An in-vivo microfluidic assay reveals cardiac toxicity of heavy metals and the protective effect of metal responsive transcription factor (MTF-1) in model.

Unlabelled: Previous toxicity assessments of heavy metals on are limited to investigating the survival, development rate, and climbing behaviour by oral administration while cardiac toxicity of these elements have not been investigated. We utilized a microfluidic device to inject known dosages of zinc (Zn) or cadmium (Cd) into the larvae's hemolymph to expose their heart directly and study their heart rate and arrhythmicity. The effect of heart-specific overexpression of metal responsive transcription factor (MTF-1) on different heartbeat parameters and survival of larvae was investigated.

Characterization of () and , Essential Paralogous Genes Which Encode the Enzymes That Catalyze the Rate-Limiting Step in the Hexosamine Biosynthetic Pathway in .

The () locus is known for its essential role in the development of the embryonic cuticle of . We show here that encodes (; ), the enzyme that catalyzes the rate-limiting step in the hexosamine biosynthesis pathway (HBP). This conserved pathway diverts 2%-5% of cellular glucose from glycolysis and is a nexus of sugar (fructose-6-phosphate), amino acid (glutamine), fatty acid [acetyl-coenzymeA (CoA)], and nucleotide/energy (UDP) metabolism.

Open access tool and microfluidic devices for phenotypic quantification of heart function of intact fruit fly and zebrafish larvae.

In this paper, the heartbeat parameters of small model organisms, i.e. Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish), were quantified in-vivo in intact larvae using microfluidics and a novel MATLAB-based software.

Fly-on-a-Chip: Microfluidics for Drosophila melanogaster Studies.

The fruit fly or Drosophila melanogaster has been used as a promising model organism in genetics, developmental and behavioral studies as well as in the fields of neuroscience, pharmacology, and toxicology. Not only all the developmental stages of Drosophila, including embryonic, larval, and adulthood stages, have been used in experimental in vivo biology, but also the organs, tissues, and cells extracted from this model have found applications in in vitro assays. However, the manual manipulation, cellular investigation and behavioral phenotyping techniques utilized in conventional Drosophila-based in vivo and in vitro assays are mostly time-consuming, labor-intensive, and low in throughput.

Localized microinjection of intact Drosophila melanogaster larva to investigate the effect of serotonin on heart rate.

In this paper, we present a novel hybrid microfluidic device for localized microinjection and heart monitoring of intact Drosophila melanogaster larvae at different developmental stages. Drosophila heart at the larval stage has been used as a model for cardiac disorder studies. However, previous pharmacological and toxicological cardiac studies are limited to dissected (semi-intact) Drosophila larvae which cannot be used for post-treatment studies.

Genetic and Molecular Analysis of Essential Genes in Centromeric Heterochromatin of the Left Arm of Chromosome 3 in .

A large portion of the genome is contained within heterochromatic regions of chromosomes, predominantly at centromeres and telomeres. The remaining euchromatic portions of the genome have been extensively characterized with respect to gene organization, function and regulation. However, it has been difficult to derive similar data for sequences within centromeric (centric) heterochromatin because these regions have not been as amenable to analysis by standard genetic and molecular tools.

Generation and characterization of new alleles of quiver (qvr) that encodes an extracellular modulator of the Shaker potassium channel.

Our earlier genetic screen uncovered a paraquat-sensitive leg-shaking mutant quiver (qvr), whose gene product interacts with the Shaker (Sh) K channel. We also mapped the qvr locus to EY04063 and noticed altered day-night activity patterns in these mutants. Such circadian behavioral defects were independently reported by another group, who employed the qvr allele we supplied them, and attributed the extreme restless phenotype of EY04063 to the qvr gene.

Meiotic recombination is suppressed near the histone-defined border of euchromatin and heterochromatin on chromosome 2L of Drosophila melanogaster.

In Drosophila melanogaster, the borders between pericentric heterochromatin and euchromatin on the major chromosome arms have been defined in various ways, including chromatin-specific histone modifications, the binding patterns of heterochromatin-enriched chromosomal proteins, and various cytogenetic techniques. Elucidation of the genetic properties that independently define the different chromatin states associated with heterochromatin and euchromatin should help refine the boundary. Since meiotic recombination is present in euchromatin, but absent in heterochromatin, it constitutes a key genetic property that can be observed transitioning between chromatin states.

An integrated hybrid microfluidic device for oviposition-based chemical screening of adult Drosophila melanogaster.

Chemical screening using Drosophila melanogaster (the fruit fly) is vital in drug discovery, agricultural, and toxicological applications. Oviposition (egg laying) on chemically-doped agar plates is an important read-out metric used to quantitatively assess the biological fitness and behavioral responses of Drosophila. Current oviposition-based chemical screening studies are inaccurate, labor-intensive, time-consuming, and inflexible due to the manual chemical doping of agar.

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster.

Drosophila melanogaster (fruit fly) is a model organism and its behaviours including oviposition (egg-laying) on agar substrates have been widely used for assessment of a variety of biological processes in flies. Physical and chemical properties of the substrate are the dominant factors affecting Drosophila's oviposition, but they have not been investigated precisely and parametrically with the existing manual approaches. As a result, many behavioral questions about Drosophila oviposition, such as the combined effects of the aforementioned substrate properties (e.

In vivo interaction proteomics reveal a novel p38 mitogen-activated protein kinase/Rack1 pathway regulating proteostasis in Drosophila muscle.

Several recent studies suggest that systemic aging in metazoans is differentially affected by functional decline in specific tissues, such as skeletal muscle. In Drosophila, longevity appears to be tightly linked to myoproteostasis, and the formation of misfolded protein aggregates is a hallmark of senescence in aging muscle. Similarly, defective myoproteostasis is described as an important contributor to the pathology of several age-related degenerative muscle diseases in humans, e.

Expression of zinc-deficient human superoxide dismutase in Drosophila neurons produces a locomotor defect linked to mitochondrial dysfunction.

More than 130 different mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been associated with amyotrophic lateral sclerosis but the mechanism of this toxicity remains controversial. To gain insight into the importance of the zinc site in the pathogenesis of SOD1 in vivo, we generated a Drosophila model with transgenic expression of a zinc-deficient human SOD1. Expression of zinc-deficient SOD1 in Drosophila resulted in a progressive movement defect with associated mitochondrial cristae vacuolization and reductions in adenosine triphosphate (ATP) levels.

Essential loci in centromeric heterochromatin of Drosophila melanogaster. I: the right arm of chromosome 2.

With the most recent releases of the Drosophila melanogaster genome sequences, much of the previously absent heterochromatic sequences have now been annotated. We undertook an extensive genetic analysis of existing lethal mutations, as well as molecular mapping and sequence analysis (using a candidate gene approach) to identify as many essential genes as possible in the centromeric heterochromatin on the right arm of the second chromosome (2Rh) of D. melanogaster.

A Drosophila model of Menkes disease reveals a role for DmATP7 in copper absorption and neurodevelopment.

Human Menkes disease is a lethal neurodegenerative disorder of copper metabolism that is caused by mutations in the ATP7A copper-transporting gene. In the present study, we attempted to construct a Drosophila model of Menkes disease by RNA interference (RNAi)-induced silencing of DmATP7, the Drosophila orthologue of mammalian ATP7A, in the digestive tract. Here, we show that a lowered level of DmATP7 mRNA in the digestive tract results in a reduced copper content in the head and the rest of the body of surviving adults, presumably owing to copper entrapment in the gut.

Cocoa confers life span extension in Drosophila melanogaster.

Cocoa is thought to be an excellent source of antioxidants. Here, we investigated the effects of cocoa supplementation on Drosophila melanogaster life span under different oxidative stress conditions. Our results illustrate that a moderate supplementation of cocoa under normoxia increases the average life span, whereas, at higher concentrations, average life span is normal.

Hypoxia rescues early mortality conferred by superoxide dismutase deficiency.

Oxidative stress is widely associated with disease and aging but the underlying mechanisms are incompletely understood. Here we show that the premature mortality of Drosophila deficient in superoxide scavengers, superoxide dismutase (SOD) 1 or SOD2, is rescued by chronic hypoxia. Strikingly, switching moribund SOD2-deficient adults from normoxia into hypoxia abruptly arrests their impending premature mortality and endows the survivors with a near-normal life span.

Instability of superoxide dismutase 1 of Drosophila in mutants deficient for its cognate copper chaperone.

Copper,zinc superoxide dismutase (SOD1) in mammals is activated principally via a copper chaperone (CCS) and to a lesser degree by a CCS-independent pathway of unknown nature. In this study, we have characterized the requirement for CCS in activating SOD1 from Drosophila. A CCS-null mutant (Ccs(n)(29)(E)) of Drosophila was created and found to phenotypically resemble Drosophila SOD1-null mutants in terms of reduced adult life span, hypersensitivity to oxidative stress, and loss of cytosolic aconitase activity.

Overexpression of metal-responsive transcription factor (MTF-1) in Drosophila melanogaster ameliorates life-span reductions associated with oxidative stress and metal toxicity.

Heavy metals are essential components of many biological processes but are toxic at high concentrations. Our results illustrate that when metal homeostasis is compromised by a mutation in the metal-responsive transcription factor (MTF-1), the life-span is shortened. In contrast, MTF-1 overexpression results in resistant flies with prolonged longevity on iron or cadmium-supplemented media but shortened life-span on zinc-supplemented medium.

Mutations of the withered (whd) gene in Drosophila melanogaster confer hypersensitivity to oxidative stress and are lesions of the carnitine palmitoyltransferase I (CPT I) gene.

Since some oxygen defense mutants of Drosophila melanogaster exhibit a crinkled wing phenotype, a screen was performed on strains bearing mutant alleles conferring a visible wing phenotype to determine whether any were hypersensitive to oxidative stress. One mutant, withered (whd), was found to be sensitive to both dietary paraquat and hyperoxia. New alleles of whd were induced on a defined genetic background and strains carrying these alleles were also found to be sensitive to oxidative stress.

Antioxidants cannot suppress the lethal phenotype of a Drosophila melanogaster model of Huntington's disease.

Substantial evidence suggests that antioxidants may play a major role in delaying the progress of Huntington's disease (HD). Here we investigated the effects of superoxide dismutase (cytoplasmic Cu/ZnSOD and mitochondrial MnSOD) and supplementation with dietary antioxidants (alpha-tocopherol and coenzyme Q10) on survival to adulthood in a Drosophila melanogaster model of HD. Our results illustrate that neither overexpression of superoxide dismutase nor supplementation of dietary antioxidants can rescue the lethal phenotype of HD flies.

The effects of vitamin supplementation on Drosophila life span under normoxia and under oxidative stress.

Vitamin A (retinol), vitamin C (ascorbic acid), and vitamin E (alpha-tocopherol) are each thought to play an important role in the aging process. Here, we investigated the effects of these vitamins on Drosophila melanogaster life span under different oxidative stress conditions. Among the vitamins tested, alpha-tocopherol exhibited the strongest antioxidant activity, extending average and maximum life span for wild-type flies under hyperoxia and for Cu/Zn superoxide dismutase-deficient (SOD1-deficient) flies under normoxia.

Hydrogen peroxide scavenging rescues frataxin deficiency in a Drosophila model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a neurodegenerative disorder arising from a deficit of the mitochondrial iron chaperone, frataxin. Evidence primarily from yeast and mammalian cells is consistent with the hypothesis that a toxic hydroxyl radical generated from hydrogen peroxide (H2O2) via iron-catalyzed Fenton chemistry at least partially underlies the pathology associated with this disease. However, no whole-organism studies have been presented that directly test this hypothesis.

Genomic analysis of nucleoside transporters in Diptera and functional characterization of DmENT2, a Drosophila equilibrative nucleoside transporter.

The recent completion of genome sequencing projects in a number of eukaryotes allows comparative analysis of orthologs, which can aid in identifying evolutionary constraints on protein structure and function. Nucleoside transporters (NTs) are present in a diverse array of organisms and previous studies have suggested that there is low protein sequence similarity but conserved structure in invertebrate and vertebrate NT orthologs. In addition, most taxa possess multiple NT isoforms but their respective roles in the physiology of the organism are not clear.

RNAi-mediated suppression of the mitochondrial iron chaperone, frataxin, in Drosophila.

The mitochondrial iron chaperone, frataxin, plays a critical role in cellular iron homeostasis and the synthesis and regeneration of Fe-S centers. Genetic insufficiency for frataxin is associated with Friedreich's Ataxia in humans and confers loss of function of Fe-containing proteins including components of the respiratory chain and mitochondrial and cytosolic aconitases. Here, we report the use of RNA-interference (RNAi) to suppress frataxin in the multicellular eukaryote, Drosophila.