila Kc cells grow at room temperature, the optimal enzyme activity assay conditions parallel those of the mammalian IDE: 37/sup 0/C and a pH range of 7-8. The Drosophila IDE activity, like the mammalian enzymes, is inhibited by bacitracin and sulfhydryl-specific reagents. Similarly, the Drosophila IDE activity is insensitive to glutathione as well as protease inhibitors such as aprotinin and leupeptin. Insulin-like growth factor II, equine insulin, and porcine insulin compete for degradation of (/sup 125/I)insulin at comparable concentrations (approximately 10/sup -6/ M), whereas insulin-like growth factor I and the individual A and B chains of insulin are less effective. The high degree of evolutionary conservation between the Drosophila and mammalian IDE suggest an important role for this enzyme in the metabolism of insulin and also provides further evidence for the existence of a complete insulin-like system in invertebrate organisms such as Drosophila.

  12. Medically relevant assays with a simple smartphone and tablet based fluorescence detection system.

    PubMed

    Wargocki, Piotr; Deng, Wei; Anwer, Ayad G; Goldys, Ewa M

    2015-01-01

    Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera) and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis. PMID:26007723

  13. Medically Relevant Assays with a Simple Smartphone and Tablet Based Fluorescence Detection System

    PubMed Central

    Wargocki, Piotr; Deng, Wei; Anwer, Ayad G.; Goldys, Ewa M.

    2015-01-01

    Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera) and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis. PMID:26007723

  14. Sexual conflict is not counterbalanced by good genes in the laboratory Drosophila melanogaster model system.

    PubMed

    Stewart, A D; Hannes, A M; Mirzatuny, A; Rice, W R

    2008-11-01

    Sexual conflict theory is based on the observation that females of many species are harmed through their interactions with males. Direct harm to females, however, can potentially be counterbalanced by indirect genetic benefits, where females make up for a reduction in offspring quantity by an increase in offspring quality through a generic increase in offspring fitness (good genes) and/or one restricted to the context of sexual selection (sexy sons). Here, we quantify the magnitude of the good genes mechanism of indirect benefits in a laboratory-adapted population of Drosophila melanogaster. We find that despite high-standing genetic variance for fitness, females gain at most only a modest benefit through the good genes form of indirect benefits--far too little to counterbalance the direct cost of male-induced harm. PMID:18681915

  15. Genetic-molecular basis for a simple Drosophila melanogaster somatic system that detects environmental mutagens

    SciTech Connect

    Green, M.M.; Todo, T.; Ryo, H.; Fujikawa, K.

    1986-09-01

    We have developed a simple, objectively scorable test for the mutagenicity of chemical compounds which can be fed Drosophila melanogaster. The test depends upon the somatic reversion of the X chromosome, recessive eye color mutation, white-ivory (wi) to wild type (w+). Reversions are scored as clones of w+ facets in the wi eyes of eclosing adults. To increase the sensitivity, a tandem quadruplication containing four wi mutations was synthesized. Thus, in homozygous females eight wi mutations are potentially revertible. Six mutagenic compounds, all alkylating agents, all gave positive results at several concentrations tested. Molecular analysis demonstrates that the induced reversions, germinal and somatic, are associated with the loss of 2.9-kilobase DNA duplicated in the wi mutation.

  16. Compact upconverting phosphor detection system for wick assays

    NASA Astrophysics Data System (ADS)

    Riris, Haris; Van der Laan, Jan E.; Cooper, David E.; Nashold, Karen M.; Carlisle, Clinton B.; Schneider, Luke V.

    1997-05-01

    We describe an integrated detection system based on upconverting phosphor particles bound to capture sites on the inside surfaces of rectangular wick capillaries. This deice can be used with either antibody or nucleic acid to detect specific micro-organisms. The system uses a high- power, 980 nm, semiconductor diode laser to illuminate 200 X 300 X 20 micrometers capture surfaces. The rectangular capillary wicks are held in a tray that is inserted into the detection system, positioning the capture surface at the object plane of the optical system. Phosphorescent light emitted from the capture surface is collected by a high numerical aperture microscope objective and directed through a series of filters onto either a CCD camera or a photomultiplier. A combination of band-reject filters attenuates the 980 nm laser excitation light and its harmonic at 490 nm, and a tunable liquid crystal filter provides for rapid scanning from 400 to 750 nm. The data acquisition and control is controlled by a laptop PC with a custom GUI interface developed using LabWindows/CVI. The system can detect a single phosphor particle bound to a capture surface.

  17. An expert system framework for nondestructive waste assay

    SciTech Connect

    Becker, G.K.

    1996-10-01

    Management and disposition of transuranic (RU) waste forms necessitates determining entrained RU and associated radioactive material quantities as per National RU Waste Characterization Program requirements. Technical justification and demonstration of a given NDA method used to determine RU mass and uncertainty in accordance with program quality assurance is difficult for many waste forms. Difficulties are typically founded in waste NDA methods that employ standards compensation and/or employment of simplifying assumptions on waste form configurations. Capability to determine and justify RU mass and mass uncertainty can be enhanced through integration of waste container data/information using expert system and empirical data-driven techniques with conventional data acquisition and analysis. Presented is a preliminary expert system framework that integrates the waste form data base, alogrithmic techniques, statistical analyses, expert domain knowledge bases, and empirical artificial intelligence modules into a cohesive system. The framework design and bases in addition to module development activities are discussed.

  18. The expression of CG9940 affects the adaptation of cardiac function, mobility, and lifespan to exercise in aging Drosophila.

    PubMed

    Wen, Deng-Tai; Zheng, Lan; Ni, Liu; Wang, Hui; Feng, Yue; Zhang, Min

    2016-10-01

    The CG9940 gene, which encodes the NAD(+) synthase protein in Drosophila, is conserved in human, zebra fish, and mosquito. NAD(+) synthase is a homodimer, which catalyzes the final step in de novo nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, an amide transfer from either ammonia or glutamine to nicotinic acid adenine dinucleotide (NaAD). Both the CG9940 and exercise are closely relative to NAD(+) level, and NAD(+) plays important roles not only in energy metabolism and mitochondrial functions but also in aging. In our study, the expression of CG9940 was changed by UAS/GAL4 system in Drosophila. Flies were trained by a training device. Cardiac function was analyzed by M-mode traces, climbing index was measured through negative geotaxis assay, and lifespan was measured via lifespan assays. The important new findings from our present study included the following: (1) the expression of the CG9940 could affect cardiac function, mobility, and lifespan in Drosophila. Over-expression of the CG9940 gene had positive effects on Drosophila, such as enhanced aging cardiac output, reduced heart failure, delayed age-related mobility decline, and prolonged lifespan, but lower-expression of the CG9940 had negative effects on them. (2) Different expressions of the CG9940 resulted in different influences on the adaptation of cardiac function, mobility, and lifespan to exercise in aging Drosophila. Both normal-expression and over-expression of the CG9940 resulted in positive influences on the adaptation of cardiac functions, mobility, and lifespan to exercise in aging Drosophila such as exercise slowed age-related decline of cardiac function, mobility and extent of lifespan in these flies, while lower-expression of the CG9940 led to negative impacts on the adaptation of mobility and lifespan to exercise in Drosophila. PMID:27448710

  19. GENETOX MANAGER SYSTEM - BACTERIAL MUTAGENESIS ASSAYS: USER'S GUIDE

    EPA Science Inventory

    This user's guide is the reference manual describing use of the EPA Genetox Manager System. eneTox Manager is a data capture, data management, and statistical analysis program used for microbial mutagenicity data. ts main purpose is to provide a homogeneous environment for the co...

  20. INEL test plan for evaluating waste assay systems

    SciTech Connect

    Mandler, J.W.; Becker, G.K.; Harker, Y.D.; Menkhaus, D.E.; Clements, T.L. Jr.

    1996-09-01

    A test bed is being established at the Idaho National Engineering Laboratory (INEL) Radioactive Waste Management Complex (RWMC). These tests are currently focused on mobile or portable radioassay systems. Prior to disposal of TRU waste at the Waste Isolation Pilot Plant (WIPP), radioassay measurements must meet the quality assurance objectives of the TRU Waste Characterization Quality Assurance Program Plan. This test plan provides technology holders with the opportunity to assess radioassay system performance through a three-tiered test program that consists of: (a) evaluations using non-interfering matrices, (b) surrogate drums with contents that resemble the attributes of INEL-specific waste forms, and (c) real waste tests. Qualified sources containing a known mixture and range of radionuclides will be used for the non-interfering and surrogate waste tests. The results of these tests will provide technology holders with information concerning radioassay system performance and provide the INEL with data useful for making decisions concerning alternative or improved radioassay systems that could support disposal of waste at WIPP.

  1. Performance evaluation of the PROCLEIX West Nile virus assay on semi-automated and automated systems.

    PubMed

    Linnen, Jeffrey M; Deras, Michael L; Cline, Janice; Wu, Wen; Broulik, Amy S; Cory, Robin E; Knight, James L; Cass, Michelle M J; Collins, Cynthia S; Giachetti, Cristina

    2007-09-01

    The PROCLEIX West Nile virus assay (WNV assay) is a qualitative nucleic acid test based on transcription-mediated amplification (TMA). The assay was used under an investigational protocol in the United States to screen blood donations for West Nile virus (WNV) RNA starting in the summer of 2003, and was licensed by the FDA in December 2005 for use on the PROCLEIX System, also known as the enhanced semi-automated system (eSAS). Performance characteristics for the assay were determined on both eSAS and the fully automated PROCLEIX TIGRIS (TIGRIS) System. Detection of both lineage 1 and lineage 2 WNV was demonstrated on both systems. For lineage 1, the 95% detection limit was 8.2 copies/ml for eSAS and 9.8 copies/ml for the TIGRIS system. For lineage 2, > or =95% detection was seen at > or =30 copies/ml on both systems. The overall specificity of the assay was >99.9% in fresh and frozen plasma specimens. Reproducibility studies on the TIGRIS system yielded > or =99.1% agreement with expected results for the 3-member panel tested (0, 30, and 100 copies/ml). The WNV assay exhibited robust performance in cadaveric specimens and specimens representing various donor and donation conditions, including specimens from different plasma collection tubes that were subjected to multiple freeze/thaw cycles; specimens with elevated levels of endogenous substances; specimens containing other viruses and microorganisms; and specimens from patients with autoimmune and other diseases. Overall, these studies demonstrate high sensitivity, specificity, and reproducibility of the WNV assay on both the semi-automated and automated systems. PMID:17607786

  2. A baculoviral display system to assay viral entry.

    PubMed

    Iida, Manami; Yoshida, Takeshi; Watari, Akihiro; Yagi, Kiyohito; Hamakubo, Takao; Kondoh, Masuo

    2013-01-01

    In this study, we evaluated a baculoviral display system for analysis of viral entry by using a recombinant adenovirus (Ad) carrying a luciferase gene and budded baculovirus (BV) that displays the adenoviral receptor, coxsackievirus and adenovirus receptor (CAR). CAR-expressing B16 cells (B16-CAR cells) were infected with luciferase-expressing Ad vector in the presence of BV that expressed or lacked CAR (CAR-BV and mock-BV, respectively). Treatment with mock-BV even at doses as high as 5 µg/mL failed to attenuate the luciferase activity of B16-CAR cells. In contrast, treatment with CAR-BV with doses as low as 0.5 µg/mL significantly decreased the luciferase activity of infected cells, which reached 65% reduction at 5 µg/mL. These findings suggest that a receptor-displaying BV system could be used to evaluate viral infection. PMID:24189431

  3. Enrichment Assay Methods Development for the Integrated Cylinder Verification System

    SciTech Connect

    Smith, Leon E.; Misner, Alex C.; Hatchell, Brian K.; Curtis, Michael M.

    2009-10-22

    International Atomic Energy Agency (IAEA) inspectors currently perform periodic inspections at uranium enrichment plants to verify UF6 cylinder enrichment declarations. Measurements are typically performed with handheld high-resolution sensors on a sampling of cylinders taken to be representative of the facility's entire product-cylinder inventory. Pacific Northwest National Laboratory (PNNL) is developing a concept to automate the verification of enrichment plant cylinders to enable 100 percent product-cylinder verification and potentially, mass-balance calculations on the facility as a whole (by also measuring feed and tails cylinders). The Integrated Cylinder Verification System (ICVS) could be located at key measurement points to positively identify each cylinder, measure its mass and enrichment, store the collected data in a secure database, and maintain continuity of knowledge on measured cylinders until IAEA inspector arrival. The three main objectives of this FY09 project are summarized here and described in more detail in the report: (1) Develop a preliminary design for a prototype NDA system, (2) Refine PNNL's MCNP models of the NDA system, and (3) Procure and test key pulse-processing components. Progress against these tasks to date, and next steps, are discussed.

  4. Cadmium zinc telluride detector system for nuclear material assay

    SciTech Connect

    Lavietes, A.D.; McQuaid, J.H.; Paulus, T.J.

    1997-07-15

    Three tools were developed towards design of an ambient temperature radiometric instrument, namely the CZT Probe--a cadmium zinc telluride based gamma and x ray detector probe, the MicroNOMAD--a low power, portable multichannel analyzed, and CZTU--spectral analysis software that provides uranium enrichment analysis. The combination of these three tools with an optimal sodium iodide (NaI) detector provides the ability to search for and then analyze uranium as well as other radionuclides in the field. Several national and international organizations including the International Atomic Energy Agency, the European Communities Safeguards Directorate, US Customs, and US DOE have expressed interest and are currently evaluating these systems.

  5. Development of a RapidFire mass spectrometry assay and a fluorescence assay for the discovery of kynurenine aminotransferase II inhibitors to treat central nervous system disorders.

    PubMed

    Lu, Hao; Kopcho, Lisa; Ghosh, Kaushik; Witmer, Mark; Parker, Michael; Gupta, Sumit; Paul, Marilyn; Krishnamurthy, Prasad; Laksmaiah, Basanth; Xie, Dianlin; Tredup, Jeffrey; Zhang, Litao; Abell, Lynn M

    2016-05-15

    Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free RapidFire mass spectrometry assay has been developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies. PMID:26874021

  6. Clenbuterol Assay by Spectral Imaging Surface Plasmon Resonance Biosensor System.

    PubMed

    Wu, Yichuan; Yao, Manwen; Fang, Xiangyi; Yang, Yucong; Cheng, Xiaoli

    2015-11-01

    To prevent illegal use of clenbuterol and for quality control in the food industry, more efficient and reliable methods for clenbuterol detection are needed. In this study, clenbuterol was detected using a spectral imaging surface plasmon resonance sensor system via two inhibition methods: (1) the target site compensation method, in which anti-clenbuterol antibody was immobilized on the sensor chip as a bioprobe and (2) the solution competition method in which a clenbuterol-BSA conjugate was immobilized on the sensor chip as the bioprobe. The detectable clenbuterol concentration ranged between 6.25 and 100 μg/mL for both methods. The clenbuterol limit of detection for the target site compensation method and solution competition method are estimated to be 6.7 and 4.5 μg/mL, respectively. The proposed methods were successfully applied to the detection of clenbuterol molecules and were found to have high specificity and high-throughput and were label free and operationally convenient. PMID:26319570

  7. First-year evaluation of a nondestructive assay system for the examination of ORNL TRU waste

    SciTech Connect

    Schultz, F.J.; Haff, K.W.; Coffey, D.E.; Norris, L.B.; Caldwell, J.T.; Close, D.A.; Kuckertz, T.H.; Kunz, W.E.; Pratt, J.C.

    1984-04-01

    The Oak Ridge National Laboratory has been selected as the demonstration site for a new transuranic neutron assay system (NAS) developed at the Los Alamos National Laboratory. In addition, in order to meet specific ORNL program objectives, an upgraded segmented gamma-ray drum scanner has been integrated into the nondestructive assay (NDA) system to serve as a radioisotope identifier and as a quantitative assay backup to the NAS. A verification study, wherein selected waste drums will be emptied into glove boxes and their contents sampled and subsequently gamma-ray assayed, will take place in FY 1984. Results will be compared to those obtained from the NDA techniques. The NAS uses pulsed-neutron interrogation (differential- dieaway technique) and passive neutron measurements to determine fissile component and an upper-limit estimate of the total TRU activity contained in each waste drum. Of the 171 waste drums assayed to date, nine drums were determined to contain less than 10 nCi/g TRU isotopes. An additional number of drums (approximately 20%) are expected to be categorized as non-TRU, which is presently defined as less than 100 nCi/g TRU concentration. This requires a detailed analysis of the data which includes waste matrix compensation, systematic qualitative and quantitative gamma-ray analyses, and interpretation of neutron multiplicity data. Reproducibility of the active assay measurements on a single waste drum indicate agreement to +-3% relative error. 14 references, 24 figures, 8 tables.

  8. Genotoxicity testing with the somatic white-ivory system in the eye of Drosophila melanogaster.

    PubMed

    Würgler, F E; Kägi, A

    1991-05-01

    The white-ivory test in Drosophila melanogaster is designed to detect chemically induced reversions of the sex-linked, recessive unstable eye-color mutation white-ivory to the wild-type form. After exposure of larvae reversions are detectable as clones of red facets in the eye of newly enclosed adult flies. Tester strains containing a quadruplication of the white-ivory gene on the X-chromosome(s) were used. In a strain with males carrying 4 copies of the gene and females carrying 8 copies of the gene, spontaneous reversions occurred proportional to the gene copy number. In contrast to this, chemically induced reversions occurred only 1.36 times more frequently in females (carrying 8 copies of the gene) than in males (carrying 4 copies). Since chemicals inducing different lesions in DNA (bleomycin, cyclophosphamide, daunomycin, diethyl sulfate and 7,12-dimethylbenz[a]anthracene) did induce statistically significant frequencies of reversions the test appears to be capable of detecting a wide variety of genotoxic chemicals with different modes of action. The recombinogen strychnine did not induce reversions. PMID:1903508

  9. A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system

    PubMed Central

    Apitz, Holger; Salecker, Iris

    2014-01-01

    Brain areas each generate specific neuron subtypes during development. However, underlying regional variations in neurogenesis strategies and regulatory mechanisms remain poorly understood. In Drosophila, neurons in four optic lobe ganglia originate from two neuroepithelia, the outer (Opc) and inner (Ipc) proliferation centers. Using genetic manipulations, we demonstrate that one Ipc neuroepithelial domain progressively transforms into migratory progenitors that mature into neural stem cells/neuroblasts within a second domain. Progenitors emerge by an epithelial-mesenchymal transition-like mechanism, requiring the Snail-family member Escargot and, in subdomains, Decapentaplegic signaling. The proneural factors Lethal of scute and Asense differentially control progenitor supply and maturation into neuroblasts. These switch expression from Asense to a third proneural protein, Atonal. Dichaete and Tailless mediate this transition essential for generating two neuron populations at defined positions. We propose that this neurogenesis mode is central for setting-up a new proliferative zone to facilitate spatio-temporal matching of neurogenesis and connectivity across ganglia. PMID:25501037

  10. Neuron hemilineages provide the functional ground plan for the Drosophila ventral nervous system

    PubMed Central

    Harris, Robin M; Pfeiffer, Barret D; Rubin, Gerald M; Truman, James W

    2015-01-01

    Drosophila central neurons arise from neuroblasts that generate neurons in a pair-wise fashion, with the two daughters providing the basis for distinct A and B hemilineage groups. 33 postembryonically-born hemilineages contribute over 90% of the neurons in each thoracic hemisegment. We devised genetic approaches to define the anatomy of most of these hemilineages and to assessed their functional roles using the heat-sensitive channel dTRPA1. The simplest hemilineages contained local interneurons and their activation caused tonic or phasic leg movements lacking interlimb coordination. The next level was hemilineages of similar projection cells that drove intersegmentally coordinated behaviors such as walking. The highest level involved hemilineages whose activation elicited complex behaviors such as takeoff. These activation phenotypes indicate that the hemilineages vary in their behavioral roles with some contributing to local networks for sensorimotor processing and others having higher order functions of coordinating these local networks into complex behavior. DOI: http://dx.doi.org/10.7554/eLife.04493.001 PMID:26193122

  11. Drosophila melanogaster brain invasion: pathogenic Wolbachia in central nervous system of the fly.

    PubMed

    Strunov, Anton; Kiseleva, Elena

    2016-04-01

    The pathogenic Wolbachia strain wMelPop rapidly over-replicates in the brain, muscles, and retina of Drosophila melanogaster, causing severe tissue degeneration and premature death of the host. The unique features of this endosymbiont make it an excellent tool to be used for biological control of insects, pests, and vectors of human diseases. To follow the dynamics of bacterial morphology and titer in the nerve cells we used transmission electron microscopy of 3-d-old female brains. The neurons and glial cells from central brain of the fly had different Wolbachia titers ranging from single bacteria to large accumulations, tearing cell apart and invading extracellular space. The neuropile regions of the brain were free of wMelPop. Wolbachia tightly interacted with host cell organelles and underwent several morphological changes in nerve cells. Based on different morphological types of bacteria described we propose for the first time a scheme of wMelPop dynamics within the somatic tissue of the host. PMID:25394184

  12. Genetic dissection of the photoreceptor system in the compound eye of Drosophila melanogaster

    PubMed Central

    Stark, William S.; Walker, John A.; Harris, William A.

    1976-01-01

    1. Three mutations which eliminate specific types of photoreceptors in Drosophila were characterized. 2. Of the eight photoreceptors in each facet, two mutations delete the outer six (R 1-6). The third eliminates R 7, one of the two central photoreceptors. Double mutants can be constructed in which only photoreceptor R 8 is present. 3. The spectral sensitivities, photopigments, and behavioural properties of these mutants were investigated. 4. R 1-6 have two sensitivity peaks, near 350 and 470 nm. These receptors contain a rhodopsin with these absorption peaks. It interconverts with a metarhodopsin that absorbs around 570 nm. 5. R 7 is a U.V.-receptor, containing rhodopsin that absorbs around 370 nm and interconverts with a metarhodopsin which absorbs around 470 nm. 6. R 8 is a non-adapting blue-receptor with a third type of rhodopsin. 7. The properties of these photopigments explain the different sensitivities and spectral adaptation phenomena of the various photoreceptors. 8. All the photoreceptors have input into phototaxis. Spectral analysis of this behaviour provides evidence for integration of the input from the different photoreceptors. ImagesPlate 6Plate 1Plate 2Plate 3Plate 4Plate 5 PMID:16992509

  13. A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system.

    PubMed

    Apitz, Holger; Salecker, Iris

    2015-01-01

    Brain areas each generate specific neuron subtypes during development. However, underlying regional variations in neurogenesis strategies and regulatory mechanisms remain poorly understood. In Drosophila, neurons in four optic lobe ganglia originate from two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers. Using genetic manipulations, we found that one IPC neuroepithelial domain progressively transformed into migratory progenitors that matured into neural stem cells (neuroblasts) in a second domain. Progenitors emerged by an epithelial-mesenchymal transition-like mechanism that required the Snail-family member Escargot and, in subdomains, Decapentaplegic signaling. The proneural factors Lethal of scute and Asense differentially controlled progenitor supply and maturation into neuroblasts. These switched expression from Asense to a third proneural protein, Atonal. Dichaete and Tailless mediated this transition, which was essential for generating two neuron populations at defined positions. We propose that this neurogenesis mode is central for setting up a new proliferative zone to facilitate spatio-temporal matching of neurogenesis and connectivity across ganglia. PMID:25501037

  14. Wnt-mediated repression via bipartite DNA recognition by TCF in the Drosophila hematopoietic system.

    PubMed

    Zhang, Chen U; Blauwkamp, Timothy A; Burby, Peter E; Cadigan, Ken M

    2014-08-01

    The Wnt/β-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/β-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/β-catenin signaling and allosteric regulation of a transcription factor by DNA. PMID:25144371

  15. Wnt-Mediated Repression via Bipartite DNA Recognition by TCF in the Drosophila Hematopoietic System

    PubMed Central

    Zhang, Chen U.; Blauwkamp, Timothy A.; Burby, Peter E.; Cadigan, Ken M.

    2014-01-01

    The Wnt/β-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/β-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/β-catenin signaling and allosteric regulation of a transcription factor by DNA. PMID:25144371

  16. Conserved genetic pathways controlling the development of the diffuse endocrine system in vertebrates and Drosophila.

    PubMed

    Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina L

    2010-05-01

    The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type. PMID:20005229

  17. A novel tissue in an established model system: the Drosophila pupal midgut.

    PubMed

    Takashima, Shigeo; Younossi-Hartenstein, Amelia; Ortiz, Paola A; Hartenstein, Volker

    2011-06-01

    The Drosophila larval and adult midguts are derived from two populations of endodermal progenitors that separate from each other in the early embryo. As larval midgut cells differentiate into an epithelial layer, adult midgut progenitors (AMPs) remain as small clusters of proliferating, undifferentiated cells attached to the basal surface of the larval gut epithelium. During the first few hours of metamorphosis, AMPs merge into a continuous epithelial tube that overgrows the larval layer and differentiates into the adult midgut; at the same time, the larval midgut degenerates. As shown in this paper, there is a second, transient pupal midgut that develops from the AMPs at the beginning of metamorphosis and that intercalates between the adult and larval midgut epithelia. Cells of the transient pupal midgut form a multilayered tube that exhibits signs of differentiation, in the form of septate junctions and rudimentary apical microvilli. Some cells of the pupal midgut develop as endocrine cells. The pupal midgut remains closely attached to the degenerating larval midgut cells. Along with these cells, pupal midgut cells are sequestered into the lumen where they form the compact "yellow body." The formation of a pupal midgut has been reported from several other species and may represent a general feature of intestinal metamorphosis in insects. PMID:21556856

  18. A novel tissue in an established model system: the Drosophila pupal midgut

    PubMed Central

    Takashima, Shigeo; Younossi-Hartenstein, Amelia; Ortiz, Paola A.; Hartenstein, Volker

    2014-01-01

    The Drosophila larval and adult midgut are derived from two populations of endodermal progenitors that separate from each other in the early embryo. As larval midgut cells differentiate into an epithelial layer, adult midgut progenitors (AMPs) remain as small clusters of proliferating, undifferentiated cells attached to the basal surface of the larval gut epithelium. During the first few hours of metamorphosis, AMPs merge into a continuous epithelial tube that overgrows the larval layer and differentiates into the adult midgut; at the same time, the larval midgut degenerates. As shown in this paper, there is a second, transient pupal midgut that develops from the AMPs at the beginning of metamorphosis, and that intercalates between the adult and larval midgut epithelia. Cells of the transient pupal midgut form a multilayered tube that exhibits signs of differentiation, in the form of septate junctions and rudimentary apical microvilli. Some cells of the pupal midgut develop as endocrine cells. The pupal midgut remains closely attached to the degenerating larval midgut cells. Along with these cells, pupal midgut cells are sequestered into the lumen where they form the compact “yellow body”. The formation of a pupal midgut has been reported from several other species, and may represent a general feature of intestinal metamorphosis in insects. PMID:21556856

  19. Conserved Genetic Pathways Controlling the Development of the Diffuse Endocrine System in Vertebrates and Drosophila

    PubMed Central

    Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina

    2014-01-01

    The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type. PMID:20005229

  20. Dissection of the peripheral motion channel in the visual system of Drosophila melanogaster.

    PubMed

    Rister, Jens; Pauls, Dennis; Schnell, Bettina; Ting, Chun-Yuan; Lee, Chi-Hon; Sinakevitch, Irina; Morante, Javier; Strausfeld, Nicholas J; Ito, Kei; Heisenberg, Martin

    2007-10-01

    In the eye, visual information is segregated into modalities such as color and motion, these being transferred to the central brain through separate channels. Here, we genetically dissect the achromatic motion channel in the fly Drosophila melanogaster at the level of the first relay station in the brain, the lamina, where it is split into four parallel pathways (L1-L3, amc/T1). The functional relevance of this divergence is little understood. We now show that the two most prominent pathways, L1 and L2, together are necessary and largely sufficient for motion-dependent behavior. At high pattern contrast, the two pathways are redundant. At intermediate contrast, they mediate motion stimuli of opposite polarity, L2 front-to-back, L1 back-to-front motion. At low contrast, L1 and L2 depend upon each other for motion processing. Of the two minor pathways, amc/T1 specifically enhances the L1 pathway at intermediate contrast. L3 appears not to contribute to motion but to orientation behavior. PMID:17920022

  1. Comparison of various assay systems for prostate-specific antigen standardization.

    PubMed

    Kuriyama, M; Akimoto, S; Akaza, H; Arai, Y; Usami, M; Imai, K; Tanaka, Y; Yamazaki, H; Kawada, Y; Koiso, K

    1992-12-01

    To avoid confusion between serum prostate-specific antigen (PSA) values among various assay systems, clinical studies on the possibility of conversion among detection values were performed. The assay kits used for the PSA comparisons were MARKIT-F PA, MARKIT-M PA, EIKEN PA, PA test WAKO, Ball ELSA PSA, E-Test Tosoh II PA, PROS-CHECK PSA, DELFIA PSA and TANDEM-R PSA. Using each kit, the standards attached to each assay system were detected, and 142 sera samples from benign hypertrophies or prostate cancers were assayed for serum PSA values. By detecting the standards for each kit, slopes were obtained which were almost identical to those obtained from original assay system. The coefficients of correlation among the PSA detection systems, using patients' sera, were very high, and linear regression lines were also obtained. The results suggest that almost identical serum PSA values may be detected either by multiplying by a coefficient to bring it to the standard or using the conversion formula. PMID:1283993

  2. Insulin- and warts-dependent regulation of tracheal plasticity modulates systemic larval growth during hypoxia in Drosophila melanogaster.

    PubMed

    Wong, Daniel M; Shen, Zhouyang; Owyang, Kristin E; Martinez-Agosto, Julian A

    2014-01-01

    Adaptation to dynamic environmental cues during organismal development requires coordination of tissue growth with available resources. More specifically, the effects of oxygen availability on body size have been well-documented, but the mechanisms through which hypoxia restricts systemic growth have not been fully elucidated. Here, we characterize the larval growth and metabolic defects in Drosophila that result from hypoxia. Hypoxic conditions reduced fat body opacity and increased lipid droplet accumulation in this tissue, without eliciting lipid aggregation in hepatocyte-like cells called oenocytes. Additionally, hypoxia increased the retention of Dilp2 in the insulin-producing cells of the larval brain, associated with a reduction of insulin signaling in peripheral tissues. Overexpression of the wildtype form of the insulin receptor ubiquitously and in the larval trachea rendered larvae resistant to hypoxia-induced growth restriction. Furthermore, Warts downregulation in the trachea was similar to increased insulin receptor signaling during oxygen deprivation, which both rescued hypoxia-induced growth restriction, inhibition of tracheal molting, and developmental delay. Insulin signaling and loss of Warts function increased tracheal growth and augmented tracheal plasticity under hypoxic conditions, enhancing oxygen delivery during periods of oxygen deprivation. Our findings demonstrate a mechanism that coordinates oxygen availability with systemic growth in which hypoxia-induced reduction of insulin receptor signaling decreases plasticity of the larval trachea that is required for the maintenance of systemic growth during times of limiting oxygen availability. PMID:25541690

  3. Insulin- and Warts-Dependent Regulation of Tracheal Plasticity Modulates Systemic Larval Growth during Hypoxia in Drosophila melanogaster

    PubMed Central

    Wong, Daniel M.; Shen, Zhouyang; Owyang, Kristin E.; Martinez-Agosto, Julian A.

    2014-01-01

    Adaptation to dynamic environmental cues during organismal development requires coordination of tissue growth with available resources. More specifically, the effects of oxygen availability on body size have been well-documented, but the mechanisms through which hypoxia restricts systemic growth have not been fully elucidated. Here, we characterize the larval growth and metabolic defects in Drosophila that result from hypoxia. Hypoxic conditions reduced fat body opacity and increased lipid droplet accumulation in this tissue, without eliciting lipid aggregation in hepatocyte-like cells called oenocytes. Additionally, hypoxia increased the retention of Dilp2 in the insulin-producing cells of the larval brain, associated with a reduction of insulin signaling in peripheral tissues. Overexpression of the wildtype form of the insulin receptor ubiquitously and in the larval trachea rendered larvae resistant to hypoxia-induced growth restriction. Furthermore, Warts downregulation in the trachea was similar to increased insulin receptor signaling during oxygen deprivation, which both rescued hypoxia-induced growth restriction, inhibition of tracheal molting, and developmental delay. Insulin signaling and loss of Warts function increased tracheal growth and augmented tracheal plasticity under hypoxic conditions, enhancing oxygen delivery during periods of oxygen deprivation. Our findings demonstrate a mechanism that coordinates oxygen availability with systemic growth in which hypoxia-induced reduction of insulin receptor signaling decreases plasticity of the larval trachea that is required for the maintenance of systemic growth during times of limiting oxygen availability. PMID:25541690

  4. A novel reporter system for neutralizing and enhancing antibody assay against dengue virus

    PubMed Central

    2014-01-01

    Background Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies. Results In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals. Conclusions This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies. PMID:24548533

  5. The FlyCatwalk: A High-Throughput Feature-Based Sorting System for Artificial Selection in Drosophila

    PubMed Central

    Medici, Vasco; Vonesch, Sibylle Chantal; Fry, Steven N.; Hafen, Ernst

    2015-01-01

    Experimental evolution is a powerful tool for investigating complex traits. Artificial selection can be applied for a specific trait and the resulting phenotypically divergent populations pool-sequenced to identify alleles that occur at substantially different frequencies in the extreme populations. To maximize the proportion of loci that are causal to the phenotype among all enriched loci, population size and number of replicates need to be high. These requirements have, in fact, limited evolution studies in higher organisms, where the time investment required for phenotyping is often prohibitive for large-scale studies. Animal size is a highly multigenic trait that remains poorly understood, and an experimental evolution approach may thus aid in gaining new insights into the genetic basis of this trait. To this end, we developed the FlyCatwalk, a fully automated, high-throughput system to sort live fruit flies (Drosophila melanogaster) based on morphometric traits. With the FlyCatwalk, we can detect gender and quantify body and wing morphology parameters at a four-old higher throughput compared with manual processing. The phenotyping results acquired using the FlyCatwalk correlate well with those obtained using the standard manual procedure. We demonstrate that an automated, high-throughput, feature-based sorting system is able to avoid previous limitations in population size and replicate numbers. Our approach can likewise be applied for a variety of traits and experimental settings that require high-throughput phenotyping. PMID:25556112

  6. Switch and template pattern formation in a discrete reaction-diffusion system inspired by the Drosophila eye

    PubMed Central

    Pennington, Matthew W.; Lubensky, David K.

    2011-01-01

    We examine a spatially discrete reaction diffusion model based on the interactions that create a periodic pattern in the Drosophila eye imaginal disc. This model is known to be capable of generating a regular hexagonal pattern of gene expression behind a moving front, as observed in the fly system. In order to better understand the novel “switch and template” mechanism behind this pattern formation, we present here a detailed study of the model's behavior in one dimension, using a combination of analytic methods and numerical searches of parameter space. We find that patterns are created robustly provided that there is an appropriate separation of timescales and that self-activation is sufficiently strong, and we derive expressions in this limit for the front speed and the pattern wavelength. Moving fronts in pattern-forming systems near an initial linear instability generically select a unique pattern, but our model operates in a strongly nonlinear regime where the final pattern depends on the initial conditions as well as on parameter values. Our work highlights the important role that cellularization and cell-autonomous feedback can play in biological pattern formation. PMID:20862598

  7. Detection of Cell Death in Drosophila Tissues

    PubMed Central

    Vasudevan, Deepika; Ryoo, Hyung Don

    2016-01-01

    Drosophila has served as a particularly attractive model to study cell death due to the vast array of tools for genetic manipulation under defined spatial and temporal conditions in vivo as well as in cultured cells. These genetic methods have been well supplemented by enzymatic assays and a panel of antibodies recognizing cell death markers. This chapter discusses reporters, mutants and assays used by various laboratories to study cell death in the context of development and in response to external insults. PMID:27108437

  8. Evaluation of Traditional Medicines for Neurodegenerative Diseases Using Drosophila Models

    PubMed Central

    Lee, Soojin; Bang, Se Min; Lee, Joon Woo; Cho, Kyoung Sang

    2014-01-01

    Drosophila is one of the oldest and most powerful genetic models and has led to novel insights into a variety of biological processes. Recently, Drosophila has emerged as a model system to study human diseases, including several important neurodegenerative diseases. Because of the genomic similarity between Drosophila and humans, Drosophila neurodegenerative disease models exhibit a variety of human-disease-like phenotypes, facilitating fast and cost-effective in vivo genetic modifier screening and drug evaluation. Using these models, many disease-associated genetic factors have been identified, leading to the identification of compelling drug candidates. Recently, the safety and efficacy of traditional medicines for human diseases have been evaluated in various animal disease models. Despite the advantages of the Drosophila model, its usage in the evaluation of traditional medicines is only nascent. Here, we introduce the Drosophila model for neurodegenerative diseases and some examples demonstrating the successful application of Drosophila models in the evaluation of traditional medicines. PMID:24790636

  9. Functional analysis and tissue-specific expression of Drosophila Na+,K+-ATPase subunits.

    PubMed

    Sun, B; Wang, W; Salvaterra, P M

    1998-07-01

    We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the beta subunits of Na+,K+-ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+-ATPase alpha subunit cDNA clone (PSalpha; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+-ATPase activity when PSalpha mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na+,K+-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na+,K+-ATPase expression in various cell and tissue types. PMID:9648860

  10. A common evolutionary origin for the ON- and OFF-edge motion detection pathways of the Drosophila visual system

    PubMed Central

    Shinomiya, Kazunori; Takemura, Shin-ya; Rivlin, Patricia K.; Plaza, Stephen M.; Scheffer, Louis K.; Meinertzhagen, Ian A.

    2015-01-01

    Synaptic circuits for identified behaviors in the Drosophila brain have typically been considered from either a developmental or functional perspective without reference to how the circuits might have been inherited from ancestral forms. For example, two candidate pathways for ON- and OFF-edge motion detection in the visual system act via circuits that use respectively either T4 or T5, two cell types of the fourth neuropil, or lobula plate (LOP), that exhibit narrow-field direction-selective responses and provide input to wide-field tangential neurons. T4 or T5 both have four subtypes that terminate one each in the four strata of the LOP. Representatives are reported in a wide range of Diptera, and both cell types exhibit various similarities in: (1) the morphology of their dendritic arbors; (2) their four morphological and functional subtypes; (3) their cholinergic profile in Drosophila; (4) their input from the pathways of L3 cells in the first neuropil, or lamina (LA), and by one of a pair of LA cells, L1 (to the T4 pathway) and L2 (to the T5 pathway); and (5) their innervation by a single, wide-field contralateral tangential neuron from the central brain. Progenitors of both also express the gene atonal early in their proliferation from the inner anlage of the developing optic lobe, being alone among many other cell type progeny to do so. Yet T4 receives input in the second neuropil, or medulla (ME), and T5 in the third neuropil or lobula (LO). Here we suggest that these two cell types were originally one, that their ancestral cell population duplicated and split to innervate separate ME and LO neuropils, and that a fiber crossing—the internal chiasma—arose between the two neuropils. The split most plausibly occurred, we suggest, with the formation of the LO as a new neuropil that formed when it separated from its ancestral neuropil to leave the ME, suggesting additionally that ME input neurons to T4 and T5 may also have had a common origin. PMID:26217193

  11. Planar cell polarity: the Dachsous/Fat system contributes differently to the embryonic and larval stages of Drosophila

    PubMed Central

    Saavedra, Pedro; Brittle, Amy; Palacios, Isabel M.; Strutt, David; Casal, José; Lawrence, Peter A.

    2016-01-01

    ABSTRACT The epidermal patterns of all three larval instars (L1–L3) of Drosophila are made by one unchanging set of cells. The seven rows of cuticular denticles of all larval stages are consistently planar polarised, some pointing forwards, others backwards. In L1 all the predenticles originate at the back of the cells but, in L2 and L3, they form at the front or the back of the cell depending on the polarity of the forthcoming denticles. We find that, to polarise all rows, the Dachsous/Fat system is differentially utilised; in L1 it is active in the placement of the actin-based predenticles but is not crucial for the final orientation of the cuticular denticles, in L2 and L3 it is needed for placement and polarity. We find Four-jointed to be strongly expressed in the tendon cells and show how this might explain the orientation of all seven rows. Unexpectedly, we find that L3 that lack Dachsous differ from larvae lacking Fat and we present evidence that this is due to differently mislocalised Dachs. We make some progress in understanding how Dachs contributes to phenotypes of wildtype and mutant larvae and adults. PMID:26935392

  12. Drosophila CLIP-190 and mammalian CLIP-170 display reduced microtubule plus end association in the nervous system

    PubMed Central

    Beaven, Robin; Dzhindzhev, Nikola S.; Qu, Yue; Hahn, Ines; Dajas-Bailador, Federico; Ohkura, Hiroyuki; Prokop, Andreas

    2015-01-01

    Axons act like cables, electrically wiring the nervous system. Polar bundles of microtubules (MTs) form their backbones and drive their growth. Plus end–tracking proteins (+TIPs) regulate MT growth dynamics and directionality at their plus ends. However, current knowledge about +TIP functions, mostly derived from work in vitro and in nonneuronal cells, may not necessarily apply to the very different context of axonal MTs. For example, the CLIP family of +TIPs are known MT polymerization promoters in nonneuronal cells. However, we show here that neither Drosophila CLIP-190 nor mammalian CLIP-170 is a prominent MT plus end tracker in neurons, which we propose is due to low plus end affinity of the CAP-Gly domain–containing N-terminus and intramolecular inhibition through the C-terminus. Instead, both CLIP-190 and CLIP-170 form F-actin–dependent patches in growth cones, mediated by binding of the coiled-coil domain to myosin-VI. Because our loss-of-function analyses in vivo and in culture failed to reveal axonal roles for CLIP-190, even in double-mutant combinations with four other +TIPs, we propose that CLIP-190 and -170 are not essential axon extension regulators. Our findings demonstrate that +TIP functions known from nonneuronal cells do not necessarily apply to the regulation of the very distinct MT networks in axons. PMID:25694447

  13. REBOCOL (Robotic Calorimetry): An automated NDA (Nondestructive assay) calorimetry and gamma isotopic system

    SciTech Connect

    Hurd, J.R.; Bonner, C.A.; Ostenak, C.A.; Phelan, P.F.; Powell, W.D.; Sheer, N.L.; Schneider, D.N.; Staley, H.C.

    1989-01-01

    ROBOCAL, which is presently being developed and tested at Los Alamos National Laboratory, is a full-scale, prototypical robotic system, for remote calorimetric and gamma-ray analysis of special nuclear materials. It integrates a fully automated, multi-drawer, vertical stacker-retriever system for staging unmeasured nuclear materials, and a fully automated gantry robot for computer-based selection and transfer of nuclear materials to calorimetric and gamma-ray measurement stations. Since ROBOCAL is designed for minimal operator intervention, a completely programmed user interface and data-base system are provided to interact with the automated mechanical and assay systems. The assay system is designed to completely integrate calorimetric and gamma-ray data acquisition and to perform state-of-the-art analyses on both homogeneous and heterogeneous distributions of nuclear materials in a wide variety of matrices. 10 refs., 10 figs., 4 tabs.

  14. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Neuroleptic drugs radioreceptor assay test system. 862.3645 Section 862.3645 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  15. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Neuroleptic drugs radioreceptor assay test system. 862.3645 Section 862.3645 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  16. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Neuroleptic drugs radioreceptor assay test system. 862.3645 Section 862.3645 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  17. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Neuroleptic drugs radioreceptor assay test system. 862.3645 Section 862.3645 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  18. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Neuroleptic drugs radioreceptor assay test system. 862.3645 Section 862.3645 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  19. TRU Waste Assay Methodology with the Combined Thermal Epithermal Neutron (CTEN) System

    SciTech Connect

    Veilleux, J. M.; Enter, J. A.

    2003-02-27

    The CTEN assay system is designed to measure plutonium bearing 208-L waste drums and make the transuranic versus low-level waste determination. The system was certified for Waste Isolation Pilot Plant operations and the Environmental Protection Agency approved the CTEN in 2002. It is the only system capable of making the transuranic/low-level waste (TRU/LLW) determination since it can routinely assay below 100 nCi/g. The system conducts a measurement by using either (or both) an active 14 MeV neutron pulse to induce fission in 239Pu and 241Pu or measures the spontaneous fission properties of 238Pu, 240Pu and 242Pu. When the coincidence neutron signal is combined with mass fraction data from a gamma system, the result is the total plutonium mass. The system's lower limit of detection is as low as 2 mg of weapons grade plutonium, making it an ideal platform to make the TRU/LLW determination. Analysis of an assay is made with visual basic application driven subroutines and Micros oft Excel spreadsheets. Input values and calculations include: the raw neutron scaler and coincidence counts; mass fraction information; plutonium mass; alpha, total and TRU activity; thermal power, 239Pu Equivalent Curies; fissile gram equivalent mass; decay heat; and uncertainties associated with each parameter. A general diagnostic analysis is performed for each assay to facilitate a technical review of the results. The results of analysis from 372 waste drums are summarized. The results indicate that modifying current operating procedures involving the use of acceptable knowledge isotope data and use of the lower detection limit could increase the number of certifiable assays from 38% to 66%.

  20. Macrophages and cellular immunity in Drosophila melanogaster.

    PubMed

    Gold, Katrina S; Brückner, Katja

    2015-12-01

    The invertebrate Drosophila melanogaster has been a powerful model for understanding blood cell development and immunity. Drosophila is a holometabolous insect, which transitions through a series of life stages from embryo, larva and pupa to adulthood. In spite of this, remarkable parallels exist between Drosophila and vertebrate macrophages, both in terms of development and function. More than 90% of Drosophila blood cells (hemocytes) are macrophages (plasmatocytes), making this highly tractable genetic system attractive for studying a variety of questions in macrophage biology. In vertebrates, recent findings revealed that macrophages have two independent origins: self-renewing macrophages, which reside and proliferate in local microenvironments in a variety of tissues, and macrophages of the monocyte lineage, which derive from hematopoietic stem or progenitor cells. Like vertebrates, Drosophila possesses two macrophage lineages with a conserved dual ontogeny. These parallels allow us to take advantage of the Drosophila model when investigating macrophage lineage specification, maintenance and amplification, and the induction of macrophages and their progenitors by local microenvironments and systemic cues. Beyond macrophage development, Drosophila further serves as a paradigm for understanding the mechanisms underlying macrophage function and cellular immunity in infection, tissue homeostasis and cancer, throughout development and adult life. PMID:27117654

  1. Ultrastructure of the ocellar visual system in normal and mutant Drosophila melanogaster.

    PubMed

    Stark, W S; Sapp, R; Carlson, S D

    1989-05-01

    Between the two compound eyes on the vertex on the adult head are the three simple eyes, ocelli. Transmission and scanning electron microscopy were used to investigate the corneal lenses, ocellar photoreceptors, and axonal projections in normal and mutant Drosophila melanogaster. In wild type flies, the cornea consists of about 45 lamellae. It has corneal nipples distally and is underlaid with a monolayer of corneagenous cells. Retinula cells have open rhabdomeres of about 2 microns (diameter) x 7 microns (length). Rhabdomeres extend to the distal extent of the cell and do not have caps. Microvilli have a rodlet within. Retinula cells are joined by belt desmosomes on the lateral borders. Eye color pigment granules are housed within the retinula cells of normal flies, not in accessory cells. The granules do not migrate in response to light. No screening pigment granules exist in the white mutant. Each ocellus has about 80 retinula cells whose axons project to corresponding ganglia from which 4 giant afferent interneurons (per ganglion) project to the brain. receptor terminals are invested with capitate projections from glia. Receptors synapse onto dyads of follower cells, usually interneuron processes, at sites of T shaped presynaptic ribbons. These "T bars" are surrounded by indistinct flattened vesicles. Interneurons make feed back synapses onto receptor terminals at T bars clustered with distinct round vesicles. Three mutants with abnormal ocelli were investigated. The none mutant has unusual compound eye and ocellar corneas. The compound eye is devoid of differentiated photoreceptors but some axons from undifferentiated cells from synapses. No receptors were found in the ocelli of none. The oc mutant has no ocelli, although sometimes an ocellar cornea like that of none is seen; the compound eye is normal. The rdo mutant is also specific to ocelli with smaller ocelli having half the wild type allotment of receptor cells; the number of giant afferents is

  2. Systems genetics analysis of body weight and energy metabolism traits in Drosophila melanogaster

    PubMed Central

    2010-01-01

    Background Obesity and phenotypic traits associated with this condition exhibit significant heritability in natural populations of most organisms. While a number of genes and genetic pathways have been implicated to play a role in obesity associated traits, the genetic architecture that underlies the natural variation in these traits is largely unknown. Here, we used 40 wild-derived inbred lines of Drosophila melanogaster to quantify genetic variation in body weight, the content of three major metabolites (glycogen, triacylglycerol, and glycerol) associated with obesity, and metabolic rate in young flies. We chose these lines because they were previously screened for variation in whole-genome transcript abundance and in several adult life-history traits, including longevity, resistance to starvation stress, chill-coma recovery, mating behavior, and competitive fitness. This enabled us not only to identify candidate genes and transcriptional networks that might explain variation for energy metabolism traits, but also to investigate the genetic interrelationships among energy metabolism, behavioral, and life-history traits that have evolved in natural populations. Results We found significant genetically based variation in all traits. Using a genome-wide association screen for single feature polymorphisms and quantitative trait transcripts, we identified 337, 211, 237, 553, and 152 novel candidate genes associated with body weight, glycogen content, triacylglycerol storage, glycerol levels, and metabolic rate, respectively. Weighted gene co-expression analyses grouped transcripts associated with each trait in significant modules of co-expressed genes and we interpreted these modules in terms of their gene enrichment based on Gene Ontology analysis. Comparison of gene co-expression modules for traits in this study with previously determined modules for life-history traits identified significant modular pleiotropy between glycogen content, body weight, competitive

  3. Potency of Transgenic Effectors for Neurogenetic Manipulation in Drosophila Larvae

    PubMed Central

    Pauls, Dennis; von Essen, Alina; Lyutova, Radostina; van Giesen, Lena; Rosner, Ronny; Wegener, Christian; Sprecher, Simon G.

    2015-01-01

    Genetic manipulations of neuronal activity are a cornerstone of studies aimed to identify the functional impact of defined neurons for animal behavior. With its small nervous system, rapid life cycle, and genetic amenability, the fruit fly Drosophila melanogaster provides an attractive model system to study neuronal circuit function. In the past two decades, a large repertoire of elegant genetic tools has been developed to manipulate and study neural circuits in the fruit fly. Current techniques allow genetic ablation, constitutive silencing, or hyperactivation of neuronal activity and also include conditional thermogenetic or optogenetic activation or inhibition. As for all genetic techniques, the choice of the proper transgenic tool is essential for behavioral studies. Potency and impact of effectors may vary in distinct neuron types or distinct types of behavior. We here systematically test genetic effectors for their potency to alter the behavior of Drosophila larvae, using two distinct behavioral paradigms: general locomotor activity and directed, visually guided navigation. Our results show largely similar but not equal effects with different effector lines in both assays. Interestingly, differences in the magnitude of induced behavioral alterations between different effector lines remain largely consistent between the two behavioral assays. The observed potencies of the effector lines in aminergic and cholinergic neurons assessed here may help researchers to choose the best-suited genetic tools to dissect neuronal networks underlying the behavior of larval fruit flies. PMID:25359929

  4. Development of a high-throughput purification method and a continuous assay system for chlorophyllase.

    PubMed

    Arkus, Kiani A J; Jez, Joseph M

    2006-06-01

    In the degradation of chlorophyll, chlorophyllase catalyzes the initial hydrolysis of the phytol moiety from the pigment. Since chlorophyll degradation is a defining feature of plant senescence, compounds inhibiting chlorophyllase activity may delay senescence, thereby improving shelf life and appearance of plant products. Here we describe the development of a 96-well plate-based purification and assay system for measuring chlorophyllase activity. Integrated lysis and immobilized metal affinity chromatography plates were used for purifying recombinant hexahistidine-tagged Triticum aestivum (wheat) chlorophyllase from Escherichia coli. Chlorophyllase assays using chlorophyll as a substrate showed that the immobilized fusion protein displayed kinetic parameters similar to those of recombinant enzyme purified by affinity chromatography; however, the need to extract reaction products from a multiwell plate limits the value of this assay for high-throughput screening applications. Replacing chlorophyll with p-nitrophenyl-ester substrates eliminates the extraction step and allows for continuous measurement of chlorophyllase activity in a multiwell plate format. Determination of steady state kinetic constants, pH rate profile, the inhibitory effects of metal ions and esterase inhibitors, and the effect of functional group-modifying reagents validated the utility of the plate-based system. The combined purification and assay system provides a convenient and rapid method for the assessment of chlorophyllase activity. PMID:16643837

  5. Application of expert system technology to nondestructive waste assay - initial prototype model

    SciTech Connect

    Becker, G.K.; Determan, J.C.

    1997-11-01

    Expert system technology has been identified as a technique useful for filling certain types of technology/capability gaps in existing waste nondestructive assay (NDA) applications. In particular, expert system techniques are being investigated with the intent of providing on-line evaluation of acquired data and/or directed acquisition of data in a manner that mimics the logic and decision making process a waste NDA expert would employ. The space from which information and data sources utilized in this process is much expanded with respect to the algorithmic approach typically utilized in waste NDA. Expert system technology provides a mechanism to manage and reason with this expanded information/data set. The material presented in this paper concerns initial studies and a resultant prototype expert system that incorporates pertinent information, and evaluation logic and decision processes, for the purpose of validating acquired waste NDA measurement assays. 6 refs., 6 figs.

  6. Drosophila Modeling of Heritable Neurodevelopmental Disorders

    PubMed Central

    Gatto, Cheryl L.; Broadie, Kendal

    2011-01-01

    Heritable neurodevelopmental disorders are multifaceted disease conditions encompassing a wide range of symptoms including intellectual disability, cognitive dysfunction, autism and myriad other behavioral impairments. In cases where single, causative genetic defects have been identified, such as Angelman syndrome, Rett syndrome, Neurofibromatosis Type 1 and Fragile X syndrome, the classical Drosophila genetic system has provided fruitful disease models. Recent Drosophila studies have advanced our understanding of UBE3A, MECP2, NF1 and FMR1 function, respectively, in genetic, biochemical, anatomical, physiological and behavioral contexts. Investigations in Drosophila continue to provide the essential mechanistic understanding required to facilitate the conception of rational therapeutic treatments. PMID:21596554

  7. Integration of electrochemistry in micro-total analysis systems for biochemical assays: recent developments.

    PubMed

    Xu, Xiaoli; Zhang, Song; Chen, Hui; Kong, Jilie

    2009-11-15

    Micro-total analysis systems (microTAS) integrate different analytical operations like sample preparation, separation and detection into a single microfabricated device. With the outstanding advantages of low cost, satisfactory analytical efficiency and flexibility in design, highly integrated and miniaturized devices from the concept of microTAS have gained widespread applications, especially in biochemical assays. Electrochemistry is shown to be quite compatible with microanalytical systems for biochemical assays, because of its attractive merits such as simplicity, rapidity, high sensitivity, reduced power consumption, and sample/reagent economy. This review presents recent developments in the integration of electrochemistry in microdevices for biochemical assays. Ingenious microelectrode design and fabrication methods, and versatility of electrochemical techniques are involved. Practical applications of such integrated microsystem in biochemical assays are focused on in situ analysis, point-of-care testing and portable devices. Electrochemical techniques are apparently suited to microsystems, since easy microfabrication of electrochemical elements and a high degree of integration with multi-analytical functions can be achieved at low cost. Such integrated microsystems will play an increasingly important role for analysis of small volume biochemical samples. Work is in progress toward new microdevice design and applications. PMID:19782186

  8. A new in vitro hemagglutinin inhibitor screening system based on a single-vesicle fusion assay.

    PubMed

    Lee, Hanki; Jin, Wook; Jeong, Byeong-Chul; Suh, Joo-Won

    2016-01-01

    Hemagglutinin (HA) from the influenza virus plays a pivotal role in the infection of host mammalian cells and is, therefore, a druggable target, similar to neuraminidase. However, research involving the influenza virus must be conducted in facilities certified at or above Biosafety Level 2 because of the potential threat of the contagiousness of this virus. To develop a new HA inhibitor screening system without intact influenza virus, we conceived a single-vesicle fusion assay using full-length recombinant HA. In this study, we first showed that full-length recombinant HA can mediate membrane fusion in ensemble and single-vesicle fusion assays. The fluorescence resonance energy transfer (FRET) frequency pattern of single-vesicle complexes completely differed when the inhibitors targeted the HA1 or HA2 domain of HA. This result indicates that analysing the FRET patterns in this assay can provide information regarding the domains of HA inhibited by compounds and compounds' inhibitory activities. Therefore, our results suggest that the assay developed here is a promising tool for the discovery of anti-influenza virus drug candidates as a new in vitro inhibitor screening system against HA from the influenza virus. PMID:27469068

  9. A new in vitro hemagglutinin inhibitor screening system based on a single-vesicle fusion assay

    PubMed Central

    Lee, Hanki; Jin, Wook; Jeong, Byeong-Chul; Suh, Joo-Won

    2016-01-01

    Hemagglutinin (HA) from the influenza virus plays a pivotal role in the infection of host mammalian cells and is, therefore, a druggable target, similar to neuraminidase. However, research involving the influenza virus must be conducted in facilities certified at or above Biosafety Level 2 because of the potential threat of the contagiousness of this virus. To develop a new HA inhibitor screening system without intact influenza virus, we conceived a single-vesicle fusion assay using full-length recombinant HA. In this study, we first showed that full-length recombinant HA can mediate membrane fusion in ensemble and single-vesicle fusion assays. The fluorescence resonance energy transfer (FRET) frequency pattern of single-vesicle complexes completely differed when the inhibitors targeted the HA1 or HA2 domain of HA. This result indicates that analysing the FRET patterns in this assay can provide information regarding the domains of HA inhibited by compounds and compounds’ inhibitory activities. Therefore, our results suggest that the assay developed here is a promising tool for the discovery of anti-influenza virus drug candidates as a new in vitro inhibitor screening system against HA from the influenza virus. PMID:27469068

  10. Mutations affecting the development of the peripheral nervous system in Drosophila: a molecular screen for novel proteins.

    PubMed Central

    Prokopenko, S N; He, Y; Lu, Y; Bellen, H J

    2000-01-01

    In our quest for novel genes required for the development of the embryonic peripheral nervous system (PNS), we have performed three genetic screens using MAb 22C10 as a marker of terminally differentiated neurons. A total of 66 essential genes required for normal PNS development were identified, including 49 novel genes. To obtain information about the molecular nature of these genes, we decided to complement our genetic screens with a molecular screen. From transposon-tagged mutations identified on the basis of their phenotype in the PNS we selected 31 P-element strains representing 26 complementation groups on the second and third chromosomes to clone and sequence the corresponding genes. We used plasmid rescue to isolate and sequence 51 genomic fragments flanking the sites of these P-element insertions. Database searches using sequences derived from the ends of plasmid rescues allowed us to assign genes to one of four classes: (1) previously characterized genes (11), (2) first mutations in cloned genes (1), (3) P-element insertions in genes that were identified, but not characterized molecularly (1), and (4) novel genes (13). Here, we report the cloning, sequence, Northern analysis, and the embryonic expression pattern of candidate cDNAs for 10 genes: astray, chrowded, dalmatian, gluon, hoi-polloi, melted, pebble, skittles, sticky ch1, and vegetable. This study allows us to draw conclusions about the identity of proteins required for the development of the nervous system in Drosophila and provides an example of a molecular approach to characterize en masse transposon-tagged mutations identified in genetic screens. PMID:11102367

  11. Neurodegeneration in drop-dead mutant drosophila melanogaster is associated with the respiratory system but not with Hypoxia.

    PubMed

    Sansone, Christine Lynn; Blumenthal, Edward M

    2013-01-01

    Mutations in the gene drop-dead (drd) cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ) were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia. PMID:23874488

  12. The use of a mutationally unstable X-chromosome in Drosophila melanogaster for mutagenicity testing.

    PubMed

    Rasmuson, B; Svahlin, H; Rasmuson, A; Montell, I; Olofsson, H

    1978-08-01

    Somatic eye-colour mutations in an unstable genetic system, caused by a transposable element in the white locus of the X-chromosome in Drosophila melanogaster, is suggested as an assay system for mutagenicity testing. The system is evaluated by comparison with a corresponding system in a stable X-chromosome. Its sensitivity is confirmed with X-ray and EMS treatment, and it is found to be confined to the specific segment of the X-chromosome where the transposable element is localized. PMID:97525

  13. Prototype Radiation Detector Positioning System For The Automated Nondestructive Assay Of Uf6 Cylinders

    SciTech Connect

    Hatchell, Brian K.; Valdez, Patrick LJ; Orton, Christopher R.; Mace, Emily K.

    2011-08-07

    International Atomic Energy Agency (IAEA) inspectors currently perform periodic inspections at uranium enrichment plants to verify UF6 cylinder enrichment declarations. Measurements are typically performed with handheld high-resolution sensors on a sampling of cylinders taken to be representative of the facility’s entire cylinder inventory. These measurements are time-consuming, expensive, and assay only a small fraction of the total cylinder volume. An automated nondestructive assay system capable of providing enrichment measurements over the full volume of the cylinder could improve upon current verification practices in terms of efficiency and assay accuracy. This paper describes an approach denoted the Integrated Cylinder Verification Station (ICVS) that supports 100% cylinder verification, provides volume-averaged cylinder enrichment assay, and reduces inspector manpower needs. To allow field measurements to be collected to validate data collection algorithms, a prototype radiation detector positioning system was constructed. The system was designed to accurately position an array of radiation detectors along the length of a cylinder to measure UF6 enrichment. A number of alternative radiation shields for the detectors were included with the system. A collimated gamma-ray spectrometer module that allows translation of the detectors in the surrounding shielding to adjust the field of view, and a collimating plug in the end to further reduce the low-energy field of view, were also developed. Proof-of-principle measurements of neutron and high-energy gamma-ray signatures, using moderated neutron detectors and large-volume spectrometers in a fixed-geometry, portal-like configuration, supported an early assessment of the viability of the concept. The system has been used successfully on two testing campaigns at an AREVA fuel fabrication plant to scan over 30 product cylinders. This paper will describe the overall design of the detector positioning system and

  14. Quantitative Assessment of Eye Phenotypes for Functional Genetic Studies Using Drosophila melanogaster.

    PubMed

    Iyer, Janani; Wang, Qingyu; Le, Thanh; Pizzo, Lucilla; Grönke, Sebastian; Ambegaokar, Surendra S; Imai, Yuzuru; Srivastava, Ashutosh; Troisí, Beatriz Llamusí; Mardon, Graeme; Artero, Ruben; Jackson, George R; Isaacs, Adrian M; Partridge, Linda; Lu, Bingwei; Kumar, Justin P; Girirajan, Santhosh

    2016-01-01

    About two-thirds of the vital genes in the Drosophila genome are involved in eye development, making the fly eye an excellent genetic system to study cellular function and development, neurodevelopment/degeneration, and complex diseases such as cancer and diabetes. We developed a novel computational method, implemented as Flynotyper software (http://flynotyper.sourceforge.net), to quantitatively assess the morphological defects in the Drosophila eye resulting from genetic alterations affecting basic cellular and developmental processes. Flynotyper utilizes a series of image processing operations to automatically detect the fly eye and the individual ommatidium, and calculates a phenotypic score as a measure of the disorderliness of ommatidial arrangement in the fly eye. As a proof of principle, we tested our method by analyzing the defects due to eye-specific knockdown of Drosophila orthologs of 12 neurodevelopmental genes to accurately document differential sensitivities of these genes to dosage alteration. We also evaluated eye images from six independent studies assessing the effect of overexpression of repeats, candidates from peptide library screens, and modifiers of neurotoxicity and developmental processes on eye morphology, and show strong concordance with the original assessment. We further demonstrate the utility of this method by analyzing 16 modifiers of sine oculis obtained from two genome-wide deficiency screens of Drosophila and accurately quantifying the effect of its enhancers and suppressors during eye development. Our method will complement existing assays for eye phenotypes, and increase the accuracy of studies that use fly eyes for functional evaluation of genes and genetic interactions. PMID:26994292

  15. Quantitative Assessment of Eye Phenotypes for Functional Genetic Studies Using Drosophila melanogaster

    PubMed Central

    Iyer, Janani; Wang, Qingyu; Le, Thanh; Pizzo, Lucilla; Grönke, Sebastian; Ambegaokar, Surendra S.; Imai, Yuzuru; Srivastava, Ashutosh; Troisí, Beatriz Llamusí; Mardon, Graeme; Artero, Ruben; Jackson, George R.; Isaacs, Adrian M.; Partridge, Linda; Lu, Bingwei; Kumar, Justin P.; Girirajan, Santhosh

    2016-01-01

    About two-thirds of the vital genes in the Drosophila genome are involved in eye development, making the fly eye an excellent genetic system to study cellular function and development, neurodevelopment/degeneration, and complex diseases such as cancer and diabetes. We developed a novel computational method, implemented as Flynotyper software (http://flynotyper.sourceforge.net), to quantitatively assess the morphological defects in the Drosophila eye resulting from genetic alterations affecting basic cellular and developmental processes. Flynotyper utilizes a series of image processing operations to automatically detect the fly eye and the individual ommatidium, and calculates a phenotypic score as a measure of the disorderliness of ommatidial arrangement in the fly eye. As a proof of principle, we tested our method by analyzing the defects due to eye-specific knockdown of Drosophila orthologs of 12 neurodevelopmental genes to accurately document differential sensitivities of these genes to dosage alteration. We also evaluated eye images from six independent studies assessing the effect of overexpression of repeats, candidates from peptide library screens, and modifiers of neurotoxicity and developmental processes on eye morphology, and show strong concordance with the original assessment. We further demonstrate the utility of this method by analyzing 16 modifiers of sine oculis obtained from two genome-wide deficiency screens of Drosophila and accurately quantifying the effect of its enhancers and suppressors during eye development. Our method will complement existing assays for eye phenotypes, and increase the accuracy of studies that use fly eyes for functional evaluation of genes and genetic interactions. PMID:26994292

  16. Iron Absorption in Drosophila melanogaster

    PubMed Central

    Mandilaras, Konstantinos; Pathmanathan, Tharse; Missirlis, Fanis

    2013-01-01

    The way in which Drosophila melanogaster acquires iron from the diet remains poorly understood despite iron absorption being of vital significance for larval growth. To describe the process of organismal iron absorption, consideration needs to be given to cellular iron import, storage, export and how intestinal epithelial cells sense and respond to iron availability. Here we review studies on the Divalent Metal Transporter-1 homolog Malvolio (iron import), the recent discovery that Multicopper Oxidase-1 has ferroxidase activity (iron export) and the role of ferritin in the process of iron acquisition (iron storage). We also describe what is known about iron regulation in insect cells. We then draw upon knowledge from mammalian iron homeostasis to identify candidate genes in flies. Questions arise from the lack of conservation in Drosophila for key mammalian players, such as ferroportin, hepcidin and all the components of the hemochromatosis-related pathway. Drosophila and other insects also lack erythropoiesis. Thus, systemic iron regulation is likely to be conveyed by different signaling pathways and tissue requirements. The significance of regulating intestinal iron uptake is inferred from reports linking Drosophila developmental, immune, heat-shock and behavioral responses to iron sequestration. PMID:23686013

  17. Iron absorption in Drosophila melanogaster.

    PubMed

    Mandilaras, Konstantinos; Pathmanathan, Tharse; Missirlis, Fanis

    2013-05-01

    The way in which Drosophila melanogaster acquires iron from the diet remains poorly understood despite iron absorption being of vital significance for larval growth. To describe the process of organismal iron absorption, consideration needs to be given to cellular iron import, storage, export and how intestinal epithelial cells sense and respond to iron availability. Here we review studies on the Divalent Metal Transporter-1 homolog Malvolio (iron import), the recent discovery that Multicopper Oxidase-1 has ferroxidase activity (iron export) and the role of ferritin in the process of iron acquisition (iron storage). We also describe what is known about iron regulation in insect cells. We then draw upon knowledge from mammalian iron homeostasis to identify candidate genes in flies. Questions arise from the lack of conservation in Drosophila for key mammalian players, such as ferroportin, hepcidin and all the components of the hemochromatosis-related pathway. Drosophila and other insects also lack erythropoiesis. Thus, systemic iron regulation is likely to be conveyed by different signaling pathways and tissue requirements. The significance of regulating intestinal iron uptake is inferred from reports linking Drosophila developmental, immune, heat-shock and behavioral responses to iron sequestration. PMID:23686013

  18. Drosophila Photoreceptors and Signaling Mechanisms

    PubMed Central

    Katz, Ben; Minke, Baruch

    2009-01-01

    Fly eyes have been a useful biological system in which fundamental principles of sensory signaling have been elucidated. The physiological optics of the fly compound eye, which was discovered in the Musca, Calliphora and Drosophila flies, has been widely exploited in pioneering genetic and developmental studies. The detailed photochemical cycle of bistable photopigments has been elucidated in Drosophila using the genetic approach. Studies of Drosophila phototransduction using the genetic approach have led to the discovery of novel proteins crucial to many biological processes. A notable example is the discovery of the inactivation no afterpotential D scaffold protein, which binds the light-activated channel, its activator the phospholipase C and it regulator protein kinase C. An additional protein discovered in the Drosophila eye is the light-activated channel transient receptor potential (TRP), the founding member of the diverse and widely spread TRP channel superfamily. The fly eye has thus played a major role in the molecular identification of processes and proteins with prime importance. PMID:19623243

  19. Mutagenicity study of fried sausages in Salmonella, Drosophila and mammalian cells in vitro and in vivo.

    PubMed

    Gocke, E; Eckhardt, K; King, M T; Wild, D

    1982-06-01

    The basic extract of pan-fried sausages was studied for mutagenic potential in seven test systems. Mutagenic activity was high in the standard Ames assay in the Salmonella typhimurium strains TA1538 and TA98 in presence of S9 mix. In vivo, in the intrasanguine host-mediated assay with strain TA98 on Aroclor-pretreated mice, the mutagenic activity of the extract was low. A borderline activity was seen in the SCE assay in vitro with V79 Chinese hamster cells in presence of S9 mix. No significant mutagenic action was found in the gene-mutation assay for thioguanine resistance with V79 cells, the Drosophila sex-linked recessive lethal test, the micronucleus test and the mammalian spot test. PMID:6810162

  20. Initial neurogenesis in Drosophila

    PubMed Central

    Hartenstein, Volker; Wodarz, Andreas

    2014-01-01

    Early neurogenesis comprises the phase of nervous system development during which neural progenitor cells are born. In early development, the embryonic ectoderm is subdivided by a conserved signaling mechanism into two main domains, the epidermal ectoderm and the neurectoderm. Subsequently, cells of the neurectoderm are internalized and form a cell layer of proliferating neural progenitors. In vertebrates, the entire neurectoderm folds into the embryo to give rise to the neural tube. In Drosophila and many other invertebrates, a subset of neurectodermal cells, called neuroblasts (NBs), delaminates and forms the neural primordium inside the embryo where they divide in an asymmetric, stem cell-like mode. The remainder of the neuroectodermal cells that stay behind at the surface loose their neurogenic potential and later give rise to the ventral part of the epidermis. The genetic and molecular analysis of the mechanisms controlling specification and proliferation of NBs in the Drosophila embryo, which played a significant part in pioneering the field of modern developmental neurobiology, represents the topic of this review. PMID:24014455

  1. Initial neurogenesis in Drosophila.

    PubMed

    Hartenstein, Volker; Wodarz, Andreas

    2013-01-01

    Early neurogenesis comprises the phase of nervous system development during which neural progenitor cells are born. In early development, the embryonic ectoderm is subdivided by a conserved signaling mechanism into two main domains, the epidermal ectoderm and the neurectoderm. Subsequently, cells of the neurectoderm are internalized and form a cell layer of proliferating neural progenitors. In vertebrates, the entire neurectoderm folds into the embryo to give rise to the neural tube. In Drosophila and many other invertebrates, a subset of neurectodermal cells, called neuroblasts (NBs), delaminates and forms the neural primordium inside the embryo where they divide in an asymmetric, stem cell-like mode. The remainder of the neurectodermal cells that stay behind at the surface loose their neurogenic potential and later give rise to the ventral part of the epidermis. The genetic and molecular analysis of the mechanisms controlling specification and proliferation of NBs in the Drosophila embryo, which played a significant part in pioneering the field of modern developmental neurobiology, represents the topic of this review. PMID:24014455

  2. Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells

    PubMed Central

    Ramirez, Juanma; Martinez, Aitor; Lectez, Benoit; Lee, So Young; Franco, Maribel; Barrio, Rosa; Dittmar, Gunnar; Mayor, Ugo

    2015-01-01

    Background Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo. Methodology/Principal Findings Using an in vivo biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination. Conclusions/Significance These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system. PMID:26460970

  3. A state-of-the-art passive gamma-ray assay system

    SciTech Connect

    Sampson, T.E.; Parker, J.L.; Cowder, L.R.; Kern, E.A.; Garcia, D.L.; Ensslin, N.

    1987-01-01

    We report details of the development of a high-accuracy, high-precision system for the non-destructive assay of /sup 235/U in solution. The system can measure samples with concentrations ranging from 0.0001 to 500 g /sup 235/U/l using 200-ml samples at low concentrations, 30-ml samples at high concentrations, and 1000-s measurement times. The accuracy and precision goals of 0.1% were essentially attained for concentrations above 100 g/l. This at-line system, designed for a production plant environment, represents a significant improvement in the state of the art.

  4. Effects of Spaceflight on Drosophila Neural Development

    NASA Technical Reports Server (NTRS)

    Keshishian, Haig S.

    1997-01-01

    The major goal from the animal side, however, has been achieved, namely to develop Drosophila lines where we can assay individual neuromuscular endings directly without dissection. This was achieved by means of using the GAL4-UAS system, where we have succeeded in establishing stocks of flies where the key neuromuscular connections can be assayed directly in undissected larvae by means of the expression of endogenously fluorescent reporters in the specific motor endings. The green fluorescent protein (GFP) as a reporter allows scoring of neural anatomy en-masse in whole mount using fluorescent microscopy without the need for either dissection or specific labeling. Two stocks have been developed. The first, which we developed first, uses the S65T mutant form, which has a dramatically brighter expression than the native protein. This animal will use GAL4 drivers with expression under the control of the elav gene, and which will ensure expression in all neurons of the embryo and larva. The second transgenic animal we have developed is of a novel kind, and makes use of dicistronic design, so that two copies of the protein will be expressed per insert. We have also developed a tricistronic form, but this has not yet been transformed into flies, and we do not imagine that this third line will be ready in time for the flight.

  5. Development of an integrated, unattended assay system for LWR-MOX fuel pellet trays

    SciTech Connect

    Stewart, J.E.; Hatcher, C.R.; Pollat, L.L.

    1994-08-01

    Four identical unattended plutonium assay systems have been developed for use at the new light-water-reactor mixed oxide (LWR-MOX) fuel fabrication facility at Hanau, Germany. The systems provide quantitative plutonium verification for all MOX pellet trays entering or leaving a large, intermediate store. Pellet-tray transport and storage systems are highly automated. Data from the ``I-Point`` (information point) assay systems will be shared by the Euratom and International Atomic Energy Agency (IAEA) Inspectorates. The I-Point system integrates, for the first time, passive neutron coincidence counting (NCC) with electro-mechanical sensing (EMS) in unattended mode. Also, provisions have been made for adding high-resolution gamma spectroscopy. The system accumulates data for every tray entering or leaving the store between inspector visits. During an inspection, data are analyzed and compared with operator declarations for the previous inspection period, nominally one month. Specification of the I-point system resulted from a collaboration between the IAEA, Euratom, Siemens, and Los Alamos. Hardware was developed by Siemens and Los Alamos through a bilateral agreement between the German Federal Ministry of Research and Technology (BMFT) and the US DOE. Siemens also provided the EMS subsystem, including software. Through the USSupport Program to the IAEA, Los Alamos developed the NCC software (NCC COLLECT) and also the software for merging and reviewing the EMS and NCC data (MERGE/REVIEW). This paper describes the overall I-Point system, but emphasizes the NCC subsystem, along with the NCC COLLECT and MERGE/REVIEW codes. We also summarize comprehensive testing results that define the quality of assay performance.

  6. Total Measurement Uncertainty for the Plutonium Finishing Plant (PFP) Segmented Gamma Scan Assay System

    SciTech Connect

    WESTSIK, G.A.

    2001-06-06

    This report presents the results of an evaluation of the Total Measurement Uncertainty (TMU) for the Canberra manufactured Segmented Gamma Scanner Assay System (SGSAS) as employed at the Hanford Plutonium Finishing Plant (PFP). In this document, TMU embodies the combined uncertainties due to all of the individual random and systematic sources of measurement uncertainty. It includes uncertainties arising from corrections and factors applied to the analysis of transuranic waste to compensate for inhomogeneities and interferences from the waste matrix and radioactive components. These include uncertainty components for any assumptions contained in the calibration of the system or computation of the data. Uncertainties are propagated at 1 sigma. The final total measurement uncertainty value is reported at the 95% confidence level. The SGSAS is a gamma assay system that is used to assay plutonium and uranium waste. The SGSAS system can be used in a stand-alone mode to perform the NDA characterization of a container, particularly for low to medium density (0-2.5 g/cc) container matrices. The SGSAS system provides a full gamma characterization of the container content. This document is an edited version of the Rocky Flats TMU Report for the Can Scan Segment Gamma Scanners, which are in use for the plutonium residues projects at the Rocky Flats plant. The can scan segmented gamma scanners at Rocky Flats are the same design as the PFP SGSAS system and use the same software (with the exception of the plutonium isotopics software). Therefore, all performance characteristics are expected to be similar. Modifications in this document reflect minor differences in the system configuration, container packaging, calibration technique, etc. These results are supported by the Quality Assurance Objective (QAO) counts, safeguards test data, calibration data, etc. for the PFP SGSAS system. Other parts of the TMU analysis utilize various modeling techniques such as Monte Carlo N

  7. Drosophila RNAi screening in a postgenomic world

    PubMed Central

    2011-01-01

    Drosophila melanogaster has a long history as a model organism with several unique features that make it an ideal research tool for the study of the relationship between genotype and phenotype. Importantly fundamental genetic principles as well as key human disease genes have been uncovered through the use of Drosophila. The contribution of the fruit fly to science and medicine continues in the postgenomic era as cell-based Drosophila RNAi screens are a cost-effective and scalable enabling technology that can be used to quantify the contribution of different genes to diverse cellular processes. Drosophila high-throughput screens can also be used as integral part of systems-level approaches to describe the architecture and dynamics of cellular networks. PMID:21752787

  8. Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

    SciTech Connect

    Koopman, R P; Langlois, R G; Nasarabadi, S; Benett, W J; Colston, B W; Johnson, D C; Brown, S B; Stratton, P L; Milanovich, F P

    2002-04-17

    This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flow cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.

  9. Bacterial regrowth in water reclamation and distribution systems revealed by viable bacterial detection assays.

    PubMed

    Lin, Yi-wen; Li, Dan; Gu, April Z; Zeng, Si-yu; He, Miao

    2016-02-01

    Microbial regrowth needs to be managed during water reclamation and distribution. The aim of present study was to investigate the removal and regrowth of Escherichia coli (E. coli) and Salmonella in water reclamation and distribution system by using membrane integrity assay (PMA-qPCR), reverse transcriptional activity assay (Q-RT-PCR) and culture-based assay, and also to evaluate the relationships among bacterial regrowth, and environmental factors in the distribution system. The results showed that most of the water reclamation processes potentially induced bacteria into VBNC state. The culturable E. coli and Salmonella regrew 1.8 and 0.7 log10 in distribution system, which included reactivation of bacteria in the viable but non-culturable (VBNC) state and reproduction of culturable bacteria. The regrowth of culturable E. coli and Salmonella in the distribution system mainly depended on the residual chlorine levels, with correlations (R(2)) of -0.598 and -0.660. The abundances of membrane integrity and reverse transcriptional activity bacteria in reclamation effluents had significant correlations with the culturable bacteria at the end point of the distribution system, demonstrating that PMA-qPCR and Q-RT-PCR are sensitive and accurate tools to determine and predict bacterial regrowth in water distribution systems. This study has improved our understanding of microbial removal and regrowth in reclaimed water treatment and distribution systems. And the results also recommended that more processes should be equipped to remove viable bacteria in water reclamation plants for the sake of inhibition microbial regrowth during water distribution and usages. PMID:26595310

  10. Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

    SciTech Connect

    Taxvig, Camilla Olesen, Pelle Thonning; Nellemann, Christine

    2011-02-01

    Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.

  11. Thyroid endocrine system disruption by pentachlorophenol: an in vitro and in vivo assay.

    PubMed

    Guo, Yongyong; Zhou, Bingsheng

    2013-10-15

    The present study aimed to evaluate the disruption caused to the thyroid endocrine system by pentachlorophenol (PCP) using in vitro and in vivo assays. In the in vitro assay, rat pituitary GH3 cells were exposed to 0, 0.1, 0.3, and 1.0 μM PCP. PCP exposure significantly downregulated basal and triiodothyronine (T3)-induced Dio 1 transcription, indicating the antagonistic activity of PCP in vitro. In the in vivo assay, zebrafish embryos were exposed to 0, 1, 3, and 10 μg/L of PCP until 14 days post-fertilization. PCP exposure resulted in decreased thyroxine (T4) levels, but elevated contents of whole-body T3. PCP exposure significantly upregulated the mRNA expression of genes along hypothalamic-pituitary-thyroid (HPT) axis, including those encoding thyroid-stimulating hormone, sodium/iodide symporter, thyroglobulin, Dio 1 and Dio 2, alpha and beta thyroid hormone receptor, and uridinediphosphate-glucuronosyl-transferase. PCP exposure did not influence the transcription of the transthyretin (TTR) gene. The results indicate that PCP potentially disrupts the thyroid endocrine system both in vitro and in vivo. PMID:24001430

  12. Devices, systems, and methods for conducting assays with improved sensitivity using sedimentation

    DOEpatents

    Schaff, Ulrich Y.; Koh, Chung-Yan; Sommer, Gregory J.

    2016-04-05

    Embodiments of the present invention are directed toward devices, systems, and method for conducting assays using sedimentation. In one example, a method includes layering a mixture on a density medium, subjecting sedimentation particles in the mixture to sedimentation forces to cause the sedimentation particles to move to a detection area through a density medium, and detecting a target analyte in a detection region of the sedimentation channel. In some examples, the sedimentation particles and labeling agent may have like charges to reduce non-specific binding of labeling agent and sedimentation particles. In some examples, the density medium is provided with a separation layer for stabilizing the assay during storage and operation. In some examples, the sedimentation channel may be provided with a generally flat sedimentation chamber for dispersing the particle pellet over a larger surface area.

  13. Devices, systems, and methods for conducting assays with improved sensitivity using sedimentation

    DOEpatents

    Schaff, Ulrich Y; Koh, Chung-Yan; Sommer, Gregory J

    2015-02-24

    Embodiments of the present invention are directed toward devices, systems, and method for conducting assays using sedimentation. In one example, a method includes layering a mixture on a density medium, subjecting sedimentation particles in the mixture to sedimentation forces to cause the sedimentation particles to move to a detection area through a density medium, and detecting a target analyte in a detection region of the sedimentation channel. In some examples, the sedimentation particles and labeling agent may have like charges to reduce non-specific binding of labeling agent and sedimentation particles. In some examples, the density medium is provided with a separation layer for stabilizing the assay during storage and operation. In some examples, the sedimentation channel may be provided with a generally flat sedimentation chamber for dispersing the particle pellet over a larger surface area.

  14. Drosophila sec10 is required for hormone secretion but not general exocytosis or neurotransmission.

    PubMed

    Andrews, Hillary K; Zhang, Yong Q; Trotta, Nick; Broadie, Kendal

    2002-12-01

    The sec6/8, or exocyst, complex is implicated in trafficking of secretory vesicles to fusion sites in the plasma membrane. Genetic analyses have been done primarily in yeast, where mutation of the eight protein subunits similarly disrupts polarized vesicle fusion. The goal of this study was to assay the sec6/8 complex in Drosophila, and specifically to test its widely hypothesized functions in synaptogenesis and neurotransmission. We used a transgenic RNAi approach to remove the most highly conserved complex component, Drosophila sec10 (dSec10). Ubiquitous dSec10 RNAi resulted in early postembryonic lethality, demonstrating that dSec10 is essential. Surprisingly, tissue-specific dSec10 RNAi revealed no essential requirement in nervous system, musculature, gut or epidermis. Assays of polarized secretion in all these tissues failed to reveal any role for dSec10. In particular, the neuromuscular synapse showed no defects in morphogenesis or vesicle trafficking/fusion underlying neurotransmission. The essential requirement for dSec10 was restricted to the ring gland, the Drosophila organ specialized for endocrine function. The developmental arrest of dSec10 RNAi animals was partially rescued by feeding ecdysone, suggesting dSec10 mediates steroid hormone secretion. We conclude that dSec10 has no detectable role in most forms of polarized trafficking/exocytosis, including neurotransmission, but rather is essential for endocrine secretion. PMID:12453153

  15. A Soil-free System for Assaying Nematicidal Activity of Chemicals.

    PubMed

    Preiser, F A; Babu, J R; Haidri, A A

    1981-10-01

    A biological assay system for studying the nematicidal activity of chemicals has been devised using a model consisting of cucumber (Cucumis sativus L. cv. Long Marketer) seedlings growing in the diSPo(R) growth-pouch apparatus. Meloidogyne incognita was used as the test organism. The response was quantified in terms of the numbers of galls produced. Statistical procedures were applied to estimate the ED(50) values of currently available nematicides. This system permits accurate quantification of galling and requires much less space and effort than the currently used methods. PMID:19300800

  16. Somatic-cell mutation induced by short exposures to cigarette smoke in urate-null, oxidative stress-sensitive Drosophila.

    PubMed

    Uchiyama, Tomoyo; Koike, Ryota; Yuma, Yoko; Okamoto, Keinosuke; Arimoto-Kobayashi, Sakae; Suzuki, Toshinori; Negishi, Tomoe

    2016-01-01

    We previously reported that a urate-null strain of Drosophila is hypersensitive to cigarette smoke (CS), and we suggested that CS induces oxidative stress in Drosophila because uric acid is a potent antioxidant. Although the carcinogenic risk of CS exposure is widely recognized; documentation of in vivo genotoxic activity of environmental CS, especially gaseous-phase CS, remains inconclusive. To date, somatic-cell mutations in Drosophila resulting from exposure to CS have not been detected via the somatic mutation and recombination test (wing spot test) with wild-type flies, a widely used Drosophila assay for the detection of somatic-cell mutation; moreover, genotoxicity has not been documented via a DNA repair test that involves DNA repair-deficient Drosophila. In this study, we used a new Drosophila strain (y v ma-l; mwh) to examine the mutagenicity induced by gaseous-phase CS; these flies are urate-null due to a mutation in ma-l, and they are heterozygous for multiple wing hair (mwh), a mutation that functions as a marker for somatic-cell mutation. In an assay with this newly developed strain, a superoxide anion-producing weed-killer, paraquat, exhibited significant mutagenicity; in contrast, paraquat was hardly mutagenic with a wild-type strain. Drosophila larvae were exposed to CS for 2, 4 or 6h, and then kept at 25°C on instant medium until adulthood. After eclosion, mutant spots, which consisted of mutant hairs on wings, were scored. The number of mutant spots increased significantly in an exposure time-dependent manner in the urate-null females (ma-l (-/-)), but not in the urate-positive females (ma-l (+/-)). In this study, we showed that short-term exposure to CS was mutagenic in this in vivo system. In addition, we obtained suggestive data regarding reactive oxygen species production in larva after CS exposure using the fluorescence probe H2DCFDA. These results suggest that oxidative damage, which might be countered by uric acid, was partly responsible

  17. Proficiency test for non-destructive assay of 220 liter radioactive waste drums by gamma assay systems

    SciTech Connect

    Van Velzen, L.P.M.; Bruggeman, M.; Botte, J.

    2007-07-01

    The European Network of Testing Facilities for the Quality Checking of Radioactive Waste Packages (ENTRAP) initiated a feasibility study on how to organize in the most cost effective way an international proficiency tests for non-destructive, gamma-ray based, assay of 220 liter radioactive waste drums in the European Union at a regular time interval of 2 or 3 years. This feasibility study addresses all aspects of proficiency testing on radioactive waste packages including the design of a commonly accepted reference 220 liter drum. This design, based on the international response on a send out questionnaire, includes matrixes, radioactive sources; a solution to overcome the tedious and expensive international transport costs of real or even simulated waste packages, general cost estimation for the organization of, and the participation in the proficiency test. The proposed concept for the proficiency testing and the estimated costs are presented. The participation costs of the first proficiency test are mainly determined by the manufacturing of the non-radioactive 220 liter drum ({+-} 55%). Applied reference sources, transport of the drum and reference sources and participation costs in the proficiency test contribute each about {+-} 15%. (authors)

  18. LANL`s mobile nondestructive assay and examination systems for radioactive wastes

    SciTech Connect

    Taggart, D.P. Betts, S.E.; Vigil, J.J.

    1996-04-09

    The ability to accurately and rapidly measure nuclear material within drums and examine their contents without having to unpack the drums saves time, reduces characterization costs and minimizes radiation exposure. Over the past two years, Los Alamos National Laboratory (LANL) has developed and fielded a suite of mobile nondestructive assay and examination systems for use primarily on its own transuranic (TRU) waste but that also have application to low level, mixed and hazardous wastes. It has become obvious that systems like these are generally useful and have applications at other Department of Energy (DOE) production and environmental technology sites. Mobile capabilities present a potential cost savings where waste drums have to be transported to a fixed NDA facility. In other cases they fill a void where there is no fixed facility available because construction costs are prohibitive (as in the case of small quantity sites) or the available facilities may not meet current or evolving safety standards. Rather than bringing waste to a facility to be characterized, one can bring the characterization capability to the waste. The three systems described are: (1) mobile radiography system; (2) mobile segmented/tomographic gamma scanner; and (3) mobile passive/active neutron assay system.

  19. Dietary sugar promotes systemic TOR activation in Drosophila through AKH-dependent selective secretion of Dilp3

    PubMed Central

    Kim, Jung; Neufeld, Thomas P.

    2015-01-01

    Secreted ligands of the insulin family promote cell growth and maintain sugar homeostasis. Insulin release is tightly regulated in response to dietary conditions, but how insulin producing cells (IPCs) coordinate their responses to distinct nutrient signals is unclear. Here, we show that regulation of insulin secretion in Drosophila larvae has been segregated into distinct branches: whereas amino acids promote secretion of Drosophila insulin-like peptide 2 (Dilp2), circulating sugars promote selective release of Dilp3. Dilp3 is uniquely required for sugar-mediated activation of TOR signaling and suppression of autophagy in the larval fat body. Sugar levels are not sensed directly by the IPCs, but rather by the adipokinetic hormone (AKH)-producing cells of the corpora cardiaca, and we demonstrate that AKH signaling is required in the IPCs for sugar-dependent Dilp3 release. Thus, IPCs integrate multiple cues to regulate secretion of distinct insulin subtypes under varying nutrient conditions. PMID:25882208

  20. The skeletomuscular system of the larva of Drosophila melanogaster (Drosophilidae, Diptera): a contribution to the morphology of a model organism.

    PubMed

    Wipfler, Benjamin; Schneeberg, Katharina; Löffler, Andreas; Hünefeld, Frank; Meier, Rudolf; Beutel, Rolf G

    2013-01-01

    The morphological features of the third instar larva of the most important insect model, Drosophila melanogaster, are documented for the first time using a broad spectrum of modern morphological techniques. External structures of the body wall, the cephaloskeleton, and the musculature are described and illustrated. Additional information about other internal organs is provided. The systematic implications of the findings are discussed briefly. Internal apomorphic features of Brachycera and Cyclorrhapha are confirmed for Drosophila. Despite the intensive investigations of the phylogeny of the megadiverse Diptera, evolutionary reconstructions are still impeded by the scarcity of anatomical data for brachyceran larvae. The available morphological information for the life stages of three insect model organisms -D. melanogaster (Diptera, Drosophilidae), Manduca sexta (Lepidoptera, Sphingidae) and Tribolium castaneum (Coleoptera, Tenebrionidae) - is addressed briefly. The usefulness of a combination of traditional and innovative techniques for an optimized acquisition of anatomical data for different life stages is highlighted. PMID:23010508

  1. β-D-Glucan Assay in Diagnosis and Monitoring the Systemic Candidiasis in a Rat Model

    PubMed Central

    Khodadadi, Hossein; Mirhendi, Hossein; Makimura, Koichi; Satoh, Kazuo; Karimi, Ladan; Izadi, Shahrokh

    2014-01-01

    Background: Determination of β-D-Glucan (BDG) in the serum aids to diagnose the invasive fungal infections. The current study evaluated the diagnostic potential value of BDG assay in monitoring the disease in experimental systemic candidiasis in a rat model. The results can provide a useful preliminary data to improve this approach in developing countries. Objectives: The present study aimed to evaluate β-D-Glucan assay in diagnosis and monitoring the systemic candidiasis in a rat model. Materials and Methods: Twenty one rats were infected with 106 Candida albicans blastospore per rat. Twelve rats were considered as the negative controls (six immunocompromised rats without infection and six intact rats). During a week, every 24 hours the BDG sera level was determined by both Fungitell and Wako kits. To confirm the systemic infection in each rat, the suspensions of their internal organs were cultivated on agar plates and the number of colony forming units (CFU) of C. albicans was counted. Results: All the infected rats were positive with BDG tests. An increasing level of BDG was observed during early days after injection. The cutoff value for discrimination of BDG positive sera was obtained from the negative sera by the Fungitell kit. The sensitivity, specificity, positive and negative predictive values assessed for the Fungitell kit were 95%, 66.6%, 90.47% and 80%, respectively. These criteria for those of Wako were 90%, 83.3%, 94.7% and 71.4%, respectively. Conclusions: While BDG assay seems to be a sensitive and specific adjunctive tool to diagnose and monitor the experimental systemic candidiasis, it seems that measuring the positive cutoff value in different laboratory conditions is necessary for favorable establishment of these tests. PMID:25371794

  2. Monitoring of cell viability and proliferation in hydrogel-encapsulated system by resazurin assay.

    PubMed

    Xiao, Jing; Zhang, Ying; Wang, Jianzheng; Yu, Weiting; Wang, Wei; Ma, Xiaojun

    2010-11-01

    Cell microencapsulation is a promising approach for cell implantation, cell-based gene therapy and large-scale cell culture. For better quality control, it is important to accurately measure the microencapsulated cell viability and proliferation in the culture. A number of assays have been used for this purpose, but limitations arise. In this study, we investigated the feasibility and reliability of resazurin as a cell growth indicator in microencapsulated culture system. According to the experiment data, there was a reversible, time- and dose-dependent growth inhibition as observed for resazurin application in encapsulated cells. A positive relationship was observed between reduction of resazurin and CHO cell number in microcapsule. Moreover, the resazurin assay provided an equivalent result to the commonly used MTT method in determining CHO cell proliferation in APA microcapsule with no notable influence on cell distribution and organization pattern. In conclusion, resazurin assay is offered as a simple, rapid and non-invasive method for in vitro microencapsulated cell viability and proliferation measurement. PMID:20437208

  3. The Drosophila CPEB Protein Orb2 Has a Novel Expression Pattern and Is Important for Asymmetric Cell Division and Nervous System Function

    PubMed Central

    Hafer, Nathaniel; Xu, Shuwa; Bhat, Krishna Moorthi; Schedl, Paul

    2011-01-01

    Cytoplasmic polyadenylation element binding (CPEB) proteins bind mRNAs to regulate their localization and translation. While the first CPEBs discovered were germline specific, subsequent studies indicate that CPEBs also function in many somatic tissues including the nervous system. Drosophila has two CPEB family members. One of these, orb, plays a key role in the establishment of polarity axes in the developing egg and early embryo, but has no known somatic functions or expression outside of the germline. Here we characterize the other Drosophila CPEB, orb2. Unlike orb, orb2 mRNA and protein are found throughout development in many different somatic tissues. While orb2 mRNA and protein of maternal origin are distributed uniformly in early embryos, this pattern changes as development proceeds and by midembryogenesis the highest levels are found in the CNS and PNS. In the embryonic CNS, Orb2 appears to be concentrated in cell bodies and mostly absent from the longitudinal and commissural axon tracts. In contrast, in the adult brain, the protein is seen in axonal and dendritic terminals. Lethal effects are observed for both RNAi knockdowns and orb2 mutant alleles while surviving adults display locomotion and behavioral defects. We also show that orb2 funtions in asymmetric division of stem cells and precursor cells during the development of the embryonic nervous system and mesoderm. PMID:21900268

  4. Neutron fluence rate measurements in a PGNAA 208-liter drum assay system using silicon carbide detectors

    NASA Astrophysics Data System (ADS)

    Dulloo, A. R.; Ruddy, F. H.; Seidel, J. G.; Lee, S.; Petrović, B.; McIlwain, M. E.

    2004-01-01

    Pulsed prompt gamma neutron activation analysis (PGNAA) is being implemented for the nondestructive assay (NDA) of mercury, cadmium and lead in containers of radioactive waste. A PGNAA prototype system capable of assaying 208-liter (55-gallon) drums has already been built and demonstrated. As part of the evaluation of this system, the thermal neutron fluence rate distribution in a drum containing a combustible waste surrogate was measured during PGNAA runs using a silicon carbide neutron detector. The fast charge-collection time of this detector type enabled the investigation of the neutron kinetics at various locations within the matrix during and between pulses of the system's 14-MeV neutron source. As expected, the response of a SiC detector equipped with a lithium-6 fluoride layer is dominated by thermal neutron-induced events between pulses. The measurement results showed that the thermal neutron fluence rate is relatively uniform over a radial depth of several centimeters in the matrix region that contributes a significant fraction of the prompt gamma radiation incident on the system's photon detector.

  5. The Drosophila larval visual system: high-resolution analysis of a simple visual neuropil.

    PubMed

    Sprecher, Simon G; Cardona, Albert; Hartenstein, Volker

    2011-10-01

    The task of the visual system is to translate light into neuronal encoded information. This translation of photons into neuronal signals is achieved by photoreceptor neurons (PRs), specialized sensory neurons, located in the eye. Upon perception of light the PRs will send a signal to target neurons, which represent a first station of visual processing. Increasing complexity of visual processing stems from the number of distinct PR subtypes and their various types of target neurons that are contacted. The visual system of the fruit fly larva represents a simple visual system (larval optic neuropil, LON) that consists of 12 PRs falling into two classes: blue-senstive PRs expressing Rhodopsin 5 (Rh5) and green-sensitive PRs expressing Rhodopsin 6 (Rh6). These afferents contact a small number of target neurons, including optic lobe pioneers (OLPs) and lateral clock neurons (LNs). We combine the use of genetic markers to label both PR subtypes and the distinct, identifiable sets of target neurons with a serial EM reconstruction to generate a high-resolution map of the larval optic neuropil. We find that the larval optic neuropil shows a clear bipartite organization consisting of one domain innervated by PRs and one devoid of PR axons. The topology of PR projections, in particular the relationship between Rh5 and Rh6 afferents, is maintained from the nerve entering the brain to the axon terminals. The target neurons can be subdivided according to neurotransmitter or neuropeptide they use as well as the location within the brain. We further track the larval optic neuropil through development from first larval instar to its location in the adult brain as the accessory medulla. PMID:21781960

  6. The Drosophila larval visual system: high-resolution analysis of a simple visual neuropil

    PubMed Central

    Sprecher, Simon G.; Cardona, Albert; Hartenstein, Volker

    2012-01-01

    The task of the visual system is to translate light into neuronal encoded information. This translation of photons into neuronal signals is achieved by photoreceptor neurons (PRs), specialized sensory neurons, located in the eye. Upon perception of light the PRs will send a signal to target neurons, which represent a first station of visual processing. Increasing complexity of visual processing stems from the number of distinct PR-subtypes and their various types of target neurons that are contacted. The visual system of the fruit fly larva represents a simple visual system (larval optic neuropil, LON) that consists of 12 PRs falling into two classes: blue-senstive PRs expressing Rhodopsin 5 (Rh5) and green-sensitive PRs expressing Rhodopsin 6 (Rh6). These afferents contact a small number of target neurons, including optic lobe pioneers (OLPs) and lateral clock neurons (LNs). We combine the use of genetic markers to label both PR-subtypes and the distinct, identifiable sets of target neurons with a serial EM reconstruction to generate a high-resolution map of the larval optic neuropil. We find that the larval optic neuropil shows a clear bipartite organization consisting of one domain innervated by PRs and one devoid of PR axons. The topology of PR projections, in particular the relationship between Rh5 and Rh6 afferents, is maintained from the nerve entering the brain to the axon terminals. The target neurons can be subdivided according to neurotransmitter or neuropeptide they use as well as the location within the brain. We further track the larval optic neuropil through development from first larval instar to its location in the adult brain as the accessory medulla. PMID:21781960

  7. Determining Airborne Concentrations of Spatial Repellent Chemicals in Mosquito Behavior Assay Systems

    PubMed Central

    Martin, Nicholas J.; Smith, Philip A.; Achee, Nicole L.; DeLong, Gerald T.

    2013-01-01

    Background Mosquito behavior assays have been used to evaluate the efficacy of vector control interventions to include spatial repellents (SR). Current analytical methods are not optimized to determine short duration concentrations of SR active ingredients (AI) in air spaces during entomological evaluations. The aim of this study was to expand on our previous research to further validate a novel air sampling method to detect and quantitate airborne concentrations of a SR under laboratory and field conditions. Methodology/Principal Findings A thermal desorption (TD) gas chromatography-mass spectrometry (GC-MS) method was used to determine the amount of dichlorodiphenyltrichloroethane (DDT) in samples of air. During laboratory experiments, 1 L volumes of air were collected over 10 min intervals from a three-chamber mosquito behavior assay system. Significantly higher levels of airborne DDT were measured in the chamber containing textiles treated with DDT compared to chambers free of AI. In the field, 57 samples of air were collected from experimental huts with and without DDT for onsite analysis. Airborne DDT was detected in samples collected from treated huts. The mean DDT air concentrations in these two huts over a period of four days with variable ambient temperature were 0.74 µg/m3 (n = 17; SD = 0.45) and 1.42 µg/m3 (n = 30; SD = 0.96). Conclusions/Significance The results from laboratory experiments confirmed that significantly different DDT exposure conditions existed in the three-chamber system establishing a chemical gradient to evaluate mosquito deterrency. The TD GC-MS method addresses a need to measure short-term (<1 h) SR concentrations in small volume (<100 L) samples of air and should be considered for standard evaluation of airborne AI levels in mosquito behavior assay systems. Future studies include the use of TD GC-MS to measure other semi-volatile vector control compounds. PMID:24015195

  8. The development of an expert system for the characterization of waste assay data

    SciTech Connect

    Bridges, S.; Hodges, J.; Sparrow, C.

    1997-11-01

    Containers of transuranic and low-level alpha contaminated waste generated as a byproduct of Department of Energy defense-related programs must be characterized before their proper disposition can be determined. Nondestructive assay methods are the most desirable means for assessing the mass and activity of the entrained transuranic radionuclides. However, there are other sources of information that may be useful in the characterization of the entrained waste (e.g., container manifests, information about the generation process, and destructive assay techniques performed on representative samples). This paper describes initial work on an expert system being developed to analyze and characterize containerized radiological waste. This system is being developed by scientists at the Mississippi State University Diagnostic and Instrumentation Laboratory (DIAL) in collaboration with scientists at the Idaho National Engineering Laboratory. The DIAL scientists are responsible for (1) the development of techniques to represent and reason with evidence from a variety of sources, and (2) the development of appropriate method(s) to represent and reason with confidence levels associated with that evidence. This paper describes exploratory versions of the expert system developed to evaluate four techniques for representing and reasoning with the confidence in the evidence: MYCIN-style certainty factors, Dempster-Shafer Theory, Bayesian networks, and fuzzy logic. 16 refs., 8 figs., 4 tabs.

  9. Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system.

    PubMed

    Hjerrild, Kathryn A; Jin, Jing; Wright, Katherine E; Brown, Rebecca E; Marshall, Jennifer M; Labbé, Geneviève M; Silk, Sarah E; Cherry, Catherine J; Clemmensen, Stine B; Jørgensen, Thomas; Illingworth, Joseph J; Alanine, Daniel G W; Milne, Kathryn H; Ashfield, Rebecca; de Jongh, Willem A; Douglas, Alexander D; Higgins, Matthew K; Draper, Simon J

    2016-01-01

    The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has recently emerged as a leading candidate antigen against the blood-stage human malaria parasite. However it has proved challenging to identify a heterologous expression platform that can produce a soluble protein-based vaccine in a manner compliant with current Good Manufacturing Practice (cGMP). Here we report the production of full-length PfRH5 protein using a cGMP-compliant platform called ExpreS(2), based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system. Five sequence variants of PfRH5 were expressed that differed in terms of mutagenesis strategies to remove potential N-linked glycans. All variants bound the PfRH5 receptor basigin and were recognized by a panel of monoclonal antibodies. Analysis following immunization of rabbits identified quantitative and qualitative differences in terms of the functional IgG antibody response against the P. falciparum parasite. The antibodies induced by one protein variant were shown to be qualitatively similar to responses induced by other vaccine platforms. This work identifies Drosophila S2 cells as a clinically-relevant platform suited for the production of 'difficult-to-make' proteins from Plasmodium parasites, and identifies a PfRH5 sequence variant that can be used for clinical production of a non-glycosylated, soluble full-length protein vaccine immunogen. PMID:27457156

  10. Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system

    PubMed Central

    Hjerrild, Kathryn A.; Jin, Jing; Wright, Katherine E.; Brown, Rebecca E.; Marshall, Jennifer M.; Labbé, Geneviève M.; Silk, Sarah E.; Cherry, Catherine J.; Clemmensen, Stine B.; Jørgensen, Thomas; Illingworth, Joseph J.; Alanine, Daniel G. W.; Milne, Kathryn H.; Ashfield, Rebecca; de Jongh, Willem A.; Douglas, Alexander D.; Higgins, Matthew K.; Draper, Simon J.

    2016-01-01

    The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has recently emerged as a leading candidate antigen against the blood-stage human malaria parasite. However it has proved challenging to identify a heterologous expression platform that can produce a soluble protein-based vaccine in a manner compliant with current Good Manufacturing Practice (cGMP). Here we report the production of full-length PfRH5 protein using a cGMP-compliant platform called ExpreS2, based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system. Five sequence variants of PfRH5 were expressed that differed in terms of mutagenesis strategies to remove potential N-linked glycans. All variants bound the PfRH5 receptor basigin and were recognized by a panel of monoclonal antibodies. Analysis following immunization of rabbits identified quantitative and qualitative differences in terms of the functional IgG antibody response against the P. falciparum parasite. The antibodies induced by one protein variant were shown to be qualitatively similar to responses induced by other vaccine platforms. This work identifies Drosophila S2 cells as a clinically-relevant platform suited for the production of ‘difficult-to-make’ proteins from Plasmodium parasites, and identifies a PfRH5 sequence variant that can be used for clinical production of a non-glycosylated, soluble full-length protein vaccine immunogen. PMID:27457156

  11. Identification and affinity of very high affinity binding sites for the phenylalkylamine series of Ca/sup +/ channel blockers in the Drosophila nervous system

    SciTech Connect

    Pauron, D.; Qar, J.; Barhanin, J.; Fournier, D.; Cuany, A.; Pralavorio, M.; Berge, J.B.; Lazdunski, M.

    1987-10-06

    The interaction of putative Ca/sup 2 +/ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-(/sup 3/H)D888 and (+/-)-(/sup 3/H)verapamil. These ligands recognize a single class of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a K/sub d/ value as exceptional low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca/sup 2 +/ channel blockers as well as bepridil inhibited (-)-(/sup 3/H)D888 binding in a competitive way with K/sub d/ values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor was used in photoaffinity experiments. A protein of M/sub r/ 135,000 +/- 5000 was specifically labeled after ultraviolet irradiation.

  12. Optogenetics in Drosophila Neuroscience.

    PubMed

    Riemensperger, Thomas; Kittel, Robert J; Fiala, André

    2016-01-01

    Optogenetic techniques enable one to target specific neurons with light-sensitive proteins, e.g., ion channels, ion pumps, or enzymes, and to manipulate their physiological state through illumination. Such artificial interference with selected elements of complex neuronal circuits can help to determine causal relationships between neuronal activity and the effect on the functioning of neuronal circuits controlling animal behavior. The advantages of optogenetics can best be exploited in genetically tractable animals whose nervous systems are, on the one hand, small enough in terms of cell numbers and to a certain degree stereotypically organized, such that distinct and identifiable neurons can be targeted reproducibly. On the other hand, the neuronal circuitry and the behavioral repertoire should be complex enough to enable one to address interesting questions. The fruit fly Drosophila melanogaster is a favorable model organism in this regard. However, the application of optogenetic tools to depolarize or hyperpolarize neurons through light-induced ionic currents has been difficult in adult flies. Only recently, several variants of Channelrhodopsin-2 (ChR2) have been introduced that provide sufficient light sensitivity, expression, and stability to depolarize central brain neurons efficiently in adult Drosophila. Here, we focus on the version currently providing highest photostimulation efficiency, ChR2-XXL. We exemplify the use of this optogenetic tool by applying it to a widely used aversive olfactory learning paradigm. Optogenetic activation of a population of dopamine-releasing neurons mimics the reinforcing properties of a punitive electric shock typically used as an unconditioned stimulus. In temporal coincidence with an odor stimulus this artificially induced neuronal activity causes learning of the odor signal, thereby creating a light-induced memory. PMID:26965122

  13. Microchip-based enzyme-linked immunosorbent assay (microELISA) system with thermal lens detection.

    PubMed

    Sato, Kiichi; Yamanaka, Maho; Hagino, Tomokazu; Tokeshi, Manabu; Kimura, Hiroko; Kitamori, Takehiko

    2004-12-01

    A microchip-based enzyme-linked immunosorbent assay (microELISA) system was developed and interferon-gamma was successfully determined. The system was composed of a microchip with a Y-shaped microchannel and a dam structure, polystyrene microbeads, and a thermal lens microscope (TLM). All reactions required for the immunoassay were done in the microchannel by successive introduction of a sample and regents. The enzyme reaction product, in a liquid phase, was detected downstream in the channel using the TLM as substrate solution was injected. The antigen-antibody reaction time was shortened by the microchip integration. The limit of the determination was improved by adopting the enzyme label. Moreover, detection procedures were greatly simplified and required time for the detection was significantly cut. The system has good potential to be developed as a small and automated high throughput analyzer. PMID:15570367

  14. Genotoxicity of nano/microparticles in in vitro micronuclei, in vivo comet and mutation assay systems

    PubMed Central

    Totsuka, Yukari; Higuchi, Takashi; Imai, Toshio; Nishikawa, Akiyoshi; Nohmi, Takehiko; Kato, Tatsuya; Masuda, Shuich; Kinae, Naohide; Hiyoshi, Kyoko; Ogo, Sayaka; Kawanishi, Masanobu; Yagi, Takashi; Ichinose, Takamichi; Fukumori, Nobutaka; Watanabe, Masatoshi; Sugimura, Takashi; Wakabayashi, Keiji

    2009-01-01

    Background Recently, manufactured nano/microparticles such as fullerenes (C60), carbon black (CB) and ceramic fiber are being widely used because of their desirable properties in industrial, medical and cosmetic fields. However, there are few data on these particles in mammalian mutagenesis and carcinogenesis. To examine genotoxic effects by C60, CB and kaolin, an in vitro micronuclei (MN) test was conducted with human lung cancer cell line, A549 cells. In addition, DNA damage and mutations were analyzed by in vivo assay systems using male C57BL/6J or gpt delta transgenic mice which were intratracheally instilled with single or multiple doses of 0.2 mg per animal of particles. Results In in vitro genotoxic analysis, increased MN frequencies were observed in A549 cells treated with C60, CB and kaolin in a dose-dependent manner. These three nano/microparticles also induced DNA damage in the lungs of C57BL/6J mice measured by comet assay. Moreover, single or multiple instillations of C60 and kaolin, increased either or both of gpt and Spi- mutant frequencies in the lungs of gpt delta transgenic mice. Mutation spectra analysis showed transversions were predominant, and more than 60% of the base substitutions occurred at G:C base pairs in the gpt genes. The G:C to C:G transversion was commonly increased by these particle instillations. Conclusion Manufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems. PMID:19725983

  15. An inventory of peroxisomal proteins and pathways in Drosophila melanogaster

    PubMed Central

    Faust, Joseph E.; Verma, Avani; Peng, Chengwei; McNew, James A.

    2012-01-01

    Peroxisomes are ubiquitous organelles housing a variety of essential biochemical pathways. Peroxisome dysfunction causes a spectrum of human diseases known as peroxisome biogenesis disorders (PBD). While much is known regarding the mechanism of peroxisome biogenesis, it is still unclear how peroxisome dysfunction leads to the disease state. Several recent studies have shown that mutations in Drosophila peroxin genes cause phenotypes similar to those seen in humans with PBDs suggesting that Drosophila might be a useful system to model PBDs. We have analyzed the proteome of Drosophila to identify the proteins involved in peroxisomal biogenesis and homeostasis as well as metabolic enzymes that function within the organelle. The subcellular localization of five of these predicted peroxisomal proteins was confirmed. Similar to C. elegans, Drosophila appears to only utilize the peroxisome targeting signal (PTS) type 1 system for matrix protein import. This work will further our understanding of peroxisomes in Drosophila and add to the usefulness of this emerging model system. PMID:22758915

  16. Optogenetic pacing in Drosophila melanogaster

    PubMed Central

    Alex, Aneesh; Li, Airong; Tanzi, Rudolph E.; Zhou, Chao

    2015-01-01

    Electrical stimulation is currently the gold standard for cardiac pacing. However, it is invasive and nonspecific for cardiac tissues. We recently developed a noninvasive cardiac pacing technique using optogenetic tools, which are widely used in neuroscience. Optogenetic pacing of the heart provides high spatial and temporal precisions, is specific for cardiac tissues, avoids artifacts associated with electrical stimulation, and therefore promises to be a powerful tool in basic cardiac research. We demonstrated optogenetic control of heart rhythm in a well-established model organism, Drosophila melanogaster. We developed transgenic flies expressing a light-gated cation channel, channelrhodopsin-2 (ChR2), specifically in their hearts and demonstrated successful optogenetic pacing of ChR2-expressing Drosophila at different developmental stages, including the larva, pupa, and adult stages. A high-speed and ultrahigh-resolution optical coherence microscopy imaging system that is capable of providing images at a rate of 130 frames/s with axial and transverse resolutions of 1.5 and 3.9 μm, respectively, was used to noninvasively monitor Drosophila cardiac function and its response to pacing stimulation. The development of a noninvasive integrated optical pacing and imaging system provides a novel platform for performing research studies in developmental cardiology. PMID:26601299

  17. Application of microchip assay system for the measurement of C-reactive protein in human saliva.

    PubMed

    Christodoulides, Nicolaos; Mohanty, Sanghamitra; Miller, Craig S; Langub, M Chris; Floriano, Pierre N; Dharshan, Priya; Ali, Mehnaaz F; Bernard, Bruce; Romanovicz, Dwight; Anslyn, Eric; Fox, Philip C; McDevitt, John T

    2005-03-01

    In the last decade, saliva has been advocated as a non-invasive alternative to blood as a diagnostic fluid. However, use of saliva has been hindered by the inadequate sensitivity of current methods to detect the lower salivary concentrations of many constituents compared to serum. Furthermore, developments in the areas related to lab-on-a-chip systems for saliva-based point of care diagnostics are complicated by the high viscosity and heterogeneous properties associated with this diagnostic fluid. The biomarker C-reactive protein (CRP) is an acute phase reactant and a well-accepted indicator of inflammation. Numerous clinical studies have established elevated serum CRP as a strong, independent risk factor for the development of cardiovascular disease (CVD). CVD has also been associated with oral infections (i.e. periodontal diseases) and there is evidence that systemic CRP may be a link between the two. Clinical measurements of CRP in serum are currently performed with "high sensitivity" CRP (hsCRP) enzyme-linked immunosorbent assay (ELISA) tests that lack the sensitivity for the detection of this important biomarker in saliva. Because measurement of salivary CRP may represent a novel approach for diagnosing and monitoring chronic inflammatory disease, including CVD and periodontal diseases, the objective of this study was to apply an ultra-sensitive microchip assay system for the measurement of CRP in human saliva. Here, we describe this novel lab-on-a-chip system in its first application for the measurement of CRP in saliva and demonstrate its advantages over the traditional ELISA method. The increased sensitivity of the microchip system (10 pg ml(-1) of CRP with 1000-fold dilution of saliva sample) is attributed to its inherent increased signal to noise ratio, resulting from the higher bead surface area available for antigen/antibody interactions and the high stringency washes associated with this approach. Finally, the microchip assay system was utilized in

  18. Drosophila glia use a conserved cotransporter mechanism to regulate extracellular volume

    PubMed Central

    Leiserson, William M.; Forbush, Biff; Keshishian, Haig

    2010-01-01

    The nervous system is protected by blood barriers that use multiple systems to control extracellular solute composition, osmotic pressure, and fluid volume. In the human nervous system, misregulation of the extracellular volume poses serious health threats. Here we show that the glial cells that form the Drosophila blood-nerve barrier have a conserved molecular mechanism that regulates extracellular volume: the Serine/Threonine kinase Fray, which we previously showed is an ortholog of mammalian PASK/SPAK; and the Na-K-Cl cotransporter NCC69, which we show is an ortholog of human NKCC1. In mammals, PASK/SPAK binds to NKCC1 and regulates its activity. In Drosophila, larvae mutant for NCC69 develop a peripheral neuropathy, where fluid accumulates between glia and axons. The accumulation of fluid has no detectable impact on action potential conduction, suggesting that the role of NCC69 is to maintain volume or osmotic homeostasis. Drosophila NCC69 has kinetics similar to human NKCC1, and NKCC1 can rescue NCC69, suggesting that they function in a conserved physiological mechanism. We show that fray and NCC69 are coexpressed in nerve glia, interact in a yeast-two-hybrid assay, and have an essentially identical bulging nerve phenotype. We propose that normally functioning nerves generate extracellular solutes that are removed by NCC69 under the control of Fray. This mechanism may perform a similar role in humans, given that NKCC1 is expressed at the blood-brain barrier. PMID:21125654

  19. Staphylococcus aureus Cell Extract Transcription-Translation Assay: Firefly Luciferase Reporter System for Evaluating Protein Translation Inhibitors

    PubMed Central

    Murray, Robert W.; Melchior, Earline P.; Hagadorn, Jeanne C.; Marotti, Keith R.

    2001-01-01

    The promoter for the Staphylococcus aureus capsule polysaccharide synthesis gene (cap1A) was cloned in front of the firefly luciferase gene for use in a cell extract S. aureus transcription-translation system. The assay is rapid, reproducible, and sensitive and has a lower background level than the radiolabeled amino acid incorporation translation assays. We present data evaluating a transcription inhibitor and a number of protein translation inhibitors in this system. PMID:11353649

  20. Conserved Mechanisms of Tumorigenesis in the Drosophila Adult Midgut

    PubMed Central

    Martorell, Òscar; Merlos-Suárez, Anna; Campbell, Kyra; Barriga, Francisco M.; Christov, Christo P.; Miguel-Aliaga, Irene; Batlle, Eduard; Casanova, Jordi; Casali, Andreu

    2014-01-01

    Whereas the series of genetic events leading to colorectal cancer (CRC) have been well established, the precise functions that these alterations play in tumor progression and how they disrupt intestinal homeostasis remain poorly characterized. Activation of the Wnt/Wg signaling pathway by a mutation in the gene APC is the most common trigger for CRC, inducing benign lesions that progress to carcinomas due to the accumulation of other genetic alterations. Among those, Ras mutations drive tumour progression in CRC, as well as in most epithelial cancers. As mammalian and Drosophila's intestines share many similarities, we decided to explore the alterations induced in the Drosophila midgut by the combined activation of the Wnt signaling pathway with gain of function of Ras signaling in the intestinal stem cells. Here we show that compound Apc-Ras clones, but not clones bearing the individual mutations, expand as aggressive intestinal tumor-like outgrowths. These lesions reproduce many of the human CRC hallmarks such as increased proliferation, blockade of cell differentiation and cell polarity and disrupted organ architecture. This process is followed by expression of tumoral markers present in human lesions. Finally, a metabolic behavioral assay shows that these flies suffer a progressive deterioration in intestinal homeostasis, providing a simple readout that could be used in screens for tumor modifiers or therapeutic compounds. Taken together, our results illustrate the conservation of the mechanisms of CRC tumorigenesis in Drosophila, providing an excellent model system to unravel the events that, upon mutation in Apc and Ras, lead to CRC initiation and progression. PMID:24516653

  1. Evidence for an Inducible Repair-Recombination System in the Female Germ Line of Drosophila Melanogaster. II. Differential Sensitivity to Gamma Rays

    PubMed Central

    Laurencon, A.; Bregliano, J. C.

    1995-01-01

    In a previous paper, we reported that the reactivity level, which regulates the frequency of transposition of I factor, a LINE element-like retrotransposon, is enhanced by the same agents that induce the SOS response in Escherichia coli. In this report, we describe experimental evidence that, for identical genotypes, the reactivity levels correlate with the sensitivity of oogenesis to gamma rays, measured by the number of eggs laid and by frequency of dominant lethals. This strongly supports the hypothesis that the reactivity level is one manifestation of an inducible DNA repair system taking place in the female germ line of Drosophila melanogaster. The implications of this finding for the understanding of the regulation of I factor are discussed and some other possible biological roles of this system are outlined. PMID:8647394

  2. Waste assay and mass balance for the decontamination and volume reduction system at LANL

    SciTech Connect

    Gruetzmacher, Kathleen M.; Ferran, Scott G.; Garner, Scott E.; Romero, Mike J.; Christensen, Davis V.; Bustos, Roland M.

    2003-07-01

    The Decontamination and Volume Reduction System (DVRS) operated by the Solid Waste Operations (SWO) Group at Los Alamos National Laboratory (LANL) processes large volume, legacy radioactive waste items. Waste boxes, in sizes varying from 4 ft x 4 ft x 8 ft to 10 ft x 12 ft x 40 ft, are assayed prior to entry into the processing building. Inside the building, the waste items are removed from their container, decontaminated and/or size reduced if necessary, and repackaged for shipment to the Waste Isolation Pilot Plant (WIPP) or on-site low-level waste disposal. The repackaged items and any secondary waste produced (e.g., personal protective equipment) are assayed again at the end of the process and a mass balance is done to determine whether there is any significant hold-up material left in the DVRS building. The DVRS building is currently classed as a radiological facility, with a building limit of 0.52 Ci of Pu239 and Am241, and 0.62 Ci of Pu238, the most common radionuclides processed. This requires tight controls on the flow of nuclear material. The large volume of the initial waste packages, the (relatively) small amounts of radioactive material in them, and the tight ceiling on the building inventory require accurate field measurements of the nuclear material. This paper describes the radioactive waste measurement techniques, the computer modeling used to determine the amount of nuclear material present in a waste package, the building inventory database, and the DVRS process itself. Future plans include raising the limit on the nuclear material inventory allowed in the building to accommodate higher activity waste packages. All DOE sites performing decontamination and decommissioning of radioactive process equipment face challenges related to waste assay and inventory issues. This paper describes an ongoing operation, incorporating lessons learned over the life of the project to date.

  3. Limitations of the ferrozine method for quantitative assay of mineral systems for ferrous and total iron

    NASA Astrophysics Data System (ADS)

    Anastácio, Alexandre S.; Harris, Brittany; Yoo, Hae-In; Fabris, José Domingos; Stucki, Joseph W.

    2008-10-01

    The quantitative assay of clay minerals, soils, and sediments for Fe(II) and total Fe is fundamental to understanding biogeochemical cycles occurring therein. The commonly used ferrozine method was originally designed to assay extracted forms of Fe(II) from non-silicate aqueous systems. It is becoming, however, increasingly the method of choice to report the total reduced state of Fe in soils and sediments. Because Fe in soils and sediments commonly exists in the structural framework of silicates, extraction by HCl, as used in the ferrozine method, fails to dissolve all of the Fe. The phenanthroline (phen) method, on the other hand, was designed to assay silicate minerals for Fe(II) and total Fe and has been proven to be highly reliable. In the present study potential sources of error in the ferrozine method were evaluated by comparing its results to those obtained by the phen method. Both methods were used to analyze clay mineral and soil samples for Fe(II) and total Fe. Results revealed that the conventional ferrozine method under reports total Fe in samples containing Fe in silicates and gives erratic results for Fe(II). The sources of error in the ferrozine method are: (1) HCl fails to dissolve silicates and (2) if the analyte solution contains Fe 3+, the analysis for Fe 2+ will be photosensitive, and reported Fe(II) values will likely be greater than the actual amount in solution. Another difficulty with the ferrozine method is that it is tedious and much more labor intensive than the phen method. For these reasons, the phen method is preferred and recommended. Its procedure is simpler, takes less time, and avoids the errors found in the ferrozine method.

  4. Genotoxicity of organic extracts of airborne particles in somatic cells of Drosophila melanogaster.

    PubMed

    Delgado-Rodríguez, A; Ortíz-Marttelo, R; Villalobos-Pietrini, R; Gómez-Arroyo, S; Graf, U

    1999-07-01

    Complex mixtures extracted from air filters exposed for 24 h in two sessions (27 July and 02 August 1991) and at two locations (Merced, downtown, and Pedregal de San Angel, south-west) in Mexico City were analysed. The organic extracts were from airborne particles equal or smaller than 10 microns (PM10), and from total suspended particles (TSP). These organic extracts were assayed in the somatic mutation and recombination test (SMART) in wings of Drosophila melanogaster using two different crosses as well as in the Salmonella/microsome assay using strain TA98 with and without S9 fraction. The presence of polycyclic aromatic hydrocarbons (PAH) in the extracts was determined by gas chromatography. The genotoxic activities observed in the two test systems were comparable with the indirect mutagens producing greater response than the direct mutagens. The quantities of particulate matter as well as the genotoxic activities were higher on 02 August than on 27 July 1991 for both locations. The amounts of airborne particles and the resulting genotoxic activities were higher at Merced than at Pedregal. In both biological systems, PM10 were more genotoxic than TSP. These results demonstrate the sensitivity of the Drosophila wing SMART-which is an in vivo eukaryotic genotoxicity assay-as a biological monitor of environmental pollution related to airborne particles. PMID:10377966

  5. Drosophila Blastorderm Analysis Software

    SciTech Connect

    2006-10-25

    PointCloudMake analyzes 3D fluorescent images of whole Drosophila embryo and produces a table-style "PointCloud" file which contains the coordinates and volumes of all the nuclei, cells, their associated relative gene expression levels along with morphological features of the embryo. See: Luengo Hendrix et at 2006 3D Morphology and Gene Expression in the Drosophila Blastoderm at Cellular Resolution manuscript submitted LBNL # LBNL-60178 Knowles DW, Keranen SVE, Biggin M. Sudar S (2002) Mapping organism expression levels at cellular resolution in developing Drosophila. In: Conchello JA, Cogswell CJ, Wilson T, editors. Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX. pp. 57-64

  6. TIF-IA-Dependent Regulation of Ribosome Synthesis in Drosophila Muscle Is Required to Maintain Systemic Insulin Signaling and Larval Growth

    PubMed Central

    Ghosh, Abhishek; Rideout, Elizabeth J.; Grewal, Savraj S.

    2014-01-01

    The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis—a limiting step in ribosome biogenesis—via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2—a secreted factor that binds and inhibits dILP activity—from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis. PMID:25356674

  7. Leigh Syndrome in Drosophila melanogaster

    PubMed Central

    Da-Rè, Caterina; von Stockum, Sophia; Biscontin, Alberto; Millino, Caterina; Cisotto, Paola; Zordan, Mauro A.; Zeviani, Massimo; Bernardi, Paolo; De Pittà, Cristiano; Costa, Rodolfo

    2014-01-01

    Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LSSurf1 patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. We established a Drosophila melanogaster model of LS based on the post-transcriptional silencing of CG9943, the Drosophila homolog of SURF1. Knockdown of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knockdown individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R+ cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. We conclude that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS. PMID:25164807

  8. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    SciTech Connect

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.

    2005-10-14

    Cell communication modulates numerous biological processes including proliferation, apoptosis, motility, invasion and differentiation. Correspondingly, there has been significant interest in the development of surface display strategies for the presentation of signaling molecules to living cells. This effort has primarily focused on naturally surface-bound ligands, such as extracellular matrix components and cell membranes. Soluble ligands (e.g. growth factors and cytokines) play an important role in intercellular communications, and their display in a surface-bound format would be of great utility in the design of array-based live cell assays. Recently, several cell microarray systems that display cDNA, RNAi, or small molecules in a surface array format were proven to be useful in accelerating high-throughput functional genetic studies and screening therapeutic agents. These surface display methods provide a flexible platform for the systematic, combinatorial investigation of genes and small molecules affecting cellular processes and phenotypes of interest. In an analogous sense, it would be an important advance if one could display soluble signaling ligands in a surface assay format that allows for systematic, patterned presentation of soluble ligands to live cells. Such a technique would make it possible to examine cellular phenotypes of interest in a parallel format with soluble signaling ligands as one of the display parameters. Herein we report a ligand-modified fluid supported lipid bilayer (SLB) assay system that can be used to functionally display soluble ligands to cells in situ (Figure 1A). By displaying soluble ligands on a SLB surface, both solution behavior (the ability to become locally enriched by reaction-diffusion processes) and solid behavior (the ability to control the spatial location of the ligands in an open system) could be combined. The method reported herein benefits from the naturally fluid state of the supported membrane, which allows

  9. Dictyostelium discoideum Ax2 as an Assay System for Screening of Pharmacological Chaperones for Phenylketonuria Mutations.

    PubMed

    Kim, Yu-Min; Yang, Yun Gyeong; Kim, Hye-Lim; Park, Young Shik

    2015-06-01

    In this study, we developed an assay system for missense mutations in human phenylalanine hydroxylases (hPAHs). To demonstrate the reliability of the system, eight mutant proteins (F39L, K42I, L48S, I65T, R252Q, L255V, S349L, and R408W) were expressed in a mutant strain (pah(-)) of Dictyostelium discoideum Ax2 disrupted in the indigenous gene encoding PAH. The transformed pah- cells grown in FM minimal medium were measured for growth rate and PAH activity to reveal a positive correlation between them. The protein level of hPAH was also determined by western blotting to show the impact of each mutation on protein stability and catalytic activity. The result was highly compatible with the previous ones obtained from other expression systems, suggesting that Dictyostelium is a dependable alternative to other expression systems. Furthermore, we found that both the protein level and activity of S349L and R408W, which were impaired severely in protein stability, were rescued in HL5 nutrient medium. Although the responsible component(s) remains unidentified, this unexpected finding showed an important advantage of our expression system for studying unstable proteins. As an economic and stable cell-based expression system, our development will contribute to mass-screening of pharmacological chaperones for missense PAH mutations as well as to the in-depth characterization of individual mutations. PMID:25563416

  10. Mutagenicity of Tween 80-solvated mild gasification products in the Ames salmonella microsomal assay system

    SciTech Connect

    Not Available

    1992-01-13

    The results of the Tween 80-solvated Ames testing of six mild gasification samples indicate significant mutagenic activity only in the composite materials (MG-119 and MG-120), previously suspected from the DMSO-solvated assays, which had shown some variable but ultimately insignificant mutagenic responses. The activity of these samples from the Tween 80-solvated assays was quite low when compared to either the positive controls or the SRC-II HD coal-liquefaction reference material. The class of mutagenic activity expressed by these samples solvated in Tween 80 was that of an indirect-acting, frameshift mutagen(s) since significant activity was found only on tester strain TA98 in the presence of the metabolic activation fraction (S9). Because DMSO and other solvents have been shown to affect the mutagenic activity of certain pure chemicals, the possibility of solvent/mutagen interactions in complex mixtures such as coal-derived liquids exists. Thus, the testing of the genotoxic activity of undefined, chemically complex compounds may require the use of at least two solvent systems to reduce the possibility of artifactual findings. 10 refs., 4 tabs.

  11. Evaluation of a CLEIA automated assay system for the detection of a panel of tumor markers.

    PubMed

    Falzarano, Renato; Viggiani, Valentina; Michienzi, Simona; Longo, Flavia; Tudini, Silvestra; Frati, Luigi; Anastasi, Emanuela

    2013-10-01

    Tumor markers are commonly used to detect a relapse of disease in oncologic patients during follow-up. It is important to evaluate new assay systems for a better and more precise assessment, as a standardized method is currently lacking. The aim of this study was to assess the concordance between an automated chemiluminescent enzyme immunoassay system (LUMIPULSE® G1200) and our reference methods using seven tumor markers. Serum samples from 787 subjects representing a variety of diagnoses, including oncologic, were analyzed using LUMIPULSE® G1200 and our reference methods. Serum values were measured for the following analytes: prostate-specific antigen (PSA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 19-9 (CA19-9), and cytokeratin 19 fragment (CYFRA 21-1). For the determination of CEA, AFP, and PSA, an automatic analyzer based on chemiluminescence was applied as reference method. To assess CYFRA 21-1, CA125, CA19-9, and CA15-3, an immunoradiometric manual system was employed. Method comparison by Passing-Bablok analysis resulted in slopes ranging from 0.9728 to 1.9089 and correlation coefficients from 0.9977 to 0.9335. The precision of each assay was assessed by testing six serum samples. Each sample was analyzed for all tumor biomarkers in duplicate and in three different runs. The coefficients of variation were less than 6.3 and 6.2 % for within-run and between-run variation, respectively. Our data suggest an overall good interassay agreement for all markers. The comparison with our reference methods showed good precision and reliability, highlighting its usefulness in clinical laboratory's routine. PMID:23775009

  12. Dietary sugar promotes systemic TOR activation in Drosophila through AKH-dependent selective secretion of Dilp3.

    PubMed

    Kim, Jung; Neufeld, Thomas P

    2015-01-01

    Secreted ligands of the insulin family promote cell growth and maintain sugar homeostasis. Insulin release is tightly regulated in response to dietary conditions, but how insulin-producing cells (IPCs) coordinate their responses to distinct nutrient signals is unclear. Here we show that regulation of insulin secretion in Drosophila larvae has been segregated into distinct branches-whereas amino acids promote the secretion of Drosophila insulin-like peptide 2 (Dilp2), circulating sugars promote the selective release of Dilp3. Dilp3 is uniquely required for the sugar-mediated activation of TOR signalling and suppression of autophagy in the larval fat body. Sugar levels are not sensed directly by the IPCs, but rather by the adipokinetic hormone (AKH)-producing cells of the corpora cardiaca, and we demonstrate that AKH signalling is required in the IPCs for sugar-dependent Dilp3 release. Thus, IPCs integrate multiple cues to regulate the secretion of distinct insulin subtypes under varying nutrient conditions. PMID:25882208

  13. Genotoxicity studies with the unstable Zeste-White (UZ) system of Drosophila melanogaster: Results with ten carcinogenic compounds

    SciTech Connect

    Batiste-Alentorn, M.; Xamena, N.; Creus, A.; Marcos, R. )

    1991-01-01

    To increase the number of chemicals tested using the Zeste-White (UZ) somatic mutation assay, ten selected carcinogens (acetamide, acrylamide, benzo({alpha})pyrene, cyclophosphamide, diethylstilbestrol, 4-nitroquinoline N-oxide, propyleneimine, safrole, thiourea, and o-toluidine) have been evaluated in this assay. The results show that all the compounds tested produce significant increases in the eye spot frequency at, at least, one of the concentrations assayed, indicating that the Zeste-White assay appears to be highly sensitive to these carcinogenic compounds. That is in agreement with data previously reported by other authors.

  14. Development of a neutron measurement system in unified non-destructive assay for the PRIDE facility

    NASA Astrophysics Data System (ADS)

    Seo, Hee; Park, Se-Hwan; Won, Byung-Hee; Ahn, Seong-Kyu; Shin, Hee-Sung; Na, Sang-Ho; Song, Dae-Yong; Kim, Ho-Dong; Lee, Seung Kyu

    2013-12-01

    The Korea Atomic Energy Research Institute (KAERI) has made an effort to develop pyroprocessing technology to resolve an on-going problem in Korea, i.e., the management of spent nuclear fuels. To this end, a test-bed facility for pyroprocessing, called PRIDE (PyRoprocessing Integrated inactive DEmonstration facility), is being constructed at KAERI. The main objective of PRIDE is to evaluate the performance of the unit processes, remote operation, maintenance, and proliferation resistance. In addition, integrating all unit processes into a one-step process is also one of the main goals. PRIDE can also provide a good opportunity to test safeguards instrumentations for a pyroprocessing facility such as nuclear material accounting devices, surveillance systems, radiation monitoring systems, and process monitoring systems. In the present study, a non-destructive assay (NDA) system for the testing of nuclear material accountancy of PRIDE was designed by integrating three different NDA techniques, i.e., neutron, gamma-ray, and mass measurements. The developed neutron detection module consists of 56 3He tubes and 16 AMPTEK A111 signal processing circuits. The amplifiers were matched in terms of the gain and showed good uniformity after a gain-matching procedure (%RSD=0.37%). The axial and the radial efficiency distributions within the cavity were then measured using a 252Cf neutron source and were compared with the MCNPX calculation results. The measured efficiency distributions showed excellent agreement with the calculations, which confirmed the accuracy of the MCNPX model of the system.

  15. Meiosis in male Drosophila

    PubMed Central

    McKee, Bruce D.; Yan, Rihui; Tsai, Jui-He

    2012-01-01

    Meiosis entails sorting and separating both homologous and sister chromatids. The mechanisms for connecting sister chromatids and homologs during meiosis are highly conserved and include specialized forms of the cohesin complex and a tightly regulated homolog synapsis/recombination pathway designed to yield regular crossovers between homologous chromatids. Drosophila male meiosis is of special interest because it dispenses with large segments of the standard meiotic script, particularly recombination, synapsis and the associated structures. Instead, Drosophila relies on a unique protein complex composed of at least two novel proteins, SNM and MNM, to provide stable connections between homologs during meiosis I. Sister chromatid cohesion in Drosophila is mediated by cohesins, ring-shaped complexes that entrap sister chromatids. However, unlike other eukaryotes Drosophila does not rely on the highly conserved Rec8 cohesin in meiosis, but instead utilizes two novel cohesion proteins, ORD and SOLO, which interact with the SMC1/3 cohesin components in providing meiotic cohesion. PMID:23087836

  16. Examination of the auxin hypothesis of phytomelatonin action in classical auxin assay systems in maize.

    PubMed

    Kim, Minjae; Seo, Hyesu; Park, Chanwoo; Park, Woong June

    2016-01-15

    Melatonin has been found in a wide range of plant groups. Its physiological roles have been suggested to be diverse in stress protection and plant growth regulation. An attractive hypothesis is that phytomelatonin acts as an auxin to regulate plant development. However, the auxin hypothesis is controversial, since both supporting and contradictory evidence has been reported. We systematically investigated whether melatonin fulfilled the definition for auxin in maize (Zea mays). Melatonin did not affect coleoptile elongation, root growth or ACC synthase gene expression, contrary to 10(-5)M IAA in our assay system. The auxin hypothesis of phytomelatonin action is not supported in maize, because melatonin appeared inactive in all of the auxin activity tests. On the other hand, melatonin was active in the protection of maize growth against salt stress, suggesting its importance in another context. PMID:26681269

  17. Kinetic Analyses of Data from a Human Serum Albumin Assay Using the liSPR System

    PubMed Central

    Henseleit, Anja; Pohl, Carolin; Kaltenbach, Hans-Michael; Hettwer, Karina; Simon, Kirsten; Uhlig, Steffen; Haustein, Natalie; Bley, Thomas; Boschke, Elke

    2015-01-01

    We used the interaction between human serum albumin (HSA) and a high-affinity antibody to evaluate binding affinity measurements by the bench-top liSPR system (capitalis technology GmbH). HSA was immobilized directly onto a carboxylated sensor layer, and the mechanism of interaction between the antibody and HSA was investigated. The bivalence and heterogeneity of the antibody caused a complex binding mechanism. Three different interaction models (1:1 binding, heterogeneous analyte, bivalent analyte) were compared, and the bivalent analyte model best fit the curves obtained from the assay. This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA). The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step. PMID:25607476

  18. In focus: spotted wing drosophila, Drosophila suzukii, across perspectives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An effective response to the invasion of spotted wing Drosophila (SWD), Drosophila suzukii, requires proper taxonomic identification at the initial phase, understanding its basic biology and phenology, developing management tools, transferring information and technology quickly to user groups, and e...

  19. Advanced material accountancy glove box assay system for process material at PFPF.

    SciTech Connect

    Maruyama, H.; Fujiwara, S.; Takahashi, S.; Kuno, K.; Menlove, Howard O.; Wenz, T. R.

    2002-01-01

    The Material Accountancv Glove Box Assay System (MAGB) is a neutron coincidence counting system that has been developed under the akeement between Japan Nuclear Cycle Development Institute (JNC) and Los Alamos National Laboratory (LANL) in order to measure plutonium in the MOX transfer container in the glove box at Plutonium Fuel Production Facility (PFPF) in Japan. The system was installed at PFPF in August 1989 and afterwards it has been using for verification ofplutonium in the transfer container by inspectorate during inspection Process equipment to improve its performance has been installed in the PFPF fabrication line. As a result the number of sample requiredfor inspection increased. Since MAGBs were only used, it was anticipated that the reduction of the time of sampling and movement time of nuclear materialfor verification would be difficult. Furthermore, the sample has to be taken from the transfer containers that contain a large amount of plutonium. Therefore, in order to reduce the time for sampling, time required for the transfer container to arrive at sampling point, and personal radiation exposure, the integrated MAGB system combined with High resolution gamma-ray spectroscopy (HRGS), called M G B, was developed. This system was installed at PFPF in August 2000, and then, the functional test and the calibration were carried out in the presence of inspectorate. In the course of these activities, the data that is necessary for evaluating the system were fully acquired The system is now being in the process of evaluation whether it can be used as an attended safeguards system for International Atomic Energy Agency (IAEA) and Japan Safeguards Office (JSGO).

  20. Discovery of Trypanosomatid Parasites in Globally Distributed Drosophila Species

    PubMed Central

    Chandler, James Angus; James, Pamela M.

    2013-01-01

    Microbial parasites of animals include bacteria, viruses, and various unicellular eukaryotes. Because of the difficulty in studying these microorganisms in both humans and disease vectors, laboratory models are commonly used for experimental analysis of host-parasite interactions. Drosophila is one such model that has made significant contributions to our knowledge of bacterial, fungal, and viral infections. Despite this, less is known about other potential parasites associated with natural Drosophila populations. Here, we surveyed sixteen Drosophila populations comprising thirteen species from four continents and Hawaii and found that they are associated with an extensive diversity of trypanosomatids (Euglenozoa, Kinetoplastea). Phylogenetic analysis finds that Drosophila-associated trypanosomatids are closely related to taxa that are responsible for various types of leishmaniases and more distantly related to the taxa responsible for human African trypanosomiasis and Chagas disease. We suggest that Drosophila may provide a powerful system for studying the interactions between trypanosomatids and their hosts. PMID:23658617

  1. Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays

    PubMed Central

    VanDussen, Kelli L; Marinshaw, Jeffrey M; Shaikh, Nurmohammad; Miyoshi, Hiroyuki; Moon, Clara; Tarr, Phillip I; Ciorba, Matthew A; Stappenbeck, Thaddeus S

    2014-01-01

    Objective The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualized medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (i.e., barrier function and host-microbial interactions). Design We created a large panel of human gastrointestinal epithelial cell lines (n = 65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays. Results We obtained epithelial lines from all accessible tissue sites within two weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3–1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarized monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli. Conclusion This culture system will facilitate the study of inter-individual, functional studies of human intestinal epithelial cells, including host-microbial interactions. PMID:25007816

  2. Three-wavelength light control of freely moving Drosophila Melanogaster for less perturbation and efficient social-behavioral studies

    PubMed Central

    Lin, Yen-Yin; Wu, Ming-Chin; Hsiao, Po-Yen; Chu, Li-An; Yang, Mei-Mei; Fu, Chien-Chung; Chiang, Ann-Shyn

    2015-01-01

    We developed a real-time automated laser-tracking system combined with continuous wave 1064-nm infrared or 473-nm blue lasers to provide punishment for studying memory in Drosophila Melanogaster. Combining optogenetic tools with laser properties, such as 473-nm and 593-nm lasers that activate light sensitive proteins in artificial transgenic flies, we can manipulate the specific neuron of an assigned fly among multiple flies to investigate neuron circuit relationships in social interactions. In restraining condition assay or optogenetic experiments, a ventral irradiated system would be more efficient due to higher ventral cuticle transmissions and neuron ganglia locations. Therefore, ventral irradiated systems cause less perturbation during behavior studies. PMID:25780741

  3. Gut-associated microbes of Drosophila melanogaster

    PubMed Central

    Broderick, Nichole; Lemaitre, Bruno

    2012-01-01

    There is growing interest in using Drosophila melanogaster to elucidate mechanisms that underlie the complex relationships between a host and its microbiota. In addition to the many genetic resources and tools Drosophila provides, its associated microbiota is relatively simple (1–30 taxa), in contrast to the complex diversity associated with vertebrates (> 500 taxa). These attributes highlight the potential of this system to dissect the complex cellular and molecular interactions that occur between a host and its microbiota. In this review, we summarize what is known regarding the composition of gut-associated microbes of Drosophila and their impact on host physiology. We also discuss these interactions in the context of their natural history and ecology and describe some recent insights into mechanisms by which Drosophila and its gut microbiota interact. “Workers with Drosophila have been considered fortunate in that they deal with the first multicellular invertebrate to be cultured monoxenically (Delcourt and Guyenot, 1910); the first to be handled axenically on a semisynthetic diet (Guyenot, 1917); and the first to be grown on a defined diet (Schultz et al., 1946). This list of advantages is somewhat embarrassing, since it implies an interest in nutrition that, in reality, was only secondary. The very first studies were concerned with the reduction of variability in genetic experiments (Delcourt and Guyenot, 1910) and standardization of the nutritional environment.” -James Sang, 1959 Ann NY Acad 1 PMID:22572876

  4. P element excision in drosophila melanogaster and related drosophilids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species, Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlik...

  5. Quantifying and predicting Drosophila larvae crawling phenotypes.

    PubMed

    Günther, Maximilian N; Nettesheim, Guilherme; Shubeita, George T

    2016-01-01

    The fruit fly Drosophila melanogaster is a widely used model for cell biology, development, disease, and neuroscience. The fly's power as a genetic model for disease and neuroscience can be augmented by a quantitative description of its behavior. Here we show that we can accurately account for the complex and unique crawling patterns exhibited by individual Drosophila larvae using a small set of four parameters obtained from the trajectories of a few crawling larvae. The values of these parameters change for larvae from different genetic mutants, as we demonstrate for fly models of Alzheimer's disease and the Fragile X syndrome, allowing applications such as genetic or drug screens. Using the quantitative model of larval crawling developed here we use the mutant-specific parameters to robustly simulate larval crawling, which allows estimating the feasibility of laborious experimental assays and aids in their design. PMID:27323901

  6. Quantifying and predicting Drosophila larvae crawling phenotypes

    PubMed Central

    Günther, Maximilian N.; Nettesheim, Guilherme; Shubeita, George T.

    2016-01-01

    The fruit fly Drosophila melanogaster is a widely used model for cell biology, development, disease, and neuroscience. The fly’s power as a genetic model for disease and neuroscience can be augmented by a quantitative description of its behavior. Here we show that we can accurately account for the complex and unique crawling patterns exhibited by individual Drosophila larvae using a small set of four parameters obtained from the trajectories of a few crawling larvae. The values of these parameters change for larvae from different genetic mutants, as we demonstrate for fly models of Alzheimer’s disease and the Fragile X syndrome, allowing applications such as genetic or drug screens. Using the quantitative model of larval crawling developed here we use the mutant-specific parameters to robustly simulate larval crawling, which allows estimating the feasibility of laborious experimental assays and aids in their design. PMID:27323901

  7. Development and Implementation of an Assay System for Rapid Screening of Transuranic Waste in Highly Contaminated Environments

    SciTech Connect

    Douglas Akers; Hopi Salomon; Lyle Robal

    2010-08-01

    An overview of the Fissile Material Monitor Waste Screener (FMM-WS) System is presented. This system is a multifunctional radioactive waste assay system suitable for the rapid assay of highly contaminated transuranic wastes immediately after retrieval, prior to packaging. The FMM-WS was developed for use at the Accelerated Cleanup Project (ARP) and began initial testing and operation in April 2008. The FMM-WS is currently in use and is providing needed data on transuranic (TRU) wastes with a range of material types, volumes, and densities from the Accelerated Retrieval Project (ARP).

  8. Multilayer-based lab-on-a-chip systems for perfused cell-based assays

    NASA Astrophysics Data System (ADS)

    Klotzbach, Udo; Sonntag, Frank; Grünzner, Stefan; Busek, Mathias; Schmieder, Florian; Franke, Volker

    2014-12-01

    A novel integrated technology chain of laser-microstructured multilayer foils for fast, flexible, and low-cost manufacturing of lab-on-a-chip devices especially for complex cell and tissue culture applications, which provides pulsatile fluid flow within physiological ranges at low media-to-cells ratio, was developed and established. Initially the microfluidic system is constructively divided into individual layers, which are formed by separate foils or plates. Based on the functional boundary conditions and the necessary properties of each layer, their corresponding foils and plates are chosen. In the third step, the foils and plates are laser microstructured and functionalized from both sides. In the fourth and last manufacturing step, the multiple plates and foils are joined using different bonding techniques like adhesive bonding, welding, etc. This multilayer technology together with pneumatically driven micropumps and valves permits the manufacturing of fluidic structures and perfusion systems, which spread out above multiple planes. Based on the established lab-on-a-chip platform for perfused cell-based assays, a multilayer microfluidic system with two parallel connected cell culture chambers was successfully implemented.

  9. In Utero Electroporation: Assay System for Migration of Cerebral Cortical Neurons

    NASA Astrophysics Data System (ADS)

    Tabata, Hidenori; Nakajima, Kazunori

    During the development of the cerebral cortex, the majority of cortical neurons are generated in the ventricular zone (VZ) facing the lateral ventricle and then migrate toward the pial surface to form the highly organized 6-layered cerebral cortex. Detailed profiles of these processes and their molecular mechanisms had been largely unknown because of the absence of an efficient assay system. The in vivo electroporation system was initially devised for use within chick embryos (Funahashi et al., 1999; Itasaki et al., 1999; Momose et al., 1999; Muramatsu et al., 1997), and we and other groups have used that system as a basis for developing an in utero electroporation system, which allows plasmid DNA to be introduced into cortical progenitor cells in developing mouse embryos in the uterus (Fukuchi-Shimogori and Grove, 2001; Saito and Nakatsuji, 2001; Tabata and Nakajima, 2001; Takahashi et al., 2002). In utero electroporation of other sites in the brain, including the hippocampus (Navarro-Quiroga et al., 2007), cerebral basal ganglia (Borrell et al., 2005; Nakahira et al., 2006), cortical hem (Takiguchi-Hayashi et al., 2004), and dorsal thalamus (Bonnin et al., 2007), has recently been reported. Introducing green fluorescent protein (GFP) enables the entire processes of migration and layer formation to be visualized (Ajioka and Nakajima, 2005; Sasaki et al., 2008; Tabata and Nakajima, 2002, 2003), and the role of any gene involved in these processes can be easily assessed by overexpressing the proteins or their mutants (Ohshima et al., 2007), or by knocking down the genes by the RNA interference technique (Bai et al., 2003). Furthermore, the Tet-On/Off system and/or other plasmid- vector-based technologies will expand the potential of the analyses. In this section we review the principles and methods of gene transfer into the cortical wall of mouse embryos by means of the in utero electroporation system.

  10. Investigating the Interplay between Sister Chromatid Cohesion and Homolog Pairing in Drosophila Nuclei

    PubMed Central

    Senaratne, T. Niroshini; Joyce, Eric F.; Wu, C.-ting

    2016-01-01

    Following DNA replication, sister chromatids must stay connected for the remainder of the cell cycle in order to ensure accurate segregation in the subsequent cell division. This important function involves an evolutionarily conserved protein complex known as cohesin; any loss of cohesin causes premature sister chromatid separation in mitosis. Here, we examined the role of cohesin in sister chromatid cohesion prior to mitosis, using fluorescence in situ hybridization (FISH) to assay the alignment of sister chromatids in interphase Drosophila cells. Surprisingly, we found that sister chromatid cohesion can be maintained in G2 with little to no cohesin. This capacity to maintain cohesion is widespread in Drosophila, unlike in other systems where a reduced dependence on cohesin for sister chromatid segregation has been observed only at specific chromosomal regions, such as the rDNA locus in budding yeast. Additionally, we show that condensin II antagonizes the alignment of sister chromatids in interphase, supporting a model wherein cohesin and condensin II oppose each other’s functions in the alignment of sister chromatids. Finally, because the maternal and paternal homologs are paired in the somatic cells of Drosophila, and because condensin II has been shown to antagonize this pairing, we consider the possibility that condensin II-regulated mechanisms for aligning homologous chromosomes may also contribute to sister chromatid cohesion. PMID:27541002

  11. Investigating the Interplay between Sister Chromatid Cohesion and Homolog Pairing in Drosophila Nuclei.

    PubMed

    Senaratne, T Niroshini; Joyce, Eric F; Nguyen, Son C; Wu, C-Ting

    2016-08-01

    Following DNA replication, sister chromatids must stay connected for the remainder of the cell cycle in order to ensure accurate segregation in the subsequent cell division. This important function involves an evolutionarily conserved protein complex known as cohesin; any loss of cohesin causes premature sister chromatid separation in mitosis. Here, we examined the role of cohesin in sister chromatid cohesion prior to mitosis, using fluorescence in situ hybridization (FISH) to assay the alignment of sister chromatids in interphase Drosophila cells. Surprisingly, we found that sister chromatid cohesion can be maintained in G2 with little to no cohesin. This capacity to maintain cohesion is widespread in Drosophila, unlike in other systems where a reduced dependence on cohesin for sister chromatid segregation has been observed only at specific chromosomal regions, such as the rDNA locus in budding yeast. Additionally, we show that condensin II antagonizes the alignment of sister chromatids in interphase, supporting a model wherein cohesin and condensin II oppose each other's functions in the alignment of sister chromatids. Finally, because the maternal and paternal homologs are paired in the somatic cells of Drosophila, and because condensin II has been shown to antagonize this pairing, we consider the possibility that condensin II-regulated mechanisms for aligning homologous chromosomes may also contribute to sister chromatid cohesion. PMID:27541002

  12. A Drosophila model for age-associated changes in sleep:wake cycles.

    PubMed

    Koh, Kyunghee; Evans, Joshua M; Hendricks, Joan C; Sehgal, Amita

    2006-09-12

    One of the most consistent behavioral changes that occurs with age in humans is the loss of sleep consolidation. This can be quite disruptive and yet little is known about its underlying basis. To better understand the effects of aging on sleep:wake cycles, we sought to study this problem in Drosophila melanogaster, a powerful system for research on aging and behavior. By assaying flies of different ages as well as monitoring individual flies constantly over the course of their lifetime, we found that the strength of sleep:wake cycles decreased and that sleep became more fragmented with age in Drosophila. These changes in sleep:wake cycles became faster or slower with manipulations of ambient temperature that decreased or increased lifespan, respectively, demonstrating that they are a function of physiological rather than chronological age. The effect of temperature on lifespan was not mediated by changes in overall activity level or sleep amount. Flies treated with the oxidative stress-producing reagent paraquat showed a breakdown of sleep:wake cycles similar to that seen with aging, leading us to propose that the accumulation of oxidative damage with age contributes to the changes in rhythm and sleep. Together, these findings establish Drosophila as a valuable model for studying age-associated sleep fragmentation and breakdown of rhythm strength, and indicate that these changes in sleep:wake cycles are an integral part of the physiological aging process. PMID:16938867

  13. Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

    PubMed Central

    Liao, Jennifer; Morin, Laura W.; Ahmad, S. Tariq

    2014-01-01

    Parkinson’s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, primarily in the substantia nigra. The disease causes motor deficiencies, which present as rigidity, tremors and dementia in humans. Rotenone is an insecticide that causes oxidative damage by inhibiting the function of the electron transport chain in mitochondria. It is also used to model Parkinson’s disease in the Drosophila. Flies have an inherent negative geotactic response, which compels them to climb upwards upon being startled. It has been established that rotenone causes early mortality and locomotion defects that disrupt the flies’ ability to climb after they have been tapped downwards. However, the effect of rotenone on spontaneous movement is not well documented. This study outlines two sensitive, reproducible, and high throughput assays to characterize rotenone-induced deficiencies in short-term startle-induced locomotion and long-term spontaneous locomotion in Drosophila. These assays can be conveniently adapted to characterize other Drosophila models of locomotion defects and efficacy of therapeutic agents. PMID:25178101

  14. Xotch, the Xenopus homolog of Drosophila notch.

    PubMed

    Coffman, C; Harris, W; Kintner, C

    1990-09-21

    During the development of a vertebrate embryo, cell fate is determined by inductive signals passing between neighboring tissues. Such determinative interactions have been difficult to characterize fully without knowledge of the molecular mechanisms involved. Mutations of Drosophila and the nematode Caenorhabditis elegans have been isolated that define a family of related gene products involved in similar types of cellular inductions. One of these genes, the Notch gene from Drosophila, is involved with cell fate choices in the neurogenic region of the blastoderm, in the developing nervous system, and in the eye-antennal imaginal disc. Complementary DNA clones were isolated from Xenopus embryos with Notch DNA in order to investigate whether cell-cell interactions in vertebrate embryos also depend on Notch-like molecules. This approach identified a Xenopus molecule, Xotch, which is remarkably similar to Drosophila Notch in both structure and developmental expression. PMID:2402639

  15. Live cell imaging in Drosophila melanogaster.

    PubMed

    Parton, Richard M; Vallés, Ana Maria; Dobbie, Ian M; Davis, Ilan

    2010-04-01

    Although many of the techniques of live cell imaging in Drosophila melanogaster are also used by the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties with regard to keeping the cells alive, introducing fluorescent probes, and imaging through thick, hazy cytoplasm. This article outlines the major tissue types amenable to study by time-lapse cinematography and different methods for keeping the cells alive. It describes various imaging and associated techniques best suited to following changes in the distribution of fluorescently labeled molecules in real time in these tissues. Imaging, in general, is a rapidly developing discipline, and recent advances in imaging technology are able to greatly extend what can be achieved with live cell imaging of Drosophila tissues. As far as possible, this article includes the latest technical developments and discusses likely future developments in imaging methods that could have an impact on research using Drosophila. PMID:20360379

  16. 2mit, an Intronic Gene of Drosophila melanogaster timeless2, Is Involved in Behavioral Plasticity

    PubMed Central

    Benna, Clara; Leonardi, Emanuela; Romoli, Ottavia; Cognolato, Moira; Tosatto, Silvio C. E.; Costa, Rodolfo; Sandrelli, Federica

    2013-01-01

    Background Intronic genes represent ~6% of the total gene complement in Drosophila melanogaster and ~85% of them encode for proteins. We recently characterized the D. melanogaster timeless2 (tim2) gene, showing its active involvement in chromosomal stability and light synchronization of the adult circadian clock. The protein coding gene named 2mit maps on the 11th tim2 intron in the opposite transcriptional orientation. Methodology/Principal Findings Here we report the molecular and functional characterization of 2mit. The 2mit gene is expressed throughout Drosophila development, localizing mainly in the nervous system during embryogenesis and mostly in the mushroom bodies and ellipsoid body of the central complex in the adult brain. In silico analyses revealed that 2mit encodes a putative leucine-Rich Repeat transmembrane receptor with intrinsically disordered regions, harboring several fully conserved functional interaction motifs in the cytosolic side. Using insertional mutations, tissue-specific over-expression, and down-regulation approaches, it was found that 2mit is implicated in adult short-term memory, assessed by a courtship conditioning assay. In D. melanogaster, tim2 and 2mit do not seem to be functionally related. Bioinformatic analyses identified 2MIT orthologs in 21 Drosophilidae, 4 Lepidoptera and in Apis mellifera. In addition, the tim2-2mit host-nested gene organization was shown to be present in A. mellifera and maintained among Drosophila species. Within the Drosophilidae 2mit-hosting tim2 intron, in silico approaches detected a neuronal specific transcriptional binding site which might have contributed to preserve the specific host-nested gene association across Drosophila species. Conclusions/Significance Taken together, these results indicate that 2mit, a gene mainly expressed in the nervous system, has a role in the behavioral plasticity of the adult Drosophila. The presence of a putative 2mit regulatory enhancer within the 2mit-hosting tim2

  17. A High Throughput Screening Assay System for the Identification of Small Molecule Inhibitors of gsp

    PubMed Central

    Bhattacharyya, Nisan; Hu, Xin; Chen, Catherine Z.; Mathews Griner, Lesley A.; Zheng, Wei; Inglese, James; Austin, Christopher P.; Marugan, Juan J.; Southall, Noel; Neumann, Susanne; Northup, John K.; Ferrer, Marc; Collins, Michael T.

    2014-01-01

    Mis-sense mutations in the α-subunit of the G-protein, Gsα, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active Gsα and increased levels of cAMP. The aim of this study was to develop an assay system that would allow the identification of small molecule inhibitors specific for the mutant Gsα protein, the so-called gsp oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant Gsα proteins (R201C and R201H). Stable cell lines with equivalent transfected Gsα protein expression that had relatively lower (WT) or higher (R201C and R201H) cAMP levels were generated. These cell lines were used to develop a fluorescence resonance energy transfer (FRET)–based cAMP assay in 1536-well microplate format for high throughput screening of small molecule libraries. A small molecule library of 343,768 compounds was screened to identify modulators of gsp activity. A total of 1,356 compounds with inhibitory activity were initially identified and reconfirmed when tested in concentration dose responses. Six hundred eighty-six molecules were selected for further analysis after removing cytotoxic compounds and those that were active in forskolin-induced WT cells. These molecules were grouped by potency, efficacy, and structural similarities to yield 22 clusters with more than 5 of structurally similar members and 144 singleton molecules. Seven chemotypes of the major clusters were identified for further testing and analyses. PMID:24667240

  18. Developmental roles of Drosophila tRNA processing endonuclease RNase ZL as revealed with a conditional rescue system

    PubMed Central

    Xie, Xie; Dubrovskaya, Veronica; Yacoub, Nancy; Walska, Joanna; Gleason, Tara; Reid, Katherine; Dubrovsky, Edward B.

    2013-01-01

    Drosophila RNase ZL (dRNaseZ) belongs to a family of endoribonucleases with a major role in tRNA 3′-end processing. The biochemical function of RNase ZL is conserved from yeast to human. Here we present a study of its biological function during Drosophila development. In flies, dRNaseZ provides a non-redundant function, as the RNZED24 knockout (KO) mutation causes early larval lethality. Mosaic and conditional rescue techniques were employed to determine dRNaseZ requirements at later stages. We found that dRNaseZ activity is essential for all phases of fly development that involve cell division, including growth of adult tissue progenitors during larval and metamorphic stages, and gametogenesis in adults. At the cellular level, two major phenotypes were identified – cell growth deficiency in endoreplicating tissues and cell cycle arrest in mitotic tissues. While cell growth and proliferation are both dependant on protein synthesis, the two phenotypes displayed reliance on different dRNaseZ functions. We found that dRNaseZ KO completely blocks tRNA maturation without diminishing the abundance of mature tRNA molecules. Our data indicate that growth arrest of endoreplicating cells is primarily attributed to the relocation of the pool of mature tRNAs into the nuclei causing a decrease in translation efficiency. Mitotically dividing cells appear to be less dependent on translation machinery as they maintain their normal size when deprived of dRNaseZ activity, but rather display a cell cycle arrest at the G2-M transition. PMID:23867108

  19. Induced Fungal Resistance to Insect Grazing: Reciprocal Fitness Consequences and Fungal Gene Expression in the Drosophila-Aspergillus Model System

    PubMed Central

    Caballero Ortiz, Silvia; Trienens, Monika; Rohlfs, Marko

    2013-01-01

    Background Fungi are key dietary resources for many animals. Fungi, in consequence, have evolved sophisticated physical and chemical defences for repelling and impairing fungivores. Expression of such defences may entail costs, requiring diversion of energy and nutrients away from fungal growth and reproduction. Inducible resistance that is mounted after attack by fungivores may allow fungi to circumvent the potential costs of defence when not needed. However, no information exists on whether fungi display inducible resistance. We combined organism and fungal gene expression approaches to investigate whether fungivory induces resistance in fungi. Methodology/Principal Findings Here we show that grazing by larval fruit flies, Drosophila melanogaster, induces resistance in the filamentous mould, Aspergillus nidulans, to subsequent feeding by larvae of the same insect. Larval grazing triggered the expression of various putative fungal resistance genes, including the secondary metabolite master regulator gene laeA. Compared to the severe pathological effects of wild type A. nidulans, which led to 100% insect mortality, larval feeding on a laeA loss-of-function mutant resulted in normal insect development. Whereas the wild type fungus recovered from larval grazing, larvae eradicated the chemically deficient mutant. In contrast, mutualistic dietary yeast, Saccharomyces cerevisiae, reached higher population densities when exposed to Drosophila larval feeding. Conclusions/Significance Our study presents novel evidence that insect grazing is capable of inducing resistance to further grazing in a filamentous fungus. This phenotypic shift in resistance to fungivory is accompanied by changes in the expression of genes involved in signal transduction, epigenetic regulation and secondary metabolite biosynthesis pathways. Depending on reciprocal insect-fungus fitness consequences, fungi may be selected for inducible resistance to maintain high fitness in fungivore-rich habitats

  20. Drosophila Models of Cardiac Disease

    PubMed Central

    Piazza, Nicole; Wessells, R.J.

    2013-01-01

    The fruit fly Drosophila melanogaster has emerged as a useful model for cardiac diseases, both developmental abnormalities and adult functional impairment. Using the tools of both classical and molecular genetics, the study of the developing fly heart has been instrumental in identifying the major signaling events of cardiac field formation, cardiomyocyte specification, and the formation of the functioning heart tube. The larval stage of fly cardiac development has become an important model system for testing isolated preparations of living hearts for the effects of biological and pharmacological compounds on cardiac activity. Meanwhile, the recent development of effective techniques to study adult cardiac performance in the fly has opened new uses for the Drosophila model system. The fly system is now being used to study long-term alterations in adult performance caused by factors such as diet, exercise, and normal aging. The fly is a unique and valuable system for the study of such complex, long-term interactions, as it is the only invertebrate genetic model system with a working heart developmentally homologous to the vertebrate heart. Thus, the fly model combines the advantages of invertebrate genetics (such as large populations, facile molecular genetic techniques, and short lifespan) with physiological measurement techniques that allow meaningful comparisons with data from vertebrate model systems. As such, the fly model is well situated to make important contributions to the understanding of complicated interactions between environmental factors and genetics in the long-term regulation of cardiac performance. PMID:21377627

  1. An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.

    PubMed

    King, Brian C; Donnelly, Marie K; Bergstrom, Gary C; Walker, Larry P; Gibson, Donna M

    2009-03-01

    Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose- or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production. PMID:18973283

  2. Src family kinases are required for WNT5 signaling through the Derailed/RYK receptor in the Drosophila embryonic central nervous system.

    PubMed

    Wouda, Rene R; Bansraj, Monique R K S; de Jong, Anja W M; Noordermeer, Jasprina N; Fradkin, Lee G

    2008-07-01

    Members of the RYK/Derailed family have recently been shown to regulate axon guidance in both Drosophila and mammals by acting as Wnt receptors. Little is known about how the kinase activity-deficient RYKs transduce Wnt signals. Here, we show that the non-receptor Src family tyrosine kinases, SRC64B and SRC42A, are involved in WNT5-mediated signaling through Derailed in the Drosophila embryonic central nervous system. Analysis of animals lacking SRC64B and SRC42A reveals defects in commissure formation similar to those observed in Wnt5 and derailed mutants. Reductions in SRC64B expression levels suppress a Wnt5/derailed-dependent dominant gain-of-function phenotype, and increased levels of either SRC64B or SRC42A enhance Wnt5/derailed-mediated axon commissure switching. Derailed and SRC64B form a complex, which contains catalytically active SRC64B, the formation or stability of which requires SRC64B kinase activity. Furthermore, Derailed is phosphorylated in a SRC64B-dependent manner and coexpression of Derailed and SRC64B results in the activation of SRC64B. The mammalian orthologs of Derailed and SRC64B also form complexes, suggesting that Src roles in RYK signaling are conserved. Finally, we show that coexpression of WNT5 and Derailed has no apparent effect upon TCF/LEF-dependent transcription, suggesting that the WNT5/Derailed signaling pathway is unlikely to directly regulate canonical Wnt pathway targets. Together, these findings indicate that the Src family kinases play novel roles in WNT5/Derailed-mediated signaling. PMID:18539923

  3. Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System.

    PubMed

    Akama, Kenji; Shirai, Kentaro; Suzuki, Seigo

    2016-07-19

    Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers. PMID:27322525

  4. Clinical performance evaluation of the BD Veritor System Flu A+B assay.

    PubMed

    Nam, Myung-Hyun; Jang, Jin Woo; Lee, Jong-Han; Cho, Chi Hyun; Lim, Chae Seung; Kim, Woo Joo

    2014-08-01

    Early identification of influenza is important for optimal patient management and infection control. Rapid influenza antigen tests have been used routinely in clinical settings to confirm clinical suspicion, despite their low sensitivity. To improve sensitivity, various influenza point-of-care test reader systems have been developed. This study evaluated the clinical performance of a digital readout rapid influenza diagnostic test (RIDT), the BD Veritor™ System Flu A+B assay (BD). Nasopharyngeal swabs taken from 250 patients (influenza A positive, n=75; influenza B positive, n=75; and influenza negative, n=100) were analyzed using the BinaxNOW® Influenza A/B antigen kit (BN), SD Influenza Ag A/B kit (SD), BD, real-time reverse transcriptase polymerase chain reaction (RT-PCR), and an influenza virus culture. Compared to RT-PCR, the sensitivities of BN, SD, and BD were 56.0, 53.3, and 72.0%, respectively, for influenza A and 57.3, 65.3, and 69.3%, respectively, for influenza B. No false-positive results were noted with the three rapid antigen tests. For influenza A, the average RT-PCR threshold cycle (Ct) for specimens that tested positive using BD was higher than that for specimens that tested positive using BN and SD. BD is a sensitive and easy method for the early detection of influenza A and B. PMID:24747589

  5. Micromechanics of Drosophila Audition

    NASA Astrophysics Data System (ADS)

    Göpfert, M. C.; Robert, D.

    2003-02-01

    An analysis is presented of the auditory micromechanics of the fruit fly Drosophila melanogaster. In this animal, the distal part of the antenna constitutes a resonantly tuned sound receiver, the vibrations of which are transduced by a chordotonal sense organ in the antenna's base. Analyzing the mechanical behavior of the antennal receiver by means of microscanning laser Doppler vibrometry, we show that the auditory system of wild-type flies exhibits a hardening stiffness nonlinearity and spontaneously generates oscillations in the absence of external stimuli. According to the deprivation of these mechanical properties in mechanosensory mutants, the receiver's nonlinearity and oscillation activity are introduced by chordotonal auditory neurons. Requiring the mechanoreceptor-specific extracellular linker protein No-mechanoreceptor-potential-A (NompA), NompC mechanosensory transduction channels, Beethoven (Btv), and Touch-insensitive-larva-B (TilB), nonlinearity and oscillation activity of the fly's antennal receiver depend on prominent components of the auditory transduction machinery and seem to originate from motility of auditory receptor cilia.

  6. CYTOGENETIC ANALYSIS OF THE L5178Y/TK+/- YIELDS TK-/- MOUSE LYMPHOMA MUTAGENESIS ASSAY SYSTEM

    EPA Science Inventory

    The L5178Y/TK t/- TK-/- mouse lymphoma mutagen assay, which allows selection of forward mutations at the autosomal thymidine kinase (TK) locus, uses a TK t/- heterozygous cell line, TK t/- 3.7.2C Quantitation of colonies of mutant TK-/- cells in the assay forms the basis for calc...

  7. Infection Dynamics and Immune Response in a Newly Described Drosophila-Trypanosomatid Association

    PubMed Central

    Votýpka, Jan; Dostálová, Anna; Yurchenko, Vyacheslav; Bird, Nathan H.; Lukeš, Julius; Lemaitre, Bruno

    2015-01-01

    ABSTRACT Trypanosomatid parasites are significant causes of human disease and are ubiquitous in insects. Despite the importance of Drosophila melanogaster as a model of infection and immunity and a long awareness that trypanosomatid infection is common in the genus, no trypanosomatid parasites naturally infecting Drosophila have been characterized. Here, we establish a new model of trypanosomatid infection in Drosophila—Jaenimonas drosophilae, gen. et sp. nov. As far as we are aware, this is the first Drosophila-parasitic trypanosomatid to be cultured and characterized. Through experimental infections, we find that Drosophila falleni, the natural host, is highly susceptible to infection, leading to a substantial decrease in host fecundity. J. drosophilae has a broad host range, readily infecting a number of Drosophila species, including D. melanogaster, with oral infection of D. melanogaster larvae resulting in the induction of numerous immune genes. When injected into adult hemolymph, J. drosophilae kills D. melanogaster, although interestingly, neither the Imd nor the Toll pathway is induced and Imd mutants do not show increased susceptibility to infection. In contrast, mutants deficient in drosocrystallin, a major component of the peritrophic matrix, are more severely infected during oral infection, suggesting that the peritrophic matrix plays an important role in mediating trypanosomatid infection in Drosophila. This work demonstrates that the J. drosophilae-Drosophila system can be a powerful model to uncover the effects of trypanosomatids in their insect hosts. PMID:26374124

  8. Milk progesterone radioimmunoassay using radioiodinated tracers: a rapid and reliable assay system

    SciTech Connect

    Espinosa, J.; Botana, L.M.; Puentes, E.; Regueiro, B.J.

    1984-09-01

    Both the validity and practicability of a direct progesterone radioimmunoassay based on radioiodinated progesterone tracers were studied. The results obtained show the reliability of the assay; when compared with assays based on /sup 3/H-progesterone tracers there are fewer steps for assay execution, saving time and reducing the number of reagents used. Various commercially available /sup 125/I-progesterone tracers were assayed, and only those with an 11 alpha-hemisuccinate bridge were suitably bound by antisera raised against progesterone-bovine serum albumin conjugates having identical bridge structure. The bridge effect caused no observable alteration in validity parameters. Finally, the results support the utility of this assay as a practical method of early diagnosis of pregnancy and as a reliable experimental technique to monitor cow ovarian function.

  9. Taste processing in Drosophila larvae

    PubMed Central

    Apostolopoulou, Anthi A.; Rist, Anna; Thum, Andreas S.

    2015-01-01

    The sense of taste allows animals to detect chemical substances in their environment to initiate appropriate behaviors: to find food or a mate, to avoid hostile environments and predators. Drosophila larvae are a promising model organism to study gustation. Their simple nervous system triggers stereotypic behavioral responses, and the coding of taste can be studied by genetic tools at the single cell level. This review briefly summarizes recent progress on how taste information is sensed and processed by larval cephalic and pharyngeal sense organs. The focus lies on several studies, which revealed cellular and molecular mechanisms required to process sugar, salt, and bitter substances. PMID:26528147

  10. Drosophila Melanogaster as an Experimental Organism.

    ERIC Educational Resources Information Center

    Rubin, Gerald M.

    1988-01-01

    Discusses the role of the fruit fly in genetics research requiring a multidisciplinary approach. Describes embryological and genetic methods used in the experimental analysis of this organism. Outlines the use of Drosophila in the study of the development and function of the nervous system. (RT)

  11. Dscam2 affects visual perception in Drosophila melanogaster

    PubMed Central

    Bosch, Danny S.; van Swinderen, Bruno; Millard, S. Sean

    2015-01-01

    Dscam2, a cell surface protein that mediates cellular repulsion, plays a crucial role in the development of the Drosophila melanogaster visual system. Dscam2 generates boundaries between neighboring modules in the fly optic lobe; in Dscam2 mutants this visual system modularity is compromised. Although developmental wiring defects have been well described in the Dscam2 mutant, behavioral consequences have not been investigated. To address this, we examined the visual behavior of Dscam2 mutant flies. Using a phototaxis assay, we ascertained that these flies are not blind, but have a reduced phototaxic response. Through population-based and single fly optomotor assays, we found that Dscam2 mutant flies can track motion but that their response is opposite to control flies under defined experimental conditions. In a fixation paradigm, which allows tethered flies to control the angular position of a visual stimulus, mutant flies' responses were diametrically opposed to those seen in control flies. These data suggest that modest changes in the modularity of the fly visual system in the Dscam2 mutant can dramatically change the perception of specific visual cues and modify behavior. PMID:26106310

  12. Drosophila Blastorderm Analysis Software

    Energy Science and Technology Software Center (ESTSC)

    2006-10-25

    PointCloudMake analyzes 3D fluorescent images of whole Drosophila embryo and produces a table-style "PointCloud" file which contains the coordinates and volumes of all the nuclei, cells, their associated relative gene expression levels along with morphological features of the embryo. See: Luengo Hendrix et at 2006 3D Morphology and Gene Expression in the Drosophila Blastoderm at Cellular Resolution manuscript submitted LBNL # LBNL-60178 Knowles DW, Keranen SVE, Biggin M. Sudar S (2002) Mapping organism expression levelsmore » at cellular resolution in developing Drosophila. In: Conchello JA, Cogswell CJ, Wilson T, editors. Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX. pp. 57-64« less

  13. Establishment of a gastric cell-based assay system for exploring inhibitors of octanoylated ghrelin production.

    PubMed

    Oiso, Shigeru; Nobe, Miyuki; Yamaguchi, Yuhei; Umemoto, Shigeru; Nakamura, Kazuo; Kariyazono, Hiroko

    2013-10-01

    Ghrelin, a gastric hormone, is a growth hormone-releasing peptide. Its serine-3 acylation with octanoic acid is essential for its orexigenic activity, and therefore, inhibition of the acylation of ghrelin may help in decreasing appetite and preventing obesity. This study aimed to establish a human gastric cell-based assay system to evaluate candidate inhibitors of octanoylated ghrelin production. In human gastric carcinoma AGS cells, obligatory factors for the posttranslational modification of ghrelin, such as certain prohormone convertases responsible for processing of proghrelin to the mature ghrelin and the enzyme-catalyzing acyl-modification of ghrelin, were well expressed, but ghrelin was expressed at low levels. Accordingly, we transfected a ghrelin-expressing vector into AGS cells and isolated a stable ghrelin-expressing cell line (AGS-GHRL8). AGS-GHRL8 cells secreted octanoylated ghrelin in accordance with the concentrations of octanoic acid in the culture medium. Given that ingested heptanoic acid is used for the acyl-modification of ghrelin, we evaluated whether heptanoic acid inhibits production of octanoylated ghrelin in AGS-GHRL8 cells. Butyric acid was used as a control. Indeed, heptanoic acid predictably decreased the secretion of octanoylated ghrelin, whereas butyric acid did not. The AGS-GHRL8 line established in this study will facilitate the screening of inhibitors of octanoylated ghrelin production. PMID:23704134

  14. A system for assaying homologous recombination at the endogenous human thymidine kinase gene

    SciTech Connect

    Benjamin, M.B.; Little, J.B. ); Potter, H. ); Yandell, D.W. Massachusetts Eye and Ear Infirmary, Boston Harvard Medical School, Boston, MA )

    1991-08-01

    A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK{sup +/+} parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or {minus}1 frameshifts. Resulting TK{sup {minus}/{minus}} mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by {approx}8 kilobases. These lines undergo spontaneous reversion to TK{sup +} at a frequency of < 10{sup {minus}7}, and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK{sup +}. The mode of reversion to TK{sup +} was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. The data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.

  15. Clues from wildlife to create an assay for thyroid system disruption.

    PubMed Central

    Colborn, Theo

    2002-01-01

    In 1996 the U.S. Congress charged the U.S. Environmental Protection Agency to develop a screening program to test chemicals for their possible estrogenic and other endocrine effects. Shortly thereafter, the Chemical Guidelines Program of the Organisation for Economic Co-operation and Development's (OECD) Environmental Directorate organized a Task Force on Endocrine Disruption Testing and Assessment to coordinate development of internationally harmonized screening and testing protocols. Most of the research devoted to this effort has focused on detecting impaired estrogenicity, androgenicity, and/or steroidogenesis, with little progress toward developing assays to detect chemicals that might interfere with thyroid function. Despite the fact that wildlife biologists have been reporting abnormal thyroid gland development and unusual thyroid hormone (TH) and retinoid ratios in fish and birds since the early 1960s, few studies have demonstrated an association between an environmental contaminant and a particular health end point other than reduced reproductive success at the population level. This article is a review of the literature that specifically examines THs and their role in normal behavior and development in wildlife. It presents several studies that associated changes in the thyroid gland, TH concentrations, and behavior with contaminant exposure. The goal of this article is to provide fodder for the creation of simple screens to detect possible thyroid system agonists and antagonists. PMID:12060830

  16. Spectrofluorimetric methods of stability-indicating assay of certain drugs affecting the cardiovascular system

    NASA Astrophysics Data System (ADS)

    Moussa, B. A.; Mohamed, M. F.; Youssef, N. F.

    2011-01-01

    Two stability-indicating spectrofluorimetric methods have been developed for the determination of ezetimibe and olmesartan medoxomil, drugs affecting the cardiovascular system, and validated in the presence of their degradation products. The first method, for ezetimibe, is based on an oxidative coupling reaction of ezetimibe with 3-methylbenzothiazolin-2-one hydrazone hydrochloride in the presence of cerium (IV) ammonium sulfate in an acidic medium. The quenching effect of ezetimibe on the fluorescence of excess cerous ions is measured at the emission wavelength, λem, of 345 nm with the excitation wavelength, λex, of 296 nm. Factors affecting the reaction were carefully studied and optimized. The second method, for olmesartan medoxomil, is based on measuring the native fluorescence intensity of olmesartan medoxomil in methanol at λem = 360 nm with λex = 286 nm. Regression plots revealed good linear relationships in the assay limits of 10-120 and 8-112 g/ml for ezetimibe and olmesartan medoxomil, respectively. The validity of the methods was assessed according to the United States Pharmacopeya guidelines. Statistical analysis of the results exposed good Student's t-test and F-ratio values. The introduced methods were successfully applied to the analysis of ezetimibe and olmesartan medoxomil in drug substances and drug products as well as in the presence of their degradation products.

  17. Advanced Non-Destructive Assay Systems and Special Instrumentation Requirements for Spent Nuclear Fuel Recycling Facilities

    SciTech Connect

    Simpson, A.P.; Clapham, M.J.; Swinson, B.

    2008-07-01

    The safe and efficient operation of the next generation of Spent Nuclear Fuel (SNF) recycling / reprocessing facilities is dependent upon the availability of high performance real time Non- Destructive Assay (NDA) systems at key in-line points. A diverse variety of such special instrument systems have been developed and commissioned at reprocessing plants worldwide over the past fifty years.. The measurement purpose, technique and plant performance for selected key systems have been reviewed. Obsolescence issues and areas for development are identified in the context of the measurements needs of future recycling facilities and their associated waste treatment plants. Areas of concern include (i) Materials Accountancy and Safeguards, (ii) Head End process control and feed envelope verification, (iii) Real-time monitoring at the Product Finishing Stages, (iv) Criticality safety and (v) Radioactive waste characterization. Common characteristics of the traditional NDA systems in historical recycling facilities are (i) In-house development of bespoke instruments resulting in equipment that if often unique to a given facility and generally not commercially available, (ii) Use of 'novel' techniques - not widely deployed in other applications, (iii) Design features that are tailored to the specific plant requirements of the facility operator, (iv) Systems and software implementation that was not always carried out to modern industry standards and (v) A tendency to be overly complex - refined by on-plant operational usage and experience. Although these systems were 'validated in use' and are generally fit for purpose, there are a number of potential problems in transferring technology that was developed ten or more years ago to the new build SNF recycling facilities of the future. These issues include (i) Obsolescence of components - particularly with respect to computer hardware and data acquisition electronics, (ii) Availability of Intellectual Property and design

  18. A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents.

    PubMed

    Jia, Nan; Nakazawa, Yuka; Guo, Chaowan; Shimada, Mayuko; Sethi, Mieran; Takahashi, Yoshito; Ueda, Hiroshi; Nagayama, Yuji; Ogi, Tomoo

    2015-01-01

    DNA repair systems protect cells from genomic instability and carcinogenesis. Therefore, assays for measuring DNA repair activity are valuable, not only for clinical diagnoses of DNA repair deficiency disorders but also for basic research and anticancer drug development. Two commonly used assays are UDS (unscheduled DNA synthesis, requiring a precise measurement of an extremely small amount of repair DNA synthesis) and RRS (recovery of RNA synthesis after DNA damage). Both UDS and RRS are major endpoints for assessing the activity of nucleotide excision repair (NER), the most versatile DNA repair process. Conventional UDS and RRS assays are laborious and time-consuming, as they measure the incorporation of radiolabeled nucleosides associated with NER. Here we describe a comprehensive protocol for monitoring nonradioactive UDS and RRS by studying the incorporation of alkyne-conjugated nucleoside analogs followed by a fluorescent azide-coupling click-chemistry reaction. The system is also suitable for quick measurement of cell sensitivity to DNA-damaging reagents and for lentivirus-based complementation assays, which can be used to systematically determine the pathogenic genes associated with DNA repair deficiency disorders. A typical UDS or RRS assay using primary fibroblasts, including a virus complementation test, takes 1 week to complete. PMID:25474029

  19. Establishing an in vivo assay system to identify components involved in environmental RNA interference in the western corn rootworm.

    PubMed

    Miyata, Keita; Ramaseshadri, Parthasarathy; Zhang, Yuanji; Segers, Gerrit; Bolognesi, Renata; Tomoyasu, Yoshinori

    2014-01-01

    The discovery of environmental RNA interference (RNAi), in which gene expression is suppressed via feeding with double-stranded RNA (dsRNA) molecules, opened the door to the practical application of RNAi-based techniques in crop pest management. The western corn rootworm (WCR, Diabrotica virgifera virgifera) is one of the most devastating corn pests in North America. Interestingly, WCR displays a robust environmental RNAi response, raising the possibility of applying an RNAi-based pest management strategy to this pest. Understanding the molecular mechanisms involved in the WCR environmental RNAi process will allow for determining the rate limiting steps involved with dsRNA toxicity and potential dsRNA resistance mechanisms in WCR. In this study, we have established a two-step in vivo assay system, which allows us to evaluate the involvement of genes in environmental RNAi in WCR. We show that laccase 2 and ebony, critical cuticle pigmentation/tanning genes, can be used as marker genes in our assay system, with ebony being a more stable marker to monitor RNAi activity. In addition, we optimized the dsRNA dose and length for the assay, and confirmed that this assay system is sensitive to detect well-known RNAi components such as Dicer-2 and Argonaute-2. We also evaluated two WCR sid1- like (sil) genes with this assay system. This system will be useful to quickly survey candidate systemic RNAi genes in WCR, and also will be adaptable for a genome-wide RNAi screening to give us an unbiased view of the environmental/systemic RNAi pathway in WCR. PMID:25003334

  20. The fruitless gene is required for the proper formation of axonal tracts in the embryonic central nervous system of Drosophila.

    PubMed Central

    Song, Ho-Juhn; Billeter, Jean-Christophe; Reynaud, Enrique; Carlo, Troy; Spana, Eric P; Perrimon, Norbert; Goodwin, Stephen F; Baker, Bruce S; Taylor, Barbara J

    2002-01-01

    The fruitless (fru) gene in Drosophila melanogaster is a multifunctional gene that has sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, less is known about its sex-nonspecific functions. We have examined fru's sex-nonspecific role in embryonic neural development. fru transcripts from sex-nonspecific promoters are expressed beginning at the earliest stages of neurogenesis, and Fru proteins are present in both neurons and glia. In embryos that lack most or all fru function, FasII- and BP102-positive axons have defasciculation defects and grow along abnormal pathways in the CNS. These defects in axonal projections in fru mutants were rescued by the expression of specific UAS-fru transgenes under the control of a pan-neuronal scabrous-GAL4 driver. Our results suggest that one of fru's sex-nonspecific roles is to regulate the pathfinding ability of axons in the embryonic CNS. PMID:12524343

  1. Hypomorphic Mutations in the Larval Photokinesis a (Lpha) Gene Have Stage-Specific Effects on Visual System Function in Drosophila Melanogaster

    PubMed Central

    Gordesky-Gold, B.; Warrick, J. M.; Bixler, A.; Beasley, J. E.; Tompkins, L.

    1995-01-01

    Of the many genes that are expressed in the visual system of Drosophila melanogaster adults, some affect larval vision. However, with the exception of one X-linked mutation, no genes that have larval-specific effects on visual system structure or function have previously been reported. We describe the isolation and characterization of two mutant alleles that define the larval photokinesis A (lphA) gene, one allele of which is associated with a P-element insertion at cytogenetic locus 8E1-10. Larvae that express lphA mutations are, like normal animals, negatively photokinetic, but they are less responsive to white light than lphA+ controls. Larvae that are heterozygous in trans for a mutant lphA allele and a deficiency that uncovers the lphA locus are blind, which indicates that the mutant allele is hypomorphic. lphA larvae respond normally to odorants and taste stimuli. Moreover, the lphA mutations do not affect adult flies' fast phototaxis or visually driven aspects of male sexual behavior, and electroretinograms recorded from the compound eyes of lphA/deficiency heterozygotes and lphA(1)/lphA(2) females are normal. These observations suggest that the lphA gene affects a larval-specific aspect of visual system function. PMID:7789764

  2. Control of adult stem cells in vivo by a dynamic physiological environment: diet-dependent systemic factors in Drosophila and beyond

    PubMed Central

    Ables, Elizabeth T.; Laws, Kaitlin M.; Drummond-Barbosa, Daniela

    2012-01-01

    Adult stem cells are inextricably linked to whole-body physiology and nutrient availability through complex systemic signaling networks. A full understanding of how stem cells sense and respond to dietary fluctuations will require identifying key systemic mediators, as well as elucidating how they are regulated and integrated with local and intrinsic factors across multiple tissues. Studies focused on the Drosophila germline have generated valuable insights into how stem cells are controlled by diet-dependent pathways, and increasing evidence suggests that diverse adult stem cell populations respond to nutrients through similar mechanisms. Systemic signals, including nutrients themselves and diet-regulated hormones such as Insulin/Insulin-like growth factor or steroid hormones, can directly or indirectly affect stem cell behavior by modifying local cell-cell communication or intrinsic factors. The physiological regulation of stem cells in response to nutritional status not only is a fascinating biological problem, but also has clinical implications, as research in this field holds the key to non-invasive approaches for manipulating stem cells in vivo. In addition, given the known associations between diet, stem cells, and cancer risk, this research may inspire novel anti-cancer therapies. PMID:23799567

  3. Hypomorphic mutations in the larval photokinesis A (lphA) gene have stage-specific effects on visual system function in Drosophila melanogaster

    SciTech Connect

    Gordesky-Gold, B.; Warrick, J.M.; Bixler, A.

    1995-04-01

    Of the many genes that are expressed in the visual system of Drosophila melanogaster adults, some affect larval vision. However, with the exception of one X-linked mutation, no genes that have larval-specific effects on visual system structure or function have previously been reported. We describe the isolation and characterization of two mutant alleles that define the larval photokinesis A (lphA) gene, one allele of which is associated with a P-element insertion at cytogenetic locus 8E1-10. Larvae that express lphA mutations are, like normal animals, negatively photokinetic, but they are less responsive to white light than lphA+ controls. Larvae that are heterozygous in trans for a mutant allele and a deficiency that uncovers the lphA locus are blind, which indicates that the mutant allele is hypomorphic. lphA larvae respond normally to odorants and taste stimuli. Moreover, the lphA mutations do not affect adult flies` fast phototaxis or visually driven aspects of male sexual behavior, and electroretinograms recorded from the compound eyes of lphA/deficiency heterozygotes and lphA{sup 1}/lphA{sup 2} females are normal. These observations suggest that the lphA gene affects a larval-specific aspect of visual system function. 27 refs., 2 figs., 4 tabs.

  4. Automated Tracking of Drosophila Specimens

    PubMed Central

    Chao, Rubén; Macía-Vázquez, Germán; Zalama, Eduardo; Gómez-García-Bermejo, Jaime; Perán, José-Ramón

    2015-01-01

    The fruit fly Drosophila Melanogaster has become a model organism in the study of neurobiology and behavior patterns. The analysis of the way the fly moves and its behavior is of great scientific interest for research on aspects such as drug tolerance, aggression or ageing in humans. In this article, a procedure for detecting, identifying and tracking numerous specimens of Drosophila by means of computer vision-based sensing systems is presented. This procedure allows dynamic information about each specimen to be collected at each moment, and then for its behavior to be quantitatively characterized. The proposed algorithm operates in three main steps: a pre-processing step, a detection and segmentation step, and tracking shape. The pre-processing and segmentation steps allow some limits of the image acquisition system and some visual artifacts (such as shadows and reflections) to be dealt with. The improvements introduced in the tracking step allow the problems corresponding to identity loss and swaps, caused by the interaction between individual flies, to be solved efficiently. Thus, a robust method that compares favorably to other existing methods is obtained. PMID:26258779

  5. Automated Tracking of Drosophila Specimens.

    PubMed

    Chao, Rubén; Macía-Vázquez, Germán; Zalama, Eduardo; Gómez-García-Bermejo, Jaime; Perán, José-Ramón

    2015-01-01

    The fruit fly Drosophila Melanogaster has become a model organism in the study of neurobiology and behavior patterns. The analysis of the way the fly moves and its behavior is of great scientific interest for research on aspects such as drug tolerance, aggression or ageing in humans. In this article, a procedure for detecting, identifying and tracking numerous specimens of Drosophila by means of computer vision-based sensing systems is presented. This procedure allows dynamic information about each specimen to be collected at each moment, and then for its behavior to be quantitatively characterized. The proposed algorithm operates in three main steps: a pre-processing step, a detection and segmentation step, and tracking shape. The pre-processing and segmentation steps allow some limits of the image acquisition system and some visual artifacts (such as shadows and reflections) to be dealt with. The improvements introduced in the tracking step allow the problems corresponding to identity loss and swaps, caused by the interaction between individual flies, to be solved efficiently. Thus, a robust method that compares favorably to other existing methods is obtained. PMID:26258779

  6. Investigating Spermatogenesis in Drosophila melanogaster

    PubMed Central

    Demarco, Rafael S.; Eikenes, Åsmund H.; Haglund, Kaisa; Jones, D. Leanne

    2014-01-01

    The process of spermatogenesis in Drosophila melanogaster provides a powerful model system to probe a variety of developmental and cell biological questions, such as the characterization of mechanisms that regulate stem cell behavior, cytokinesis, meiosis, and mitochondrial dynamics. Classical genetic approaches, together with binary expression systems, FRT-mediated recombination, and novel imaging systems to capture single cell behavior, are rapidly expanding our knowledge of the molecular mechanisms regulating all aspects of spermatogenesis. This methods chapter provides a detailed description of the system, a review of key questions chapter that have been addressed or remain unanswered thus far, and an introduction to tools and techniques available to probe each stage of spermatogenesis. PMID:24798812

  7. Identification of a second homolog of N-ethylmaleimide-sensitive fusion protein that is expressed in the nervous system and secretory tissues of Drosophila.

    PubMed Central

    Boulianne, G L; Trimble, W S

    1995-01-01

    N-Ethylmaleimide-sensitive fusion protein (NSF) is an ATPase known to have an essential role in intracellular membrane transport events. Recently, cDNA clones encoding a Drosophila melanogaster homolog of this protein, named dNSF, were characterized and found to be expressed in the nervous system. We now report the identification of a second homolog of NSF, called dNSF-2 within this species and report evidence that this ubiquitous and widely utilized fusion protein belongs to a multigene family. The predicted amino acid sequence of dNSF-2 is 84.5% identical to dNSF (hereafter named dNSF-1), 59% identical to NSF from Chinese hamster, and 38.5% identical to the yeast homolog SEC18. The highest similarity was found in a region of dNSF-2 containing one of two ATP-binding sites; this region is most similar to members of a superfamily of ATPases. dNSF-2 is localized to a region between bands 87F12 and 88A3 on chromosome 3, and in situ hybridization techniques revealed expression in the nervous system during embryogenesis and in several imaginal discs and secretory structures in the larvae. Developmental modulation of dNSF-2 expression suggests that quantitative changes in the secretory apparatus are important in histogenesis. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7624376

  8. Genotoxicity studies of methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary cells

    SciTech Connect

    Mason, J.M.; Zeiger, E.; Haworth, S.; Ivett, J.; Valencia, R.

    1987-01-01

    The genotoxic effects of methyl isocyanate (MIC) were investigated using four short-term tests: the Salmonella reversion assay (Ames test), the Drosophila sex-linked recessive lethal assay, and the sister chromatic exchange (SCE) and chromosomal aberration assays in cultured Chinese hamster ovary (CHO) cells. No evidence was found for the induction of mutations in either Salmonella or Drosophila. MIC did, however, induce SCEs and chromosomal aberrations in CHO cells both in the presence and absence of Aroclor-induced rat liver S-9.

  9. Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in Drosophila melanogaster

    PubMed Central

    Scialo, Filippo; Sriram, Ashwin; Stefanatos, Rhoda; Sanz, Alberto

    2016-01-01

    Drosophila melanogaster is a popular research model organism thanks to its’ powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates “the background advantage”, but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail. PMID:27570965

  10. Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in Drosophila melanogaster.

    PubMed

    Scialo, Filippo; Sriram, Ashwin; Stefanatos, Rhoda; Sanz, Alberto

    2016-01-01

    Drosophila melanogaster is a popular research model organism thanks to its' powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates "the background advantage", but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail. PMID:27570965

  11. Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system.

    PubMed

    Huber, Warren J; Marohnic, Christopher C; Peters, Michelle; Alam, Jawed; Reed, James R; Masters, Bettie Sue Siler; Backes, Wayne L

    2009-04-01

    Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K(m) for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025-0.05 microM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment. PMID:19131520

  12. [Evaluation of D10 hemoglobin testing system for hemoglobin A1C assay].

    PubMed

    Marzullo, C; Minery, M

    2008-01-01

    Bio-Rad D10 hemoglobin testing system with rack loader for hemoglobinA1C assay was evaluated. Analytical qualities were satisfactory. Imprecision was good (within-run cv was 0,5% for 4,5% of HBA(1C), 0,63% for 7,4% of HBA1C, 0,46% for 11,1% of HBA1C, between-run cv was 1,16% for 4,7% of HBA1C, 1,01% for 7,6% of HBA1C, 1,04% for 11,2% of HBA1C). Results were very well correlated with those obtained on Bio-Rad Variant II (r = 0,998). Bland and Altman graph showed good agreement between the two methods for HbA1C under 15%. The measuring range was up to 18,3% of HBA1C. There was no specimen related carry over. Triglycerides under 5,5 mmol/L and bilirubin under 734 mumol/L did not interfere. Carbamylation of HBA1C did not interfere for urea concentration under 14 mmol/L. Practicability was very good. Detection of common hemoglobin variants (HbS, C, D, E, O) is available. Fast and easy switching between short and long program allows to perform HBA1C determination for patients with hemoglobin variants. So, D10 is an interesting and easy to use small HPLC automate witch offers accurate HBA1C quantification certified by NGSP. PMID:18227011

  13. Pathfast Presepsin Assay for Early Diagnosis of Systemic Inflammatory Response Syndrome in Patients with Nephrolithiasis

    PubMed Central

    Hou, Yan-song; Wang, Hua; Chen, Hao; Wu, Ling-feng; Lu, Lin-feng; He, Yi

    2015-01-01

    It is relatively difficult to diagnose bacterial sepsis in nephrolithiasis patients. The aim of the study is to evaluate the diagnostic ability of presepsin in the differential diagnosis including SIRS, infection, or sepsis and to compare its diagnostic value with other markers, mainly as CRP, procalcitonin (PCT), and white blood cell (WBC) in patients of nephrolithiasis presenting with SIRS. 39 patients of nephrolithiasis who were diagnosed as SIRS were prospectively investigated. Plasma presepsin was detected by Pathfast presepsin assay system; CRP and PCT were measured as well. Additionally, 25 nephrolithiasis patients without SIRS were included. At all timing samples, patients were classified as SIRS or non-SIRS group. Median plasma presepsin levels were significantly increased in the SIRS group compared with non-SIRS group (452 pg/mL versus 178 ng/mL, P < 0.001), and presepsin was markedly elevated even in the early stage of SIRS (584 pg/mL 6 h, 660 pg/mL 24 h versus 452 pg/mL, P < 0.001). According to the receiver-operating characteristic (ROC) analysis, presepsin demonstrated a high diagnostic value compared with either PCT or CRP. In the early stage of SIRS, presepsin remained a highly sensitive (74.7%) and specific (88.4%) diagnostic marker compared with either PCT, CRP, or WBC. Moreover, the areas under the curve (AUCs) of presepsin (84.6%) were also superior to those seen in either PCT (79.6%) or CRP (71.8%). Thus plasma presepsin levels have comparable performance in SIRS for patients with nephrolithiasis. PMID:25722986

  14. Paper-based acetylcholinesterase inhibition assay combining a wet system for organophosphate and carbamate pesticides detection

    PubMed Central

    Apilux, Amara; Isarankura-Na-Ayudhya, Chartchalerm; Tantimongcolwat, Tanawut; Prachayasittikul, Virapong

    2015-01-01

    A dramatic increase in pesticide usage in agriculture highlights the need for on-site monitoring for public health and safety. Here, a paper-based sensor combined with a wet system was developed for the simple and rapid screening of organophosphate (OP) and carbamate (CM) pesticides based on the inhibition of acetylcholinesterase (AChE). The paper-based sensor was designed as a foldable device consisting of a cover and detection sheets pre-prepared with indoxyl acetate and AChE, respectively. The paper-based sensor requires only the incubation of a sample on the test zone for 10 minutes, followed by closing of the foldable sheet to initiate the enzymatic reaction. Importantly, the buffer loading hole was additionally designed on the cover sheet to facilitate the interaction of the coated substrate and the immobilized enzyme. This subsequently facilitates the mixing of indoxyl acetate with AChE, resulting in the improved analytical performance of the sensor. The absence or decrease in blue color produced by the AChE hydrolysis of indoxyl acetate can be observed in the presence of OPs and CMs. Under optimized conditions and using image analysis, the limit of detection (LOD) of carbofuran, dichlorvos, carbaryl, paraoxon, and pirimicarb are 0.003, 0.3, 0.5, 0.6, and 0.6 ppm, respectively. The assay could be applied to determine OP and CM residues in spiked food samples. Visual interpretation of the color signal was clearly observed at the concentration of 5 mg/kg. Furthermore, a self-contained sample pre-concentration approach greatly enhanced the detection sensitivity. The paper-based device developed here is low-cost, requires minimal reagents and is easy to handle. As such, it would be practically useful for pesticide screening by non-professional end-users. PMID:26417364

  15. Heritable Endosymbionts of Drosophila

    PubMed Central

    Mateos, Mariana; Castrezana, Sergio J.; Nankivell, Becky J.; Estes, Anne M.; Markow, Therese A.; Moran, Nancy A.

    2006-01-01

    Although heritable microorganisms are increasingly recognized as widespread in insects, no systematic screens for such symbionts have been conducted in Drosophila species (the primary insect genetic models for studies of evolution, development, and innate immunity). Previous efforts screened relatively few Drosophila lineages, mainly for Wolbachia. We conducted an extensive survey of potentially heritable endosymbionts from any bacterial lineage via PCR screens of mature ovaries in 181 recently collected fly strains representing 35 species from 11 species groups. Due to our fly sampling methods, however, we are likely to have missed fly strains infected with sex ratio-distorting endosymbionts. Only Wolbachia and Spiroplasma, both widespread in insects, were confirmed as symbionts. These findings indicate that in contrast to some other insect groups, other heritable symbionts are uncommon in Drosophila species, possibly reflecting a robust innate immune response that eliminates many bacteria. A more extensive survey targeted these two symbiont types through diagnostic PCR in 1225 strains representing 225 species from 32 species groups. Of these, 19 species were infected by Wolbachia while only 3 species had Spiroplasma. Several new strains of Wolbachia and Spiroplasma were discovered, including ones divergent from any reported to date. The phylogenetic distribution of Wolbachia and Spiroplasma in Drosophila is discussed. PMID:16783009

  16. A Drosophila model to investigate the neurotoxic side effects of radiation exposure.

    PubMed

    Sudmeier, Lisa J; Howard, Steven P; Ganetzky, Barry

    2015-07-01

    Children undergoing cranial radiation therapy (CRT) for pediatric central nervous system malignancies are at increased risk for neurological deficits later in life. We have developed a model of neurotoxic damage in adult Drosophila following irradiation during the juvenile stages with the goal of elucidating underlying neuropathological mechanisms and of ultimately identifying potential therapeutic targets. Wild-type third-instar larvae were irradiated with single doses of γ-radiation, and the percentage that survived to adulthood was determined. Motor function of surviving adults was examined with a climbing assay, and longevity was assessed by measuring lifespan. Neuronal cell death was assayed by using immunohistochemistry in adult brains. We also tested the sensitivity at different developmental stages by irradiating larvae at various time points. Irradiating late third-instar larvae at a dose of 20 Gy or higher impaired the motor activity of surviving adults. A dose of 40 Gy or higher resulted in a precipitous reduction in the percentage of larvae that survive to adulthood. A dose-dependent decrease in adult longevity was paralleled by a dose-dependent increase in activated Death caspase-1 (Dcp1) in adult brains. Survival to adulthood and adult lifespan were more severely impaired with decreasing larval age at the time of irradiation. Our initial survey of the Drosophila Genetic Reference Panel demonstrated that differences in genotype can confer phenotypic differences in radio-sensitivity for developmental survival and motor function. This work demonstrates the usefulness of Drosophila to model the toxic effects of radiation during development, and has the potential to unravel underlying mechanisms and to facilitate the discovery of novel therapeutic interventions. PMID:26092528

  17. Towards a Molecular Understanding of Drosophila Hearing

    PubMed Central

    Caldwell, Jason C.; Eberl, Daniel F.

    2007-01-01

    The Drosophila auditory system is presented as a powerful new genetic model system for understanding the molecular aspects of development and physiology of hearing organs. The fly’s ear resides in the antenna, with Johnston’s organ serving as the mechanoreceptor. New approaches using electrophysiology and laser vibrometry have provided useful tools to apply to the study of mutations that disrupt hearing. The fundamental developmental processes that generate the peripheral nervous system are fairly well understood, although specific variations of these processes for chordotonal organs (CHO) and especially for Johnston’s organ require more scrutiny. In contrast, even the fundamental physiologic workings of mechanosensitive systems are still poorly understood, but rapid recent progress is beginning to shed light. The identification and analysis of mutations that affect auditory function are summarized here, and prospects for the role of the Drosophila auditory system in understanding both insect and vertebrate hearing are discussed. PMID:12382274

  18. Chemical mutagenesis testing in Drosophila. VII. Results of 22 coded compounds tested in larval feeding experiments

    SciTech Connect

    Zimmering, S.; Mason, J.M.; Valencia, R. )

    1989-01-01

    Twenty-two chemicals were tested for mutagenicity in the sex-linked recessive lethal (SLRL) mutation assay after being fed to Drosophila melanogaster larvae. One compound, maleic hydrazide, was found to be mutagenic. It was tested for the ability to produce reciprocal translocations (RTs) and was positive in that assay as well.

  19. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes

    PubMed Central

    2015-01-01

    In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems. PMID:25802491

  20. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes.

    PubMed

    Savoian, Matthew S

    2015-07-01

    In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems. PMID:25802491

  1. Angiogenesis Assays.

    PubMed

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis. PMID:26608294

  2. Multiple redundant medulla projection neurons mediate color vision in Drosophila.

    PubMed

    Melnattur, Krishna V; Pursley, Randall; Lin, Tzu-Yang; Ting, Chun-Yuan; Smith, Paul D; Pohida, Thomas; Lee, Chi-Hon

    2014-01-01

    The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons, we have uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. We developed a novel aversive operant conditioning assay for intensity-independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs (light-emitting diodes). The flies' optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow-spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad-spectrum photoreceptors R1-R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. We hypothesize that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate ultraviolet (UV) spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons. PMID:24766346

  3. A Drosophila Model of Epidermolysis Bullosa Simplex.

    PubMed

    Bohnekamp, Jens; Cryderman, Diane E; Paululat, Achim; Baccam, Gabriel C; Wallrath, Lori L; Magin, Thomas M

    2015-08-01

    The blistering skin disorder epidermolysis bullosa simplex (EBS) results from dominant mutations in keratin 5 (K5) or keratin 14 (K14) genes, encoding the intermediate filament (IF) network of basal epidermal keratinocytes. The mechanisms governing keratin network formation and collapse due to EBS mutations remain incompletely understood. Drosophila lacks cytoplasmic IFs, providing a 'null' environment to examine the formation of keratin networks and determine mechanisms by which mutant keratins cause pathology. Here, we report that ubiquitous co-expression of transgenes encoding wild-type human K14 and K5 resulted in the formation of extensive keratin networks in Drosophila epithelial and non-epithelial tissues, causing no overt phenotype. Similar to mammalian cells, treatment of transgenic fly tissues with phosphatase inhibitors caused keratin network collapse, validating Drosophila as a genetic model system to investigate keratin dynamics. Co-expression of K5 and a K14(R125C) mutant that causes the most severe form of EBS resulted in widespread formation of EBS-like cytoplasmic keratin aggregates in epithelial and non-epithelial fly tissues. Expression of K14(R125C)/K5 caused semi-lethality; adult survivors developed wing blisters and were flightless due to a lack of intercellular adhesion during wing heart development. This Drosophila model of EBS is valuable for the identification of pathways altered by mutant keratins and for the development of EBS therapies. PMID:25830653

  4. A model system for pathogen detection using a two-component bacteriophage/bioluminescent signal amplification assay

    NASA Astrophysics Data System (ADS)

    Bright, Nathan G.; Carroll, Richard J.; Applegate, Bruce M.

    2004-03-01

    Microbial contamination has become a mounting concern the last decade due to an increased emphasis of minimally processed food products specifically produce, and the recognition of foodborne pathogens such as Campylobacter jejuni, Escherichia coli O157:H7, and Listeria monocytogenes. This research investigates a detection approach utilizing bacteriophage pathogen specificity coupled with a bacterial bioluminescent bioreporter utilizing the quorum sensing molecule from Vibrio fischeri, N-(3-oxohexanoyl)-homoserine lactone (3-oxo-C6-HSL). The 3-oxo-C6-HSL molecules diffuse out of the target cell after infection and induce bioluminescence from a population of 3-oxo-C6-HSL bioreporters (ROLux). E. coli phage M13, a well-characterized bacteriophage, offers a model system testing the use of bacteriophage for pathogen detection through cell-to-cell communication via a LuxR/3-oxo-C6-HSL system. Simulated temperate phage assays tested functionality of the ROLux reporter and production of 3-oxo-C6-HSL by various test strains. These assays showed detection limits of 102cfu after 24 hours in a varietry of detection formats. Assays incorporating the bacteriophage M13-luxI with the ROLux reporter and a known population of target cells were subsequently developed and have shown consistent detection limits of 105cfu target organisms. Measurable light response from high concentrations of target cells was almost immediate, suggesting an enrichment step to further improve detection limits and reduce assay time.

  5. Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.

    PubMed

    Wu, Qing-Ping; Zhang, Lei; Shao, Xiao-Xia; Wang, Jia-Hui; Gao, Yu; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-04-01

    Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1. PMID:26767372

  6. Development of a fluorescent microsphere-based multiplexed high-throughput assay system for profiling of transcription factor activation.

    PubMed

    Yaoi, Takuro; Jiang, Xin; Li, Xianqiang

    2006-06-01

    Transcription factors (TFs), which play crucial roles in the regulation of gene expression in the human genome, are highly regulated by a variety of mechanisms. A single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate the inducible expression of target genes. Alterations in the activities of TFs are often associated with human diseases, such as altered activating factor 1, estrogen receptor, and p53 function in cancer, nuclear factor kappaB in inflammatory diseases, and peroxisome proliferator-activated receptor gamma in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases, and aid in developing assays for drug discovery. Here, we developed a 24-plex fluorescent microsphere-based TF activation assay system with a 96-well plate format. The assay system enabled high-throughput profiling of the DNA binding activity of TFs in multiple samples with high sensitivity. PMID:16834534

  7. Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9–Mediated Protein Tagging

    PubMed Central

    Li, Qingyun; Barish, Scott; Okuwa, Sumie; Volkan, Pelin C.

    2015-01-01

    The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc. PMID:26497147

  8. Homeotic function of Drosophila Bithorax-Complex miRNAs mediates fertility by restricting multiple Hox genes and TALE cofactors in the central nervous system

    PubMed Central

    Garaulet, Daniel L.; Castellanos, Monica; Bejarano, Fernando; Sanfilippo, Piero; Tyler, David M.; Allan, Douglas W.; Sánchez-Herrero, Ernesto; Lai, Eric C.

    2014-01-01

    The Drosophila Bithorax-Complex (BX-C) Hox cluster contains a bidirectionally-transcribed miRNA locus, and a deletion mutant (∆mir) lays no eggs and is completely sterile. We show these miRNAs are expressed and active in distinct spatial registers along the anterior-posterior axis in the central nervous system. ∆mir larvae derepress a network of direct homeobox gene targets in the posterior ventral nerve cord (VNC), including BX-C genes and their TALE cofactors. These are phenotypically critical targets, since sterility of ∆mir mutants was substantially rescued by heterozygosity of these genes. The posterior VNC contains Ilp7+ oviduct motoneurons, whose innervation and morphology are defective in ∆mir females, and substantially rescued by heterozygosity of ∆mir targets, especially within the BX-C. Collectively, we reveal (1) critical roles for Hox miRNAs that determine segment-specific expression of homeotic genes, which are not masked by transcriptional regulation, and (2) that BX-C miRNAs are essential for neural patterning and reproductive behavior. PMID:24909902

  9. Spontaneous mitotic recombination and evidence for an x-ray-inducible system for the repair of DNA damage in Drosophila melanogaster

    SciTech Connect

    Kennison, J.A.; Ripoll, P.

    1981-05-01

    Spontaneous mitotic recombination in the left arm of chromosome 3 was examined in both unirradiated control flies and sibs irradiated early in development by determining the sizes and frequencies of multiple-wing-hair (mwh) clones in the wing blade of heterozygous mwh/+ flies. Approximately 16% of the spontaneous mwh clones arise from events generating cells with an average cell division rate one-third that of the surrounding cells;these are thought to result from events that generate aneuploid cells. Such clones probably arise from a failure correctly to repair spontaneous DNA damage. The frequency of spontaneous events late in development decreases significantly after irradiation as much as 150 hours earlier in development. The suppression of spontaneous events decreases with a longer period of time between irradiation and the final cell divisions in the wing blade. These results suggest the existence of a repair system for DNA damage in Drosophila that is induced by irradiation. The decrease in effect with time following irradiation could result from slow degradation or dilution by subsequent cell growth and division.

  10. Radiation survival of murine and human melanoma cells utilizing two assay systems: monolayer and soft agar.

    PubMed Central

    Yohem, K. H.; Slymen, D. J.; Bregman, M. D.; Meyskens, F. L.

    1988-01-01

    The radiation response of murine and human melanoma cells assayed in bilayer soft agar and monolayer was examined. Cells from the murine melanoma Cloudman S91 CCL 53.1 cell line and three human melanoma cell strains (C8146C, C8161, and R83-4) developed in our laboratory were irradiated by single dose X-rays and plated either in agar or on plastic. D0 values were the same within 95% confidence intervals for cells from the human melanoma cell strains C8146C, C8161, and R83-4 but were dissimilar for the murine cell line CCL 53.1 Dq values were different for all cells studied. The shape of the survival curve for all four melanomas was not identical for cells assayed in soft agar versus cells grown on plastic. This would indicate that apparent radiosensitivity was influenced by the method of assay although there were no apparent consistent differences between the curves generated by monolayer or bilayer soft agar assays. PMID:3348949

  11. GENOTOXICITY OF TEN CIGARETTE SMOKE CONDENSATES IN FOUR TEST SYSTEMS: COMPARISONS BETWEEN ASSAYS AND CONDENSATES

    EPA Science Inventory

    What is the study?
    This the first assessment of a set of cigarette smoke condensates from a range of cigarette types in a variety (4) of short-term genotoxicity assays.
    Why was it done?
    No such comparative study of cigarette smoke condensates has been reported. H...

  12. Optimized DPPH assay in a detergent-based buffer system for measuring antioxidant activity of proteins

    PubMed Central

    Nicklisch, Sascha C.T.; Waite, J. Herbert

    2014-01-01

    The free radical method using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) is a well established assay for the in vitro determination of antioxidant activity in food and biological extracts. The standard DPPH assay uses methanol or ethanol as solvents, or buffered alcoholic solutions in a ratio of 40%/60% (buffer/alcohol, v/v) to keep the hydrophobic hydrazyl radical and phenolic test compounds soluble while offering sufficient buffering capacity at different pHs tested. Following this protocol, we were unable to keep proteinaceous antioxidants soluble at different pHs to test for their antioxidant activity. Thus, the assay protocol was modified as follows to improve its utility:•Non-ionic detergents were added to keep the DPPH radical soluble and to provide a mild and non-denaturing environment for the antioxidant protein.•Maximal concentration of DPPH was limited to 100 μM to stay within the sensitivity range of the detector at the given wavelength (515 nm) and to increase the dynamic range of the assay.•0.1 M citrate phosphate buffer was introduced to prevent experimental artifacts due to changing buffer compositions at different pHs. PMID:25530949

  13. Genetic Analysis of Olfactory Behavior in Drosophila: A New Screen Yields the Ota Mutants

    PubMed Central

    Woodard, C.; Huang, T.; Sun, H.; Helfand, S. L.; Carlson, J.

    1989-01-01

    A simple means of measuring Drosophila olfactory response is described, and the behavior which it measures is characterized. The assay was used to screen for X-linked mutants defective in olfactory function. Six ota mutants were isolated and characterized (ota = olfactory trap abnormal). Four of the mutants were found to be abnormal in another chemosensory behavior as well. Two of the mutant phenotypes extend to include another sensory system: they are defective in visual system physiology. All were normal, however, in a test of giant fiber system physiology. Two of the mutations are dominant, and the recessive mutations define two complementation groups. Mutations representing each complementation group, as well as one of the dominant mutations, were mapped. For the mutants with defective visual system physiology, the visual defects were shown to cosegregate with olfactory phenotypes. PMID:2511068

  14. Functional genomic analysis of the Drosophila immune response.

    PubMed

    Valanne, Susanna

    2014-01-01

    Drosophila melanogaster has been widely used as a model organism for over a century now, and also as an immunological research model for over 20 years. With the emergence of RNA interference (RNAi) in Drosophila as a robust tool to silence genes of interest, large-scale or genome-wide functional analysis has become a popular way of studying the Drosophila immune response in cell culture. Drosophila immunity is composed of cellular and humoral immunity mechanisms, and especially the systemic, humoral response pathways have been extensively dissected using the functional genomic approach. Although most components of the main immune pathways had already been found using traditional genetic screening techniques, important findings including pathway components, positive and negative regulators and modifiers have been made with RNAi screening. Additionally, RNAi screening has produced new information on host-pathogen interactions related to the pathogenesis of many microbial species. PMID:23707784

  15. Insights From Natural Host-Parasite Interactions: The Drosophila Model

    PubMed Central

    Keebaugh, Erin S.; Schlenke, Todd A.

    2013-01-01

    Immune responses against opportunistic pathogens have been extensively studied in Drosophila, leading to a detailed map of the genetics behind innate immunity networks including the Toll, Imd, Jak-Stat, and JNK pathways. However, immune mechanisms of other organisms, particularly plants, have primarily been investigated using natural pathogens. It was the use of natural pathogens in plant research that revealed the plant R/Avr system, a specialized immune response derived from antagonistic coevolution between plant immune proteins and their natural pathogens’ virulence proteins. Thus, we recommend that researchers begin to use natural Drosophila pathogens to identify novel immune mechanisms that may have arisen through antagonistic coevolution with common natural pathogens. In this review, we address the benefits of using natural pathogens in research, describe the known natural pathogens of Drosophila, and discuss exciting prospects for future research on select natural pathogens of Drosophila. PMID:23764256

  16. Optogenetic pacing in Drosophila melanogaster (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Alex, Aneesh; Li, Airong; Men, Jing; Jerwick, Jason; Tanzi, Rudolph E.; Zhou, Chao

    2016-03-01

    A non-invasive, contact-less cardiac pacing technology can be a powerful tool in basic cardiac research and in clinics. Currently, electrical pacing is the gold standard for cardiac pacing. Although highly effective in controlling the cardiac function, the invasive nature, non-specificity to cardiac tissues and possible tissue damage limits its capabilities. Optical pacing of heart is a promising alternative, which is non-invasive and more specific, has high spatial and temporal precision, and avoids shortcomings in electrical stimulation. Optical coherence tomography has been proved to be an effective technique in non-invasive imaging in vivo with ultrahigh resolution and imaging speed. In the last several years, non-invasive specific optical pacing in animal hearts has been reported in quail, zebrafish, and rabbit models. However, Drosophila Melanogaster, which is a significant model with orthologs of 75% of human disease genes, has rarely been studied concerning their optical pacing in heart. Here, we combined optogenetic control of Drosophila heartbeat with optical coherence microscopy (OCM) technique for the first time. The light-gated cation channel, channelrhodopsin-2 (ChR2) was specifically expressed by transgene as a pacemaker in drosophila heart. By stimulating the pacemaker with 472 nm pulsed laser light at different frequencies, we achieved non-invasive and more specific optical control of the Drosophila heart rhythm, which demonstrates the wide potential of optical pacing for studying cardiac dynamics and development. Imaging capability of our customized OCM system was also involved to observe the pacing effect visually. No tissue damage was found after long exposure to laser pulses, which proved the safety of optogenetic control of Drosophila heart.

  17. Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation.

    PubMed

    Shinde, Vaibhav; Klima, Stefanie; Sureshkumar, Perumal Srinivasan; Meganathan, Kesavan; Jagtap, Smita; Rempel, Eugen; Rahnenführer, Jörg; Hengstler, Jan Georg; Waldmann, Tanja; Hescheler, Jürgen; Leist, Marcel; Sachinidis, Agapios

    2015-01-01

    Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically. PMID:26132533

  18. Automation of an in vitro cytotoxicity assay used to estimate starting doses in acute oral systemic toxicity tests.

    PubMed

    Bouhifd, Mounir; Bories, Gilles; Casado, Juan; Coecke, Sandra; Norlén, Hedvig; Parissis, Nicholaos; Rodrigues, Robim M; Whelan, Maurice P

    2012-06-01

    Application of High Throughput Screening (HTS) to the regulatory safety assessment of chemicals is still in its infancy but shows great promise in terms of facilitating better understanding of toxicological modes-of-action, reducing the reliance on animal testing, and allowing more data-poor chemicals to be assessed at a reasonable cost. To promote the uptake and acceptance of HTS approaches, we describe in a stepwise manner how a well known cytotoxicity assay can be automated to increase throughput while maintaining reliability. Results generated with selected reference chemicals compared very favourably with data obtained from a previous international validation study concerning the prediction of acute systemic toxicity in rodents. The automated assay was then included in a formal ECVAM validation study to determine if the assay could be used for binary classification of chemicals with respect to their acute oral toxicity, using a threshold equivalent to a dose of 2000 mg/kg b.w. in a rodent bioassay (LD50). This involved the blind-testing of 56 reference chemicals on the HTS platform to produce concentration-response and IC50 data. Finally, the assay was adapted to a format more suited to higher throughput testing without compromising the quality of the data obtained. PMID:22465836

  19. Programmable flow system for automation of oxygen radical absorbance capacity assay using pyrogallol red for estimation of antioxidant reactivity.

    PubMed

    Ramos, Inês I; Gregório, Bruno J R; Barreiros, Luísa; Magalhães, Luís M; Tóth, Ildikó V; Reis, Salette; Lima, José L F C; Segundo, Marcela A

    2016-04-01

    An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90 ± 5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 μM. RSD values were <3.4 and <4.9%, for intra and inter-assay precision, respectively. Compared to previous batch automated ORAC assays, the developed system also accounted for high sampling frequency (29 h(-1)), low operating costs and low generation of waste. PMID:26838448

  20. Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

    NASA Astrophysics Data System (ADS)

    Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

    2012-06-01

    There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

  1. Identification and Functional Analysis of Healing Regulators in Drosophila

    PubMed Central

    Álvarez-Fernández, Carmen; Tamirisa, Srividya; Prada, Federico; Chernomoretz, Ariel; Podhajcer, Osvaldo; Blanco, Enrique; Martín-Blanco, Enrique

    2015-01-01

    Wound healing is an essential homeostatic mechanism that maintains the epithelial barrier integrity after tissue damage. Although we know the overall steps in wound healing, many of the underlying molecular mechanisms remain unclear. Genetically amenable systems, such as wound healing in Drosophila imaginal discs, do not model all aspects of the repair process. However, they do allow the less understood aspects of the healing response to be explored, e.g., which signal(s) are responsible for initiating tissue remodeling? How is sealing of the epithelia achieved? Or, what inhibitory cues cancel the healing machinery upon completion? Answering these and other questions first requires the identification and functional analysis of wound specific genes. A variety of different microarray analyses of murine and humans have identified characteristic profiles of gene expression at the wound site, however, very few functional studies in healing regulation have been carried out. We developed an experimentally controlled method that is healing-permissive and that allows live imaging and biochemical analysis of cultured imaginal discs. We performed comparative genome-wide profiling between Drosophila imaginal cells actively involved in healing versus their non-engaged siblings. Sets of potential wound-specific genes were subsequently identified. Importantly, besides identifying and categorizing new genes, we functionally tested many of their gene products by genetic interference and overexpression in healing assays. This non-saturated analysis defines a relevant set of genes whose changes in expression level are functionally significant for proper tissue repair. Amongst these we identified the TCP1 chaperonin complex as a key regulator of the actin cytoskeleton essential for the wound healing response. There is promise that our newly identified wound-healing genes will guide future work in the more complex mammalian wound healing response. PMID:25647511

  2. Deletion analysis of a phytochrome-regulated monocot rbcS promoter in a transient assay system.

    PubMed Central

    Rolfe, S A; Tobin, E M

    1991-01-01

    We have developed a transient gene expression assay system in the aquatic monocot Lemna gibba in which DNA was introduced into intact tissue by particle bombardment. Constructs based on the Lemna rbcS gene SSU5B, which is positively regulated by phytochrome in vivo, also showed phytochrome regulation in the transient assay system. Reporter gene expression increased 12-fold over dark levels in response to a single treatment with red light. This increase was not observed if far-red light was immediately followed by the red light. A 5' deletion analysis of the promoter defined a region from position -205 to position -83 relative to the start of transcription as necessary to observe the phytochrome response. This region contains the binding site for the light-induced binding activity (LRF-1) found in Lemna nuclear extracts. Upstream of position -205, we found evidence for the presence of at least two upstream activating sequences and a silencer. Images PMID:2011579

  3. A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion.

    PubMed Central

    Pan, David A; Hardie, D Grahame

    2002-01-01

    We have identified single genes encoding homologues of the alpha, beta and gamma subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila melanogaster. Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast. Expression of double stranded (ds) RNAs targeted at any of the putative alpha, beta or gamma subunits ablated this activity, and abolished expression of the alpha subunit. The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells. Using a phosphospecific antibody, we showed that activation was associated with phosphorylation of a threonine residue (Thr-184) within the 'activation loop' of the alpha subunit. We also identified a homologue of acetyl-CoA carboxylase (DmACC) in Drosophila and, using a phosphospecific antibody, showed that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia. By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK alpha subunit was mainly detected in the nucleus. Our results show that the AMPK system is highly conserved between insects and mammals. Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs. PMID:12093363

  4. Studies on three structurally related phenylenediamines with the mouse micronucleus assay system.

    PubMed

    Soler-Niedziela, L; Shi, X; Nath, J; Ong, T

    1991-01-01

    Three structurally related compounds, 4-chloro-o-phenylenediamine (COP), 4-nitro-o-phenylenediamine (NOP) and p-phenylenediamine dihydrochloride (PPD), are used in fur dyes, inks and hair coloring formulations. COP has been reported to be carcinogenic in both rats and mice. NOP and PPD are non-carcinogens, but have consistently tested positive in short-term in vitro genotoxicity assays. Studies were undertaken to evaluate their genotoxicity with the in vivo mouse bone-marrow micronucleus assay. Five CD-1 male mice per dose were injected i.p. with the compounds and sacrificed at intervals of 24, 48 and 72 h. 2000 cells were scored per animal to determine the frequency of micronucleated-polychromatic erythrocytes (MPCE). COP induced significant dose-related increases in MPCE over the 3 doses tested at each of the sampling intervals. The peak response occurred at 24 h. No response was observed in animals treated with PPD or NOP. PMID:1703280

  5. Final Report Nucleic Acid System - PCR, Multiplex Assays and Sample Preparation Project

    SciTech Connect

    Koopman, R.P.; Langlois, R.G.; Nasarabadi, S.; Benett, W.J.; Richards, J.B.; Hadley, D.R.; Miles, R.R.; Brown, S.B.; Stratton, P.L.; Milanovich, F.P.

    2001-04-20

    The objective of this project was to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction). This entailed not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This project had two principal deliverables: (1) design, construct, test and deliver a 24 chamber, multiplex capable suitcase sized PCR instrument, and (2) develop and reduce to practice a multiplex assay for the detection of PCR product by flow cytometry. In addition, significant resources were allocated to test and evaluation of the Hand-held Advanced Nucleic Acid Analyzer (HANAA). This project helps provide the signature and intelligence gathering community the ability to perform, on-site or remote, rapid analysis of environmental or like samples for the presence of a suite of biological warfare pathogens.

  6. Fabrication of comb interdigitated electrodes array (IDA) for a microbead-based electrochemical assay system.

    PubMed

    Kim, Sang Kyung; Hesketh, Peter J; Li, Changming; Thomas, Jennifer H; Halsall, H Brian; Heineman, William R

    2004-11-01

    This research is directed towards developing a more sensitive and rapid electrochemical sensor for enzyme labeled immunoassays by coupling redox cycling at interdigitated electrode arrays (IDA) with the enzyme label beta-galactosidase. Coplanar and comb IDA electrodes with a 2.4 microm gap were fabricated and their redox cycling currents were measured. ANSYS was used to model steady state currents for electrodes with different geometries. Comb IDA electrodes enhanced the signal about three times more than the coplanar IDAs, which agreed with the results of the simulation. Magnetic microbead-based enzyme assay, as a typical example of biochemical detection, was done using the comb and coplanar IDAs. The enzymes could be placed close to the sensing electrodes (approximately 10 microm for the comb IDAs) and detection took less than 1 min with a limit of detection of 70 amol of beta-galactosidase. We conclude that faster and more sensitive assays can be achieved with the comb IDA. PMID:15522606

  7. Drosophila by the dozen

    SciTech Connect

    Celniker, Susan E.; Hoskins, Roger A.

    2007-07-13

    This year's conference on Drosophila research illustratedwell the current focus of Drosophila genomics on the comprehensiveidentification of functional elements in the genome sequence, includingmRNA transcripts arising from multiple alternative start sites and splicesites, a multiplicity of noncoding transcripts and small RNAs,identification of binding sites for transcription factors, sequenceconservation in related species and sequence variation within species.Resources and technologies for genetics and functional genomics aresteadily being improved, including the building of collections oftransposon insertion mutants and hairpin constructs for RNA interference(RNAi). The conference also highlighted progress in the use of genomicinformation by many laboratories to study diverse aspects of biology andmodels of human disease. Here we will review a few highlights of especialinterest to readers of Genome Biology.

  8. Automation of cell-based drug absorption assays in 96-well format using permeable support systems.

    PubMed

    Larson, Brad; Banks, Peter; Sherman, Hilary; Rothenberg, Mark

    2012-06-01

    Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound's absorption characteristics. PMID:22357561

  9. Hardware and software system for automatic microemulsion assay evaluation by analysis of optical properties

    NASA Astrophysics Data System (ADS)

    Maeder, Ulf; Schmidts, Thomas; Burg, Jan-Michael; Heverhagen, Johannes T.; Runkel, Frank; Fiebich, Martin

    2010-03-01

    A new hardware device called Microemulsion Analyzer (MEA), which facilitates the preparation and evaluation of microemulsions, was developed. Microemulsions, consisting of three phases (oil, surfactant and water) and prepared on deep well plates according to the PDMPD method can be automatically evaluated by means of the optical properties. The ratio of ingredients to form a microemulsion strongly depends on the properties and the amounts of the used ingredients. A microemulsion assay is set up on deep well plates to determine these ratios. The optical properties of the ingredients change from turbid to transparent as soon as a microemulsion is formed. The MEA contains a frame and an imageprocessing and analysis algorithm. The frame itself consists of aluminum, an electro luminescent foil (ELF) and a camera. As the frame keeps the well plate at the correct position and angle, the ELF provides constant illumination of the plate from below. The camera provides an image that is processed by the algorithm to automatically evaluate the turbidity in the wells. Using the determined parameters, a phase diagram is created that visualizes the information. This build-up can be used to analyze microemulsion assays and to get results in a standardized way. In addition, it is possible to perform stability tests of the assay by creating special differential stability diagrams after a period of time.

  10. The growing promise of Toll-deficient Drosophila melanogaster as a model for studying Aspergillus pathogenesis and treatment.

    PubMed

    Lionakis, Michail S; Kontoyiannis, Dimitrios P

    2010-01-01

    Despite considerable progress over recent years, the prognosis of invasive aspergillosis (IA) remains unfavorable, reflecting an incomplete understanding of Aspergillus pathogenesis and suboptimal antifungal efficacy in vivo. Mammalian host systems including rodents and rabbits are important tools in elucidating antifungal drug activity and the immunopathogenesis of IA. Nonetheless, they are hampered by limitations that impose a "bottleneck" in mass screening of novel antifungal compounds and putative Aspergillus virulence factors including their cost, labor intensity and ethical constraints. Drosophila melanogaster is an invertebrate host with a long tract record of genetic studies and a simple, yet highly conserved innate immune system. Herein, we describe our experience using this fly model as a facile, non-laborious, inexpensive pathosystem for high-throughput screening of novel antifungal compounds and putative Aspergillus mutants, and studying antifungal innate immunity. We present three infection protocols (i.e., injection, rolling, ingestion) that introduce Aspergillus either directly into the hemolymph or at different epithelial surfaces of Toll-deficient Drosophila flies. As a proof of principle, we demonstrate attenuated virulence of known hypovirulent Aspergillus strains and protection of Aspergillus-infected flies given oral Aspergillus-active agents such is voriconazole. These protocols can be adapted for similar studies of other fungal pathogens. Crossing and generation of Toll-deficient Drosophila flies takes 3 weeks; Aspergillus conidial preparation takes 3 days; fly inoculation depending on the infection assay takes 1 to 6-8 hours; and assessment of fly survival, Aspergillus strain virulence, Drosophila innate host parameters and/or drug activity takes 4-8 days. PMID:21178494

  11. Ribosomal S6 Kinase Cooperates with Casein Kinase 2 to Modulate the Drosophila Circadian Molecular Oscillator

    PubMed Central

    Akten, Bikem; Tangredi, Michelle M.; Jauch, Eike; Roberts, Mary A.; Ng, Fanny; Raabe, Thomas; Jackson, F. Rob

    2009-01-01

    There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the MAPK pathway, but there is no direct evidence that it functions as a component of the circadian system. Here, we show that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, we demonstrate a physical interaction between S6KII and the Casein Kinase 2 regulatory subunit (CK2β), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. We propose that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism. PMID:19144847

  12. Functional dissection of the Hox protein Abdominal-B in Drosophila cell culture

    SciTech Connect

    Zhai, Zongzhao; Yang, Xingke; Lohmann, Ingrid

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer ct340 CRM was identified to be the posterior spiracle enhancer of gene cut. Black-Right-Pointing-Pointer ct340 is under the direct transcriptional control of Hox protein Abd-B. Black-Right-Pointing-Pointer An efficient cloning system was developed to assay protein-DNA interaction. Black-Right-Pointing-Pointer New features of Abd-B dependent target gene regulation were detected. -- Abstract: Hox transcription factors regulate the morphogenesis along the anterior-posterior (A/P) body axis through the interaction with small cis-regulatory modules (CRMs) of their target gene, however so far very few Hox CRMs are known and have been analyzed in detail. In this study we have identified a new Hox CRM, ct340, which guides the expression of the cell type specification gene cut (ct) in the posterior spiracle under the direct control of the Hox protein Abdominal-B (Abd-B). Using the ct340 enhancer activity as readout, an efficient cloning system to generate VP16 activation domain fusion protein was developed to unambiguously test protein-DNA interaction in Drosophila cell culture. By functionally dissecting the Abd-B protein, new features of Abd-B dependent target gene regulation were detected. Due to its easy adaptability, this system can be generally used to map functional domains within sequence-specific transcriptional factors in Drosophila cell culture, and thus provide preliminary knowledge of the protein functional domain structure for further in vivo analysis.

  13. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  14. Measurement of Larval Activity in the Drosophila Activity Monitor

    PubMed Central

    McParland, Aidan L.; Follansbee, Taylor L.; Ganter, Geoffrey K.

    2016-01-01

    Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals’ positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish. PMID:25993121

  15. A bacterial type III secretion-based delivery system for functional assays of fungal effectors in cereals.

    PubMed

    Upadhyaya, Narayana M; Ellis, Jeffery G; Dodds, Peter N

    2014-01-01

    Large numbers of candidate effectors are being identified by genome sequencing of fungal pathogens and in planta expression studies. These effectors are both a boon and a curse for pathogens as they modulate the host cellular environment or suppress defense response to allow fungal growth as well as become targets of plant resistance (R) proteins. Recognition of a fungal effector by a plant R protein triggers a hypersensitive reaction (HR) leading to death of plant cells in and around the infection site, thus preventing further proliferation of the pathogen. Such HR induction has been used as an indicator of effector activity in functional assays of candidate effectors in dicots based on Agrobacterium-mediated transient expression. However, the Agrobacterium assay is not functional in cereal leaves. We therefore have adapted an alternative assay based on effector protein delivery using the type III secretion system (T3SS) of a non-pathogenic Pseudomonas spp. for use in wheat and other cereals. Here, we describe protocols for delivery of effector proteins into wheat and barley cells using the AvrRpm1 T3SS signal in the engineered non-pathogenic Pseudomonas fluorescens strain Effector-to-Host Analyzer (EtHAn). For ease of making expression clones we have generated the GATEWAY cloning compatible vectors. A calmodulin-dependent adenylate cyclase (Cya) reporter protein can be used as an effective marker for fusion protein delivery into wheat and barley by this system. PMID:24643568

  16. Quantitation, with a new assay, and Theiler's virus capsid protein in the central nervous system of mice

    SciTech Connect

    Cash, E.; Chamorro, M.; Brahic, M.

    1986-11-01

    The authors developed a quantitative assay for antigens at the single-cell level. Tissue sections were reacted with (i) a primarily antibody , (ii) a biotinylated secondary antibody, or (iii) /sup 35/S-streptavidin. Binding of streptavidin to cells was quantitated by microscopic autoradiography. They showed that the number of autoradiographic grains was proportional to the amount of antigen per cell. With this assay, they studied the synthesis of Theiler's virus capsid proteins VP1, VP2, and VP3 in permissive BHK cells grown in vitro and in mouse central nervous system (CNS) cells during a persistent infection. They found that synthesis of the three capsid proteins was restricted in mouse CNS cells. Restricted virus replication could play a major role in the persistence of Theiler's virus in mouse CNS cells.

  17. Experimental verification of modeling results for a PGNAA system for nondestructive assay of RCRA metals in drums

    PubMed

    Dulloo; Ruddy; Congedo; Seidel; McIlwain

    2000-10-01

    The use of pulsed gamma neutron activation analysis (PGNAA) for the nondestructive assay of mercury, cadmium and lead in a concrete matrix has been demonstrated. Calculations have also been performed to study interference with PGNAA detection sensitivity resulting from the presence of 238U, 235U, and 239Pu at levels expected in mixed waste. Analyses based on transport calculations examined the effects of these nuclides on neutron kinetics, and the contribution from the capture, fission and decay gamma-rays emitted by the nuclides to the gamma-ray spectrum. These calculations predicted insignificant effects on the system's detection limits (115, 9 and 4400 ppm for Hg, Cd and Pb, respectively, for a 2000 s assay). This paper describes verification measurements recently conducted using samples of 235U and 231U in the test drum geometry, which have provided experimental confirmation of the modeling results. PMID:11003484

  18. The development of the Drosophila larval brain.

    PubMed

    Hartenstein, Volker; Spindler, Shana; Pereanu, Wayne; Fung, Siaumin

    2008-01-01

    In this chapter we will start out by describing in more detail the progenitors of the nervous system, the neuroblasts and ganglion mother cells. Subsequently we will survey the generic cell types that make up the developing Drosophila brain, namely neurons, glial cells and tracheal cells. Finally, we will attempt a synopsis of the neuronal connectivity of the larval brain that can be deduced from the analysis of neural lineages and their relationship to neuropile compartments. PMID:18683635

  19. A plasmacytoid dendritic cell (CD123+/CD11c-) based assay system to predict contact allergenicity of chemicals

    PubMed Central

    Ayehunie, Seyoum; Snell, Maureen; Child, Matthew; Klausner, Mitchell

    2009-01-01

    A predictive allergenicity test system for assessing the contact allergenicity of chemicals is needed by the cosmetic and pharmaceutical industry to monitor product safety in the marketplace. Development of such non-animal alternative assay systems for skin sensitization and hazard identification has been pursued by policy makers and regulatory agencies. We investigated whether phenotypic and functional changes to a subset of dendritic cells (DC), plasmacytoid DC (pDC), could be used to identify contact allergens. To achieve this goal, normal human DC were generated from CD34+ progenitor cells and cryopreserved. Frozen DC were thawed and the pDC fraction (CD123+/CD11c-) was harvested using FACS sorting. The pDC were cultured, expanded, and exposed to chemical allergens (N=26) or non-allergens (N=22). Concentrations of each chemical that resulted in >50% viability was determined using FACS analysis of propidium iodide stained cells using pDC from 2-5 donors. Expression of the surface marker, CD86, which has been implicated in dendritic cell maturation, was used as a marker of allergenicity. CD86 expression increased (≥ 1.5 fold) for 25 of 26 allergens (sensitivity = 96%) but did not increase for 19 of 22 non-allergens (specificity = 86%). In a direct comparison to historical data for the regulatory approved, mouse local lymph node assay (LLNA) for 23 allergens and 22 non-allergens, the pDC method had sensitivity and specificity of 96% and 86%, respectively, while the sensitivity and specificity of the LLNA assay was 83% and 82%, respectively. In conclusion, CD86 expression in pDC appears to be a sensitive and specific indicator to identify contact allergenicity. Such an assay method utilizing normal human cells will be useful for high throughput screening of chemicals for allergenicity. PMID:19665512

  20. Uncoupling neuronal death and dysfunction in Drosophila models of neurodegenerative disease.

    PubMed

    Chouhan, Amit K; Guo, Caiwei; Hsieh, Yi-Chen; Ye, Hui; Senturk, Mumine; Zuo, Zhongyuan; Li, Yarong; Chatterjee, Shreyasi; Botas, Juan; Jackson, George R; Bellen, Hugo J; Shulman, Joshua M

    2016-01-01

    Common neurodegenerative proteinopathies, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by the misfolding and aggregation of toxic protein species, including the amyloid beta (Aß) peptide, microtubule-associated protein Tau (Tau), and alpha-synuclein (αSyn) protein. These factors also show toxicity in Drosophila; however, potential limitations of prior studies include poor discrimination between effects on the adult versus developing nervous system and neuronal versus glial cell types. In addition, variable expression paradigms and outcomes hinder systematic comparison of toxicity profiles. Using standardized conditions and medium-throughput assays, we express human Tau, Aß or αSyn selectively in neurons of the adult Drosophila retina and monitor age-dependent changes in both structure and function, based on tissue histology and recordings of the electroretinogram (ERG), respectively. We find that each protein causes a unique profile of neurodegenerative pathology, demonstrating distinct and separable impacts on neuronal death and dysfunction. Strikingly, expression of Tau leads to progressive loss of ERG responses whereas retinal architecture and neuronal numbers are largely preserved. By contrast, Aß induces modest, age-dependent neuronal loss without degrading the retinal ERG. αSyn expression, using a codon-optimized transgene, is characterized by marked retinal vacuolar change, progressive photoreceptor cell death, and delayed-onset but modest ERG changes. Lastly, to address potential mechanisms, we perform transmission electron microscopy (TEM) to reveal potential degenerative changes at the ultrastructural level. Surprisingly, Tau and αSyn each cause prominent but distinct synaptotoxic profiles, including disorganization or enlargement of photoreceptor terminals, respectively. Our findings highlight variable and dynamic properties of neurodegeneration triggered by these disease-relevant proteins in vivo, and suggest

  1. Transcription of a Drosophila tRNAArg gene in yeast extract: 5'-flanking sequence dependence for transcription in a heterologous system.

    PubMed Central

    Schaack, J; Söll, D

    1985-01-01

    The Drosophila tRNA gene encoded on pArg is efficiently transcribed in extracts of Saccharomyces cerevisiae, but the efficiency is 5'-flanking sequence dependent: deletion to between positions -21 and -17 (relative to position +1 of the mature coding sequence) reduces transcription to a very low level. This demonstrates that requirement for wild-type 5'-flanking sequence exists in the case of a heterologous combination of a tRNA gene and transcription extract. Expression of pArg in vivo in S. cerevisiae is also dependent on the wild-type 5'-flanking sequence, but only with deletion to between -17 and -11 is the steady-state level of pArg transcripts reduced to near zero. The 5'-flanking sequence requirement in S. cerevisiae extract is similar to that found in Drosophila Kc cell extract. However, transcription kinetics distinguish S. cerevisiae extract from that of Drosophila Kc cells. tRNA genes added to S. cerevisiae extract exhibit a lag phase before initiation of active transcription, but this lag is much shorter and much less temperature dependent than is the lag phase in Drosophila Kc cell extract. Images PMID:3889849

  2. Serum Amyloid P Component Ameliorates Neurological Damage Caused by Expressing a Lysozyme Variant in the Central Nervous System of Drosophila melanogaster.

    PubMed

    Helmfors, Linda; Bergkvist, Liza; Brorsson, Ann-Christin

    2016-01-01

    Lysozyme amyloidosis is a hereditary disease in which mutations in the gene coding for lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT) lysozyme and the disease-associated variant F57I in the central nervous system (CNS) of a Drosophila melanogaster model of lysozyme amyloidosis, with and without co-expression of serum amyloid p component (SAP). SAP is known to be a universal constituent of amyloid deposits and to associate with lysozyme fibrils. There are clear indications that SAP may play an important role in lysozyme amyloidosis, which requires further elucidation. We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan than flies expressing WT lysozyme. We also identified apoptotic cells in the brains of F57I flies demonstrating that the flies' neurological functions are impaired when F57I is expressed in the nerve cells. However, co-expression of SAP in the CNS prevented cell death and restored the F57I flies' lifespan. Thus, SAP has the apparent ability to protect nerve cells from damage caused by F57I. Furthermore, it was found that co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies. Our findings suggest that the F57I mutation affects the aggregation process of lysozyme resulting in the formation of cytotoxic species and that SAP is able to prevent cell death in the F57I flies by preventing accumulation of toxic F57I structures. PMID:27428539

  3. Serum Amyloid P Component Ameliorates Neurological Damage Caused by Expressing a Lysozyme Variant in the Central Nervous System of Drosophila melanogaster

    PubMed Central

    2016-01-01

    Lysozyme amyloidosis is a hereditary disease in which mutations in the gene coding for lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT) lysozyme and the disease-associated variant F57I in the central nervous system (CNS) of a Drosophila melanogaster model of lysozyme amyloidosis, with and without co-expression of serum amyloid p component (SAP). SAP is known to be a universal constituent of amyloid deposits and to associate with lysozyme fibrils. There are clear indications that SAP may play an important role in lysozyme amyloidosis, which requires further elucidation. We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan than flies expressing WT lysozyme. We also identified apoptotic cells in the brains of F57I flies demonstrating that the flies’ neurological functions are impaired when F57I is expressed in the nerve cells. However, co-expression of SAP in the CNS prevented cell death and restored the F57I flies’ lifespan. Thus, SAP has the apparent ability to protect nerve cells from damage caused by F57I. Furthermore, it was found that co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies. Our findings suggest that the F57I mutation affects the aggregation process of lysozyme resulting in the formation of cytotoxic species and that SAP is able to prevent cell death in the F57I flies by preventing accumulation of toxic F57I structures. PMID:27428539

  4. Postembryonic lineages of the Drosophila ventral nervous system: Neuroglian expression reveals the adult hemilineage associated fiber tracts in the adult thoracic neuromeres.

    PubMed

    Shepherd, David; Harris, Robin; Williams, Darren W; Truman, James W

    2016-09-01

    During larval life most of the thoracic neuroblasts (NBs) in Drosophila undergo a second phase of neurogenesis to generate adult-specific neurons that remain in an immature, developmentally stalled state until pupation. Using a combination of MARCM and immunostaining with a neurotactin antibody, Truman et al. (2004; Development 131:5167-5184) identified 24 adult-specific NB lineages within each thoracic hemineuromere of the larval ventral nervous system (VNS), but because of the neurotactin labeling of lineage tracts disappearing early in metamorphosis, they were unable extend the identification of these lineages into the adult. Here we show that immunostaining with an antibody against the cell adhesion molecule neuroglian reveals the same larval secondary lineage projections through metamorphosis and bfy identifying each neuroglian-positive tract at selected stages we have traced the larval hemilineage tracts for all three thoracic neuromeres through metamorphosis into the adult. To validate tract identifications we used the genetic toolkit developed by Harris et al. (2015; Elife 4) to preserve hemilineage-specific GAL4 expression patterns from larval into the adult stage. The immortalized expression proved a powerful confirmation of the analysis of the neuroglian scaffold. This work has enabled us to directly link the secondary, larval NB lineages to their adult counterparts. The data provide an anatomical framework that 1) makes it possible to assign most neurons to their parent lineage and 2) allows more precise definitions of the neuronal organization of the adult VNS based in developmental units/rules. J. Comp. Neurol. 524:2677-2695, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:26878258

  5. Development of a screening system for cystic fibrosis: meconium or blood spot trypsin assay or both?

    PubMed

    Pederzini, F; Faraguna, D; Giglio, L; Pedrotti, D; Perobelli, L; Mastella, G

    1990-10-01

    High blood trypsin levels during early days of life are found in newborns subsequently diagnosed to be affected by cystic fibrosis. The authors compared the validity of the traditional meconium test with the blood immunoreactive trypsin (IRT) assay, carried out in parallel on 113,302 neonates from three regions of North-eastern Italy. The meconium test showed a sensitivity of 57.7%. The sensitivity of the IRT test was higher (96.1%). It was possible to identify by IRT 10 out of 11 false negative CFs at the meconium test. A shortcoming of neonatal IRT, however, is its low specificity; 1.6% of the newborns had to be retested. A new screening policy was therefore proposed and carried out on 69,640 newborns: the Lactase test (LACT) on meconium was introduced as a complementary assay in IRT positive newborns. If LACT exceeded 2 U/g dry meconium, a confirmatory sweat test was immediately requested; if LACT test was negative and IRT exceeded 85 micrograms/l, IRT was repeated. Postneonatal retesting values above 25 micrograms/l required a sweat test. As a result, the estimated prevalence of CF was 1:4,352, the sensitivity was 93.3%; the specificity turned out to be 99.6%, considering all false positive newborns investigated with retesting and/or direct sweat test. PMID:2124772

  6. Biotesting of wastewater: Comparative study using the Salmonella and CHO assay systems

    SciTech Connect

    Waters, L.C.; Schenley, R.L.; Owen, B.A.; Jolley, R.L.; Buchanan, M.V. ); Walsh, P.J. ); Hsie, A.W. ); Condie, L.W. )

    1989-01-01

    Means to assess the toxicity of wastewaters are essential to implementing the Federal Clean Water Act. Health risk assessment based on single chemicals is limited by the number of chemicals that can be identified and to those chemicals for which toxicity data are available. Long-term whole animal tests on large numbers of waste-water samples are not practical. In this study, two short-term tests, the Salmonella mutagenicity assay and the Chinese hamster ovary (CHO) cell assay for mutagenicity and cytotoxicity, were evaluated as potentially useful biomonitors of wastewaters. Cytotoxicity and mutagenicity were detected in some unconcentrated wastewater samples using these modifications. Data on eight wastewater samples, representing five different sites, indicated that the Salmonella test is the more sensitive indicator of mutagenic activity in those samples, whereas the CHO test is a sensitive indicator of the presence of cytotoxic components. Wastewater concentrates, prepared by adsorption onto XAD-2 and blue cotton, were compared in the two bioassays. In a single concentrate, the two short-term tests detected distinctly different mutagens. Advantages of using the CHO-AS52 cell line instead of the CHO-K{sub 1}BH{sub 4} line for detecting wastewater mutagens were indicated. This study illustrates the complementary use of multiple bioassays and concentration methods to detect and characterize toxic components in wastewater.

  7. Genetic effects of ethylene dibromide in Drosophila melanogaster

    SciTech Connect

    Kale, P.; Baum, J.W.

    1981-01-01

    Drosophila, an organism known to be one of the best among the available systems for mutation detection, can be used for detecting very low concentrations of airborne mutagens. Using ethylene dibromide (EDB), it was demonstrated that Drosophila could detect concentrations as low as 0.2 ppM in air when exposed for a relatively short period of 11 hrs. The exposure period can be prolonged to as many as 700 hrs using Drosophila. Response was proportional to integrated exposure in ppM-hr at low exposure values. It is, therefore, possible to detect airborne mutagens in the parts per billion range using proper germ cell stages in this system. Drosophila may, therefore, be used as a biological monitor to detect mutagenicity of air in polluted areas. Thus, the system may complement Tradescantia, another sensitive system being used for this purpose. Ethylene dibromide was selected as a test chemical in these studies since its mutagenic properties had previously been demonstrated in Drosophila and in Tradescantia. In Tradescantia, extensive data on the dose vs. mutation relation were available and these data were considered useful in evaluating the comparative sensitivity of the two systems for detecting airborne mutagens.

  8. An electrochemical one-step system for assaying methyltransferase activity based on transport of a quantum dot signaling tracer.

    PubMed

    Baek, Songyi; Won, Byoung Yeon; Park, Ki Soo; Park, Hyun Gyu

    2013-11-15

    A one-step, electrochemical method for assaying methyltransferase (MTase) activity, based on the convective transport of a quantum dot (QD) signaling tracer, has been developed. The assay chip used in this system was prepared by modifying a gold matrix with CdSe/ZnS QD-tagged dsDNA, which contains a specific methylation site (5'-GATC-3') recognized by MTase. Treatment of the chip with DNA adenine methylation (Dam) MTase, generates a methylated sequence (5'-GAmTC-3') within the dsDNA. The methylated dsDNA is then subjected to a cleavage reaction, induced by DpnI, which leads to release from the gold matrix of a DNA fragment tethered to a QD. Detection of the released QD, using square wave anodic stripping voltammetry (SWASV) on a glassy carbon (GC) electrode, enables the reliable quantitation of the methylated DNA. Because it is accomplished in a simple and convenient one step and does not require any complicated secondary or tedious washing steps, the new assay method holds great promise for epigenetic analysis in facility-limited environments or point-of-care testing (POCT) applications. PMID:23777705

  9. The genome sequence of Drosophila melanogaster.

    SciTech Connect

    2000-03-24

    The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the {approximately}120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes {approximately}13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

  10. Imaging Calcium in Drosophila at Egg Activation.

    PubMed

    Derrick, Christopher J; York-Andersen, Anna H; Weil, Timothy T

    2016-01-01

    Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca(2+)) often termed a 'Ca(2+) wave'. While the speed and number of oscillations of the Ca(2+) wave varies between species, the change in intracellular Ca(2+) is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton. In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca(2+)-dependent events. Here we present a protocol for imaging the Ca(2+) wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca(2+) wave and the downstream changes associated with it. PMID:27584955

  11. Flightless Flies: Drosophila models of neuromuscular disease

    PubMed Central

    Lloyd, Thomas E.; Taylor, J. Paul

    2010-01-01

    The fruit fly, Drosophila melanogaster, has a long and rich history as an important model organism for biologists. In particular, study of the fruit fly has been essential to much of our fundamental understanding of the development and function of the nervous system. In recent years, studies using fruit flies have provided important insights into the pathogenesis of neurodegenerative and neuromuscular diseases. Fly models of spinal muscular atrophy, spinobulbar muscular atrophy, myotonic dystrophy, dystrophinopathies and other inherited neuromuscular diseases recapitulate many of the key pathologic features of the human disease. The ability to perform genetic screens holds promise for uncovering the molecular mechanisms of disease, and indeed, for identifying novel therapeutic targets. This review will summarize recent progress in developing fly models of neuromuscular diseases and will emphasize the contribution that Drosophila has made to our understanding of these diseases. PMID:20329357

  12. Validation of Non-Invasive Waste Assay System (Gamma Box Counter) Performance at AECL Whiteshell Laboratories - 13136

    SciTech Connect

    Attas, E.M.; Bialas, E.; Rhodes, M.J.

    2013-07-01

    Low-level radioactive waste (LLW) in solid form, resulting from decommissioning and operations activities at AECL's Whiteshell Laboratories (WL), is packaged in B-25 and B-1000 standard waste containers and characterized before it is shipped to an on-site interim storage facility, pending AECL decisions on long term management of its LLW. Assay of the waste packages before shipment contributes to an inventory of the interim storage facility and provides data to support acceptance at a future repository. A key characterization step is a gamma spectrometric measurement carried out under standard conditions using an automated, multi-detector Waste Assay System (WAS), purchased from Antech Corporation. A combination of ORTEC gamma acquisition software and custom software is used in this system to incorporate multiple measurements from two collimated high-resolution detectors. The software corrects the intensities of the gamma spectral lines for geometry and attenuation, and generates a table of calculated activities or limits of detection for a user-defined list of radioisotopes that may potentially be present. Validation of WAS performance was a prerequisite to routine operation. Documentation of the validation process provides assurance of the quality of the results produced, which may be needed one or two decades after they were generated. Aspects of the validation included setting up a quality control routine, measurements of standard point sources in reproducible positions, study of the gamma background, optimization of user-selectable software parameters, investigation of the effect of non-uniform distribution of materials and radionuclides, and comparison of results with measurements made using other gamma detector systems designed to assay bulk materials. The following key components of the validation process have been established. A daily quality control routine has been instituted, to verify stability of the gamma detector operation and the background levels

  13. SPARC–Dependent Cardiomyopathy in Drosophila

    PubMed Central

    Motamedchaboki, Khatereh; Bodmer, Rolf

    2016-01-01

    Background— The Drosophila heart is an important model for studying the genetics underpinning mammalian cardiac function. The system comprises contractile cardiomyocytes, adjacent to which are pairs of highly endocytic pericardial nephrocytes that modulate cardiac function by uncharacterized mechanisms. Identifying these mechanisms and the molecules involved is important because they may be relevant to human cardiac physiology. Methods and Results— This work aimed to identify circulating cardiomodulatory factors of potential relevance to humans using the Drosophila nephrocyte–cardiomyocyte system. A Kruppel-like factor 15 (dKlf15) loss-of-function strategy was used to ablate nephrocytes and then heart function and the hemolymph proteome were analyzed. Ablation of nephrocytes led to a severe cardiomyopathy characterized by a lengthening of diastolic interval. Rendering adult nephrocytes dysfunctional by disrupting their endocytic function or temporally conditional knockdown of dKlf15 led to a similar cardiomyopathy. Proteomics revealed that nephrocytes regulate the circulating levels of many secreted proteins, the most notable of which was the evolutionarily conserved matricellular protein Secreted Protein Acidic and Rich in Cysteine (SPARC), a protein involved in mammalian cardiac function. Finally, reducing SPARC gene dosage ameliorated the cardiomyopathy that developed in the absence of nephrocytes. Conclusions— The data implicate SPARC in the noncell autonomous control of cardiac function in Drosophila and suggest that modulation of SPARC gene expression may ameliorate cardiac dysfunction in humans. PMID:26839388

  14. Tilting the balance between facilitatory and inhibitory functions of mammalian and Drosophila Complexins orchestrates synaptic vesicle exocytosis

    PubMed Central

    Xue, Mingshan; Lin, Yong Qi; Pan, Hongling; Reim, Kerstin; Deng, Hui; Bellen, Hugo J.; Rosenmund, Christian

    2009-01-01

    Summary SNARE-mediated synaptic exocytosis is orchestrated by facilitatory and inhibitory mechanisms. Genetic ablations of Complexins, a family of SNARE complex–binding proteins, in mice and Drosophila cause apparently opposite effects on neurotransmitter release, leading to contradictory hypotheses of Complexin function. Reconstitution experiments with different fusion assays and Complexins also yield conflicting results. We therefore performed cross-species rescue experiments to compare the functions of murine and Drosophila Complexins in both mouse and fly synapses. We found that murine and Drosophila Complexins employ conserved mechanisms to regulate exocytosis despite their strikingly different overall effects on neurotransmitter release. Both Complexins contain distinct domains that facilitate or inhibit synaptic vesicle fusion, and the strength of each facilitatory or inhibitory function differs significantly between murine and Drosophila Complexins. Our results show that a relative shift in the balance of facilitatory and inhibitory functions results in differential regulation of neurotransmitter release by murine and Drosophila Complexins in vivo, reconciling previous incompatible findings. PMID:19914185

  15. The Drosophila anatomy ontology

    PubMed Central

    2013-01-01

    Background Anatomy ontologies are query-able classifications of anatomical structures. They provide a widely-used means for standardising the annotation of phenotypes and expression in both human-readable and programmatically accessible forms. They are also frequently used to group annotations in biologically meaningful ways. Accurate annotation requires clear textual definitions for terms, ideally accompanied by images. Accurate grouping and fruitful programmatic usage requires high-quality formal definitions that can be used to automate classification and check for errors. The Drosophila anatomy ontology (DAO) consists of over 8000 classes with broad coverage of Drosophila anatomy. It has been used extensively for annotation by a range of resources, but until recently it was poorly formalised and had few textual definitions. Results We have transformed the DAO into an ontology rich in formal and textual definitions in which the majority of classifications are automated and extensive error checking ensures quality. Here we present an overview of the content of the DAO, the patterns used in its formalisation, and the various uses it has been put to. Conclusions As a result of the work described here, the DAO provides a high-quality, queryable reference for the wild-type anatomy of Drosophila melanogaster and a set of terms to annotate data related to that anatomy. Extensive, well referenced textual definitions make it both a reliable and useful reference and ensure accurate use in annotation. Wide use of formal axioms allows a large proportion of classification to be automated and the use of consistency checking to eliminate errors. This increased formalisation has resulted in significant improvements to the completeness and accuracy of classification. The broad use of both formal and informal definitions make further development of the ontology sustainable and scalable. The patterns of formalisation used in the DAO are likely to be useful to developers of other

  16. Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors.

    PubMed

    Perret, Bénédicte G; Wagner, Renaud; Lecat, Sandra; Brillet, Karl; Rabut, Gwénaël; Bucher, Bernard; Pattus, Franc

    2003-09-01

    The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [35S]GTPgammaS incorporation. The EGFPhMOR protein had the expected molecular weight (70kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken. PMID:12963349

  17. Cell-based assay system for high-throughput screening of anti-photo-aging agents in fibroblast transfectants.

    PubMed

    Lee, S; Shin, S; Jung, E; Park, D

    2016-08-01

    The matricellular protein CCN1 is significantly elevated in acutely ultraviolet-irradiated human skin and negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation. In this study, we established a stable cell line, termed CCN1-GFs, by transfection of the pAcGFP1-1-CCN1 promoter plasmid and examined its usefulness as a cell-based assay system for screening anti-aging ingredients. The promoter of the reporter plasmid pAcGFP1-1-CCN1 promoter was transfected into NIH3T3 cells using the Lipofectamine reagent. G418-resistant cells were selected and further cloned. To confirm whether AcGFP1-1-CCN1 promoter plasmid recombined in the NIH3T3 cells, the level of AcGFP1-1-CCN1 was measured by PCR analysis. To determine if NIH3T3 cells expressed the gene encoding green fluorescence protein in a CCN1 promoter-dependent manner, the reporter enzyme activities were assayed using a fluorimeter and flow cytometer. To determine if CCN1 inhibitor, which was selected through this system, exerted a direct effect on the downstream signal, mRNA expression of collagen1 and MMP1A was checked by using real-time PCR. UVB irradiation of CCN1-GFs resulted in increased CCN1 promoter activity. Treatment with retinoic acid, a CCN1 inhibitor, inhibited UV-induced CCN1 promoter activity. Subsequent use of this assay system to screen anti-aging ingredients revealed that CCN1-GFs treated with sclareol showed decreased levels of UVB-induced CCN1 expression. Sclareol attenuated UVB-induced photo-aging by an increase in collagen synthesis and decrease in MMP-1 activity. PMID:26281901

  18. Sexual circuitry in Drosophila.

    PubMed

    Auer, Thomas O; Benton, Richard

    2016-06-01

    The sexual behavior of Drosophila melanogaster is an outstanding paradigm to understand the molecular and neuronal basis of sophisticated animal actions. We discuss recent advances in our knowledge of the genetic hardwiring of the underlying neuronal circuitry, and how pertinent sensory cues are differentially detected and integrated in the male and female brain. We also consider how experience influences these circuits over short timescales, and the evolution of these pathways over longer timescales to endow species-specific sexual displays and responses. PMID:26851712

  19. A Transcriptional Reporter of Intracellular Ca2+ in Drosophila

    PubMed Central

    Riabinina, Olena; Li, Jiefu; Potter, Christopher J

    2015-01-01

    Intracellular Ca2+ is a widely used neuronal activity indicator. Here we describe a transcriptional reporter of intracellular Ca2+ (TRIC) in Drosophila, which uses a binary expression system to report Ca2+-dependent interactions between calmodulin and its target peptide. We show that in vitro assays predict in vivo properties of TRIC, and that TRIC signals in sensory systems depend on neuronal activity. TRIC can quantitatively monitor neuronal responses that change slowly, such as those of neuropeptide F-expressing neurons to sexual deprivation and neuroendocrine pars intercerebralis (PI) cells to food and arousal. Furthermore, TRIC-induced expression of a neuronal silencer in nutrient activated cells enhanced stress resistance, providing proof-of-principle that TRIC can be used for circuit manipulation. Thus, TRIC facilitates the monitoring and manipulation of neuronal activity, especially those reflecting slow changes in physiological states that are poorly captured by existing methods. TRIC’s modular design should enable optimization and adaptation to other organisms. PMID:25961791

  20. Technical note: A portable on-chip assay system for absorbance and plasmonic detection of protein hormone in milk.

    PubMed

    Ozhikandathil, Jayan; Badilescu, Simona; Packirisamy, Muthukumaran

    2015-07-01

    This paper reports a portable device and method to extract and detect protein hormone in milk samples. Recombinant protein hormone spiked into milk samples was extracted by solid-phase extraction, and detection was carried out using the plasmonic property of gold nanoislands deposited on a glass substrate. Trace levels of hormone spiked in milk were analyzed by their optical absorbance property using a microfluidic chip. We built a portable assay system using disposable lab-on-chip devices. The proposed method is able to detect spiked recombinant protein hormone in milk at concentrations as low as 5ng/mL. PMID:25497819

  1. Nutritional regulation of stem and progenitor cells in Drosophila

    PubMed Central

    Shim, Jiwon; Gururaja-Rao, Shubha; Banerjee, Utpal

    2013-01-01

    Stem cells and their progenitors are maintained within a microenvironment, termed the niche, through local cell-cell communication. Systemic signals originating outside the niche also affect stem cell and progenitor behavior. This review summarizes studies that pertain to nutritional effects on stem and progenitor cell maintenance and proliferation in Drosophila. Multiple tissue types are discussed that utilize the insulin-related signaling pathway to convey nutritional information either directly to these progenitors or via other cell types within the niche. The concept of systemic control of these cell types is not limited to Drosophila and may be functional in vertebrate systems, including mammals. PMID:24255094

  2. Bioimage Informatics in the context of Drosophila research.

    PubMed

    Jug, Florian; Pietzsch, Tobias; Preibisch, Stephan; Tomancak, Pavel

    2014-06-15

    Modern biological research relies heavily on microscopic imaging. The advanced genetic toolkit of Drosophila makes it possible to label molecular and cellular components with unprecedented level of specificity necessitating the application of the most sophisticated imaging technologies. Imaging in Drosophila spans all scales from single molecules to the entire populations of adult organisms, from electron microscopy to live imaging of developmental processes. As the imaging approaches become more complex and ambitious, there is an increasing need for quantitative, computer-mediated image processing and analysis to make sense of the imagery. Bioimage Informatics is an emerging research field that covers all aspects of biological image analysis from data handling, through processing, to quantitative measurements, analysis and data presentation. Some of the most advanced, large scale projects, combining cutting edge imaging with complex bioimage informatics pipelines, are realized in the Drosophila research community. In this review, we discuss the current research in biological image analysis specifically relevant to the type of systems level image datasets that are uniquely available for the Drosophila model system. We focus on how state-of-the-art computer vision algorithms are impacting the ability of Drosophila researchers to analyze biological systems in space and time. We pay particular attention to how these algorithmic advances from computer science are made usable to practicing biologists through open source platforms and how biologists can themselves participate in their further development. PMID:24732429

  3. Early Olfactory Processing in Drosophila: Mechanisms and Principles

    PubMed Central

    Wilson, Rachel I.

    2014-01-01

    In the olfactory system of Drosophila melanogaster, it is relatively straightforward to make in vivo measurements of activity in neurons corresponding to targeted processing. This, together with the numerical simplicity of the Drosophila olfactory system, has produced rapid gains in our understanding of Drosophila olfaction. This review summarizes the neurophysiology of the first two layers of this system: the peripheral olfactory receptor neurons and their postsynaptic targets in the antennal lobe. We now understand in some detail the cellular and synaptic mechanisms that shape odor representations in these neurons. Together, these mechanisms imply that interesting neural adaptations to environmental statistics have occurred and place some fundamental constraints on early sensory processing that pose challenges for higher brain regions. These findings suggest some general principles with broad relevance to early sensory processing in other modalities. PMID:23841839

  4. Peptidoglycan recognition by the Drosophila Imd pathway.

    PubMed

    Kaneko, Takashi; Golenbock, Douglas; Silverman, Neal

    2005-01-01

    The structural requirements for recognition of peptidoglycan (PGN) by PGRP-LC and activation of the Drosophila IMD pathway are not yet clear. In order to examine this question more carefully, the activity of peptidoglycan from different types of bacteria was compared in cell-based and whole animal assays. Drosophila S2* cells, but not adult flies, responded to Lys-type Micrococcus luteus PGN, but with significantly less potency compared to Dap-type Escherichia coli PGN, while intact Lys-type PGN from Staphylococcus aureus was inactive. After treatment with lysostaphin, which digests the cross-bridging peptides, S. aureus PGN weakly stimulated the IMD pathway, similar to M. luteus PGN. Further digestion with mutanolysin, which creates monomeric PGN fragments, abolished the activity of S. aureus PGN. On the other hand, monomeric E. coli PGN, generated by mutanolysin digestion, was still active but required different isoforms of PGRP-LC for recognition. Polymeric PGN required only PGRP-LCx, while monomeric E. coli PGN required both the PGRP-LCa and PGRP-LCx isoforms. These results suggest that the recognition by PGRP-LCx alone requires polymeric PGN, and that polymeric Dap-type PGN is a more potent PGRP-LCx agonist, compared to Lys-type PGN. These results also suggest that the heteromeric PGRP-LCa/LCx receptor complex recognizes monomeric Dap-type, but not Lys-type, PGN. PMID:16303095

  5. Evolutionary relationships of Drosophila mojavensis geographic host races and their sister species Drosophila arizonae.

    PubMed

    Reed, L K; Nyboer, M; Markow, T A

    2007-03-01

    The cactophilic Drosophila mojavensis species group living in the deserts and dry tropical forests of the southwestern United States and Mexico provides a valuable system for studies in diversification and speciation. Rigorous studies of the relationships between host races of D. mojavensis and the relationships among the members of the species group (D. mojavensis, Drosophila arizona, and Drosophila navojoa) are lacking. We used mitochondrial CO1 sequence data to address the phylogenetics and population genetics of this species group. In this study we have found that the sister species D. mojavensis and D. arizonae share no mitochondrial haplotypes and thus show no evidence for recent introgression. We estimate the divergence time between D. mojavensis and D. arizonae to be between 1.91 and 2.97 million years ago. D. arizonae shows little structure in our population genetic analyses but there is phylogenetic differentiation between southeastern and northern populations of D. arizonae. Drosophila mojavensis shows significant population and phylogenetic structure across the four geographic regions of its distribution. The mitochondrial data support an origin of D. mojavensis on the mainland with early differentiation into the populations now found in the Mojave Desert and the Mainland Sonoran Desert and later colonization of the Baja Peninsula, in contrast to previous models. Also, the sister clade to D. mojavensis/D. arizonae includes D. navojoa and Drosophila huaylasi. By defining the genetic relationships among these populations, we provide a foundation for more sophisticated hypothesis testing regarding the timing of early speciation events and host switches in this species group. PMID:17305857

  6. [Shellfish monitoring system for paralytic shellfish toxins using enzyme-linked immunosorbent assay].

    PubMed

    Shinozaki, Takashi; Watanabe, Ryuichi; Kawatsu, Kentaro; Sakurada, Kiyonari; Takahi, Shinya; Ueno, Ken-ichi; Matsushima, Ryoji; Suzuki, Toshiyuki

    2013-01-01

    We investigated the applicability of enzyme-linked immunosorbent assay (PSP-ELISA) using a monoclonal antibody against paralytic shellfish toxins (PST) for screening oysters collected at several coastal areas in Kumamoto prefecture, Japan. Oysters collected between 2007 and 2010 were analyzed by PSP-ELISA. As an alternative calibrant, a naturally contaminated oyster extract was used to quantify toxins in the oyster samples. The toxicity of the calibrant oyster extract determined by the official testing method, mouse bioassay (MBA), was 4 MU/g. Oyster samples collected over 3 years showed a similar toxin profile to the alternative standard, resulting in good agreement between the PSP-ELISA and the MBA. The PSP-ELISA method was better than the MBA in terms of sensitivity, indicating that it may be useful for earlier warning of contamination of oysters by PST in the distinct coastal areas. To use the PSP-ELISA as a screening method prior to MBA, we finally set a screening level at 2 MU/g PSP-ELISA for oyster monitoring in Kumamoto prefecture. We confirmed that there were on samples exceeding the quarantine level (4 MU/g) in MBA among samples quantified as below the screening level by the PSP-ELISA. It was concluded that the use of PSP-ELISA could reduce the numbers of animals needed for MBA testing. PMID:24389470

  7. Calcium and egg activation in Drosophila.

    PubMed

    Sartain, Caroline V; Wolfner, Mariana F

    2013-01-01

    In many animals, a rise in intracellular calcium levels is the trigger for egg activation, the process by which an arrested mature oocyte transitions to prepare for embryogenesis. In nearly all animals studied to date, this calcium rise, and thus egg activation, is triggered by the fertilizing sperm. However in the insects that have been examined, fertilization is not necessary to activate their oocytes. Rather, these insects' eggs activate as they transit through the female's reproductive tract, regardless of male contribution. Recent studies in Drosophila have shown that egg activation nevertheless requires calcium and that the downstream events and molecules of egg activation are also conserved, despite the difference in initial trigger. Genetic studies have uncovered essential roles for the calcium-dependent enzyme calcineurin and its regulator calcipressin, and have hinted at roles for calmodulin, in Drosophila egg activation. Physiological and in vitro studies have led to a model in which mechanical forces that impact the Drosophila oocyte as it moves through the reproductive tract triggers the influx of calcium from the external environment, thereby initiating egg activation. Future research will aim to test this model, as well as to determine the spatiotemporal dynamics of cytoplasmic calcium flux and mode of signal propagation in this unique system. PMID:23218670

  8. Visualizing the spindle checkpoint in Drosophila spermatocytes

    PubMed Central

    Rebollo, Elena; González, Cayetano

    2000-01-01

    The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time-lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila. PMID:11256627

  9. The Ran Pathway in Drosophila melanogaster Mitosis

    PubMed Central

    Chen, Jack W. C.; Barker, Amy R.; Wakefield, James G.

    2015-01-01

    Over the last two decades, the small GTPase Ran has emerged as a central regulator of both mitosis and meiosis, particularly in the generation, maintenance, and regulation of the microtubule (MT)-based bipolar spindle. Ran-regulated pathways in mitosis bear many similarities to the well-characterized functions of Ran in nuclear transport and, as with transport, the majority of these mitotic effects are mediated through affecting the physical interaction between karyopherins and Spindle Assembly Factors (SAFs)—a loose term describing proteins or protein complexes involved in spindle assembly through promoting nucleation, stabilization, and/or depolymerization of MTs, through anchoring MTs to specific structures such as centrosomes, chromatin or kinetochores, or through sliding MTs along each other to generate the force required to achieve bipolarity. As such, the Ran-mediated pathway represents a crucial functional module within the wider spindle assembly landscape. Research into mitosis using the model organism Drosophila melanogaster has contributed substantially to our understanding of centrosome and spindle function. However, in comparison to mammalian systems, very little is known about the contribution of Ran-mediated pathways in Drosophila mitosis. This article sets out to summarize our understanding of the roles of the Ran pathway components in Drosophila mitosis, focusing on the syncytial blastoderm embryo, arguing that it can provide important insights into the conserved functions on Ran during spindle formation. PMID:26636083

  10. Myoblast fusion in Drosophila

    SciTech Connect

    Haralalka, Shruti; Abmayr, Susan M.

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  11. Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila

    PubMed Central

    Soldano, Alessia; Alpizar, Yeranddy A; Boonen, Brett; Franco, Luis; López-Requena, Alejandro; Liu, Guangda; Mora, Natalia; Yaksi, Emre; Voets, Thomas; Vennekens, Rudi; Hassan, Bassem A; Talavera, Karel

    2016-01-01

    Detecting pathogens and mounting immune responses upon infection is crucial for animal health. However, these responses come at a high metabolic price (McKean and Lazzaro, 2011, Kominsky et al., 2010), and avoiding pathogens before infection may be advantageous. The bacterial endotoxins lipopolysaccharides (LPS) are important immune system infection cues (Abbas et al., 2014), but it remains unknown whether animals possess sensory mechanisms to detect them prior to infection. Here we show that Drosophila melanogaster display strong aversive responses to LPS and that gustatory neurons expressing Gr66a bitter receptors mediate avoidance of LPS in feeding and egg laying assays. We found the expression of the chemosensory cation channel dTRPA1 in these cells to be necessary and sufficient for LPS avoidance. Furthermore, LPS stimulates Drosophila neurons in a TRPA1-dependent manner and activates exogenous dTRPA1 channels in human cells. Our findings demonstrate that flies detect bacterial endotoxins via a gustatory pathway through TRPA1 activation as conserved molecular mechanism. DOI: http://dx.doi.org/10.7554/eLife.13133.001 PMID:27296646

  12. Low-diversity bacterial community in the gut of the fruitfly Drosophila melanogaster.

    PubMed

    Wong, Chun Nin Adam; Ng, Patrick; Douglas, Angela E

    2011-07-01

    The bacteria in the fruitfly Drosophila melanogaster of different life stages was quantified by 454 pyrosequencing of 16S rRNA gene amplicons. The sequence reads were dominated by 5 operational taxonomic units (OTUs) at ≤ 97% sequence identity that could be assigned to Acetobacter pomorum, A. tropicalis, Lactobacillus brevis, L. fructivorans and L. plantarum. The saturated rarefaction curves and species richness indices indicated that the sampling (85,000-159,000 reads per sample) was comprehensive. Parallel diagnostic PCR assays revealed only minor variation in the complement of the five bacterial species across individual insects and three D. melanogaster strains. Other gut-associated bacteria included 6 OTUs with low %ID to previously reported sequences, raising the possibility that they represent novel taxa within the genera Acetobacter and Lactobacillus. A developmental change in the most abundant species, from L. fructivorans in young adults to A. pomorum in aged adults was identified; changes in gut oxygen tension or immune system function might account for this effect. Host immune responses and disturbance may also contribute to the low bacterial diversity in the Drosophila gut habitat. PMID:21631690

  13. Assay methods for small ubiquitin-like modifier (SUMO)-SUMO-interacting motif (SIM) interactions in vivo and in vitro using a split-luciferase complementation system.

    PubMed

    Hirohama, Mikako; Voet, Arnout R D; Ozawa, Takeaki; Saitoh, Hisato; Nakao, Yoichi; Zhang, Kam Y J; Ito, Akihiro; Yoshida, Minoru

    2014-03-01

    SUMOylation is a posttranslational process that attaches a small ubiquitin-like modifier (SUMO) to its target proteins covalently. SUMOylation controls multiple cellular processes through the recognition of SUMO by a SUMO-interacting motif (SIM). In this study, we developed assay systems for detecting noncovalent interactions between SUMO and SIM in cells using split-luciferase complementation. We applied a version of this assay to the detection of in vitro SUMO-SIM interactions using a bacterial expression system. These novel assays enable screening of inhibitors of SUMO-dependent protein-protein interactions, either in vivo or in vitro, in a high-throughput manner. PMID:24333278

  14. [Development of a Real-Time Reverse Transcription PCR Assay System for Detection of Three Subtypes of Influenza A and Influenza B Viruses].

    PubMed

    Yanagita, Mitsutoshi; Kuwamura, Yoshitaka; Kinoshita, Satoru; Nakajima, Takashi; Tomizawa, Syuichi; Ozawa, Tetsuo

    2015-12-01

    We developed the initial real-time reverse transcription PCR assay system for seasonal influenza viruses in 2011. This prototype assay system could detect and identify specific influenza A virus subtypes[H1N1, H3N2 and (H1N1) pdm09] and influenza B virus. In the 2012-2013 season, our prototype PCR assay didn't work well because of point mutations occurred in the neuraminidase (NA) gene of the A (H3N2) strain. We improved the prototype assay by changing the target gene for A (H3N2) strain (2013 improved PCR assay). Moreover, we added the measurement system for the matrix (M) gene that was well conserved and common to all influenza A subtypes. In the 2013-2014 season, point mutations in the hemagglutinin (HA) gene of the A (H1N1) pdm09 strain lowered the sensitivity of the 2013 improved PCR assay, so that we changed the target gene for A (H1N1)pdm09 strain (2014 improved PCR assay). We analyzed swab samples from 1,721 patients in total by at least one of the three PCR assays we developed, and demonstrated that the PCR assays had excellent sensitivity and specificity compared with those of the commercially available rapid immunochromatography kit we used. In this study, the M gene was positive in all patients who were finally diagnosed as influenza A positive by 2013 or 2014 improved PCR assay. Therefore, measurement of the M gene, which is hardly to be affected by antigenic drift of influenza viruses, is thought to be useful in clinical practice. PMID:27089652

  15. An in vitro assay system for studying synapse formation between nociceptive dorsal root ganglion and dorsal horn neurons

    PubMed Central

    Joseph, Donald J.; Choudhury, Papiya; MacDermott, Amy B.

    2010-01-01

    Synapses between nociceptive dorsal root ganglion (DRG) neurons and spinal cord dorsal horn neurons represent the first loci for transmission of painful stimuli. Our knowledge of the molecular organization and development of these synapses is sparse due, partly, to a lack of a reliable model system that reconstitutes synaptogenesis between these two neuronal populations. To address this issue, we have established an in vitro assay system consisting of separately purified DRG neurons and dorsal horn neurons on astrocyte micro-islands. Using immunocytochemistry, we have found that 97%, 93%, 98%, 96%, and 94% of DRG neurons on these microislands express markers often associated with nociceptive neurons including Substance P, TRPV1, calcitonin-gene related peptide (CGRP), TrKA, and peripherin, respectively. Triple labeling with these nociceptive-like markers, synaptic vesicle marker Vglut2 and using MAP2 as a dendritic marker revealed the presence of nociceptive-like markers at synaptic terminals. Using this immunocytochemical approach, we counted contact points as overlapping MAP2/Vglut2 puncta and showed that they increased with time in culture. Single and dual patch clamp recordings showed that overlapping Vglut2/MAP2 puncta observed after a few days in culture are likely to be functional synapses between DRG and dorsal horn neurons in our in vitro assay system. Taken together, these data suggest our co-culture microisland model system consists of mostly nociceptive-like DRG neurons that express presynaptic markers and form functional synapses with their dorsal horn partners. Thus, this model system may have direct application for studies on factors regulating development of nociceptive DRG/dorsal horn synapses. PMID:20385165

  16. Common genes regulate food and ethanol intake in Drosophila.

    PubMed

    Sekhon, Morgan L; Lamina, Omoteniola; Hogan, Kerry E; Kliethermes, Christopher L

    2016-06-01

    The abuse liability of alcohol (ethanol) is believed to result in part from its actions on neurobiological substrates that underlie the motivation toward food and other natural reinforcers, and a growing body of evidence indicates that these substrates are broadly conserved among animal phyla. Understanding the extent to which the substrates regulating ethanol and food intake overlap is an important step toward developing therapeutics that selectively reduce ethanol intake. In the current experiments, we measured food and ethanol intake in Recombinant Inbred (RI) lines of Drosophila melanogaster using several assays, and then calculated genetic correlations to estimate the degree to which common genes might underlie behavior in these assays. We found that food intake and ethanol intake as measured in the capillary assay are genetically correlated traits in D. melanogaster, as well as in a panel of 11 Drosophila species that we tested subsequently. RI line differences in food intake in a dyed food assay were genetically unrelated to ethanol intake in the capillary assay or to ethanol preference measured using an olfactory trap apparatus. Using publicly available gene expression data, we found that expression profiles across the RI lines of a number of genes (including the D2-like dopamine receptor, DOPA decarboxylase, and fruitless) correlated with the RI line differences in food and ethanol intake we measured, while the expression profiles of other genes, including NPF, and the NPF and 5-HT2 receptors, correlated only with ethanol intake or preference. Our results suggest that food and ethanol intake are regulated by some common genes in Drosophila, but that other genes regulate ethanol intake independently of food intake. These results have implications toward the development of therapeutics that preferentially reduce ethanol intake. PMID:27286934

  17. An integrated microfluidic cell culture system for high-throughput perfusion three-dimensional cell culture-based assays: effect of cell culture model on the results of chemosensitivity assays.

    PubMed

    Huang, Song-Bin; Wang, Shih-Siou; Hsieh, Chia-Hsun; Lin, Yung Chang; Lai, Chao-Sung; Wu, Min-Hsien

    2013-03-21

    Although microfluidic cell culture systems are versatile tools for cellular assays, their use has yet to set in motion an evolutionary shift away from conventional cell culture methods. This situation is mainly due to technical hurdles: the operational barriers to the end-users, the lack of compatible detection schemes capable of reading out the results of a microfluidic-based cellular assay, and the lack of fundamental data to bridge the gap between microfluidic and conventional cell culture models. To address these issues, we propose a high-throughput, perfusion, three-dimensional (3-D) microfluidic cell culture system encompassing 30 microbioreactors. This integrated system not only aims to provide a user-friendly cell culture tool for biologists to perform assays but also to enable them to obtain precise data. Its technical features include (i) integration of a heater chip based on transparent indium tin oxide glass, providing stable thermal conditions for cell culturing; (ii) a microscale 3-D culture sample loading scheme that is both efficient and precise; (iii) a non-mechanical pneumatically driven multiplex medium perfusion mechanism; and (iv) a microplate reader-compatible waste medium collector array for the subsequent high throughput bioassays. In this study, we found that the 3-D culture sample loading method provided uniform sample loading [coefficient of variation (CV): 3.2%]. In addition, the multiplex medium perfusion mechanism led to reasonably uniform (CV: 3.6-6.9%) medium pumping rates in the 30 microchannels. Moreover, we used the proposed system to perform a successful cell culture-based chemosensitivity assay. To determine the effects of cell culture models on the cellular proliferation, and the results of chemosensitivity assays, we compared our data with that obtained using three conventional cell culture models. We found that the nature of the cell culture format could lead to different evaluation outcomes. Consequently, when establishing a

  18. Doped colorimetric assay liposomes

    DOEpatents

    Charych, Deborah; Stevens, Raymond C.

    2001-01-01

    The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

  19. Sensorimotor structure of Drosophila larva phototaxis

    PubMed Central

    Kane, Elizabeth A.; Gershow, Marc; Afonso, Bruno; Larderet, Ivan; Klein, Mason; Carter, Ashley R.; de Bivort, Benjamin L.; Sprecher, Simon G.; Samuel, Aravinthan D. T.

    2013-01-01

    The avoidance of light by fly larvae is a classic paradigm for sensorimotor behavior. Here, we use behavioral assays and video microscopy to quantify the sensorimotor structure of phototaxis using the Drosophila larva. Larval locomotion is composed of sequences of runs (periods of forward movement) that are interrupted by abrupt turns, during which the larva pauses and sweeps its head back and forth, probing local light information to determine the direction of the successive run. All phototactic responses are mediated by the same set of sensorimotor transformations that require temporal processing of sensory inputs. Through functional imaging and genetic inactivation of specific neurons downstream of the sensory periphery, we have begun to map these sensorimotor circuits into the larval central brain. We find that specific sensorimotor pathways that govern distinct light-evoked responses begin to segregate at the first relay after the photosensory neurons. PMID:24043822

  20. A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice

    PubMed Central

    2014-01-01

    Background Microinjection of clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-related RNA and DNA into fertilized eggs is a novel approach for creating gene-modified mice. Blastocysts obtained just before implantation may be appropriate for testing the fidelity of CRIPSR/Cas9-mediated genome editing because they can be individually handled in vitro and obtained 3 days after microinjection, thus allowing researchers to check mutations rapidly. However, it is not known whether indel mutations caused by the CRISPR/Cas9 system can be reproducibly detected in embryos. In this study, we assessed the detection of CRISPR/Cas9-induced mutations in embryos. Results T7 endonuclease I was more effective than Surveyor nuclease for detecting mutations in annealed fragments derived from 2 plasmids, which contained nearly identical sequences. Mouse fertilized eggs were microinjected with CRISPR/Cas9-related RNA/DNA to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor (G protein-coupled) activity modifying protein 2 (Ramp2) gene. Individual blastocysts were lysed to obtain crude DNA solutions, which were used for polymerase chain reaction (PCR) assays. T7 endonuclease I-based PCR and sequencing analysis demonstrated that 25–100% of the embryos were knockout embryos and 7–57% of the embryos were knockin embryos. Our results also established that crude DNA from a single blastocyst was an appropriate template for Whole genome amplification and subsequent assessment by PCR and the T7 endonuclease I-based assay. Conclusions The single blastocyst-based assay was useful for determining whether CRISPR/Cas9-mediated genome editing worked in murine embryos. PMID:25042988

  1. Mechanistic investigation of adult myotube response to exercise and drug treatment in vitro using a multiplexed functional assay system

    PubMed Central

    McAleer, C. W.; Smith, A. S. T.; Najjar, S.; Pirozzi, K.; Long, C. J.

    2014-01-01

    The ability to accurately measure skeletal muscle functional performance at the single-cell level would be advantageous for exercise physiology studies and disease modeling applications. To that end, this study characterizes the functional response of individual skeletal muscle myotubes derived from adult rodent tissue to creatine treatment and chronic exercise. The observed improvements to functional performance in response to these treatments appear to correlate with alterations in hypertrophic and mitochondrial biogenesis pathways, supporting previously published in vivo and in vitro data, which highlights the role of these pathways in augmenting skeletal muscle output. The developed system represents a multiplexed functional in vitro assay capable of long-term assessment of contractile cellular outputs in real-time that is compatible with concomitant molecular biology analysis. Adoption of this system in drug toxicity and efficacy studies would improve understanding of compound activity on physical cellular outputs and provide more streamlined and predictive data for future preclinical analyses. PMID:25301895

  2. Rsu1 regulates ethanol consumption in Drosophila and humans

    PubMed Central

    Ojelade, Shamsideen A.; Jia, Tianye; Rodan, Aylin R.; Chenyang, Tao; Kadrmas, Julie L.; Cattrell, Anna; Ruggeri, Barbara; Charoen, Pimphen; Lemaitre, Hervé; Banaschewski, Tobias; Büchel, Christian; Bokde, Arun L. W.; Carvalho, Fabiana; Conrod, Patricia J.; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny A.; Heinz, Andreas; Ittermann, Bernd; Lathrop, Mark; Lubbe, Steven; Martinot, Jean-Luc; Paus, Tomás; Smolka, Michael N.; Spanagel, Rainer; O’Reilly, Paul F.; Laitinen, Jaana; Veijola, Juha M.; Feng, Jianfeng; Desrivières, Sylvane; Jarvelin, Marjo-Riitta; Schumann, Gunter; Rothenfluh, Adrian

    2015-01-01

    Alcohol abuse is highly prevalent, but little is understood about the molecular causes. Here, we report that Ras suppressor 1 (Rsu1) affects ethanol consumption in flies and humans. Drosophila lacking Rsu1 show reduced sensitivity to ethanol-induced sedation. We show that Rsu1 is required in the adult nervous system for normal sensitivity and that it acts downstream of the integrin cell adhesion molecule and upstream of the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase to regulate the actin cytoskeleton. In an ethanol preference assay, global loss of Rsu1 causes high naïve preference. In contrast, flies lacking Rsu1 only in the mushroom bodies of the brain show normal naïve preference but then fail to acquire ethanol preference like normal flies. Rsu1 is, thus, required in distinct neurons to modulate naïve and acquired ethanol preference. In humans, we find that polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption, across phyla. PMID:26170296

  3. Rsu1 regulates ethanol consumption in Drosophila and humans.

    PubMed

    Ojelade, Shamsideen A; Jia, Tianye; Rodan, Aylin R; Chenyang, Tao; Kadrmas, Julie L; Cattrell, Anna; Ruggeri, Barbara; Charoen, Pimphen; Lemaitre, Hervé; Banaschewski, Tobias; Büchel, Christian; Bokde, Arun L W; Carvalho, Fabiana; Conrod, Patricia J; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny A; Heinz, Andreas; Ittermann, Bernd; Lathrop, Mark; Lubbe, Steven; Martinot, Jean-Luc; Paus, Tomás; Smolka, Michael N; Spanagel, Rainer; O'Reilly, Paul F; Laitinen, Jaana; Veijola, Juha M; Feng, Jianfeng; Desrivières, Sylvane; Jarvelin, Marjo-Riitta; Schumann, Gunter; Rothenfluh, Adrian

    2015-07-28

    Alcohol abuse is highly prevalent, but little is understood about the molecular causes. Here, we report that Ras suppressor 1 (Rsu1) affects ethanol consumption in flies and humans. Drosophila lacking Rsu1 show reduced sensitivity to ethanol-induced sedation. We show that Rsu1 is required in the adult nervous system for normal sensitivity and that it acts downstream of the integrin cell adhesion molecule and upstream of the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase to regulate the actin cytoskeleton. In an ethanol preference assay, global loss of Rsu1 causes high naïve preference. In contrast, flies lacking Rsu1 only in the mushroom bodies of the brain show normal naïve preference but then fail to acquire ethanol preference like normal flies. Rsu1 is, thus, required in distinct neurons to modulate naïve and acquired ethanol preference. In humans, we find that polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption, across phyla. PMID:26170296

  4. Review: Thermal preference in Drosophila

    PubMed Central

    Dillon, Michael E.; Wang, George; Garrity, Paul A.; Huey, Raymond B.

    2009-01-01

    Environmental temperature strongly affects physiology of ectotherms. Small ectotherms, like Drosophila, cannot endogenously regulate body temperature so must rely on behavior to maintain body temperature within a physiologically permissive range. Here we review what is known about Drosophila thermal preference. Work on thermal behavior in this group is particularly exciting because it provides the opportunity to connect genes to neuromolecular mechanisms to behavior to fitness in the wild. PMID:20161211

  5. GERMLINE MOLECULAR MUTATION ASSAY IN DROSOPHILA MELANOGASTER. (R825810)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  6. Neurally expressed Drosophila genes encoding homologs of the NSF and SNAP secretory proteins.

    PubMed Central

    Ordway, R W; Pallanck, L; Ganetzky, B

    1994-01-01

    Several lines of investigation have now converged to indicate that the neurotransmitter release apparatus is formed by assembly of cytosolic proteins with proteins of the synaptic vesicle and presynaptic terminal membranes. We are undertaking a genetic approach in Drosophila melanogaster to investigate the functions of two types of cytosolic proteins thought to function in this complex: N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs). We have identified Drosophila homologs of the vertebrate and yeast NSF and SNAP genes. Both Drosophila genes encode polypeptides that closely resemble their vertebrate counterparts and are expressed in the nervous system; neither appears to be in a family of closely related Drosophila genes. These results indicate that the Drosophila NSF and SNAP genes are excellent candidates for mutational analysis of neurotransmitter release. Images PMID:8202553

  7. Identification and Characterization of Genes Involved in Embryonic Crystal Cell Formation During Drosophila Hematopoiesis

    PubMed Central

    Milchanowski, Allison B.; Henkenius, Amy L.; Narayanan, Maya; Hartenstein, Volker; Banerjee, Utpal

    2004-01-01

    Parallels between vertebrate and Drosophila hematopoiesis add to the value of flies as a model organism to gain insights into blood development. The Drosophila hematopoietic system is composed of at least three classes of terminally differentiated blood cells: plasmatocytes, crystal cells, and lamellocytes. Recent studies have identified transcriptional and signaling pathways in Drosophila involving proteins similar to those seen in human blood development. To identify additional genes involved in Drosophila hematopoiesis, we have conducted a P-element-based genetic screen to isolate mutations that affect embryonic crystal cell development. Using a marker of terminally differentiated crystal cells, we screened 1040 P-element-lethal lines located on the second and third chromosomes and identified 44 individual lines that affect crystal cell development. Identifying novel genes and pathways involved in Drosophila hematopoiesis is likely to provide further insights into mammalian hematopoietic development and disorders. PMID:15454546

  8. Establishment of a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the melanogenesis-related genes in human melanoma cells.

    PubMed

    Lin, Chih-Chien; Yang, Chao-Hsun; Lin, Ying-Ju; Chiu, Ya-Wen; Chen, Cheng-Yu

    2015-01-01

    There are two established depigmenting agent assays currently in use. However, these methods are unreliable and time-consuming. Therefore, it will be valuable to establish a better assay system for depigmenting agent analysis. In this study, we established a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the microphthalmia-associated transcription factor (MITF), tyrosinase (Tyr) and dopachrome tautomerase (Dct) genes in MeWo human melanoma cells. We used several melanogenesis regulators, including theophylline, hesperetin, arbutin and rottlerin, to confirm the function of this assay system. The established MeWo/pMITF-EGFP, MeWo/pTyr-EGFP and MeWo/pDct-EGFP stable cells integrated the pMITF-EGFP, pTyr-EGFP and pDct-EGFP plasmids into their genomic DNA. These stably transfected cells were used to examine alterations in the expression of the MITF, Tyr and Dct genes. All of the tested compounds, including theophylline, hesperetin, arbutin and rottlerin, could be analyzed in the stable cells, producing reliable results. Therefore, we believe that this melanogenesis regulation assay system can be used as a rapid and reliable assay system to analyze the regulation of melanogenesis by many known or unknown compounds. PMID:25435499

  9. Chromosome and genetic testing using ChIP assay.

    PubMed

    Kohzaki, Hidetsugu; Asano, Maki

    2016-01-01

    Chromatin immunoprecipitation (ChIP) assay can be used to easily visualize information about proteins, DNA, and RNA on chromosomes and is widely used for analysis of genomes, epigenomes, mRNAs, and non-coding RNAs. The ChIP assay can detect, not only DNA-binding proteins of various organisms, but also the temporal and spatial regulating mechanisms of RNA-binding proteins. Because of these features, demand for ChIP assay is expected to grow. Here, by using yeast and Drosophila as examples, we describe the superiority of the improved ChIP assay that we have developed. PMID:27100707

  10. A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.

    PubMed

    Aouida, Mustapha; Kim, Kangchang; Shaikh, Abdul Rajjak; Pardo, Jose M; Eppinger, Jörg; Yun, Dae-Jin; Bressan, Ray A; Narasimhan, Meena L

    2013-01-01

    Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to

  11. New Tools for the Analysis of Glial Cell Biology in Drosophila

    PubMed Central

    Awasaki, Takeshi; Lee, Tzumin

    2010-01-01

    Due to its genetic, molecular, and behavioral tractability, Drosophila has emerged as a powerful model system for studying molecular and cellular mechanisms underlying the development and function of nervous systems. The Drosophila nervous system has fewer neurons and exhibits a lower glia:neuron ratio than is seen in vertebrate nervous systems. Despite the simplicity of the Drosophila nervous system, glial organization in flies is as sophisticated as it is in vertebrates. Furthermore, fly glial cells play vital roles in neural development and behavior. In addition, powerful genetic tools are continuously being created to explore cell function in vivo. In taking advantage of these features, the fly nervous system serves as an excellent model system to study general aspects of glial cell development and function in vivo. In this article, we review and discuss advanced genetic tools that are potentially useful for understanding glial cell biology in Drosophila. PMID:21305614

  12. Glycoprofiling as a novel tool in serological assays of systemic sclerosis: A comparative study with three bioanalytical methods

    PubMed Central

    Klukova, Ludmila; Bertok, Tomas; Petrikova, Miroslava; Sediva, Alena; Mislovicova, Danica; Katrlik, Jaroslav; Vikartovska, Alica; Filip, Jaroslav; Kasak, Peter; Andicsová-Eckstein, Anita; Mosnáček, Jaroslav; Lukáč, Jozef; Rovenský, Jozef; Imrich, Richard; Tkac, Jan

    2016-01-01

    Systemic sclerosis (SSc) is an autoimmune disease seriously affecting patient´s quality of life. The heterogeneity of the disease also means