False negative rates in Drosophila cell-based RNAi screens: a case study

<p>Abstract</p> <p>Background</p> <p>High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention.</p> <p>Results</p> <p>We performed a meta-analysis of several genome-wide, cell-based <it>Drosophila </it>RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene.</p> <p>Conclusions</p> <p>RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.</p

The Aedes aegypti Toll Pathway Controls Dengue Virus Infection

Aedes aegypti, the mosquito vector of dengue viruses, utilizes its innate immune system to ward off a variety of pathogens, some of which can cause disease in humans. To date, the features of insects' innate immune defenses against viruses have mainly been studied in the fruit fly Drosophila melanogaster, which appears to utilize different immune pathways against different types of viruses, in addition to an RNA interference–based defense system. We have used the recently released whole-genome sequence of the Ae. aegypti mosquito, in combination with high-throughput gene expression and RNA interference (RNAi)-based reverse genetic analyses, to characterize its response to dengue virus infection in different body compartments. We have further addressed the impact of the mosquito's endogenous microbial flora on virus infection. Our findings indicate a significant role for the Toll pathway in regulating resistance to dengue virus, as indicated by an infection-responsive regulation and functional assessment of several Toll pathway–associated genes. We have also shown that the mosquito's natural microbiota play a role in modulating the dengue virus infection, possibly through basal-level stimulation of the Toll immune pathway

The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding “EGF-like” domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the “WIF” domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling

Universal three-dimensional crosslinker for all-photopatterned electronics

All-solution processing of large-area organic electronics requires multiple steps of patterning and stacking of various device components. Here, we report the fabrication of highly integrated arrays of polymer thin-film transistors and logic gates entirely through a series of solution processes. The fabrication is done using a three-dimensional crosslinker in tetrahedral geometry containing four photocrosslinkable azide moieties, referred to as 4Bx. 4Bx can be mixed with a variety of solution-processable electronic materials (polymer semiconductors, polymer insulators, and metal nanoparticles) and generate crosslinked network under exposure to UV. Fully crosslinked network film can be formed even at an unprecedentedly small loading, which enables preserving the inherent electrical and structural characteristics of host material. Because the crosslinked electronic component layers are strongly resistant to chemical solvents, micropatterning the layers at high resolution as well as stacking the layers on top of each other by series of solution processing steps is possible

The Drosophila melanogaster host model

The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed

Zinc oxide nanoparticles exhibit cytotoxicity and genotoxicity through oxidative stress responses in human lung fibroblasts and Drosophila melanogaster

Cheng Teng Ng,1,2,* Liang Qing Yong,1,* Manoor Prakash Hande,3 Choon Nam Ong,2 Liya E Yu,4 Boon Huat Bay,1 Gyeong Hun Baeg1 1Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 2Environmental Research Institute, National University of Singapore, Singapore; 3Department of Physiology, Yong Loo Lin School of Medicine, 4Department of Civil and Environmental Engineering, National University of Singapore, Singapore, Singapore *These authors contributed equally to this work Background: Although zinc oxide nanoparticles (ZnO NPs) have been widely used, there has been an increasing number of reports on the toxicity of ZnO NPs. However, study on the underlying mechanisms under in vivo conditions is insufficient.Methods: In this study, we investigated the toxicological profiles of ZnO NPs in MRC5 human lung fibroblasts in vitro and in an in vivo model using the fruit fly Drosophila melanogaster. A comprehensive study was conducted to evaluate the uptake, cytotoxicity, reactive oxygen species (ROS) formation, gene expression profiling and genotoxicity induced by ZnO NPs.Results: For in vitro toxicity, the results showed that there was a significant release of extracellular lactate dehydrogenase and decreased cell viability in ZnO NP-treated MRC5 lung cells, indicating cellular damage and cytotoxicity. Generation of ROS was observed to be related to significant expression of DNA Damage Inducible Transcript (DDIT3) and endoplasmic reticulum (ER) to nucleus signaling 1 (ERN1) genes, which are ER stress-related genes. Oxidative stress induced DNA damage was further verified by a significant release of DNA oxidation product, 8-hydroxydeoxyguanosine (8-OHdG), as well as by the Comet assay. For the in vivo study using the fruit fly D. melanogaster as a model, significant toxicity was observed in F1 progenies upon ingestion of ZnO NPs. ZnO NPs induced significant decrease in the egg-to-adult viability of the flies. We further showed that the decreased viability is closely associated with ROS induction by ZnO NPs. Removal of one copy of the D. melanogaster Nrf2 alleles further decreased the ZnO NPs-induced lethality due to increased production of ROS, indicating that nuclear factor E2-related factor 2 (Nrf2) plays important role in ZnO NPs-mediated ROS production.Conclusion: The present study suggests that ZnO NPs induced significant oxidative stress-related cytotoxicity and genotoxicity in human lung fibroblasts in vitro and in D. melanogaster in vivo. More extensive studies would be needed to verify the safety issues related to increased usage of ZnO NPs by consumers. Keywords: zinc oxide nanoparticles, ROS, oxidative stress, MRC5 cells, Drosophila&nbsp