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Sample records for quantitative rnai screen

  1. RNAi Screening in Spodoptera frugiperda.

    PubMed

    Ghosh, Subhanita; Singh, Gatikrushna; Sachdev, Bindiya; Kumar, Ajit; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K

    2016-01-01

    RNA interference is a potent and precise reverse genetic approach to carryout large-scale functional genomic studies in a given organism. During the past decade, RNAi has also emerged as an important investigative tool to understand the process of viral pathogenesis. Our laboratory has successfully generated transgenic reporter and RNAi sensor line of Spodoptera frugiperda (Sf21) cells and developed a reversal of silencing assay via siRNA or shRNA guided screening to investigate RNAi factors or viral pathogenic factors with extraordinary fidelity. Here we describe empirical approaches and conceptual understanding to execute successful RNAi screening in Spodoptera frugiperda 21-cell line.

  2. Institutional Profile: The Sheffield RNAi screening facility: a service for high-throughput, genome-wide Drosophila RNAi screens.

    PubMed

    Brown, Stephen

    2010-12-01

    The Sheffield RNAi Screening Facility (SRSF) was established in November 2008, as Britain's first Drosophila RNAi screening centre, funded by the University of Sheffield, Biomedical Sciences Department and the Wellcome Trust. The SRSF was formed to service the needs of research groups wanting to carry out high-throughput RNAi screens with Drosophila cells. The rationale for the SRSF is to provide RNAi libraries and the specialist equipment and expertise to do such screens. The facility supports both plate reader assays, high-content microscopy as well as the equipment needed to process these samples in a high-throughput fashion. The SRSF can either be used to identify genes involved in disease representing future drug targets, or to identify genes involved in drug resistance and efficacy.

  3. A Computational model for compressed sensing RNAi cellular screening

    PubMed Central

    2012-01-01

    Background RNA interference (RNAi) becomes an increasingly important and effective genetic tool to study the function of target genes by suppressing specific genes of interest. This system approach helps identify signaling pathways and cellular phase types by tracking intensity and/or morphological changes of cells. The traditional RNAi screening scheme, in which one siRNA is designed to knockdown one specific mRNA target, needs a large library of siRNAs and turns out to be time-consuming and expensive. Results In this paper, we propose a conceptual model, called compressed sensing RNAi (csRNAi), which employs a unique combination of group of small interfering RNAs (siRNAs) to knockdown a much larger size of genes. This strategy is based on the fact that one gene can be partially bound with several small interfering RNAs (siRNAs) and conversely, one siRNA can bind to a few genes with distinct binding affinity. This model constructs a multi-to-multi correspondence between siRNAs and their targets, with siRNAs much fewer than mRNA targets, compared with the conventional scheme. Mathematically this problem involves an underdetermined system of equations (linear or nonlinear), which is ill-posed in general. However, the recently developed compressed sensing (CS) theory can solve this problem. We present a mathematical model to describe the csRNAi system based on both CS theory and biological concerns. To build this model, we first search nucleotide motifs in a target gene set. Then we propose a machine learning based method to find the effective siRNAs with novel features, such as image features and speech features to describe an siRNA sequence. Numerical simulations show that we can reduce the siRNA library to one third of that in the conventional scheme. In addition, the features to describe siRNAs outperform the existing ones substantially. Conclusions This csRNAi system is very promising in saving both time and cost for large-scale RNAi screening experiments which

  4. iScreen: Image-Based High-Content RNAi Screening Analysis Tools.

    PubMed

    Zhong, Rui; Dong, Xiaonan; Levine, Beth; Xie, Yang; Xiao, Guanghua

    2015-09-01

    High-throughput RNA interference (RNAi) screening has opened up a path to investigating functional genomics in a genome-wide pattern. However, such studies are often restricted to assays that have a single readout format. Recently, advanced image technologies have been coupled with high-throughput RNAi screening to develop high-content screening, in which one or more cell image(s), instead of a single readout, were generated from each well. This image-based high-content screening technology has led to genome-wide functional annotation in a wider spectrum of biological research studies, as well as in drug and target discovery, so that complex cellular phenotypes can be measured in a multiparametric format. Despite these advances, data analysis and visualization tools are still largely lacking for these types of experiments. Therefore, we developed iScreen (image-Based High-content RNAi Screening Analysis Tool), an R package for the statistical modeling and visualization of image-based high-content RNAi screening. Two case studies were used to demonstrate the capability and efficiency of the iScreen package. iScreen is available for download on CRAN (http://cran.cnr.berkeley.edu/web/packages/iScreen/index.html). The user manual is also available as a supplementary document.

  5. Axon Regeneration Genes Identified by RNAi Screening in C. elegans

    PubMed Central

    Nix, Paola; Hammarlund, Marc; Hauth, Linda; Lachnit, Martina; Jorgensen, Erik M.

    2014-01-01

    Axons of the mammalian CNS lose the ability to regenerate soon after development due to both an inhibitory CNS environment and the loss of cell-intrinsic factors necessary for regeneration. The complex molecular events required for robust regeneration of mature neurons are not fully understood, particularly in vivo. To identify genes affecting axon regeneration in Caenorhabditis elegans, we performed both an RNAi-based screen for defective motor axon regeneration in unc-70/β-spectrin mutants and a candidate gene screen. From these screens, we identified at least 50 conserved genes with growth-promoting or growth-inhibiting functions. Through our analysis of mutants, we shed new light on certain aspects of regeneration, including the role of β-spectrin and membrane dynamics, the antagonistic activity of MAP kinase signaling pathways, and the role of stress in promoting axon regeneration. Many gene candidates had not previously been associated with axon regeneration and implicate new pathways of interest for therapeutic intervention. PMID:24403161

  6. RNAi screening reveals a large signaling network controlling the Golgi apparatus in human cells.

    PubMed

    Chia, Joanne; Goh, Germaine; Racine, Victor; Ng, Susanne; Kumar, Pankaj; Bard, Frederic

    2012-01-01

    The Golgi apparatus has many important physiological functions, including sorting of secretory cargo and biosynthesis of complex glycans. These functions depend on the intricate and compartmentalized organization of the Golgi apparatus. To investigate the mechanisms that regulate Golgi architecture, we developed a quantitative morphological assay using three different Golgi compartment markers and quantitative image analysis, and performed a kinome- and phosphatome-wide RNAi screen in HeLa cells. Depletion of 159 signaling genes, nearly 20% of genes assayed, induced strong and varied perturbations in Golgi morphology. Using bioinformatics data, a large regulatory network could be constructed. Specific subnetworks are involved in phosphoinositides regulation, acto-myosin dynamics and mitogen activated protein kinase signaling. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades can control glycosylation directly at the Golgi level. Our results provide a genetic overview of the signaling pathways that control the Golgi apparatus in human cells.

  7. Computational detection and suppression of sequence-specific off-target phenotypes from whole genome RNAi screens

    PubMed Central

    Zhong, Rui; Kim, Jimi; Kim, Hyun Seok; Kim, Minsoo; Lum, Lawrence; Levine, Beth; Xiao, Guanghua; White, Michael A.; Xie, Yang

    2014-01-01

    A challenge for large-scale siRNA loss-of-function studies is the biological pleiotropy resulting from multiple modes of action of siRNA reagents. A major confounding feature of these reagents is the microRNA-like translational quelling resulting from short regions of oligonucleotide complementarity to many different messenger RNAs. We developed a computational approach, deconvolution analysis of RNAi screening data, for automated quantitation of off-target effects in RNAi screening data sets. Substantial reduction of off-target rates was experimentally validated in five distinct biological screens across different genome-wide siRNA libraries. A public-access graphical-user-interface has been constructed to facilitate application of this algorithm. PMID:24972830

  8. Phenotypic screen for RNAi effects in the codling moth Cydia pomonella.

    PubMed

    Wang, Jinda; Gu, Liuqi; Ireland, Stephen; Garczynski, Stephen F; Knipple, Douglas C

    2015-11-10

    RNAi-based technologies have the potential to augment, or replace existing pest management strategies. However, some insect taxa are less susceptible to the induction of the post-transcriptional gene silencing effect than others, such as the Lepidoptera. Here we describe experiments to investigate the induction of RNAi in the codling moth, Cydia pomonella, a major lepidopteran pest of apple, pear, and walnut. Prior to a knockdown screen, fluorescently labeled small interfering RNA (siRNA) and double-stranded RNA (dsRNA) derived from green fluorescent protein (GFP) coding sequence were delivered to the surface of artificial diet to which neonate larvae were introduced and subsequently examined for the distribution of fluorescence in their tissues. Fluorescence was highly concentrated in the midgut but its presence in other tissues was equivocal. Next, dsRNAs were made for C. pomonella genes orthologous to those that have well defined deleterious phenotypes in Drosophila melanogaster. A screen was conducted using dsRNAs encoding cullin-1 (Cpcul1), maleless (Cpmle), musashi (Cpmsi), a homeobox gene (CpHbx), and pumilio (Cppum). The dsRNAs designed from these target genes were administered to neonate larvae by delivery to the surface of the growth medium. None of the dsRNA treatments affected larval viability, however Cpcul1-dsRNA had a significant effect on larval growth, with the average length of larvae about 3mm, compared to about 4mm in the control groups. Measurement of Cpcul1 transcript levels by quantitative real-time PCR (qRT-PCR) revealed a dose-dependent RNAi effect in response to increasing amount of Cpcul1-dsRNA. Despite their reduced size, Cpcul1-dsRNA-treated larvae molted normally and matured to adulthood in a manner similar to controls. In an additional experiment, Cpcul1-siRNA was found to induce similar stunting effect as that induced by Cpcul1-dsRNA.

  9. Genome-Wide RNAi Screens in C. elegans to Identify Genes Influencing Lifespan and Innate Immunity.

    PubMed

    Sinha, Amit; Rae, Robbie

    2016-01-01

    RNA interference is a rapid, inexpensive, and highly effective tool used to inhibit gene function. In C. elegans, whole genome screens have been used to identify genes involved with numerous traits including aging and innate immunity. RNAi in C. elegans can be carried out via feeding, soaking, or injection. Here we outline protocols used to maintain, grow, and carry out RNAi via feeding in C. elegans and determine whether the inhibited genes are essential for lifespan or innate immunity.

  10. A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia

    PubMed Central

    Marker, Simone; Carradec, Quentin; Tanty, Véronique; Arnaiz, Olivier; Meyer, Eric

    2014-01-01

    In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia. PMID:24860163

  11. A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia.

    PubMed

    Marker, Simone; Carradec, Quentin; Tanty, Véronique; Arnaiz, Olivier; Meyer, Eric

    2014-06-01

    In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia.

  12. Tribolium castaneum as a model for high-throughput RNAi screening.

    PubMed

    Knorr, Eileen; Bingsohn, Linda; Kanost, Michael R; Vilcinskas, Andreas

    2013-01-01

    Coleopteran insects are a highly diverse and successful order, and many beetle species are significant agricultural pests. New biorational strategies for managing populations of beetles and other insect species are needed as pests develop resistance to chemical insecticides and Bt toxins. There is now an opportunity to use genome sequence data to identify genes that are essential for insect growth, development, or survival as new targets for designing control technology. This goal requires a method for high-throughput in vivo screening of thousands of genes to identify candidate genes that, when their expression is disrupted, have a phenotype that may be useful in insect pest control. Tribolium castaneum, the red flour beetle, is a model organism that offers considerable advantages for such screening, including ease of rearing in large numbers, a sequenced genome, and a strong, systemic RNAi response for specific depletion of gene transcripts. The RNAi effect in T. castaneum can be elicited in any tissue and any stage by the injection of dsRNA into the hemocoel, and injection of dsRNA into adult females can even be used to identify phenotypes in offspring. A pilot RNAi screen (iBeetle) is underway. Several T. castaneum genes with promising RNAi phenotypes for further development as mechanisms for plant protection have been identified. These include heat shock protein 90, chitin synthase, the segmentation gene hairy, and a matrix metalloprotease. Candidate genes identified in T. castaneum screens can then be tested in agricultural pest species (in which screening is not feasible), to evaluate their effectiveness for use in potential plant-based RNAi control strategies. Delivery of dsRNA expressed by genetically modified crops to the midgut of phytophagous insects is under investigation as a new tool for very specific protection of plants from insect pest species. The T. castaneum screening platform offers a system for discovery of candidate genes with high potential

  13. Quantitative evaluation of first, second, and third generation hairpin systems reveals the limit of mammalian vector-based RNAi.

    PubMed

    Watanabe, Colin; Cuellar, Trinna L; Haley, Benjamin

    2016-01-01

    Incorporating miRNA-like features into vector-based hairpin scaffolds has been shown to augment small RNA processing and RNAi efficiency. Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences. Thus, we used quantitative cell-based assays to compare separate third generation artificial miRNA systems, miR-E (based on miR-30a) and miR-3G (based on miR-16-2 and first described in this study) to widely-adopted, first and second generation formats in both Pol-II and Pol-III expression vector contexts. Despite their unique structures and strandedness, and in contrast to first and second-generation RNAi triggers, the third generation formats operated with remarkable similarity to one another, and strong silencing was observed with a significant fraction of the evaluated target sequences within either promoter context. By pairing an established siRNA design algorithm with the third generation vectors we could readily identify targeting sequences that matched or exceeded the potency of those discovered through large-scale sensor-based assays. We find that third generation hairpin systems enable the maximal level of siRNA function, likely through enhanced processing and accumulation of precisely-defined guide RNAs. Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds. Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme.

  14. Quantitative evaluation of first, second, and third generation hairpin systems reveals the limit of mammalian vector-based RNAi

    PubMed Central

    Watanabe, Colin; Cuellar, Trinna L.; Haley, Benjamin

    2016-01-01

    ABSTRACT Incorporating miRNA-like features into vector-based hairpin scaffolds has been shown to augment small RNA processing and RNAi efficiency. Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences. Thus, we used quantitative cell-based assays to compare separate third generation artificial miRNA systems, miR-E (based on miR-30a) and miR-3G (based on miR-16-2 and first described in this study) to widely-adopted, first and second generation formats in both Pol-II and Pol-III expression vector contexts. Despite their unique structures and strandedness, and in contrast to first and second-generation RNAi triggers, the third generation formats operated with remarkable similarity to one another, and strong silencing was observed with a significant fraction of the evaluated target sequences within either promoter context. By pairing an established siRNA design algorithm with the third generation vectors we could readily identify targeting sequences that matched or exceeded the potency of those discovered through large-scale sensor-based assays. We find that third generation hairpin systems enable the maximal level of siRNA function, likely through enhanced processing and accumulation of precisely-defined guide RNAs. Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds. Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme. PMID:26786363

  15. A novel multiplex cell viability assay for high-throughput RNAi screening.

    PubMed

    Gilbert, Daniel F; Erdmann, Gerrit; Zhang, Xian; Fritzsche, Anja; Demir, Kubilay; Jaedicke, Andreas; Muehlenberg, Katja; Wanker, Erich E; Boutros, Michael

    2011-01-01

    Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

  16. A Multivariate Computational Method to Analyze High-Content RNAi Screening Data.

    PubMed

    Rameseder, Jonathan; Krismer, Konstantin; Dayma, Yogesh; Ehrenberger, Tobias; Hwang, Mun Kyung; Airoldi, Edoardo M; Floyd, Scott R; Yaffe, Michael B

    2015-09-01

    High-content screening (HCS) using RNA interference (RNAi) in combination with automated microscopy is a powerful investigative tool to explore complex biological processes. However, despite the plethora of data generated from these screens, little progress has been made in analyzing HC data using multivariate methods that exploit the full richness of multidimensional data. We developed a novel multivariate method for HCS, multivariate robust analysis method (M-RAM), integrating image feature selection with ranking of perturbations for hit identification, and applied this method to an HC RNAi screen to discover novel components of the DNA damage response in an osteosarcoma cell line. M-RAM automatically selects the most informative phenotypic readouts and time points to facilitate the more efficient design of follow-up experiments and enhance biological understanding. Our method outperforms univariate hit identification and identifies relevant genes that these approaches would have missed. We found that statistical cell-to-cell variation in phenotypic responses is an important predictor of hits in RNAi-directed image-based screens. Genes that we identified as modulators of DNA damage signaling in U2OS cells include B-Raf, a cancer driver gene in multiple tumor types, whose role in DNA damage signaling we confirm experimentally, and multiple subunits of protein kinase A.

  17. A multivariate computational method to analyze high-content RNAi screening data

    PubMed Central

    Rameseder, Jonathan; Krismer, Konstantin; Dayma, Yogesh; Ehrenberger, Tobias; Hwang, Mun Kyung; Airoldi, Edoardo M.; Floyd, Scott R.; Yaffe, Michael B.

    2017-01-01

    High-content screening (HCS) using RNA interference (RNAi) in combination with automated microscopy is a powerful investigative tool to explore complex biological processes. However, despite the plethora of data generated from these screens, little progress has been made in analyzing HC data using multivariate methods that exploit the full richness of multidimensional data. We developed a novel multivariate method for HCS, Multivariate Robust Analysis Method (M-RAM), integrating image feature selection with ranking of perturbations for hit identification, and applied this method to a HC RNAi screen to discover novel components of the DNA damage response in an osteosarcoma cell line. M-RAM automatically selects the most informative phenotypic readouts and time points to facilitate the more efficient design of follow-up experiments and enhance biological understanding. Our method outperforms univariate hit identification and identifies relevant genes that these approaches would have missed. We found that statistical cell-to-cell variation in phenotypic responses is an important predictor of ‘hits’ in RNAi-directed image-based screens. Genes that we identified as modulators of DNA damage signaling in U2OS cells include B-Raf, a cancer driver gene in multiple tumor types, whose role in DNA damage signaling we confirm experimentally, and multiple subunits of protein kinase A. PMID:25918037

  18. Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway.

    PubMed

    Chen, Xiaochu; Xu, Lan

    2016-01-01

    The transforming growth factor-β (TGF-β) family of cytokines figures prominently in regulation of embryonic development and adult tissue homeostasis from Drosophila to mammals. Genetic defects affecting TGF-β signaling underlie developmental disorders and diseases such as cancer in human. Therefore, delineating the molecular mechanism by which TGF-β regulates cell biology is critical for understanding normal biology and disease mechanisms. Forward genetic screens in model organisms and biochemical approaches in mammalian tissue culture were instrumental in initial characterization of the TGF-β signal transduction pathway. With complete sequence information of the genomes and the advent of RNA interference (RNAi) technology, genome-wide RNAi screening emerged as a powerful functional genomics approach to systematically delineate molecular components of signal transduction pathways. Here, we describe a protocol for image-based whole-genome RNAi screening aimed at identifying molecules required for TGF-β signaling into the nucleus. Using this protocol we examined >90 % of annotated Drosophila open reading frames (ORF) individually and successfully uncovered several novel factors serving critical roles in the TGF-β pathway. Thus cell-based high-throughput functional genomics can uncover new mechanistic insights on signaling pathways beyond what the classical genetics had revealed.

  19. Phenotype Recognition for RNAi Screening by Random Projection Forest

    NASA Astrophysics Data System (ADS)

    Zhang, Bailing

    2011-06-01

    High-content screening is important in drug discovery. The use of images of living cells as the basic unit for molecule discovery can aid the identification of small compounds altering cellular phenotypes. As such, efficient computational methods are required for the rate limiting task of cellular phenotype identification. In this paper we first investigate the effectiveness of a feature description approach by combining Haralick texture analysis with Curvelet transform and then propose a new ensemble approach for classification. The ensemble contains a set of base classifiers which are trained using random projection (RP) of original features onto higher-dimensional spaces. With Classification and Regression Tree (CART) as the base classifier, it has been empirically demonstrated that the proposed Random Projection Forest ensemble gives better classification results than those achieved by the Boosting, Bagging and Rotation Forest algorithms, offering a classification rate ˜88% with smallest standard deviation, which compares sharply with the published result of 82%.

  20. Chemical & RNAi screening at MSKCC: a collaborative platform to discover & repurpose drugs to fight disease

    PubMed Central

    Bhinder, Bhavneet; Antczak, Christophe; Shum, David; Radu, Constantin; Mahida, Jeni P.; Liu-Sullivan, Nancy; Ibáñez, Glorymar; Raja, Balajee Somalinga; Calder, Paul A.; Djaballah, Hakim

    2014-01-01

    Memorial Sloan-Kettering Cancer Center (MSKCC) has implemented the creation of a full service state-of-the-art High-throughput Screening Core Facility (HTSCF) equipped with modern robotics and custom-built screening data management resources to rapidly store and query chemical and RNAi screening data outputs. The mission of the facility is to provide oncology clinicians and researchers alike with access to cost-effective HTS solutions for both chemical and RNAi screening, with an ultimate goal of novel target identification and drug discovery. HTSCF was established in 2003 to support the institution’s commitment to growth in molecular pharmacology and in the realm of therapeutic agents to fight chronic diseases such as cancer. This endeavor required broad range of expertise in technology development to establish robust and innovative assays, large collections of diverse chemical and RNAi duplexes to probe specific cellular events, sophisticated compound and data handling capabilities, and a profound knowledge in assay development, hit validation, and characterization. Our goal has been to strive for constant innovation, and we strongly believe in shifting the paradigm from traditional drug discovery towards translational research now, making allowance for unmet clinical needs in patients. Our efforts towards repurposing FDA-approved drugs fructified when digoxin, identified through primary HTS, was administered in the clinic for treatment of stage Vb retinoblastoma. In summary, the overall aim of our facility is to identify novel chemical probes, to study cellular processes relevant to investigator’s research interest in chemical biology and functional genomics, and to be instrumental in accelerating the process of drug discovery in academia. PMID:24661215

  1. Advances in genome-wide RNAi cellular screens: a case study using the Drosophila JAK/STAT pathway

    PubMed Central

    2012-01-01

    Background Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated. Results Here we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols. Conclusions Our work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens. PMID:23006893

  2. An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock

    PubMed Central

    Agrawal, Parul; Hardin, Paul E.

    2016-01-01

    Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans. PMID:27784754

  3. An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock.

    PubMed

    Agrawal, Parul; Hardin, Paul E

    2016-12-07

    Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans.

  4. Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human.

    PubMed

    Zanotto-Filho, Alfeu; Dashnamoorthy, Ravi; Loranc, Eva; de Souza, Luis H T; Moreira, José C F; Suresh, Uthra; Chen, Yidong; Bishop, Alexander J R

    2016-01-01

    Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.

  5. An effective method for controlling false discovery and false nondiscovery rates in genome-scale RNAi screens.

    PubMed

    Zhang, Xiaohua Douglas

    2010-10-01

    In most genome-scale RNA interference (RNAi) screens, the ultimate goal is to select siRNAs with a large inhibition or activation effect. The selection of hits typically requires statistical control of 2 errors: false positives and false negatives. Traditional methods of controlling false positives and false negatives do not take into account the important feature in RNAi screens: many small-interfering RNAs (siRNAs) may have very small but real nonzero average effects on the measured response and thus cannot allow us to effectively control false positives and false negatives. To address for deficiencies in the application of traditional approaches in RNAi screening, the author proposes a new method for controlling false positives and false negatives in RNAi high-throughput screens. The false negatives are statistically controlled through a false-negative rate (FNR) or false nondiscovery rate (FNDR). FNR is the proportion of false negatives among all siRNAs examined, whereas FNDR is the proportion of false negatives among declared nonhits. The author also proposes new concepts, q*-value and p*-value, to control FNR and FNDR, respectively. The proposed method should have broad utility for hit selection in which one needs to control both false discovery and false nondiscovery rates in genome-scale RNAi screens in a robust manner.

  6. Construction of simple and efficient siRNA validation systems for screening and identification of effective RNAi-targeted sequences from mammalian genes.

    PubMed

    Tsai, Wen-Hui; Chang, Wen-Tsan

    2014-01-01

    RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing induced by double-stranded RNAs (dsRNAs). Among the widely used dsRNAs, small interfering RNAs (siRNAs) and short hairpin RNAs have evolved as extremely powerful and the most popular gene silencing reagents. The key challenge to achieving efficient gene silencing especially for the purpose of therapeutics is mainly dependent on the effectiveness and specificity of the selected RNAi-targeted sequences. Practically, only a small number of dsRNAs are capable of inducing highly effective and sequence-specific gene silencing via RNAi mechanism. In addition, the efficiency of gene silencing induced by dsRNAs can only be experimentally examined based on inhibition of the target gene expression. Therefore, it is essential to develop a fully robust and comparative validation system for measuring the efficacy of designed dsRNAs. In this chapter, we focus our discussion on a reliable and quantitative reporter-based siRNA validation system that has been previously established in our laboratory. The system consists of a short synthetic DNA fragment containing an RNAi-targeted sequence of interest and two expression vectors for targeting reporter and triggering siRNA expressions. The efficiency of siRNAs is determined by their abilities to inhibit expression of the targeting reporters with easily quantified readouts including enhanced green fluorescence protein and firefly luciferase. Since only a readily available short synthetic DNA fragment is needed for constructing this reliable and efficient reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective RNAi-targeted sequences from mammalian genes but also implicates the use of RNAi-based dsRNA reagents for reverse functional genomics and molecular therapeutics.

  7. Towards Functional Annotation of the Preimplantation Transcriptome: An RNAi Screen in Mammalian Embryos

    PubMed Central

    Cui, Wei; Dai, Xiangpeng; Marcho, Chelsea; Han, Zhengbin; Zhang, Kun; Tremblay, Kimberly D.; Mager, Jesse

    2016-01-01

    With readily available transcriptome-wide data, understanding the role of each expressed gene is an essential next step. Although RNAi technologies allow for genome-wide screens in cell culture, these approaches cannot replace strategies for discovery in the embryo. Here we present, for the first time, a knockdown screen in mouse preimplantation embryos. Early mammalian development encompasses dynamic cellular, molecular and epigenetic events that are largely conserved from mouse to man. We assayed 712 genes for requirements during preimplantation. We identified 59 genes required for successful development or outgrowth and implantation. We have characterized each phenotype and revealed cellular, molecular, and lineage specific defects following knockdown of transcript. Induced network analyses demonstrate this as a valid approach to identify networks of genes that play important roles during preimplantation. Our approach provides a robust and efficient strategy towards identification of novel phenotypes during mouse preimplantation and facilitates functional annotation of the mammalian transcriptome. PMID:27869233

  8. The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology

    PubMed Central

    Schmitt-Engel, Christian; Schultheis, Dorothea; Schwirz, Jonas; Ströhlein, Nadi; Troelenberg, Nicole; Majumdar, Upalparna; Dao, Van Anh; Grossmann, Daniela; Richter, Tobias; Tech, Maike; Dönitz, Jürgen; Gerischer, Lizzy; Theis, Mirko; Schild, Inga; Trauner, Jochen; Koniszewski, Nikolaus D. B.; Küster, Elke; Kittelmann, Sebastian; Hu, Yonggang; Lehmann, Sabrina; Siemanowski, Janna; Ulrich, Julia; Panfilio, Kristen A.; Schröder, Reinhard; Morgenstern, Burkhard; Stanke, Mario; Buchhholz, Frank; Frasch, Manfred; Roth, Siegfried; Wimmer, Ernst A.; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor

    2015-01-01

    Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila. PMID:26215380

  9. The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

    PubMed

    Schmitt-Engel, Christian; Schultheis, Dorothea; Schwirz, Jonas; Ströhlein, Nadi; Troelenberg, Nicole; Majumdar, Upalparna; Dao, Van Anh; Grossmann, Daniela; Richter, Tobias; Tech, Maike; Dönitz, Jürgen; Gerischer, Lizzy; Theis, Mirko; Schild, Inga; Trauner, Jochen; Koniszewski, Nikolaus D B; Küster, Elke; Kittelmann, Sebastian; Hu, Yonggang; Lehmann, Sabrina; Siemanowski, Janna; Ulrich, Julia; Panfilio, Kristen A; Schröder, Reinhard; Morgenstern, Burkhard; Stanke, Mario; Buchhholz, Frank; Frasch, Manfred; Roth, Siegfried; Wimmer, Ernst A; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor

    2015-07-28

    Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

  10. Identification of novel genes involved in light-dependent CRY degradation through a genome-wide RNAi screen.

    PubMed

    Sathyanarayanan, Sriram; Zheng, Xiangzhong; Kumar, Shailesh; Chen, Chun-Hong; Chen, Dechun; Hay, Bruce; Sehgal, Amita

    2008-06-01

    Circadian clocks regulate many different physiological processes and synchronize these to environmental light:dark cycles. In Drosophila, light is transmitted to the clock by a circadian blue light photoreceptor CRYPTOCHROME (CRY). In response to light, CRY promotes the degradation of the circadian clock protein TIMELESS (TIM) and then is itself degraded. To identify novel genes involved in circadian entrainment, we performed an unbiased genome-wide screen in Drosophila cells using a sensitive and quantitative assay that measures light-induced degradation of CRY. We systematically knocked down the expression of approximately 21,000 genes and identified those that regulate CRY stability. These genes include ubiquitin ligases, signal transduction molecules, and redox molecules. Many of the genes identified in the screen are specific for CRY degradation and do not affect degradation of the TIM protein in response to light, suggesting that, for the most part, these two pathways are distinct. We further validated the effect of three candidate genes on CRY stability in vivo by assaying flies mutant for each of these genes. This work identifies a novel regulatory network involved in light-dependent CRY degradation and demonstrates the power of a genome-wide RNAi approach for understanding circadian biology.

  11. Functional genomics down under: RNAi screening in the Victorian Centre for Functional Genomics.

    PubMed

    Thomas, Daniel W; Gould, Cathryn M; Handoko, Yanny; Simpson, Kaylene J

    2014-05-01

    The Victorian Centre for Functional Genomics (VCFG) is an RNAi screening facility housed at the Peter MacCallum Cancer Centre in Melbourne, Australia. The Peter Mac is Australia's largest dedicated Cancer Research Institute, home to a team of over 520 scientists that focus on understanding the genetic risk of cancer, the molecular events regulating cancer growth and dissemination and improving detection through new diagnostic tools (www.petermac.org). Peter Mac is a well recognised technology leader and established the VCFG with a view to enabling researchers Australia and New Zealand-wide access to cutting edge functional genomics technology, infrastructure and expertise. This review documents the technology platforms operated within the VCFG and provides insight into the workflows and analysis pipelines currently in operation.

  12. Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection

    PubMed Central

    de Macedo Dossin, Fernando; Choi, Seo Yeon; Kim, Nam Youl; Kim, Hi Chul; Jung, Sung Yong; Schenkman, Sergio; Almeida, Igor C.; Emans, Neil; Freitas-Junior, Lucio H.

    2011-01-01

    The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy. PMID:21625474

  13. Automated cell analysis tool for a genome-wide RNAi screen with support vector machine based supervised learning

    NASA Astrophysics Data System (ADS)

    Remmele, Steffen; Ritzerfeld, Julia; Nickel, Walter; Hesser, Jürgen

    2011-03-01

    RNAi-based high-throughput microscopy screens have become an important tool in biological sciences in order to decrypt mostly unknown biological functions of human genes. However, manual analysis is impossible for such screens since the amount of image data sets can often be in the hundred thousands. Reliable automated tools are thus required to analyse the fluorescence microscopy image data sets usually containing two or more reaction channels. The herein presented image analysis tool is designed to analyse an RNAi screen investigating the intracellular trafficking and targeting of acylated Src kinases. In this specific screen, a data set consists of three reaction channels and the investigated cells can appear in different phenotypes. The main issue of the image processing task is an automatic cell segmentation which has to be robust and accurate for all different phenotypes and a successive phenotype classification. The cell segmentation is done in two steps by segmenting the cell nuclei first and then using a classifier-enhanced region growing on basis of the cell nuclei to segment the cells. The classification of the cells is realized by a support vector machine which has to be trained manually using supervised learning. Furthermore, the tool is brightness invariant allowing different staining quality and it provides a quality control that copes with typical defects during preparation and acquisition. A first version of the tool has already been successfully applied for an RNAi-screen containing three hundred thousand image data sets and the SVM extended version is designed for additional screens.

  14. Genomic RNAi screening in Drosophila S2 cells: What have we learned about host-pathogen interactions?

    PubMed Central

    Cherry, Sara

    2008-01-01

    The détente between pathogen and host has been of keen interest to researchers in spite of being exceedingly difficult to probe. Recently, new RNA interference (RNAi) technologies, in particular in Drosophila tissue culture cells, have made it possible to interrogate the genetics of host organisms rapidly, with nearly complete genomic coverage and high fidelity. Therefore, it is not surprising that the applications of RNAi to the study of host-pathogen interactions were amongst the first to be published, and have already revealed many new insights into the hosts’ role in infection. This review will highlight the application of RNAi screening to pathogen-host interactions in Drosophila cells and will reveal some of the lessons learned from this approach. PMID:18539520

  15. Genome-wide RNAi screen identifies networks involved in intestinal stem cell regulation in Drosophila.

    PubMed

    Zeng, Xiankun; Han, Lili; Singh, Shree Ram; Liu, Hanhan; Neumüller, Ralph A; Yan, Dong; Hu, Yanhui; Liu, Ying; Liu, Wei; Lin, Xinhua; Hou, Steven X

    2015-02-24

    The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs) in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC) lineage differentiation, and ISC-to-enteroendocrine (EE) lineage differentiation. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, we found that factors related to basic stem cell cellular processes are commonly required in all stem cells, and stem-cell-specific, niche-related signals are required only in the unique stem cell type. Our findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.

  16. Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication

    PubMed Central

    Wu, Kan Xing; Phuektes, Patchara; Kumar, Pankaj; Goh, Germaine Yen Lin; Moreau, Dimitri; Chow, Vincent Tak Kwong; Bard, Frederic; Chu, Justin Jang Hann

    2016-01-01

    Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets. PMID:27748395

  17. Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

    PubMed Central

    Kraehling, Jan R.; Chidlow, John H.; Rajagopal, Chitra; Sugiyama, Michael G.; Fowler, Joseph W.; Lee, Monica Y.; Zhang, Xinbo; Ramírez, Cristina M.; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W.; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L.; Fernández-Hernando, Carlos; Sessa, William C.

    2016-01-01

    In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL. PMID:27869117

  18. Drosophila Genome-Wide RNAi Screen Identifies Multiple Regulators of HIF–Dependent Transcription in Hypoxia

    PubMed Central

    Dekanty, Andrés; Romero, Nuria M.; Bertolin, Agustina P.; Thomas, María G.; Leishman, Claudia C.; Perez-Perri, Joel I.; Boccaccio, Graciela L.; Wappner, Pablo

    2010-01-01

    Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF–dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF–related pathologies, including heart attack, cancer, and stroke. PMID:20585616

  19. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia.

    PubMed

    Zuber, Johannes; Shi, Junwei; Wang, Eric; Rappaport, Amy R; Herrmann, Harald; Sison, Edward A; Magoon, Daniel; Qi, Jun; Blatt, Katharina; Wunderlich, Mark; Taylor, Meredith J; Johns, Christopher; Chicas, Agustin; Mulloy, James C; Kogan, Scott C; Brown, Patrick; Valent, Peter; Bradner, James E; Lowe, Scott W; Vakoc, Christopher R

    2011-08-03

    Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

  20. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia

    PubMed Central

    Zuber, Johannes; Shi, Junwei; Wang, Eric; Rappaport, Amy R.; Herrmann, Harald; Sison, Edward A.; Magoon, Daniel; Qi, Jun; Blatt, Katharina; Wunderlich, Mark; Taylor, Meredith J.; Johns, Christopher; Chicas, Agustin; Mulloy, James C.; Kogan, Scott C.; Brown, Patrick; Valent, Peter; Bradner, James E.; Lowe, Scott W.; Vakoc, Christopher R.

    2012-01-01

    Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs1. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states2. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention. PMID:21814200

  1. RNAi Screen Identifies Novel Regulators of RNP Granules in the Caenorhabditis elegans Germ Line.

    PubMed

    Wood, Megan P; Hollis, Angela; Severance, Ashley L; Karrick, Megan L; Schisa, Jennifer A

    2016-08-09

    Complexes of RNA and RNA binding proteins form large-scale supramolecular structures under many cellular contexts. In Caenorhabditis elegans, small germ granules are present in the germ line that share characteristics with liquid droplets that undergo phase transitions. In meiotically-arrested oocytes of middle-aged hermaphrodites, the germ granules appear to aggregate or condense into large assemblies of RNA-binding proteins and maternal mRNAs. Prior characterization of the assembly of large-scale RNP structures via candidate approaches has identified a small number of regulators of phase transitions in the C. elegans germ line; however, the assembly, function, and regulation of these large RNP assemblies remain incompletely understood. To identify genes that promote remodeling and assembly of large RNP granules in meiotically-arrested oocytes, we performed a targeted, functional RNAi screen and identified over 300 genes that regulate the assembly of the RNA-binding protein MEX-3 into large granules. Among the most common GO classes are several categories related to RNA biology, as well as novel categories such as cell cortex, ER, and chromosome segregation. We found that arrested oocytes that fail to localize MEX-3 into cortical granules display reduced oocyte quality, consistent with the idea that the larger RNP assemblies promote oocyte quality when fertilization is delayed. Interestingly, a relatively small number of genes overlap with the regulators of germ granule assembly during normal development, or with the regulators of solid RNP granules in cgh-1 oocytes, suggesting fundamental differences in the regulation of RNP granule phase transitions during meiotic arrest.

  2. RNAi Screen Identifies Novel Regulators of RNP Granules in the Caenorhabditis elegans Germ Line

    PubMed Central

    Wood, Megan P.; Hollis, Angela; Severance, Ashley L.; Karrick, Megan L.; Schisa, Jennifer A.

    2016-01-01

    Complexes of RNA and RNA binding proteins form large-scale supramolecular structures under many cellular contexts. In Caenorhabditis elegans, small germ granules are present in the germ line that share characteristics with liquid droplets that undergo phase transitions. In meiotically-arrested oocytes of middle-aged hermaphrodites, the germ granules appear to aggregate or condense into large assemblies of RNA-binding proteins and maternal mRNAs. Prior characterization of the assembly of large-scale RNP structures via candidate approaches has identified a small number of regulators of phase transitions in the C. elegans germ line; however, the assembly, function, and regulation of these large RNP assemblies remain incompletely understood. To identify genes that promote remodeling and assembly of large RNP granules in meiotically-arrested oocytes, we performed a targeted, functional RNAi screen and identified over 300 genes that regulate the assembly of the RNA-binding protein MEX-3 into large granules. Among the most common GO classes are several categories related to RNA biology, as well as novel categories such as cell cortex, ER, and chromosome segregation. We found that arrested oocytes that fail to localize MEX-3 into cortical granules display reduced oocyte quality, consistent with the idea that the larger RNP assemblies promote oocyte quality when fertilization is delayed. Interestingly, a relatively small number of genes overlap with the regulators of germ granule assembly during normal development, or with the regulators of solid RNP granules in cgh-1 oocytes, suggesting fundamental differences in the regulation of RNP granule phase transitions during meiotic arrest. PMID:27317775

  3. An RNAi Screen for Genes Involved in Nanoscale Protrusion Formation on Corneal Lens in Drosophila melanogaster.

    PubMed

    Minami, Ryunosuke; Sato, Chiaki; Yamahama, Yumi; Kubo, Hideo; Hariyama, Takahiko; Kimura, Ken-Ichi

    2016-12-01

    The "moth-eye" structure, which is observed on the surface of corneal lens in several insects, supports anti-reflective and self-cleaning functions due to nanoscale protrusions known as corneal nipples. Although the morphology and function of the "moth-eye" structure, are relatively well studied, the mechanism of protrusion formation from cell-secreted substances is unknown. In Drosophila melanogaster, a compound eye consists of approximately 800 facets, the surface of which is formed by the corneal lens with nanoscale protrusions. In the present study, we sought to identify genes involved in "moth-eye" structure, formation in order to elucidate the developmental mechanism of the protrusions in Drosophila. We re-examined the aberrant patterns in classical glossy-eye mutants by scanning electron microscope and classified the aberrant patterns into groups. Next, we screened genes encoding putative structural cuticular proteins and genes involved in cuticular formation using eye specific RNAi silencing methods combined with the Gal4/UAS expression system. We identified 12 of 100 candidate genes, such as cuticular proteins family genes (Cuticular protein 23B and Cuticular protein 49Ah), cuticle secretion-related genes (Syntaxin 1A and Sec61 ββ subunit), ecdysone signaling and biosynthesis-related genes (Ecdysone receptor, Blimp-1, and shroud), and genes involved in cell polarity/cell architecture (Actin 5C, shotgun, armadillo, discs large1, and coracle). Although some of the genes we identified may affect corneal protrusion formation indirectly through general patterning defects in eye formation, these initial findings have encouraged us to more systematically explore the precise mechanisms underlying the formation of nanoscale protrusions in Drosophila.

  4. Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression

    PubMed Central

    Lamitina, Todd; Huang, Chunyi George; Strange, Kevin

    2006-01-01

    The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter (Pgpdh-1::GFP) is detected only during hypertonic stress but is not induced by other stressors. Using Pgpdh-1::GFP expression as a phenotype, we screened ≈16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function. PMID:16880390

  5. Identification of novel modulators of mitochondrial function by a genome-wide RNAi screen in Drosophila melanogaster

    PubMed Central

    Chen, Jian; Shi, Xiaoying; Padmanabhan, Ranjani; Wang, Qiong; Wu, Zhidan; Stevenson, Susan C.; Hild, Marc; Garza, Dan; Li, Hao

    2008-01-01

    Mitochondrial dysfunction is associated with many human diseases. There has not been a systematic genetic approach for identifying regulators of basal mitochondrial biogenesis and function in higher eukaryotes. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells using mitochondrial Citrate synthase (CS) activity as the primary readout. We screened 13,071 dsRNAs and identified 152 genes that modulate CS activity. These modulators are involved in a wide range of biological processes and pathways including mitochondrial-related functions, transcriptional and translational regulation, and signaling pathways. Selected hits among the 152 genes were further analyzed for their effect on mitochondrial CS activity in transgenic flies or fly mutants. We confirmed a number of gene hits including HDAC6, Rpd3(HDAC1), CG3249, vimar, Src42A, klumpfuss, barren, and smt3 which exert effects on mitochondrial CS activities in vivo, demonstrating the value of Drosophila genome-wide RNAi screens for identifying genes and pathways that modulate mitochondrial function. PMID:18042644

  6. Phenotypic screen for RNAi effects in the codling moth Cydia pomonella

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNAi-based technologies have the potential to augment, or replace existing pest management strategies. However, some insect taxa are less susceptible to the induction of the post-transcriptional gene silencing effect than others, such as the Lepidoptera. Here we describe experiments to investigate t...

  7. Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes.

    PubMed

    Pfender, Sybille; Kuznetsov, Vitaliy; Pasternak, Michał; Tischer, Thomas; Santhanam, Balaji; Schuh, Melina

    2015-08-13

    During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down's syndrome. Which genes safeguard accurate progression through meiosis is largely unclear. Here we develop high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNA interference within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated data set of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and allows systematic studies of meiosis in mammals.

  8. A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly.

    PubMed

    Krastev, Dragomir B; Slabicki, Mikolaj; Paszkowski-Rogacz, Maciej; Hubner, Nina C; Junqueira, Magno; Shevchenko, Andrej; Mann, Matthias; Neugebauer, Karla M; Buchholz, Frank

    2011-06-05

    TP53 (tumour protein 53) is one of the most frequently mutated genes in human cancer and its role during cellular transformation has been studied extensively. However, the homeostatic functions of p53 are less well understood. Here, we explore the molecular dependency network of TP53 through an RNAi-mediated synthetic interaction screen employing two HCT116 isogenic cell lines and a genome-scale endoribonuclease-prepared short interfering RNA library. We identify a variety of TP53 synthetic interactions unmasking the complex connections of p53 to cellular physiology and growth control. Molecular dissection of the TP53 synthetic interaction with UNRIP indicates an enhanced dependency of TP53-negative cells on small nucleolar ribonucleoprotein (snoRNP) assembly. This dependency is mediated by the snoRNP chaperone gene NOLC1 (also known as NOPP140), which we identify as a physiological p53 target gene. This unanticipated function of TP53 in snoRNP assembly highlights the potential of RNAi-mediated synthetic interaction screens to dissect molecular pathways of tumour suppressor genes.

  9. Potential Direct Regulators of the Drosophila yellow Gene Identified by Yeast One-Hybrid and RNAi Screens

    PubMed Central

    Kalay, Gizem; Lusk, Richard; Dome, Mackenzie; Hens, Korneel; Deplancke, Bart; Wittkopp, Patricia J.

    2016-01-01

    The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5′ intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming. PMID:27527791

  10. Identification of Drosophila Zfh2 as a mediator of hypercapnic immune regulation by a genome-wide RNAi screen

    PubMed Central

    Kwon, Yong-Jae; Hu, Jennifer A.; Krupinski, Thomas; Casalino-Matsuda, S. Marina; Sporn, Peter H. S.; Sznajder, Jacob I.; Beitel, Greg J.

    2015-01-01

    Hypercapnia, elevated partial pressure of carbon dioxide (PCO2) in blood and tissue, develops in many patients with chronic severe obstructive pulmonary disease and other advanced lung disorders. Patients with advanced disease frequently develop bacterial lung infections, and hypercapnia is a risk factor for mortality in such individuals. We previously demonstrated that hypercapnia suppresses induction of NF-κB-regulated innate immune response genes required for host defense in human, mouse and Drosophila cells, and increases mortality from bacterial infections in both mice and Drosophila. However, the molecular mediator(s) of hypercapnic immune suppression are undefined. Here, we report a genome-wide RNAi screen in Drosophila S2* cells stimulated with bacterial peptidoglycan (PGN). The screen identified 16 genes with human orthologs whose knockdown reduced hypercapnic suppression of the gene encoding the antimicrobial peptide (AMPs) Diptericin (Dipt), but did not increase Dipt mRNA levels in air. In vivo tests of one of the strongest screen hits, Zfh2 (mammalian orthologs ZFHX3/ATBF1 and ZFHX4), demonstrate that reducing zfh2 function using a mutation or RNAi improves survival of flies exposed to elevated CO2 and infected with S. aureus. Tissue-specific knockdown of zfh2 in the fat body, the major immune and metabolic organ of the fly, mitigates hypercapnia-induced reductions in Dipt and other AMPs and improves resistance of CO2-exposed flies to infection. Zfh2 mutations also partially rescue hypercapnia-induced delays in egg hatching, suggesting that Zfh2's role in mediating responses to hypercapnia extends beyond the immune system. Together, these results identify Zfh2 as the first in vivo mediator of hypercapnic immune suppression. PMID:26643480

  11. A systematic phenotypic screen of F-box genes through a tissue-specific RNAi-based approach in Drosophila.

    PubMed

    Dui, Wen; Lu, Wei; Ma, Jun; Jiao, Renjie

    2012-08-20

    F-box proteins are components of the SCF (SkpA-Cullin 1-F-box) E3 ligase complexes, acting as the specificity-determinants in targeting substrate proteins for ubiquitination and degradation. In humans, at least 22 out of 75 F-box proteins have experimentally documented substrates, whereas in Drosophila 12 F-box proteins have been characterized with known substrates. To systematically investigate the genetic and molecular functions of F-box proteins in Drosophila, we performed a survey of the literature and databases. We identified 45 Drosophila genes that encode proteins containing at least one F-box domain. We collected publically available RNAi lines against these genes and used them in a tissue-specific RNAi-based phenotypic screen. Here, we present our systematic phenotypic dataset from the eye, the wing and the notum. This dataset is the first of its kind and represents a useful resource for future studies of the molecular and genetic functions of F-box genes in Drosophila. Our results show that, as expected, F-box genes in Drosophila have regulatory roles in a diverse array of processes including cell proliferation, cell growth, signal transduction, and cellular and animal survival.

  12. TRAP-seq Profiling and RNAi-Based Genetic Screens Identify Conserved Glial Genes Required for Adult Drosophila Behavior

    PubMed Central

    Ng, Fanny S.; Sengupta, Sukanya; Huang, Yanmei; Yu, Amy M.; You, Samantha; Roberts, Mary A.; Iyer, Lakshmanan K.; Yang, Yongjie; Jackson, F. Rob

    2016-01-01

    Although, glial cells have well characterized functions in the developing and mature brain, it is only in the past decade that roles for these cells in behavior and plasticity have been delineated. Glial astrocytes and glia-neuron signaling, for example, are now known to have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, we have conducted cell-specific, genome-wide expression profiling of adult Drosophila astrocyte-like brain cells and performed RNA interference (RNAi)-based genetic screens to identify glial factors that regulate behavior. Importantly, our studies demonstrate that adult fly astrocyte-like cells and mouse astrocytes have similar molecular signatures; in contrast, fly astrocytes and surface glia—different classes of glial cells—have distinct expression profiles. Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). Of interest, 1 of the relevant genes encodes a vesicle recycling factor, 4 encode secreted proteins and 3 encode membrane transporters. These results strongly support the idea that glia-neuron communication is vital for adult behavior. PMID:28066175

  13. Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila

    PubMed Central

    Liu, Ying; Ge, Qinglan; Chan, Brian; Liu, Hanhan; Singh, Shree Ram; Manley, Jacob; Lee, Jae; Weideman, Ann Marie; Hou, Gerald; Hou, Steven X.

    2016-01-01

    Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. We also find that mitosis/cytokinesis proteins are especially important for male GSC maintenance. Our findings provide valuable insights and resources for studying stem cell regulation at the organismal level. PMID:27484291

  14. Second-generation sequencing supply an effective way to screen RNAi targets in large scale for potential application in pest insect control.

    PubMed

    Wang, Yubing; Zhang, Hao; Li, Haichao; Miao, Xuexia

    2011-04-11

    The key of RNAi approach success for potential insect pest control is mainly dependent on careful target selection and a convenient delivery system. We adopted second-generation sequencing technology to screen RNAi targets. Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) technologies were used to screen optimal RNAi targets from Ostrinia furnalalis. Total 14690 stage specific genes were obtained which can be considered as potential targets, and 47 were confirmed by qRT-PCR. Ten larval stage specific expression genes were selected for RNAi test. When 50 ng/µl dsRNAs of the genes DS10 and DS28 were directly sprayed on the newly hatched larvae which placed on the filter paper, the larval mortalities were around 40∼50%, while the dsRNAs of ten genes were sprayed on the larvae along with artificial diet, the mortalities reached 73% to 100% at 5 d after treatment. The qRT-PCR analysis verified the correlation between larval mortality and the down-regulation of the target gene expression. Topically applied fluorescent dsRNA confirmed that dsRNA did penetrate the body wall and circulate in the body cavity. It seems likely that the combination of DGE-tag with RNA-seq is a rapid, high-throughput, cost less and an easy way to select the candidate target genes for RNAi. More importantly, it demonstrated that dsRNAs are able to penetrate the integument and cause larval developmental stunt and/or death in a lepidopteron insect. This finding largely broadens the target selection for RNAi from just gut-specific genes to the targets in whole insects and may lead to new strategies for designing RNAi-based technology against insect damage.

  15. Identification of gene knockdown targets conferring enhanced isobutanol and 1-butanol tolerance to Saccharomyces cerevisiae using a tunable RNAi screening approach.

    PubMed

    Crook, Nathan; Sun, Jie; Morse, Nicholas; Schmitz, Alexander; Alper, Hal S

    2016-12-01

    Improving yeast tolerance to 1-butanol and isobutanol is a step toward enabling high-titer production. To identify previously unknown genetic targets leading to increased tolerance, we establish a tunable RNA interference (RNAi) screening approach. Specifically, we optimized the efficiency and tunability of RNA interference library screening in yeast, ultimately enabling downregulation efficiencies from 0 to 94 %. Using this system, we identified the Hsp70 family as a key regulator of isobutanol tolerance in a single round of screening, with downregulation of these genes conferring up to 64 % increased growth in 12 g/L isobutanol. For 1-butanol, we find through two rounds of iterative screening that the combined downregulation of alcohol dehydrogenase and enolase improves growth up to 3100 % in 10 g/L 1-butanol. Collectively, this work improves the tunability of RNAi in yeast as demonstrated by the discovery of novel effectors for these complex phenotypes.

  16. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes.

    PubMed

    Morgens, David W; Deans, Richard M; Li, Amy; Bassik, Michael C

    2016-06-01

    We compared the ability of short hairpin RNA (shRNA) and CRISPR/Cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line K562. We found that the precision of the two libraries in detecting essential genes was similar and that combining data from both screens improved performance. Notably, results from the two screens showed little correlation, which can be partially explained by the identification of distinct essential biological processes with each technology.

  17. Systematic comparison of CRISPR-Cas9 and RNAi screens for essential genes

    PubMed Central

    Morgens, David W; Deans, Richard M; Li, Amy; Bassik, Michael C

    2016-01-01

    We compare the ability of shRNA and CRISPR/Cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line K562. We find that the precision of the two libraries in detecting essential genes is similar and that combining data from both screens improves performance. Notably, results from the two screens show little correlation, which can be partially explained by identification of distinct essential biological processes with each technology. PMID:27159373

  18. A Paired RNAi and RabGAP Overexpression Screen Identifies Rab11 as a Regulator of β-Amyloid Production

    PubMed Central

    Udayar, Vinod; Buggia-Prévot, Virginie; Guerreiro, Rita L.; Siegel, Gabriele; Rambabu, Naresh; Soohoo, Amanda L.; Ponnusamy, Moorthi; Siegenthaler, Barbara; Bali, Jitin; Guerreiro, Rita; Brás, José; Sassi, Celeste; Gibbs, J. Raphael; Hernandez, Dena; Lupton, Michelle K.; Brown, Kristelle; Morgan, Kevin; Powell, John; Singleton, Andrew; Hardy, John; Simons, Mikael; Ries, Jonas; Puthenveedu, Manojkumar A.; Hardy, John; Thinakaran, Gopal; Rajendran, Lawrence

    2014-01-01

    Summary Alzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides, which are generated from amyloid precursor protein (APP) by β- and γ-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Aβ levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Aβ production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled β-secretase endosomal recycling to the plasma membrane and thus affected Aβ production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Aβ levels and show how systems biology approaches can unravel the molecular complexity underlying AD. PMID:24373285

  19. RNAi screens identify CHD4 as an essential gene in breast cancer growth

    PubMed Central

    Cicalese, Angelo; Fornasari, Lorenzo; Furia, Laura; Riva, Laura; Carugo, Alessandro; Curigliano, Giuseppe; Criscitiello, Carmen; Pruneri, Giancarlo; Pelicci, Pier Giuseppe; Faretta, Mario; Bossi, Daniela; Lanfrancone, Luisa

    2016-01-01

    Epigenetic regulation plays an essential role in tumor development and epigenetic modifiers are considered optimal potential druggable candidates. In order to identify new breast cancer vulnerabilities and improve therapeutic chances for patients, we performed in vivo and in vitro shRNA screens in a human breast cancer cell model (MCF10DCIS.com cell line) using epigenetic libraries. Among the genes identified in our screening, we deeply investigated the role of Chromodomain Helicase DNA binding Protein 4 (CHD4) in breast cancer tumorigenesis. CHD4 silencing significantly reduced tumor growth in vivo and proliferation in vitro of MCF10DCIS.com cells. Similarly, in vivo breast cancer growth was decreased in a spontaneous mouse model of breast carcinoma (MMTV-NeuT system) and in metastatic patient-derived xenograft models. Conversely, no reduction in proliferative ability of non-transformed mammary epithelial cells (MCF10A) was detected. Moreover, we showed that CHD4 depletion arrests proliferation by inducing a G0/G1 block of cell cycle associated with up-regulation of CDKN1A (p21). These results highlight the relevance of genetic screens in the identification of tumor frailties and the role of CHD4 as a potential pharmacological target to inhibit breast cancer growth. PMID:27779108

  20. Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator

    PubMed Central

    Jung, Jennifer; Nayak, Arnab; Schaeffer, Véronique; Starzetz, Tatjana; Kirsch, Achim K; Müller, Stefan; Dikic, Ivan; Mittelbronn, Michel; Behrends, Christian

    2017-01-01

    Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator. DOI: http://dx.doi.org/10.7554/eLife.23063.001 PMID:28195531

  1. RNAi screens for Rho GTPase regulators of cell shape and YAP/TAZ localisation in triple negative breast cancer

    PubMed Central

    Pascual-Vargas, Patricia; Cooper, Samuel; Sero, Julia; Bousgouni, Vicky; Arias-Garcia, Mar; Bakal, Chris

    2017-01-01

    In order to metastasise, triple negative breast cancer (TNBC) must make dynamic changes in cell shape. The shape of all eukaryotic cells is regulated by Rho Guanine Nucleotide Exchange Factors (RhoGEFs), which activate Rho-family GTPases in response to mechanical and informational cues. In contrast, Rho GTPase-activating proteins (RhoGAPs) inhibit Rho GTPases. However, which RhoGEFs and RhoGAPS couple TNBC cell shape to changes in their environment is very poorly understood. Moreover, whether the activity of particular RhoGEFs and RhoGAPs become dysregulated as cells evolve the ability to metastasise is not clear. Towards the ultimate goal of identifying RhoGEFs and RhoGAPs that are essential for TNBC metastasis, we performed an RNAi screen to isolate RhoGEFs and RhoGAPs that contribute to the morphogenesis of the highly metastatic TNBC cell line LM2, and its less-metastatic parental cell line MDA-MB-231. For ~6 million cells from each cell line, we measured 127 different features following the depletion of 142 genes. Using a linear classifier scheme we also describe the morphological heterogeneity of each gene-depleted population. PMID:28248929

  2. RNAi screen of Salmonella invasion shows role of COPI in membrane targeting of cholesterol and Cdc42

    PubMed Central

    Misselwitz, Benjamin; Dilling, Sabrina; Vonaesch, Pascale; Sacher, Raphael; Snijder, Berend; Schlumberger, Markus; Rout, Samuel; Stark, Manuel; Mering, Christian von; Pelkmans, Lucas; Hardt, Wolf-Dietrich

    2011-01-01

    The pathogen Salmonella Typhimurium is a common cause of diarrhea and invades the gut tissue by injecting a cocktail of virulence factors into epithelial cells, triggering actin rearrangements, membrane ruffling and pathogen entry. One of these factors is SopE, a G-nucleotide exchange factor for the host cellular Rho GTPases Rac1 and Cdc42. How SopE mediates cellular invasion is incompletely understood. Using genome-scale RNAi screening we identified 72 known and novel host cell proteins affecting SopE-mediated entry. Follow-up assays assigned these ‘hits' to particular steps of the invasion process; i.e., binding, effector injection, membrane ruffling, membrane closure and maturation of the Salmonella-containing vacuole. Depletion of the COPI complex revealed a unique effect on virulence factor injection and membrane ruffling. Both effects are attributable to mislocalization of cholesterol, sphingolipids, Rac1 and Cdc42 away from the plasma membrane into a large intracellular compartment. Equivalent results were obtained with the vesicular stomatitis virus. Therefore, COPI-facilitated maintenance of lipids may represent a novel, unifying mechanism essential for a wide range of pathogens, offering opportunities for designing new drugs. PMID:21407211

  3. Genome-wide RNAi screen for synthetic lethal interactions with the C. elegans kinesin-5 homolog BMK-1

    PubMed Central

    Maia, André F.; Tanenbaum, Marvin E.; Galli, Matilde; Lelieveld, Daphne; Egan, David A.; Gassmann, Reto; Sunkel, Claudio E.; van den Heuvel, Sander; Medema, René H.

    2015-01-01

    Kinesins are a superfamily of microtubule-based molecular motors that perform various transport needs and have essential roles in cell division. Among these, the kinesin-5 family has been shown to play a major role in the formation and maintenance of the bipolar mitotic spindle. Moreover, recent work suggests that kinesin-5 motors may have additional roles. In contrast to most model organisms, the sole kinesin-5 gene in Caenorhabditis elegans, bmk-1, is not required for successful mitosis and animals lacking bmk-1 are viable and fertile. To gain insight into factors that may act redundantly with BMK-1 in spindle assembly and to identify possible additional cellular pathways involving BMK-1, we performed a synthetic lethal screen using the bmk-1 deletion allele ok391. We successfully knocked down 82% of the C. elegans genome using RNAi and assayed viability in bmk-1(ok391) and wild type strains using an automated high-throughput approach based on fluorescence microscopy. The dataset includes a final list of 37 synthetic lethal interactions whose further study is likely to provide insight into kinesin-5 function. PMID:25984351

  4. RNAi screens for Rho GTPase regulators of cell shape and YAP/TAZ localisation in triple negative breast cancer.

    PubMed

    Pascual-Vargas, Patricia; Cooper, Samuel; Sero, Julia; Bousgouni, Vicky; Arias-Garcia, Mar; Bakal, Chris

    2017-03-01

    In order to metastasise, triple negative breast cancer (TNBC) must make dynamic changes in cell shape. The shape of all eukaryotic cells is regulated by Rho Guanine Nucleotide Exchange Factors (RhoGEFs), which activate Rho-family GTPases in response to mechanical and informational cues. In contrast, Rho GTPase-activating proteins (RhoGAPs) inhibit Rho GTPases. However, which RhoGEFs and RhoGAPS couple TNBC cell shape to changes in their environment is very poorly understood. Moreover, whether the activity of particular RhoGEFs and RhoGAPs become dysregulated as cells evolve the ability to metastasise is not clear. Towards the ultimate goal of identifying RhoGEFs and RhoGAPs that are essential for TNBC metastasis, we performed an RNAi screen to isolate RhoGEFs and RhoGAPs that contribute to the morphogenesis of the highly metastatic TNBC cell line LM2, and its less-metastatic parental cell line MDA-MB-231. For ~6 million cells from each cell line, we measured 127 different features following the depletion of 142 genes. Using a linear classifier scheme we also describe the morphological heterogeneity of each gene-depleted population.

  5. A simple method for analyzing actives in random RNAi screens: introducing the “H Score” for hit nomination & gene prioritization

    PubMed Central

    Bhinder, Bhavneet; Djaballah, Hakim

    2013-01-01

    Due to the numerous challenges in hit identification from random RNAi screening, we have examined current practices with a discovery of a variety of methodologies employed and published in many reports; majority of them, unfortunately, do not address the minimum associated criteria for hit nomination, as this could potentially have been the cause or may well be the explanation as to the lack of confirmation and follow up studies, currently facing the RNAi field. Overall, we find that these criteria or parameters are not well defined, in most cases arbitrary in nature, and hence rendering it extremely difficult to judge the quality of and confidence in nominated hits across published studies. For this purpose, we have developed a simple method to score actives independent of assay readout; and provide, for the first time, a homogenous platform enabling cross-comparison of active gene lists resulting from different RNAi screening technologies. Here, we report on our recently developed method dedicated to RNAi data output analysis referred to as the BDA method applicable to both arrayed and pooled RNAi technologies; wherein the concerns pertaining to inconsistent hit nomination and off-target silencing in conjugation with minimal activity criteria to identify a high value target are addressed. In this report, a combined hit rate per gene, called “H score”, is introduced and defined. The H score provides a very useful tool for stringent active gene nomination, gene list comparison across multiple studies, prioritization of hits, and evaluation of the quality of the nominated gene hits. PMID:22934950

  6. Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis

    PubMed Central

    Miyoshi, N; Wittner, B S; Shioda, K; Hitora, T; Ito, T; Ramaswamy, S; Isselbacher, K J; Sgroi, D C; Shioda, T

    2015-01-01

    Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the nodes-and-connections RNAi knockdown screening that generates a map of target interactions through systematic iterations of in silico prediction of targets and their experimental validation. An initial RNAi knockdown screening of MCF-7 human breast cancer cells targeting 6560 proteins identified four signaling molecules required for their fulvestrant-induced apoptosis. Signaling molecules physically or functionally interacting with these four primary node targets were computationally predicted and experimentally validated, resulting in identification of four second-generation nodes. Three rounds of further iterations of the prediction–validation cycle generated third, fourth and fifth generation of nodes, completing a 19-node interaction map that contained three predicted nodes but without experimental validation because of technical limitations. The interaction map involved all three members of the death-associated protein kinases (DAPKs) as well as their upstream and downstream signaling molecules (calmodulins and myosin light chain kinases), suggesting that DAPKs play critical roles in the cytocidal action of fulvestrant. The in silico Kaplan–Meier analysis of previously reported human breast cancer cohorts demonstrated significant prognostic predictive power for five of the experimentally validated nodes and for three of the prediction-only nodes. Immunohistochemical studies on the expression of 10 nodal proteins in human breast cancer tissues not only supported their prognostic prediction power but also provided statistically significant evidence of their synchronized expression, implying functional interactions among these nodal proteins. Thus, the Nodes-and-Connections approach to RNAi knockdown screening yields

  7. Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction.

    PubMed

    Mano, Miguel; Ippodrino, Rudy; Zentilin, Lorena; Zacchigna, Serena; Giacca, Mauro

    2015-09-08

    Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors' broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency.

  8. Functional screening of mammalian mechanosensitive genes using Drosophila RNAi library– Smarcd3/Bap60 is a mechanosensitive pro-inflammatory gene

    PubMed Central

    Kumar, Sandeep; Jang, In-hwan; Kim, Chan Woo; Kang, Dong-Won; Lee, Won Jae; Jo, Hanjoong

    2016-01-01

    Disturbed blood flow (d-flow) induces atherosclerosis by altering the expression of mechanosensitive genes in the arterial endothelium. Previously, we identified >580 mechanosensitive genes in the mouse arterial endothelium, but their role in endothelial inflammation is incompletely understood. From this set, we obtained 84 Drosophila RNAi lines that silences the target gene under the control of upstream activation sequence (UAS) promoter. These lines were crossed with C564-GAL4 flies expressing GFP under the control of drosomycin promoter, an NF-κB target gene and a marker of pathogen-induced inflammation. Silencing of psmd12 or ERN1 decreased infection-induced drosomycin expression, while Bap60 silencing significantly increased the drosomycin expression. Interestingly, knockdown of Bap60 in adult flies using temperature-inducible Bap60 RNAi (C564ts-GAL4-Bap60-RNAi) enhanced drosomycin expression upon Gram-positive bacterial challenge but the basal drosomycin expression remained unchanged compared to the control. In the mammalian system, smarcd3 (mammalian ortholog of Bap60) expression was reduced in the human- and mouse aortic endothelial cells exposed to oscillatory shear in vitro as well as in the d-flow regions of mouse arterial endothelium in vivo. Moreover, siRNA-mediated knockdown of smarcd3 induced endothelial inflammation. In summary, we developed an in vivo Drosophila RNAi screening method to identify flow-sensitive genes that regulate endothelial inflammation. PMID:27819340

  9. Comparative RNAi screening identifies a conserved core metazoan actinome by phenotype

    PubMed Central

    Sims, David; Liu, Tao; Fedorova, Marina; Schöck, Frieder; Dopie, Joseph; Vartiainen, Maria K.; Kiger, Amy A.; Perrimon, Norbert

    2011-01-01

    Although a large number of actin-binding proteins and their regulators have been identified through classical approaches, gaps in our knowledge remain. Here, we used genome-wide RNA interference as a systematic method to define metazoan actin regulators based on visual phenotype. Using comparative screens in cultured Drosophila and human cells, we generated phenotypic profiles for annotated actin regulators together with proteins bearing predicted actin-binding domains. These phenotypic clusters for the known metazoan “actinome” were used to identify putative new core actin regulators, together with a number of genes with conserved but poorly studied roles in the regulation of the actin cytoskeleton, several of which we studied in detail. This work suggests that although our search for new components of the core actin machinery is nearing saturation, regulation at the level of nuclear actin export, RNA splicing, ubiquitination, and other upstream processes remains an important but unexplored frontier of actin biology. PMID:21893601

  10. Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry

    PubMed Central

    Taylor, Gwen M.; Kielian, Margaret

    2013-01-01

    The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy. PMID:24367265

  11. Systematic high-content genome-wide RNAi screens of endothelial cell migration and morphology

    PubMed Central

    Williams, Steven P.; Gould, Cathryn M.; Nowell, Cameron J.; Karnezis, Tara; Achen, Marc G.; Simpson, Kaylene J.; Stacker, Steven A.

    2017-01-01

    Many cell types undergo migration during embryogenesis and disease. Endothelial cells line blood vessels and lymphatics, which migrate during development as part of angiogenesis, lymphangiogenesis and other types of vessel remodelling. These processes are also important in wound healing, cancer metastasis and cardiovascular conditions. However, the molecular control of endothelial cell migration is poorly understood. Here, we present a dataset containing siRNA screens that identify known and novel components of signalling pathways regulating migration of lymphatic endothelial cells. These components are compared to signalling in blood vascular endothelial cells. Further, using high-content microscopy, we captured a dataset of images of migrating cells following transfection with a genome-wide siRNA library. These datasets are suitable for the identification and analysis of genes involved in endothelial cell migration and morphology, and for computational approaches to identify signalling networks controlling the migratory response and integration of cell morphology, gene function and cell signaling. This may facilitate identification of protein targets for therapeutically modulating angiogenesis and lymphangiogenesis in the context of human disease. PMID:28248931

  12. Identification of Novel Genes Involved in Sarcopenia Through RNAi Screening in Caenorhabditis elegans

    PubMed Central

    Kashyap, Luv; Perera, Subashan

    2012-01-01

    Background. Aging in humans is characterized by a progressive loss of muscle mass and strength known as sarcopenia. Although considered to be a normal aspect of aging, the loss of strength can have significant effects on the health, functioning, and independence of elderly individuals. Although these aspects of sarcopenia have been well studied, the molecular mechanisms leading to its development are still unclear. The nematode Caenorhabditis elegans might be a novel animal model for sarcopenia as worms experience sarcopenia during aging and mutations affecting the daf-2/insulin-like signaling pathway are able to delay this process. Methods. Via the use of RNA interference, we screened a total of 43 genes, most of which have been shown to be required for the enhanced longevity of daf-2 mutants, to assess for the effects of these genes on muscle function and worm mobility during aging. Results. We identified 17 novel genes that are essential for the delay in the onset of sarcopenia in daf-2 mutants. The identified genes include splicing factors, vacuolar sorting proteins, transcription factors, and metabolic enzymes. Using a transgenic strain that only responds to RNA interference in the body wall muscle, we also found that most of the identified genes act in muscle to prevent the onset of sarcopenia. Conclusions. Our results demonstrate that at least in worms, specific genetic pathways that modify the development of sarcopenia can be identified. Interestingly, almost all the identified genes also have a known human homolog, and hence, our findings may offer significant leads toward the identification of genes involved in sarcopenia in people. PMID:21593014

  13. Fast-suppressor screening for new components in protein trafficking, organelle biogenesis and silencing pathway in Arabidopsis thaliana using DEX-inducible FREE1-RNAi plants.

    PubMed

    Zhao, Qiong; Gao, Caiji; Lee, PoShing; Liu, Lin; Li, Shaofang; Hu, Tangjin; Shen, Jinbo; Pan, Shuying; Ye, Hao; Chen, Yunru; Cao, Wenhan; Cui, Yong; Zeng, Peng; Yu, Sheng; Gao, Yangbin; Chen, Liang; Mo, Beixin; Liu, Xin; Xiao, Shi; Zhao, Yunde; Zhong, Silin; Chen, Xuemei; Jiang, Liwen

    2015-06-20

    Membrane trafficking is essential for plant growth and responses to external signals. The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex (endosomal sorting complex required for transport). FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body (MVB), vacuolar protein transport and vacuole biogenesis, and autophagic degradation. FREE1 knockout plants show defective MVB formation, abnormal vacuolar transport, fragmented vacuoles, accumulated autophagosomes, and seedling lethality. To further uncover the underlying mechanisms of FREE1 function in plants, we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants. The obtained mutants are termed as suppressors of free1 (sof). To date, 229 putative sof mutants have been identified. Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors. Also 145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants. Through next-generation sequencing (NGS) of bulked DNA from F2 mapping population of two RNAi-related sof mutants, FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly. These FREE1- and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis, as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

  14. Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-α protein levels and activity.

    PubMed

    Bolt, M J; Stossi, F; Callison, A M; Mancini, M G; Dandekar, R; Mancini, M A

    2015-01-08

    Estrogen receptor-α (ERα) is a central transcription factor that regulates mammary gland physiology and a key driver in breast cancer. In the present study, we aimed to identify novel modulators of ERα-mediated transcriptional regulation via a custom-built siRNA library screen. This screen was directed against a variety of coregulators, transcription modifiers, signaling molecules and DNA damage response proteins. By utilizing a microscopy-based, multi-end point, estrogen responsive biosensor cell line platform, the primary screen identified a wide range of factors that altered ERα protein levels, chromatin remodeling and mRNA output. We then focused on UBR5, a ubiquitin ligase and known oncogene that modulates ERα protein levels and transcriptional output. Finally, we demonstrated that UBR5 also affects endogenous ERα target genes and E2-mediated cell proliferation in breast cancer cells. In conclusion, our multi-end point RNAi screen identified novel modulators of ERα levels and activity, and provided a robust systems level view of factors involved in mechanisms of nuclear receptor action and pathophysiology. Utilizing a high throughput RNAi screening approach we identified UBR5, a protein commonly amplified in breast cancer, as a novel regulator of ERα protein levels and transcriptional activity.

  15. Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling.

    PubMed

    Mleczko-Sanecka, Katarzyna; Roche, Franziska; da Silva, Ana Rita; Call, Debora; D'Alessio, Flavia; Ragab, Anan; Lapinski, Philip E; Ummanni, Ramesh; Korf, Ulrike; Oakes, Christopher; Damm, Georg; D'Alessandro, Lorenza A; Klingmüller, Ursula; King, Philip D; Boutros, Michael; Hentze, Matthias W; Muckenthaler, Martina U

    2014-03-06

    The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.

  16. Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling

    PubMed Central

    Mleczko-Sanecka, Katarzyna; Roche, Franziska; Rita da Silva, Ana; Call, Debora; D’Alessio, Flavia; Ragab, Anan; Lapinski, Philip E.; Ummanni, Ramesh; Korf, Ulrike; Oakes, Christopher; Damm, Georg; D’Alessandro, Lorenza A.; Klingmüller, Ursula; King, Philip D.; Boutros, Michael; Hentze, Matthias W.

    2014-01-01

    The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the “iron-regulated” bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6–triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism. PMID:24385536

  17. Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract.

    PubMed

    Kanoh, Hirotaka; Kuraishi, Takayuki; Tong, Li-Li; Watanabe, Ryo; Nagata, Shinji; Kurata, Shoichiro

    2015-11-13

    Damage-associated molecular patterns (DAMPs), so-called "danger signals," play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. Here, we demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, we performed ex vivo genome-wide RNAi screening in Drosophila cultured cells, and identified several signaling factors that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that our larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.

  18. A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity.

    PubMed

    Ding, Li; Paszkowski-Rogacz, Maciej; Nitzsche, Anja; Slabicki, Mikolaj Michal; Heninger, Anne-Kristin; de Vries, Ingrid; Kittler, Ralf; Junqueira, Magno; Shevchenko, Andrej; Schulz, Herbert; Hubner, Norbert; Doss, Michael Xavier; Sachinidis, Agapios; Hescheler, Juergen; Iacone, Roberto; Anastassiadis, Konstantinos; Stewart, A Francis; Pisabarro, M Teresa; Caldarelli, Antonio; Poser, Ina; Theis, Mirko; Buchholz, Frank

    2009-05-08

    Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity.

  19. Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

    PubMed

    Agarwal, A; MacKenzie, R J; Eide, C A; Davare, M A; Watanabe-Smith, K; Tognon, C E; Mongoue-Tchokote, S; Park, B; Braziel, R M; Tyner, J W; Druker, B J

    2015-06-04

    To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2Rγ) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2Rγ abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2Rγ in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2Rγ completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2Rγ interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2Rγ, indicating IL2Rγ and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2Rγ in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth.

  20. RNAi screen in Drosophila larvae identifies histone deacetylase 3 as a positive regulator of the hsp70 heat shock gene expression during heat shock

    PubMed Central

    Achary, Bhavana G.; Campbell, Katie M.; Co, Ivy S.; Gilmour, David S.

    2014-01-01

    Transcription regulation of the Drosophila hsp70 gene is a complex process that involves regulation of multiple steps including establishment of paused Pol II and release of Pol II into elongation upon heat shock activation. While the major players involved in regulation of gene expression have been studied in detail, additional factors involved in this process continue to be discovered. To identify factors involved in hsp70 expression, we developed a screen that capitalizes on a visual assessment of heat shock activation using a hsp70-beta galactosidase reporter and publicly available RNAi fly lines to deplete candidate proteins. We validated the screen by showing that depletion of HSF, CycT, Cdk9, Nurf 301, or ELL prevented full induction of hsp70 by heat shock. Our screen also identified the histone deacetylase HDAC3 and its associated protein SMRTER as positive regulators of hsp70 activation. Additionally we show that HDAC3 and SMRTER contribute to hsp70 gene expression at a step subsequent to HSF-mediated activation and release of the paused Pol II that resides at the promoter prior to heat shock induction. PMID:24607507

  1. RNAi screen in Drosophila larvae identifies histone deacetylase 3 as a positive regulator of the hsp70 heat shock gene expression during heat shock.

    PubMed

    Achary, Bhavana G; Campbell, Katie M; Co, Ivy S; Gilmour, David S

    2014-05-01

    The transcription regulation of the Drosophila hsp70 gene is a complex process that involves the regulation of multiple steps, including the establishment of paused Pol II and release of Pol II into elongation upon heat shock activation. While the major players involved in the regulation of gene expression have been studied in detail, additional factors involved in this process continue to be discovered. To identify factors involved in hsp70 expression, we developed a screen that capitalizes on a visual assessment of heat shock activation using a hsp70-beta galactosidase reporter and publicly available RNAi fly lines to deplete candidate proteins. We validated the screen by showing that the depletion of HSF, CycT, Cdk9, Nurf 301, or ELL prevented the full induction of hsp70 by heat shock. Our screen also identified the histone deacetylase HDAC3 and its associated protein SMRTER as positive regulators of hsp70 activation. Additionally, we show that HDAC3 and SMRTER contribute to hsp70 gene expression at a step subsequent to HSF-mediated activation and release of the paused Pol II that resides at the promoter prior to heat shock induction.

  2. Large-Scale Functional RNAi Screen in C. elegans Identifies TGF-β and Notch Signaling Pathways as Modifiers of CACNA1A

    PubMed Central

    Pereira, Maria da Conceição; Morais, Sara; Sequeiros, Jorge

    2016-01-01

    Variants in CACNA1A that encodes the pore-forming α1-subunit of human voltage-gated Cav2.1 (P/Q-type) Ca2+ channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. To identify modifiers of incoordination in movement disorders, we performed a large-scale functional RNAi screen, using the Caenorhabditis elegans strain CB55, which carries a truncating mutation in the unc-2 gene, the worm ortholog for the human CACNA1A. The screen was carried out by the feeding method in 96-well liquid culture format, using the ORFeome v1.1 feeding library, and time-lapse imaging of worms in liquid culture was used to assess changes in thrashing behavior. We looked for genes that, when silenced, either ameliorated the slow and uncoordinated phenotype of unc-2, or interacted to produce a more severe phenotype. Of the 350 putative hits from the primary screen, 37 genes consistently showed reproducible results. At least 75% of these are specifically expressed in the C. elegans neurons. Functional network analysis and gene ontology revealed overrepresentation of genes involved in development, growth, locomotion, signal transduction, and vesicle-mediated transport. We have expanded the functional network of genes involved in neurodegeneration leading to cerebellar ataxia related to unc-2/CACNA1A, further confirming the involvement of the transforming growth factor β pathway and adding a novel signaling cascade, the Notch pathway. PMID:27005779

  3. Identification of common and cell type specific LXXLL motif EcR cofactors using a bioinformatics refined candidate RNAi screen in Drosophila melanogaster cell lines

    PubMed Central

    2011-01-01

    Background During Drosophila development, titers of the steroid ecdysone trigger and maintain temporal and tissue specific biological transitions. Decades of evidence reveal that the ecdysone response is both unique to specific tissues and distinct among developmental timepoints. To achieve this diversity in response, the several isoforms of the Ecdysone Receptor, which transduce the hormone signal to the genome level, are believed to interact with tissue specific cofactors. To date, little is known about the identity of these cofactor interactions; therefore, we conducted a bioinformatics informed, RNAi luciferase reporter screen against a subset of putative candidate cofactors identified through an in silico proteome screen. Candidates were chosen based on criteria obtained from bioinformatic consensus of known nuclear receptor cofactors and homologs, including amino acid sequence motif content and context. Results The bioinformatics pre-screen of the Drosophila melanogaster proteome was successful in identifying an enriched putative candidate gene cohort. Over 80% of the genes tested yielded a positive hit in our reporter screen. We have identified both cell type specific and common cofactors which appear to be necessary for proper ecdysone induced gene regulation. We have determined that certain cofactors act as co-repressors to reduce target gene expression, while others act as co-activators to increase target gene expression. Interestingly, we find that a few of the cofactors shared among cell types have a reversible roles to function as co-repressors in certain cell types while in other cell types they serve as co-activators. Lastly, these proteins are highly conserved, with higher order organism homologs also harboring the LXXLL steroid receptor interaction domains, suggesting a highly conserved mode of steroid cell target specificity. Conclusions In conclusion, we submit these cofactors as novel components of the ecdysone signaling pathway in order to

  4. Human Genome-Wide RNAi Screen for Host Factors That Facilitate Salmonella Invasion Reveals a Role for Potassium Secretion in Promoting Internalization

    PubMed Central

    Thornbrough, Joshua M.; Gopinath, Adarsh; Hundley, Tom; Worley, Micah J.

    2016-01-01

    Salmonella enterica can actively invade the gastro-intestinal epithelium. This frequently leads to diarrheal disease, and also gives the pathogen access to phagocytes that can serve as vehicles for dissemination into deeper tissue. The ability to invade host cells is also important in maintaining the carrier state. While much is known about the bacterial factors that promote invasion, relatively little is known about the host factors involved. To gain insight into how Salmonella enterica serovar Typhimurium is able to invade normally non-phagocytic cells, we undertook a global RNAi screen with S. Typhimurium-infected human epithelial cells. In all, we identified 633 genes as contributing to bacterial internalization. These genes fall into a diverse group of functional categories revealing that cytoskeletal regulators are not the only factors that modulate invasion. In fact, potassium ion transport was the most enriched molecular function category in our screen, reinforcing a link between potassium and internalization. In addition to providing new insights into the molecular mechanisms underlying the ability of pathogens to invade host cells, all 633 host factors identified are candidates for new anti-microbial targets for treating Salmonella infections, and may be useful in curtailing infections with other pathogens as well. PMID:27880807

  5. A Robotic Platform for Quantitative High-Throughput Screening

    PubMed Central

    Michael, Sam; Auld, Douglas; Klumpp, Carleen; Jadhav, Ajit; Zheng, Wei; Thorne, Natasha; Austin, Christopher P.; Inglese, James

    2008-01-01

    Abstract High-throughput screening (HTS) is increasingly being adopted in academic institutions, where the decoupling of screening and drug development has led to unique challenges, as well as novel uses of instrumentation, assay formulations, and software tools. Advances in technology have made automated unattended screening in the 1,536-well plate format broadly accessible and have further facilitated the exploration of new technologies and approaches to screening. A case in point is our recently developed quantitative HTS (qHTS) paradigm, which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay. The practical implementation of qHTS for cell-based and biochemical assays across libraries of > 100,000 compounds (e.g., between 700,000 and 2,000,000 sample wells tested) requires maximal efficiency and miniaturization and the ability to easily accommodate many different assay formats and screening protocols. Here, we describe the design and utilization of a fully integrated and automated screening system for qHTS at the National Institutes of Health's Chemical Genomics Center. We report system productivity, reliability, and flexibility, as well as modifications made to increase throughput, add additional capabilities, and address limitations. The combination of this system and qHTS has led to the generation of over 6 million CRCs from > 120 assays in the last 3 years and is a technology that can be widely implemented to increase efficiency of screening and lead generation. PMID:19035846

  6. RNAi-based screening identifies the Mms22L–Nfkbil2 complex as a novel regulator of DNA replication in human cells

    PubMed Central

    Piwko, Wojciech; Olma, Michael H; Held, Michael; Bianco, Julien N; Pedrioli, Patrick G A; Hofmann, Kay; Pasero, Philippe; Gerlich, Daniel W; Peter, Matthias

    2010-01-01

    Cullin 4 (Cul4)-based ubiquitin ligases emerged as critical regulators of DNA replication and repair. Over 50 Cul4-specific adaptors (DNA damage-binding 1 (Ddb1)–Cul4-associated factors; DCAFs) have been identified and are thought to assemble functionally distinct Cul4 complexes. Using a live-cell imaging-based RNAi screen, we analysed the function of DCAFs and Cul4-linked proteins, and identified specific subsets required for progression through G1 and S phase. We discovered C6orf167/Mms22-like protein (Mms22L) as a putative human orthologue of budding yeast Mms22, which, together with cullin Rtt101, regulates genome stability by promoting DNA replication through natural pause sites and damaged templates. Loss of Mms22L function in human cells results in S phase-dependent genomic instability characterised by spontaneous double-strand breaks and DNA damage checkpoint activation. Unlike yeast Mms22, human Mms22L does not stably bind to Cul4, but is degraded in a Cul4-dependent manner and upon replication stress. Mms22L physically and functionally interacts with the scaffold-like protein Nfkbil2 that co-purifies with histones, several chromatin remodelling and DNA replication/repair factors. Together, our results strongly suggest that the Mms22L–Nfkbil2 complex contributes to genome stability by regulating the chromatin state at stalled replication forks. PMID:21113133

  7. RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans

    PubMed Central

    Ossareh-Nazari, Batool; Katsiarimpa, Anthi; Merlet, Jorge; Pintard, Lionel

    2016-01-01

    Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans. Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint. PMID:27543292

  8. A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein-coupled receptors.

    PubMed

    Vaqué, Jose P; Dorsam, Robert T; Feng, Xiaodong; Iglesias-Bartolome, Ramiro; Forsthoefel, David J; Chen, Qianming; Debant, Anne; Seeger, Mark A; Ksander, Bruce R; Teramoto, Hidemi; Gutkind, J Silvio

    2013-01-10

    Activating mutations in GNAQ and GNA11, encoding members of the Gα(q) family of G protein α subunits, are the driver oncogenes in uveal melanoma, and mutations in Gq-linked G protein-coupled receptors have been identified recently in numerous human malignancies. How Gα(q) and its coupled receptors transduce mitogenic signals is still unclear because of the complexity of signaling events perturbed upon Gq activation. Using a synthetic-biology approach and a genome-wide RNAi screen, we found that a highly conserved guanine nucleotide exchange factor, Trio, is essential for activating Rho- and Rac-regulated signaling pathways acting on JNK and p38, and thereby transducing proliferative signals from Gα(q) to the nucleus independently of phospholipase C-β. Indeed, whereas many biological responses elicited by Gq depend on the transient activation of second-messenger systems, Gq utilizes a hard-wired protein-protein-interaction-based signaling circuitry to achieve the sustained stimulation of proliferative pathways, thereby controlling normal and aberrant cell growth.

  9. Illustration of SSMD, z score, SSMD*, z* score, and t statistic for hit selection in RNAi high-throughput screens.

    PubMed

    Zhang, Xiaohua Douglas

    2011-08-01

    Hit selection is the ultimate goal in many high-throughput screens. Various analytic methods are available for this purpose. Some commonly used ones are z score, z* score, strictly standardized mean difference (SSMD), SSMD*, and t statistic. It is critical to know how to use them correctly because the misusage of them can readily produce misleading results. Here, the author presents basic concepts, elaborates their commonality and difference, describes some common misusage that people should avoid, and uses simulated simple examples to illustrate how to use them correctly.

  10. A whole-genome RNAi screen identifies an 8q22 gene cluster that inhibits death receptor-mediated apoptosis.

    PubMed

    Dompe, Nicholas; Rivers, Celina Sanchez; Li, Li; Cordes, Shaun; Schwickart, Martin; Punnoose, Elizabeth A; Amler, Lukas; Seshagiri, Somasekar; Tang, Jerry; Modrusan, Zora; Davis, David P

    2011-10-25

    Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeutic agents designed to engage this pathway are undergoing clinical trials. With the aim of uncovering strategies to activate apoptosis in cancer cells, we used a pooled shRNA screen to interrogate death receptor signaling. This screening approach identified 16 genes that modulate the sensitivity to ligand induced apoptosis, with several genes exhibiting frequent overexpression and/or copy number gain in cancer. Interestingly, two of the top hits, EDD1 and GRHL2, are found 50 kb apart on chromosome 8q22, a region that is frequently amplified in many cancers. By using a series of silencing and overexpression studies, we show that EDD1 and GRHL2 suppress death-receptor expression, and that EDD1 expression is elevated in breast, pancreas, and lung cancer cell lines resistant to death receptor-mediated apoptosis. Supporting the relevance of EDD1 and GRHL2 as therapeutic candidates to engage apoptosis in cancer cells, silencing the expression of either gene sensitizes 8q22-amplified breast cancer cell lines to death receptor induced apoptosis. Our findings highlight a mechanism by which cancer cells may evade apoptosis, and therefore provide insight in the search for new targets and functional biomarkers for this pathway.

  11. Large-scale functional RNAi screen in C. elegans identifies genes that regulate the dysfunction of mutant polyglutamine neurons

    PubMed Central

    2012-01-01

    Background A central goal in Huntington's disease (HD) research is to identify and prioritize candidate targets for neuroprotective intervention, which requires genome-scale information on the modifiers of early-stage neuron injury in HD. Results Here, we performed a large-scale RNA interference screen in C. elegans strains that express N-terminal huntingtin (htt) in touch receptor neurons. These neurons control the response to light touch. Their function is strongly impaired by expanded polyglutamines (128Q) as shown by the nearly complete loss of touch response in adult animals, providing an in vivo model in which to manipulate the early phases of expanded-polyQ neurotoxicity. In total, 6034 genes were examined, revealing 662 gene inactivations that either reduce or aggravate defective touch response in 128Q animals. Several genes were previously implicated in HD or neurodegenerative disease, suggesting that this screen has effectively identified candidate targets for HD. Network-based analysis emphasized a subset of high-confidence modifier genes in pathways of interest in HD including metabolic, neurodevelopmental and pro-survival pathways. Finally, 49 modifiers of 128Q-neuron dysfunction that are dysregulated in the striatum of either R/2 or CHL2 HD mice, or both, were identified. Conclusions Collectively, these results highlight the relevance to HD pathogenesis, providing novel information on the potential therapeutic targets for neuroprotection in HD. PMID:22413862

  12. RNAi Screen for NRF2 Inducers Identifies Targets That Rescue Primary Lung Epithelial Cells from Cigarette Smoke Induced Radical Stress

    PubMed Central

    Schumacher, Frances-Rose; Schubert, Steffen; Hannus, Michael; Sönnichsen, Birte; Ittrich, Carina; Kreideweiss, Stefan; Rippmann, Jörg F.

    2016-01-01

    Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the ‘druggable’ genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism. PMID:27832175

  13. RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses

    PubMed Central

    Panda, Debasis; Das, Anshuman; Dinh, Phat X.; Subramaniam, Sakthivel; Nayak, Debasis; Barrows, Nicholas J.; Pearson, James L.; Thompson, Jesse; Kelly, David L.; Ladunga, Istvan; Pattnaik, Asit K.

    2011-01-01

    Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genome-wide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of VSV infection, such as entry/uncoating, gene expression, and assembly/release, were identified. Depletion of the proteins of the coatomer complex I or its upstream effectors ARF1 or GBF1 led to detection of reduced levels of VSV RNA. Coatomer complex I was also required for infection of lymphocytic choriomeningitis virus and human parainfluenza virus type 3. These results highlight the evolutionarily conserved requirements for gene expression of diverse families of NS RNA viruses and demonstrate the involvement of host cell secretory pathway in the process. PMID:22065774

  14. High-Content Genome-Wide RNAi Screen Reveals CCR3 as a Key Mediator of Neuronal Cell Death

    PubMed Central

    Wang, Huaishan; Sherbini, Omar; Ling-lin Pai, Emily; Kwon, Ji-Sun; He, Wei; Wang, Hong; Chi, Zhikai; Xu, Jinchong; Jiang, Haisong; Andrabi, Shaida A.

    2016-01-01

    Neuronal loss caused by ischemic injury, trauma, or disease can lead to devastating consequences for the individual. With the goal of limiting neuronal loss, a number of cell death pathways have been studied, but there may be additional contributors to neuronal death that are yet unknown. To identify previously unknown cell death mediators, we performed a high-content genome-wide screening of short, interfering RNA (siRNA) with an siRNA library in murine neural stem cells after exposure to N-methyl-N-nitroso-N′-nitroguanidine (MNNG), which leads to DNA damage and cell death. Eighty genes were identified as key mediators for cell death. Among them, 14 are known cell death mediators and 66 have not previously been linked to cell death pathways. Using an integrated approach with functional and bioinformatics analysis, we provide possible molecular networks, interconnected pathways, and/or protein complexes that may participate in cell death. Of the 66 genes, we selected CCR3 for further evaluation and found that CCR3 is a mediator of neuronal injury. CCR3 inhibition or deletion protects murine cortical cultures from oxygen-glucose deprivation–induced cell death, and CCR3 deletion in mice provides protection from ischemia in vivo. Taken together, our findings suggest that CCR3 is a previously unknown mediator of cell death. Future identification of the neural cell death network in which CCR3 participates will enhance our understanding of the molecular mechanisms of neural cell death. PMID:27822494

  15. High-Content Genome-Wide RNAi Screen Reveals CCR3 as a Key Mediator of Neuronal Cell Death.

    PubMed

    Zhang, Jianmin; Wang, Huaishan; Sherbini, Omar; Ling-Lin Pai, Emily; Kang, Sung-Ung; Kwon, Ji-Sun; Yang, Jia; He, Wei; Wang, Hong; Eacker, Stephen M; Chi, Zhikai; Mao, Xiaobo; Xu, Jinchong; Jiang, Haisong; Andrabi, Shaida A; Dawson, Ted M; Dawson, Valina L

    2016-01-01

    Neuronal loss caused by ischemic injury, trauma, or disease can lead to devastating consequences for the individual. With the goal of limiting neuronal loss, a number of cell death pathways have been studied, but there may be additional contributors to neuronal death that are yet unknown. To identify previously unknown cell death mediators, we performed a high-content genome-wide screening of short, interfering RNA (siRNA) with an siRNA library in murine neural stem cells after exposure to N-methyl-N-nitroso-N'-nitroguanidine (MNNG), which leads to DNA damage and cell death. Eighty genes were identified as key mediators for cell death. Among them, 14 are known cell death mediators and 66 have not previously been linked to cell death pathways. Using an integrated approach with functional and bioinformatics analysis, we provide possible molecular networks, interconnected pathways, and/or protein complexes that may participate in cell death. Of the 66 genes, we selected CCR3 for further evaluation and found that CCR3 is a mediator of neuronal injury. CCR3 inhibition or deletion protects murine cortical cultures from oxygen-glucose deprivation-induced cell death, and CCR3 deletion in mice provides protection from ischemia in vivo. Taken together, our findings suggest that CCR3 is a previously unknown mediator of cell death. Future identification of the neural cell death network in which CCR3 participates will enhance our understanding of the molecular mechanisms of neural cell death.

  16. A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly

    PubMed Central

    Ohn, Takbum; Kedersha, Nancy; Hickman, Tyler; Tisdale, Sarah; Anderson, Paul

    2015-01-01

    Stress granules (SGs) and processing bodies (PBs) are microscopically visible ribonucleoprotein granules that cooperatively regulate the translation and decay of messenger RNA1–3. Using an RNA-mediated interference-based screen, we identify 101 human genes required for SG assembly, 39 genes required for PB assembly, and 31 genes required for coordinate SG and PB assembly. Although 51 genes encode proteins involved in mRNA translation, splicing and transcription, most are not obviously associated with RNA metabolism. We find that several components of the hexosamine biosynthetic pathway, which reversibly modifies proteins with O-linked N-acetylglucosamine (O-GlcNAc) in response to stress, are required for SG and PB assembly. O-GlcNAc-modified proteins are prominent components of SGs but not PBs, and include RACK1 (receptor for activated C kinase 1), prohibitin-2, glyceraldehyde-3-phosphate dehydrogenase and numerous ribosomal proteins. Our results suggest that O-GlcNAc modification of the translational machinery is required for aggregation of untranslated messenger ribonucleoproteins into SGs. The lack of enzymes of the hexosamine biosynthetic pathway in budding yeast may contribute to differences between mammalian SGs and related yeast EGP (eIF4E, 4G and Pab1 containing) bodies. PMID:18794846

  17. A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia

    PubMed Central

    Słabicki, Mikołaj; Theis, Mirko; Krastev, Dragomir B.; Samsonov, Sergey; Mundwiller, Emeline; Junqueira, Magno; Paszkowski-Rogacz, Maciej; Teyra, Joan; Heninger, Anne-Kristin; Poser, Ina; Prieur, Fabienne; Truchetto, Jérémy; Confavreux, Christian; Marelli, Cécilia; Durr, Alexandra; Camdessanche, Jean Philippe; Brice, Alexis; Shevchenko, Andrej; Pisabarro, M. Teresa; Stevanin, Giovanni; Buchholz, Frank

    2010-01-01

    DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair. PMID:20613862

  18. RNAi screening of developmental toolkit genes: a search for novel wing genes in the red flour beetle, Tribolium castaneum.

    PubMed

    Linz, David M; Tomoyasu, Yoshinori

    2015-01-01

    The amazing array of diversity among insect wings offers a powerful opportunity to study the mechanisms guiding morphological evolution. Studies in Drosophila (the fruit fly) have identified dozens of genes important for wing development. These genes are often called candidate genes, serving as an ideal starting point to study wing development in other insects. However, we also need to explore beyond the candidate genes to gain a more comprehensive view of insect wing evolution. As a first step away from the traditional candidate genes, we utilized Tribolium (the red flour beetle) as a model and assessed the potential involvement of a group of developmental toolkit genes (embryonic patterning genes) in beetle wing development. We hypothesized that the highly pleiotropic nature of these developmental genes would increase the likelihood of finding novel wing genes in Tribolium. Through the RNA interference screening, we found that Tc-cactus has a less characterized (but potentially evolutionarily conserved) role in wing development. We also found that the odd-skipped family genes are essential for the formation of the thoracic pleural plates, including the recently discovered wing serial homologs in Tribolium. In addition, we obtained several novel insights into the function of these developmental genes, such as the involvement of mille-pattes and Tc-odd-paired in metamorphosis. Despite these findings, no gene we examined was found to have novel wing-related roles unique in Tribolium. These results suggest a relatively conserved nature of developmental toolkit genes and highlight the limited degree to which these genes are co-opted during insect wing evolution.

  19. A cellular high-throughput screening approach for therapeutic trans-cleaving ribozymes and RNAi against arbitrary mRNA disease targets.

    PubMed

    Yau, Edwin H; Butler, Mark C; Sullivan, Jack M

    2016-10-01

    Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N = G,C,A,U; H = C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to

  20. Quantitative High-throughput Luciferase Screening in Identifying CAR Modulators

    PubMed Central

    Lynch, Caitlin; Zhao, Jinghua; Wang, Hongbing; Xia, Menghang

    2017-01-01

    Summary The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR. PMID:27518621

  1. Evaluation of Quantitative Environmental Stress Screening (ESS) Methods. Volume 1

    DTIC Science & Technology

    1991-11-01

    muu4 The objective of this study was to evaluate Environmental Stress Screening (ESS) techniques contained in DOD-HDBK-344,’ by applying the methodology...to several electronic products during actual factor production. Validation of the techniques , the develop- ment of improved, qi•p’lified,_ad...automated procedures and subsequent revisions to the Handbook were the objectives, qf the evaluation. The Rome Laboratory has developed techniques which

  2. QUANTITATIVE CT ANALYSIS, AIRFLOW OBSTRUCTION AND LUNG CANCER IN THE PITTSBURGH LUNG SCREENING STUDY

    PubMed Central

    Wilson, David O; Leader, Joseph K; Fuhrman, Carl R; Reilly, John J; Sciurba, Frank C.; Weissfeld, Joel L

    2011-01-01

    Background To study the relationship between emphysema, airflow obstruction and lung cancer in a high risk population we performed quantitative analysis of screening computed tomography (CT) scans. Methods Subjects completed questionnaires, spirometry and low-dose helical chest CT. Analyses compared cases and controls according to automated quantitative analysis of lung parenchyma and airways measures. Results Our case-control study of 117 matched pairs of lung cancer cases and controls did not reveal any airway or lung parenchymal findings on quantitative analysis of screening CT scans that were associated with increased lung cancer risk. Airway measures including wall area %, lumen perimeter, lumen area and average wall HU, and parenchymal measures including lung fraction < −910 Hounsfield Units (HU), were not statistically different between cases and controls. Conclusions The relationship between visual assessment of emphysema and increased lung cancer risk could not be verified by quantitative analysis of low-dose screening CT scans in a high risk tobacco exposed population. PMID:21610523

  3. RNAi therapeutics for CNS disorders.

    PubMed

    Boudreau, Ryan L; Davidson, Beverly L

    2010-06-18

    RNA interference (RNAi) is a process of sequence-specific gene silencing and serves as a powerful molecular tool to manipulate gene expression in vitro and in vivo. RNAi technologies have been applied to study gene function and validate drug targets. Researchers are investigating RNAi-based compounds as novel therapeutics to treat a variety of human diseases that are currently lacking sufficient treatment. To date, numerous studies support that RNAi therapeutics can improve disease phenotypes in various rodent models of human disease. Here, we focus on the development of RNAi-based therapies aimed at treating neurological disorders for which reduction of mutant or toxic gene expression may provide clinical benefit. We review RNAi-based gene-silencing strategies, proof-of-concept studies testing therapeutic RNAi for CNS disorders, and highlight the most recent research aimed at transitioning RNAi-based therapeutics toward clinical trials.

  4. A whole-genome RNAi screen uncovers a novel role for human potassium channels in cell killing by the parasite Entamoeba histolytica

    PubMed Central

    Marie, Chelsea; Verkerke, Hans P.; Theodorescu, Dan; Petri, William A.

    2015-01-01

    The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K+) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K+ channel expression. Inhibition of human K+ channels by genetic silencing, pharmacologic inhibitors and with excess K+ protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K+ channel activation and K+ efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca2+-dependent K+ channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K+ efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K+ channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages. PMID:26346926

  5. A whole-genome RNAi screen uncovers a novel role for human potassium channels in cell killing by the parasite Entamoeba histolytica.

    PubMed

    Marie, Chelsea; Verkerke, Hans P; Theodorescu, Dan; Petri, William A

    2015-09-08

    The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K(+)) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K(+) channel expression. Inhibition of human K(+) channels by genetic silencing, pharmacologic inhibitors and with excess K(+) protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K(+) channel activation and K(+) efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca(2+)-dependent K(+) channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K(+) efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K(+) channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages.

  6. Rapid screening of triazines and quantitative determination in drinking water.

    PubMed

    Hamada, Mazen; Wintersteiger, Reinhold

    2002-01-01

    A sensitive, rapid and inexpensive analysis method has been developed for the triazines most frequently used in Palestine; the method includes fluorodensitometric screening and densitometric determination of the individual substances. Terbutryn as a model substance was derivatized with dansyl chloride in sodium hydrogen-carbonate or phosphate buffer solution to yield a green-blue fluorescent compound. Derivatization occurred at 120 degrees C within maximum of 10-min reaction time. The fluorescent compound formed was separated from excess reagent and other by-products on silica gel TLC plates and was then determined fluorodensitometrically. A linearity range between 20 and 1200 pg/spot was achieved. The method was also applied to other triazine herbicides such as ametryn, atrazine, propazine, terbuthylazine and simazine. Drinking water samples spiked with triazines were extracted using RP-C18 polar plus cartridges, and the extract could be then dansylated as a total. Recoveries were between 88% and 95%; the detection limit was 10 pg/spot and could be further improved to 2 pg/spot by a dipping solution. For quantification, each of the six triazines can be separated on one of three different stationary phases after solid phase extraction and measured densitometrically. The LOD for each individual triazine was 100 ng/l.

  7. Recombinant fungal entomopathogen RNAi target insect gene.

    PubMed

    Hu, Qiongbo; Wu, Wei

    2016-11-01

    RNA interference (RNAi) technology is considered as an alternative for control of pests. However, RNAi has not been used in field conditions yet, since delivering exogenous ds/siRNA to target pests is very difficult. The laboratory methods of introducing the ds/siRNA into insects through feeding, micro feeding / dripping and injecting cannot be used in fields. Transgenic crop is perhaps the most effective application of RNAi for pest control, but it needs long-time basic researches in order to reduce the cost and evaluate the safety. Therefore, transgenic microbe is maybe a better choice. Entomopathogenic fungi generally invade the host insects through cuticle like chemical insecticides contact insect to control sucking sap pests. Isaria fumosorosea is a common fungal entomopathogen in whitefly, Bemisia tabaci. We constructed a recombinant strain of I. fumosorosea expressing specific dsRNA of whitefly's TLR7 gene. It could silence the TLR7 gene and improve the virulence against whitefly. Transgenic fungal entomopathogen has shown great potential to attain the application of RNAi technology for pests control in fields. In the future, the research interests should be focused on the selection of susceptible target pests and their vital genes, and optimizing the methods for screening genes and recombinants as well.

  8. Antiviral RNAi: Translating Science Toward Therapeutic Success

    PubMed Central

    2016-01-01

    Viruses continuously evolve to contend with an ever-changing environment that involves transmission between hosts and sometimes species, immune responses, and in some cases therapeutic interventions. Given the high mutation rate of viruses relative to the timescales of host evolution and drug development, novel drug classes that are readily screened and translated to the clinic are needed. RNA interference (RNAi) – a natural mechanism for specific degradation of target RNAs that is conserved from plants to invertebrates and vertebrates – can potentially be harnessed to yield therapies with extensive specificity, ease of design, and broad application. In this review, we discuss basic mechanisms of action and therapeutic applications of RNAi, including design considerations and areas for future development in the field. PMID:21826573

  9. Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture.

    PubMed

    Yeung, Yik A; Wittrup, K Dane

    2002-01-01

    Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.

  10. A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair

    PubMed Central

    Herr, Patrick; Lundin, Cecilia; Evers, Bastiaan; Ebner, Daniel; Bauerschmidt, Christina; Kingham, Guy; Palmai-Pallag, Timea; Mortusewicz, Oliver; Frings, Oliver; Sonnhammer, Erik; Helleday, Thomas

    2015-01-01

    To identify new regulators of homologous recombination repair, we carried out a genome-wide short-interfering RNA screen combined with ionizing irradiation using RAD51 foci formation as readout. All candidates were confirmed by independent short-interfering RNAs and validated in secondary assays like recombination repair activity and RPA foci formation. Network analysis of the top modifiers identified gene clusters involved in recombination repair as well as components of the ribosome, the proteasome and the spliceosome, which are known to be required for effective DNA repair. We identified and characterized the RNA polymerase II-associated protein CDC73/Parafibromin as a new player in recombination repair and show that it is critical for genomic stability. CDC73 interacts with components of the SCF/Cullin and INO80/NuA4 chromatin-remodeling complexes to promote Histone ubiquitination. Our findings indicate that CDC73 is involved in local chromatin decondensation at sites of DNA damage to promote DNA repair. This function of CDC73 is related to but independent of its role in transcriptional elongation. PMID:27462432

  11. A Drosophila RNAi library modulates Hippo pathway-dependent tissue growth.

    PubMed

    Vissers, Joseph H A; Manning, Samuel A; Kulkarni, Aishwarya; Harvey, Kieran F

    2016-01-13

    Libraries of transgenic Drosophila melanogaster carrying RNA interference (RNAi) constructs have been used extensively to perform large-scale functional genetic screens in vivo. For example, RNAi screens have facilitated the discovery of multiple components of the Hippo pathway, an evolutionarily conserved growth-regulatory network. Here we investigate an important technical limitation with the widely used VDRC KK RNAi collection. We find that approximately 25% of VDRC KK RNAi lines cause false-positive enhancement of the Hippo pathway, owing to ectopic expression of the Tiptop transcription factor. Of relevance to the broader Drosophila community, ectopic tiptop (tio) expression can also cause organ malformations and mask phenotypes such as organ overgrowth. To enhance the use of the VDRC KK RNAi library, we have generated a D. melanogaster strain that will allow researchers to test, in a single cross, whether their genetic screen of interest will be affected by ectopic tio expression.

  12. Asian Citrus Psyllid RNAi Pathway – RNAi evidence

    PubMed Central

    Taning, Clauvis N. T.; Andrade, Eduardo C.; Hunter, Wayne B.; Christiaens, Olivier; Smagghe, Guy

    2016-01-01

    Diaphorina citri, known as the Asian citrus psyllid, is an important pest of citrus because it transmits a phloem-limited bacteria strongly implicated in huanglongbing (citrus greening disease). Emerging biotechnologies, such as RNA interference, could provide a new sustainable and environmentally friendly strategy for the management of this pest. In this study, genome and functional analysis were performed to verify whether the RNAi core genes are present in the Asian psyllid genome and if the RNAi machinery could be exploited to develop a management strategy for this pest. Analyses of RNAi-related genes in the Asian citrus psyllid genome showed an absence of sequences encoding R2D2, a dsRNA-binding protein that functions as a cofactor of Dicer-2 in Drosophila. Nevertheless, bioassays using an in Planta System showed that the Asian citrus psyllid was very sensitive to ingested dsRNA, demonstrating a strong RNAi response. A small dose of dsRNA administered through a citrus flush was enough to trigger the RNAi mechanism, causing significant suppression of the targeted transcript, and increased psyllid mortality. This study provides evidence of a functional RNAi machinery, which could be further exploited to develop RNAi based management strategies for the control of the Asian citrus psyllid. PMID:27901078

  13. A Genome-Wide RNAi Screen Identifies FOXO4 as a Metastasis-Suppressor through Counteracting PI3K/AKT Signal Pathway in Prostate Cancer

    PubMed Central

    Su, Bing; Gao, Lingqiu; Baranowski, Catherine; Gillard, Bryan; Wang, Jianmin; Ransom, Ryan; Ko, Hyun-Kyung; Gelman, Irwin H.

    2014-01-01

    Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness. PMID:24983969

  14. Environmental RNAi in herbivorous insects.

    PubMed

    Ivashuta, Sergey; Zhang, Yuanji; Wiggins, B Elizabeth; Ramaseshadri, Partha; Segers, Gerrit C; Johnson, Steven; Meyer, Steve E; Kerstetter, Randy A; McNulty, Brian C; Bolognesi, Renata; Heck, Gregory R

    2015-05-01

    Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism.

  15. Environmental RNAi in herbivorous insects

    PubMed Central

    Ivashuta, Sergey; Zhang, Yuanji; Wiggins, B. Elizabeth; Ramaseshadri, Partha; Segers, Gerrit C.; Johnson, Steven; Meyer, Steve E.; Kerstetter, Randy A.; McNulty, Brian C.; Bolognesi, Renata; Heck, Gregory R.

    2015-01-01

    Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism. PMID:25802407

  16. Quantitative methylene blue decolourisation assays as rapid screening tools for assessing the efficiency of catalytic reactions.

    PubMed

    Kruid, Jan; Fogel, Ronen; Limson, Janice Leigh

    2017-05-01

    Identifying the most efficient oxidation process to achieve maximum removal of a target pollutant compound forms the subject of much research. There exists a need to develop rapid screening tools to support research in this area. In this work we report on the development of a quantitative assay as a means for identifying catalysts capable of decolourising methylene blue through the generation of oxidising species from hydrogen peroxide. Here, a previously described methylene blue test strip method was repurposed as a quantitative, aqueous-based spectrophotometric assay. From amongst a selection of metal salts and metallophthalocyanine complexes, monitoring of the decolourisation of the cationic dye methylene blue (via Fenton-like and non-Fenton oxidation reactions) by the assay identified the following to be suitable oxidation catalysts: CuSO4 (a Fenton-like catalyst), iron(II)phthalocyanine (a non-Fenton oxidation catalyst), as well as manganese(II) phthalocyanine. The applicability of the method was examined for the removal of bisphenol A (BPA), as measured by HPLC, during parallel oxidation experiments. The order of catalytic activity was identified as FePc > MnPc > CuSO4 for both BPA and MB. The quantitative MB decolourisation assay may offer a rapid method for screening a wide range of potential catalysts for oxidation processes.

  17. RNAi: future in insect management.

    PubMed

    Burand, John P; Hunter, Wayne B

    2013-03-01

    RNA interference is a post- transcriptional, gene regulation mechanism found in virtually all plants and animals including insects. The demonstration of RNAi in insects and its successful use as a tool in the study of functional genomics opened the door to the development of a variety of novel, environmentally sound approaches for insect pest management. Here the current understanding of the biogenesis of the two RNAi classes in insects is reviewed. These are microRNAs (miRNAs) and short interfering RNAs (siRNAs). Several other key approaches in RNAi -based for insect control, as well as for the prevention of diseases in insects are also reviewed. The problems and prospects for the future use of RNAi in insects are presented.

  18. Development of new RNAi therapeutics.

    PubMed

    Liu, G; Wong-Staal, F; Li, Q-X

    2007-02-01

    RNAi-mediated gene inactivation has become a cornerstone of the present day gene function studies that are the foundation of mechanism and target based drug discovery and development, which could potentially shorten the otherwise long process of drug development. In particular, the coming of age of "RNAi drug" could provide new promising therapeutics bypassing traditional approaches. However, there are technological hurdles need to overcome and the biological limitations need to consider for achieving effective therapeutics. Major hurdles include the intrinsic poor pharmacokinetic property of siRNA and major biological restrictions include off-target effects, interferon response and the interference with endogenous miRNA. Recent innovations in nucleic acid chemistry, formulations and delivery methods have gradually rendered it possible to develop effective RNAi-based therapeutics. Careful design based on the newest RNAi/miRNA biology can also help to minimize the potential tissue toxicity. If successful with systemic application, RNAi drug will no doubt revolutionize the whole drug development process. This review attempts to describe the progress in this area, including applications in preclinical models and recent favorable experience in a number of human trials of local diseases, along with the discussion on the potential limitations of RNAi therapeutics.

  19. A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

    PubMed Central

    Helka, Blake-Joseph; Brennan, John D.

    2013-01-01

    Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays. PMID:24022739

  20. Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

    PubMed Central

    Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

    2015-01-01

    Objective Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. PMID:26487969

  1. Quantitative characterization of planarian wild-type behavior as a platform for screening locomotion phenotypes.

    PubMed

    Talbot, Jared; Schötz, Eva-Maria

    2011-04-01

    Changes in animal behavior resulting from genetic or chemical intervention are frequently used for phenotype characterizations. The majority of these studies are qualitative in nature, especially in systems that go beyond the classical model organisms. Here, we introduce a quantitative method to characterize behavior in the freshwater planarian Schmidtea mediterranea. Wild-type locomotion in confinement was quantified using a wide set of parameters, and the influences of intrinsic intra-worm versus inter-worm variability on our measurements was studied. We also examined the effect of substrate, confinement geometry and the interactions with the boundary on planarian behavior. The method is based on a simple experimental setup, using automated center-of-mass tracking and image analysis, making it an easily implemented alternative to current methods for screening planarian locomotion phenotypes. As a proof of principle, two drug-induced behavioral phenotypes were generated to show the capacity of this method.

  2. Quantitative high throughput screening identifies inhibitors of anthrax-induced cell death

    PubMed Central

    Zhu, Ping Jun; Hobson, Peyton; Southall, Noel; Qiu, Cunping; Thomas, Craig J.; Lu, Jiamo; Inglese, James; Zheng, Wei; Leppla, Stephen H.; Bugge, Thomas H.; Austin, Christopher P.; Liu, Shihui

    2009-01-01

    Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a β-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization. PMID:19540764

  3. QUANTITATIVE SCREENING OF SINGLE COPIES OF HUMAN PAPILLOMA VIRAL DNA WITHOUT AMPLIFICATION

    PubMed Central

    Li, Jiangwei; Lee, Ji-Young; Yeung, Edward S.

    2008-01-01

    We describe a novel quantitative viral screening method based on single-molecule detection that does not require amplification. DNA of human papilloma virus (HPV), the major etiological agent of cervical cancer, served as the screening target in this study. Eight 100-nucleotide (nt) single-stranded (ss)-DNA probes were designed complementary to the E6-E7 gene of HPV-16 DNA. The probes were covalently stained with Alexa Fluor 532 and hybridized to the target in solution. The individual hybridized molecules were imaged with an intensified charge-coupled device (ICCD) in two ways. In the single-color mode, target molecules were detected via fluorescence from hybridized probes only. This system could detect HPV-16 DNA in the presence of human genomic DNA down to 0.7 copy/cell, and had a linear dynamic range of over six orders of magnitude. In the dual-color mode, we employed fluorescence resonance energy transfer (FRET) and added YOYO-3 dye as the acceptor. The two colors from Alexa Fluor 532 and YOYO-3 were dispersed by a transmission grating located in front of the ICCD. With this reinforced criteria for identifying the hybridized molecules, zero false-positive count was achieved. We also showed that DNA extracts from Pap test specimens did not interfere with the measurements. PMID:16970325

  4. Development of Screening Method for an Frail Elderly by Measurement Quantitative Lower Limb Muscular Strength

    NASA Astrophysics Data System (ADS)

    Yamashita, Kazuhiko; Iwakami, Yumi; Imaizumi, Kazuya; Sato, Mitsuru; Nakajima, Sawako; Ino, Shuichi; Kawasumi, Masashi; Ifukube, Tohru

    Falling is one of the most serious problems for the elderly. The aim of this study was to develop a screening method for identifying factors that increase the risk of falling among the elderly, particularly with regard to lower limb muscular strength. Subjects were 48 elderly volunteers, including 25 classed as healthy and 23 classed as frail. All subjects underwent measurement of lower limb muscular strength via toe gap force and measurement of muscle strength of the hip joint adductor via knee gap force. In the frail group, toe gap force of the right foot was 20% lower than that in the healthy group; toe gap force of the left foot in the frail group was 23% lower than that in the healthy group, while knee gap force was 20% lower. Furthermore, we found that combining left toe gap force and knee gap force gave the highest odds ratio (6.05) with 82.6% sensitivity and 56.0% specificity when the toe gap force was 24 N and the knee gap force was 100 N. Thus, lower limb muscular strength can be used for simple and efficient screening, and approaches to prevent falls can be based on quantitative data such as lower limb muscular strength.

  5. Quantitative screening for anticestode drugs based on changes in baseline enzyme secretion by Taenia crassiceps.

    PubMed

    Mahanty, Siddhartha; Madrid, Elise M; Nash, Theodore E

    2013-02-01

    Neurocysticercosis (NCC), an infection of the brain with the larval stage of the Taenia solium tapeworm, is responsible for an estimated one-third of adult-onset epilepsy cases in regions of the world where it is endemic. Currently, anthelmintic drugs used for treatment of NCC are only partially effective, and there is, therefore, a pressing need for new therapeutic agents. Discovery of new anthelmintics with activity against T. solium has been limited by the lack of suitable sensitive assays that allow high-throughput screening. Using an in vitro culture system with Taenia crassiceps metacestodes, we demonstrate that changes in secretion of parasite-associated alkaline phosphatase (AP) and phosphoglucose isomerase (PGI) can be used to detect and quantify anthelmintic effects of praziquantel (PZQ), a drug with activity against T. solium. We applied two enzyme release assays to screen for anti-T. crassiceps activity in nonconventional antiparasitic drugs and demonstrate that nitazoxanide and artesunate induced release of both AP and PGI in differing time- and dose-related patterns. Furthermore, imatinib, a tyrosine kinase inhibitor previously reported to have parasiticidal activity against Schistosoma mansoni, also induced release of both AP and PGI in a dose-dependent manner, similar in pattern to that observed with the other anthelmintics. We also evaluated release of ATP into cyst supernatants as an indicator of drug effects but did not see any differences between treated and untreated cysts. These data provide the basis for rapid and quantitative screening assays for testing for anthelmintic activity in candidate anticestode agents.

  6. Quantitative High-Throughput Screen Identifies Inhibitors of the Schistosoma mansoni Redox Cascade

    PubMed Central

    Simeonov, Anton; Jadhav, Ajit; Sayed, Ahmed A.; Wang, Yuhong; Nelson, Michael E.; Thomas, Craig J.; Inglese, James; Williams, David L.; Austin, Christopher P.

    2008-01-01

    Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 µL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC50s ranging from micromolar to the assay response limit (∼25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel

  7. Quantitative high-throughput screening for chemical toxicity in a population-based in vitro model.

    PubMed

    Lock, Eric F; Abdo, Nour; Huang, Ruili; Xia, Menghang; Kosyk, Oksana; O'Shea, Shannon H; Zhou, Yi-Hui; Sedykh, Alexander; Tropsha, Alexander; Austin, Christopher P; Tice, Raymond R; Wright, Fred A; Rusyn, Ivan

    2012-04-01

    A shift in toxicity testing from in vivo to in vitro may efficiently prioritize compounds, reveal new mechanisms, and enable predictive modeling. Quantitative high-throughput screening (qHTS) is a major source of data for computational toxicology, and our goal in this study was to aid in the development of predictive in vitro models of chemical-induced toxicity, anchored on interindividual genetic variability. Eighty-one human lymphoblast cell lines from 27 Centre d'Etude du Polymorphisme Humain trios were exposed to 240 chemical substances (12 concentrations, 0.26nM-46.0μM) and evaluated for cytotoxicity and apoptosis. qHTS screening in the genetically defined population produced robust and reproducible results, which allowed for cross-compound, cross-assay, and cross-individual comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited interindividual differences in cytotoxicity. Specifically, the qHTS in a population-based human in vitro model system has several unique aspects that are of utility for toxicity testing, chemical prioritization, and high-throughput risk assessment. First, standardized and high-quality concentration-response profiling, with reproducibility confirmed by comparison with previous experiments, enables prioritization of chemicals for variability in interindividual range in cytotoxicity. Second, genome-wide association analysis of cytotoxicity phenotypes allows exploration of the potential genetic determinants of interindividual variability in toxicity. Furthermore, highly significant associations identified through the analysis of population-level correlations between basal gene expression variability and chemical-induced toxicity suggest plausible mode of action hypotheses for follow-up analyses. We conclude that as the improved resolution of genetic profiling can now be matched with high-quality in vitro screening data, the evaluation of the toxicity pathways and the effects of

  8. A systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening

    PubMed Central

    Attene-Ramos, Matias S.; Huang, Ruili; Sakamuru, Srilatha; Witt, Kristine L.; Beeson, Gyda C.; Shou, Louie; Schnellmann, Rick G.; Beeson, Craig C.; Tice, Raymond R.; Austin, Christopher P.; Xia, Menghang

    2014-01-01

    A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for one or five h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled identification of 20 compounds as uncouplers. This comprehensive approach allows for evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs. PMID:23895456

  9. RNAI: Future in insect management

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference is a post-transcriptional, gene regulation mechanism found in virtually all plants and animals including insects. The demonstration of RNAi in insects and its successful use as a tool in the study of functional genomics opened the door to the development of a variety of novel, envir...

  10. RNAi as a Routine Route Toward Breast Cancer Therapy

    DTIC Science & Technology

    2009-09-01

    a third -generation shRNA library 2. collection of cell lines for screening within the proposed program 6 3. determination that microRNAs can...08-1-0572 TITLE: RNAi as a routine route toward breast cancer therapy PRINCIPAL INVESTIGATOR: Gregory J. Hannon, Ph.D... therapy 5a. CONTRACT NUMBER W81XWH – 08 – 1 - 0572 5b. GRANT NUMBER BC076047 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S

  11. Towards the elements of successful insect RNAi.

    PubMed

    Scott, Jeffrey G; Michel, Kristin; Bartholomay, Lyric C; Siegfried, Blair D; Hunter, Wayne B; Smagghe, Guy; Zhu, Kun Yan; Douglas, Angela E

    2013-12-01

    RNA interference (RNAi), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. The accumulating body of literature on insect RNAi has revealed that the efficiency of RNAi varies between different species, the mode of RNAi delivery, and the genes being targeted. There is also variation in the duration of transcript suppression. At present, we have a limited capacity to predict the ideal experimental strategy for RNAi of a particular gene/insect because of our incomplete understanding of whether and how the RNAi signal is amplified and spread among insect cells. Consequently, development of the optimal RNAi protocols is a highly empirical process. This limitation can be relieved by systematic analysis of the molecular physiological basis of RNAi mechanisms in insects. An enhanced conceptual understanding of RNAi function in insects will facilitate the application of RNAi for dissection of gene function, and to fast-track the application of RNAi to both control pests and develop effective methods to protect beneficial insects and non-insect arthropods, particularly the honey bee (Apis mellifera) and cultured Pacific white shrimp (Litopenaeus vannamei) from viral and parasitic diseases.

  12. A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays.

    PubMed

    Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R

    2015-08-01

    A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise-filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC(50) (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds.

  13. Quantitative screening of genes regulating tryptophan hydroxylase transcription in Caenorhabditis elegans using microfluidics and an adaptive algorithm.

    PubMed

    Lee, Hyewon; Crane, Matthew M; Zhang, Yun; Lu, Hang

    2013-02-01

    Forward genetic screening via mutagenesis is a powerful method for identifying regulatory factors in target pathways in model organisms such as the soil-dwelling free-living nematode Caenorhabditis elegans (C. elegans). Currently manual microscopy is the standard technique for conducting such screens; however, it is labor-intensive and time-consuming because screening requires imaging thousands of animals. Recently microfluidic chips have been developed to increase the throughput of some of such experiments; nonetheless, most of these chips are multilayer devices and complicated to fabricate and therefore prone to failure during fabrication and operation. In addition, most sorting decisions are made manually and the criteria used for sorting are subjective. To overcome these limitations, we developed a simple single-layer microfluidic device and an adaptive algorithm to make sorting decisions. The one-layer device greatly improves the reliability, while quantitative analysis with the adaptive algorithm allows for the identification of mutations that generate subtle changes in expression, which would have been hard to detect by eye. The screening criterion is set based on the mutagenized population, not separate control populations measured prior to actual screening experiments, to account for stochasticity and day-to-day variations of gene expression in mutagenized worms. Moreover, during each experiment, the threshold is constantly updated to reflect the balance between maximizing sorting rate and minimizing false-positive rate. Using this system, we screened for mutants that have altered expression levels of tryptophan hydroxylase, a key enzyme for serotonin synthesis in a CaMKII gain-of-function background. We found several putative mutants in this screen. Furthermore, this microfluidic system and quantitative analysis can be easily adapted to study other pathways in C. elegans.

  14. The Future of Toxicity Testing: A Focus on In Vitro Methods Using a Quantitative High Throughput Screening Platform

    PubMed Central

    Shukla, Sunita J.; Huang, Ruili; Austin, Christopher P.; Xia, Menghang

    2010-01-01

    The U.S. Tox21 collaborative program represents a paradigm shift in toxicity testing of chemical compounds from traditional in vivo tests to less expensive and higher throughput in vitro methods to prioritize compounds for further study, identify mechanisms of action, and ultimately develop predictive models for adverse health effects in humans. The NIH Chemical Genomics Center (NCGC) is an integral component of the Tox21 collaboration due to its quantitative high throughput screening (qHTS) paradigm, in which titration-based screening is used to profile hundreds of thousands of compounds per week. Here, we describe the Tox21 collaboration, qHTS-based compound testing, and the various Tox21 screening assays that have been validated and tested at the NCGC to date. PMID:20708096

  15. The Mathematics of a Successful Deconvolution: A Quantitative Assessment of Mixture-Based Combinatorial Libraries Screened Against Two Formylpeptide Receptors

    PubMed Central

    Santos, Radleigh G.; Appel, Jon R.; Giulianotti, Marc A.; Edwards, Bruce S.; Sklar, Larry A.; Houghten, Richard A.; Pinilla, Clemencia

    2014-01-01

    In the past 20 years, synthetic combinatorial methods have fundamentally advanced the ability to synthesize and screen large numbers of compounds for drug discovery and basic research. Mixture-based libraries and positional scanning deconvolution combine two approaches for the rapid identification of specific scaffolds and active ligands. Here we present a quantitative assessment of the screening of 32 positional scanning libraries in the identification of highly specific and selective ligands for two formylpeptide receptors. We also compare and contrast two mixture-based library approaches using a mathematical model to facilitate the selection of active scaffolds and libraries to be pursued for further evaluation. The flexibility demonstrated in the differently formatted mixture-based libraries allows for their screening in a wide range of assays. PMID:23722730

  16. The mathematics of a successful deconvolution: a quantitative assessment of mixture-based combinatorial libraries screened against two formylpeptide receptors.

    PubMed

    Santos, Radleigh G; Appel, Jon R; Giulianotti, Marc A; Edwards, Bruce S; Sklar, Larry A; Houghten, Richard A; Pinilla, Clemencia

    2013-05-30

    In the past 20 years, synthetic combinatorial methods have fundamentally advanced the ability to synthesize and screen large numbers of compounds for drug discovery and basic research. Mixture-based libraries and positional scanning deconvolution combine two approaches for the rapid identification of specific scaffolds and active ligands. Here we present a quantitative assessment of the screening of 32 positional scanning libraries in the identification of highly specific and selective ligands for two formylpeptide receptors. We also compare and contrast two mixture-based library approaches using a mathematical model to facilitate the selection of active scaffolds and libraries to be pursued for further evaluation. The flexibility demonstrated in the differently formatted mixture-based libraries allows for their screening in a wide range of assays.

  17. A Colony Multiplex Quantitative PCR-Based 3S3DBC Method and Variations of It for Screening DNA Libraries

    PubMed Central

    An, Yang; Toyoda, Atsushi; Zhao, Chen; Fujiyama, Asao; Agata, Kiyokazu

    2015-01-01

    A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements. PMID:25646755

  18. Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem mass spectrometry.

    PubMed

    Gergov, M; Robson, J N; Ojanperä, I; Heinonen, O P; Vuori, E

    2001-09-15

    A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is presented for the simultaneous screening and quantitation of 18 antihistamine drugs in blood samples. Sample pretreatment involved liquid-liquid extraction of the basic antihistamines followed by a second extraction of the acidic antihistamines. The recoveries were 43-113% for basic drugs and 23-66% for acidic drugs. The combined extracts were run by LC on C(18) reversed phase column using acetonitrile-ammonium acetate mobile phase at pH 3.2. The mass spectrometric analysis was performed with a triple stage quadrupole mass analyzer. Screening was performed using multiple reaction monitoring (MRM) and any compounds tentatively identified as antihistamine drugs were then automatedly verified by their Product Ion Spectra in a subsequent MS/MS run. Quantitation was based on the MRM data from the screening step. In validation tests, the method showed good linearity at the relevant concentrations. The attained limits of quantitation varied between 0.0005 and 0.01mg/l in blood and were lower than the therapeutic concentrations (C(max)). The limits for identification by Product Ion Spectra were also lower than C(max), except for clemastine, which has exceptionally low concentrations in blood. The intra-assay relative standard deviations were better than 10% and the inaccuracy varied between 39% for levocabastine and 5% for cyclizine, the majority of the values being <20%.

  19. Using nuclear run-on transcription assays in RNAi studies.

    PubMed

    Khraiwesh, Basel

    2011-01-01

    RNA interference (RNAi) is a mechanism regulating gene transcript levels either by transcriptional gene silencing or by posttranscriptional gene silencing, which act in the genome maintenance and the regulation of gene expression which is typically inferred from measuring transcript abundance. Nuclear "run-on" (or "run-off") transcription assays have been used to obtain quantitative information about the relative rates of transcription of different genes in nuclei isolated from a particular tissue or organ. Basically, these assays exploit the activity of RNA polymerases to synthesize radiolabeled transcripts that then can be hybridized to filter-bound, cold, excess single-stranded DNA probes representing genes of interest. The protocol presented here streamlines, adapts, and optimizes nuclear run-on transcription assays for use in RNAi studies.

  20. Highly sensitive capillary electrophoresis-mass spectrometry for rapid screening and accurate quantitation of drugs of abuse in urine.

    PubMed

    Kohler, Isabelle; Schappler, Julie; Rudaz, Serge

    2013-05-30

    The combination of capillary electrophoresis (CE) and mass spectrometry (MS) is particularly well adapted to bioanalysis due to its high separation efficiency, selectivity, and sensitivity; its short analytical time; and its low solvent and sample consumption. For clinical and forensic toxicology, a two-step analysis is usually performed: first, a screening step for compound identification, and second, confirmation and/or accurate quantitation in cases of presumed positive results. In this study, a fast and sensitive CE-MS workflow was developed for the screening and quantitation of drugs of abuse in urine samples. A CE with a time-of-flight MS (CE-TOF/MS) screening method was developed using a simple urine dilution and on-line sample preconcentration with pH-mediated stacking. The sample stacking allowed for a high loading capacity (20.5% of the capillary length), leading to limits of detection as low as 2 ng mL(-1) for drugs of abuse. Compound quantitation of positive samples was performed by CE-MS/MS with a triple quadrupole MS equipped with an adapted triple-tube sprayer and an electrospray ionization (ESI) source. The CE-ESI-MS/MS method was validated for two model compounds, cocaine (COC) and methadone (MTD), according to the Guidance of the Food and Drug Administration. The quantitative performance was evaluated for selectivity, response function, the lower limit of quantitation, trueness, precision, and accuracy. COC and MTD detection in urine samples was determined to be accurate over the range of 10-1000 ng mL(-1) and 21-1000 ng mL(-1), respectively.

  1. Contributing Factors to Colorectal Cancer Screening among Chinese People: A Review of Quantitative Studies

    PubMed Central

    Leung, Doris Y. P.; Chow, Ka Ming; Lo, Sally W. S.; So, Winnie K. W.; Chan, Carmen W. H.

    2016-01-01

    Colorectal cancer (CRC) is a major health problem in Asia. It has been reported that the Chinese are more susceptible to CRC than many other ethnic groups. Screening for CRC is a cost-effective prevention and control strategy; however, the screening rates among the Chinese are sub-optimal. We conducted a review to identify the factors associated with CRC screening participation among Chinese people. Twenty-two studies that examined the factors related to CRC screening behaviors among the Chinese were identified through five databases. Seven factors were consistently reported to influence CRC screening behaviors in at least one of the studies: socio-demographic characteristics (educational level, health insurance, and knowledge about CRC and its screening); psychological factors (perceived severity of CRC, susceptibility of having CRC, and barriers to screening); and contact with medical provider (physician recommendation). The evidence base for many of these relationships is quite limited. Furthermore, the associations of many factors, including age, gender, income, cancer worry/fear, and self-efficacy with CRC screening behaviors, were mixed or inconsistent across these studies, thereby indicating that more studies are needed in this area. PMID:27196920

  2. Topographic quantitative EEG sequelae of chronic marihuana use: a replication using medically and psychiatrically screened normal subjects.

    PubMed

    Struve, F A; Straumanis, J J; Patrick, G; Leavitt, J; Manno, J E; Manno, B R

    1999-10-01

    In two previous studies it was reported that chronic marihuana (THC) use was associated with unique quantitative EEG features which were present in the non-intoxicated state. THC users, as contrasted with controls, had significant elevations of Absolute Power, Relative Power, and Coherence of alpha activity over the bilateral frontal cortex. Furthermore, a quantitative EEG discriminant function analyses permitted a 95% correct user versus non-user classification. However, because all of the THC users and 58% of the non-user controls were psychiatric inpatients, diagnostic and medication effects, if any, were uncontrolled. In the present study the same quantitative EEG methods were used to study daily THC users and non-user controls who underwent a rigorous screening process to insure that they were medically and psychiatrically healthy. The results of previous studies were replicated and an additional EEG correlate of chronic THC exposure (reduced alpha frequency) was identified.

  3. Predictive power of quantitative and qualitative fecal immunochemical tests for hemoglobin in population screening for colorectal neoplasm.

    PubMed

    Huang, Yanqin; Li, Qilong; Ge, Weiting; Cai, Shanrong; Zhang, Suzhan; Zheng, Shu

    2014-01-01

    The aim of this study was to evaluate the performance of qualitative and quantitative fecal immunochemical tests (FITs) in population screening for colorectal neoplasm. A total of 9000 participants aged between 40 and 74 years were enrolled in this study. Each participant received two stool sampling tubes and was asked to simultaneously submit two stool samples from the same bowel movement. The stool samples of each participant were tested using an immunogold labeling FIT dipstick (qualitative FIT) and an automated fecal blood analyzer (quantitative FIT). Colonoscopy was performed for those who test positive in either FIT. The positive predictive values and population detection rates of the FITs for predicting colorectal neoplasm were compared. A total of 6494 (72.16%) participants simultaneously submitted two stool samples. The diagnostic consistency for a positive result between quantitative and qualitative FITs was poor (κ=0.278, 95% confidence interval=0.223-0.333). The positive predictive values of the quantitative FIT were significantly higher than those of the qualitative FIT for predicting large (≥1 cm) adenomas (23 cases, 14.29% and 16 cases, 6.72%, P=0.013) and colorectal cancer (10 cases, 6.21% and 5 cases, 2.10%, P=0.034); however, the population detection rate for advanced neoplasm of the quantitative FIT was not significantly different from that of the qualitative FIT. Quantitative FIT is superior to qualitative FIT in predicting advanced colorectal neoplasm during colorectal cancer screening. Further studies are needed to elucidate the causes of the predictive superiority.

  4. Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple applications.

    PubMed

    Holt, Tom G; Choi, Bernard K; Geoghagen, Neil S; Jensen, Kristian K; Luo, Qi; LaMarr, William A; Makara, Gergely M; Malkowitz, Lorraine; Ozbal, Can C; Xiong, Yusheng; Dufresne, Claude; Luo, Ming-Juan

    2009-10-01

    Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. MS permits the selective detection of enzyme substrates or products in a wide range of biological matrices without need for derivatization, labeling, or capture technologies. As part of a cardiovascular drug discovery effort aimed at finding modulators of cystathionine beta-synthase (CBS), we used the RapidFire((R)) label-free high-throughput MS (HTMS) technology to develop a high-throughput screening (HTS) assay for CBS activity. The in vitro assay used HTMS to quantify the unlabeled product of the CBS reaction, cystathionine. Cystathionine HTMS analyses were carried out with a throughput of 7 s per sample and quantitation over a linear range of 80-10,000 nM. A compound library of 25,559 samples (or 80 384-well plates) was screened as singlets using the HTMS assay in a period of 8 days. With a hit rate of 0.32%, the actives showed a 90% confirmation rate. The in vitro assay was applied to secondary screens in more complex matrices with no additional analytical development. Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project.

  5. Development of a sensitive E-screen assay for quantitative analysis of estrogenic activity in municipal sewage plant effluents.

    PubMed

    Körner, W; Hanf, V; Schuller, W; Kempter, C; Metzger, J; Hagenmaier, H

    1999-01-12

    A simplified proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-screen assay) was optimized and validated for the sensitive quantitative determination of total estrogenic activity in effluent samples from municipal sewage plants. After solid phase extraction of 1 l sewage on either 0.2 g polystyrene copolymer (ENV+) or 1 g RP-C18 material and removal of the solvent, analysis of the extracts in the E-screen assay could be performed without any clean-up step. This was even possible with untreated sewage. Parallel extraction of four sewage samples on both different solid phase materials gave comparable quantitative results in the E-screen. A blank sample did not induce cell proliferation. As additive behaviour of the estrogenic response of single compounds was proven for two different mixtures each containing three xenoestrogens, total estrogenic activity in the sewage samples, expressed as 17 beta-estradiol equivalent concentration (EEQ), could be calculated comparing the EC50 values of the samples with those of the positive control 17 beta-estradiol. The detection limit of the E-screen method was 0.05 pmol EEQ/l (0.014 ng EEQ/l), the limit of quantification 0.25-0.5 pmol EEQ/l (0.07-0.14 ng EEQ/l). In total, extracts of nine effluent and one influent sample from five different municipal sewage plants in South Germany were analyzed in the E-screen. All samples strongly induced cell proliferation in a dose-dependent manner which was completely inhibited by coincubation with 5 nM of the estrogen receptor-antagonist ICI 182,780. The proliferative effect relative to the positive control 17 beta-estradiol (RPE) was between 30 and 101%. 17 beta-Estradiol equivalent concentrations were between 2.5 and 25 ng/l indicating a significant input of estrogenic substances via sewage treatment plants into rivers.

  6. Development and interlaboratory validation of quantitative polymerase chain reaction method for screening analysis of genetically modified soybeans.

    PubMed

    Takabatake, Reona; Onishi, Mari; Koiwa, Tomohiro; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Kurashima, Takeyo; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

    2013-01-01

    A novel real-time polymerase chain reaction (PCR)-based quantitative screening method was developed for three genetically modified soybeans: RRS, A2704-12, and MON89788. The 35S promoter (P35S) of cauliflower mosaic virus is introduced into RRS and A2704-12 but not MON89788. We then designed a screening method comprised of the combination of the quantification of P35S and the event-specific quantification of MON89788. The conversion factor (Cf) required to convert the amount of a genetically modified organism (GMO) from a copy number ratio to a weight ratio was determined experimentally. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDR), respectively. The determined RSDR values for the method were less than 25% for both targets. We consider that the developed method would be suitable for the simple detection and approximate quantification of GMO.

  7. High-content screening of drug-induced cardiotoxicity using quantitative single cell imaging cytometry on microfluidic device.

    PubMed

    Kim, Min Jung; Lee, Su Chul; Pal, Sukdeb; Han, Eunyoung; Song, Joon Myong

    2011-01-07

    Drug-induced cardiotoxicity or cytotoxicity followed by cell death in cardiac muscle is one of the major concerns in drug development. Herein, we report a high-content quantitative multicolor single cell imaging tool for automatic screening of drug-induced cardiotoxicity in an intact cell. A tunable multicolor imaging system coupled with a miniaturized sample platform was destined to elucidate drug-induced cardiotoxicity via simultaneous quantitative monitoring of intracellular sodium ion concentration, potassium ion channel permeability and apoptosis/necrosis in H9c2(2-1) cell line. Cells were treated with cisapride (a human ether-à-go-go-related gene (hERG) channel blocker), digoxin (Na(+)/K(+)-pump blocker), camptothecin (anticancer agent) and a newly synthesized anti-cancer drug candidate (SH-03). Decrease in potassium channel permeability in cisapride-treated cells indicated that it can also inhibit the trafficking of the hERG channel. Digoxin treatment resulted in an increase of intracellular [Na(+)]. However, it did not affect potassium channel permeability. Camptothecin and SH-03 did not show any cytotoxic effect at normal use (≤300 nM and 10 μM, respectively). This result clearly indicates the potential of SH-03 as a new anticancer drug candidate. The developed method was also used to correlate the cell death pathway with alterations in intracellular [Na(+)]. The developed protocol can directly depict and quantitate targeted cellular responses, subsequently enabling an automated, easy to operate tool that is applicable to drug-induced cytotoxicity monitoring with special reference to next generation drug discovery screening. This multicolor imaging based system has great potential as a complementary system to the conventional patch clamp technique and flow cytometric measurement for the screening of drug cardiotoxicity.

  8. Intelligent Interfaces for Mining Large-Scale RNAi-HCS Image Databases.

    PubMed

    Lin, Chen; Mak, Wayne; Hong, Pengyu; Sepp, Katharine; Perrimon, Norbert

    2007-11-05

    Recently, High-content screening (HCS) has been combined with RNA interference (RNAi) to become an essential image-based high-throughput method for studying genes and biological networks through RNAi-induced cellular phenotype analyses. However, a genome-wide RNAi-HCS screen typically generates tens of thousands of images, most of which remain uncategorized due to the inadequacies of existing HCS image analysis tools. Until now, it still requires highly trained scientists to browse a prohibitively large RNAi-HCS image database and produce only a handful of qualitative results regarding cellular morphological phenotypes. For this reason we have developed intelligent interfaces to facilitate the application of the HCS technology in biomedical research. Our new interfaces empower biologists with computational power not only to effectively and efficiently explore large-scale RNAi-HCS image databases, but also to apply their knowledge and experience to interactive mining of cellular phenotypes using Content-Based Image Retrieval (CBIR) with Relevance Feedback (RF) techniques.

  9. Intelligent Interfaces for Mining Large-Scale RNAi-HCS Image Databases

    PubMed Central

    Lin, Chen; Mak, Wayne; Hong, Pengyu; Sepp, Katharine; Perrimon, Norbert

    2010-01-01

    Recently, High-content screening (HCS) has been combined with RNA interference (RNAi) to become an essential image-based high-throughput method for studying genes and biological networks through RNAi-induced cellular phenotype analyses. However, a genome-wide RNAi-HCS screen typically generates tens of thousands of images, most of which remain uncategorized due to the inadequacies of existing HCS image analysis tools. Until now, it still requires highly trained scientists to browse a prohibitively large RNAi-HCS image database and produce only a handful of qualitative results regarding cellular morphological phenotypes. For this reason we have developed intelligent interfaces to facilitate the application of the HCS technology in biomedical research. Our new interfaces empower biologists with computational power not only to effectively and efficiently explore large-scale RNAi-HCS image databases, but also to apply their knowledge and experience to interactive mining of cellular phenotypes using Content-Based Image Retrieval (CBIR) with Relevance Feedback (RF) techniques. PMID:21278820

  10. Gene mutation, quantitative mutagenesis, and mutagen screening in mammalian cells: study with the CHO/HGPRT system

    SciTech Connect

    Hsie, A.W.

    1980-01-01

    We have employed CHO cells to develop and define a set of stringent conditions for studying mutation induction to TG resistance. Several lines of evidence support the CHO/HGPRT system as a specific-locus mutational assay. The system permits quantification of mutation at the HGPRT locus induced by various physical and chemical mutagens. The quantitative nature of the system provides a basis for the study of structure-function relationships of various classes of chemical mutagens. The intra- and interlaboratory reproducibility of this system suggests its potential for screening environmental agents for mutagenic activity.

  11. Simultaneous quantitative determination, identification and qualitative screening of pesticides in fruits and vegetables using LC-Q-Orbitrap™-MS.

    PubMed

    Zomer, Paul; Mol, Hans G J

    2015-01-01

    A method based on QuEChERS extraction and LC-quadrupole-Orbitrap™ MS detection was established utilising an improved fully non-targeted way of data acquisition with and without fragmentation. A full-scan acquisition event without fragmentation (resolving power 70,000) was followed by five consecutive fragmentation events (variable data independent acquisition - vDIA; resolving power 35,000) where all ions from the full-scan range are fragmented. Compared with fragmentation in a single event (all-ion fragmentation - AIF), this improves both selectivity and sensitivity for the fragment ions, which is beneficial for screening performance and identification capability. The method was validated, using the data from the same measurements, for two types of analysis: quantitation/identification and qualitative screening. The quantitative validation, performed according to the guidelines in SANCO/12571/2013, tested the performance of the method for 184 compounds in lettuce and orange at two spiking levels: 10 and 50 ng g(-1). The validation showed that the vast majority of the compounds met the criteria for trueness and precision set in the SANCO guidance document. In the qualitative validation the same 184 compounds were used to test the untargeted screening capabilities of the method. In this validation the compounds were spiked at three levels into 11 different fruit and vegetable matrices, which were measured twice on separate days. Taking all data from the qualitative validation together, an overall detection rate of 92% was achieved at the 10 ng g(-1) level, increasing to 98% at 200 ng g(-1). A screening detection limit (as defined in the SANCO guidelines) of 10 ng g(-1) could be achieved for 134 compounds. For 39 and two pesticides the SDL was 50 and 200 ng g(-1), respectively. For the other nine compounds no SDL could be established. The identification (ion ratio) criteria as recommended in the SANCO document could be met for 93% of the detected pesticide

  12. Quantitative real-time PCR eliminates false-positives in colony screening PCR.

    PubMed

    Skarratt, Kristen K; Fuller, Stephen J

    2014-01-01

    We report an alternative approach to colony screening using real-time PCR (qPCR) which can be used instead of the traditional end-point PCR to eliminate false-positives and reduce processing times. False-positive transformants can easily be distinguished from true-positives by comparing Ct values derived from qPCR amplification curves. In addition, the use of qPCR allows for more efficient processing since a gel electrophoresis step is not required and the screening process is no longer limited by the capacity of the gel apparatus.

  13. Nuclear organisation and RNAi in fission yeast.

    PubMed

    Woolcock, Katrina J; Bühler, Marc

    2013-06-01

    Over the last decade, the fission yeast Schizosaccharomyces pombe has been used extensively for investigating RNA interference (RNAi)-mediated heterochromatin assembly. However, only recently have studies begun to shed light on the 3D organisation of chromatin and the RNAi machinery in the fission yeast nucleus. These studies indicate association of repressive and active chromatin with different regions of the nuclear periphery, similar to other model organisms, and clustering of functionally related genomic features. Unexpectedly, RNAi factors were shown to associate with nuclear pores and were implicated in the regulation of genomic features outside of the well-studied heterochromatic regions. Nuclear organisation is likely to contribute to substrate specificity of the RNAi pathway. However, further studies are required to elucidate the exact mechanisms and functional importance of this nuclear organisation.

  14. Progress in RNAi-based antiviral therapeutics.

    PubMed

    Zhou, Jiehua; Rossi, John J

    2011-01-01

    RNA interference (RNAi) refers to the conserved sequence-specific degradation of message RNA mediated by small interfering (si)RNA duplexes 21-25 nucleotides in length. Given the ability to specifically silence any gene of interest, siRNAs offers several advantages over conventional drugs as potential therapeutic agents for the treatment of human maladies including cancers, genetic disorders, and infectious diseases. Antiviral RNAi strategies have received much attention and several compounds are currently being tested in clinical trials. In particular, the development of siRNA-based HIV (human immunodeficiency virus) therapeutics has progressed rapidly and many recent studies have shown that the use of RNAi could inhibit HIV-1 replication by targeting a number of viral or cellular genes. Therefore, the present chapter mainly focuses on the recent progress of RNAi-based anti-HIV gene therapeutics, with particular attention to molecular targets and delivery strategies of the siRNAs.

  15. RNAi-mediated plant protection against aphids.

    PubMed

    Yu, Xiu-Dao; Liu, Zong-Cai; Huang, Si-Liang; Chen, Zhi-Qin; Sun, Yong-Wei; Duan, Peng-Fei; Ma, You-Zhi; Xia, Lan-Qin

    2016-06-01

    Aphids (Aphididae) are major agricultural pests that cause significant yield losses of crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring harmful plant viruses. Expression of double-stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through plant-mediated RNA interference (RNAi). Thus, as a potential alternative and effective strategy for insect pest management in agricultural practice, plant-mediated RNAi for aphid control has received close attention in recent years. In this review, the mechanism of RNAi in insects and the so far explored effective RNAi target genes in aphids, their potential applications in the development of transgenic plants for aphid control and the major challenges in this regard are reviewed, and the future prospects of using plant-mediated RNAi for aphid control are discussed. This review is intended to be a helpful insight into the generation of aphid-resistant plants through plant-mediated RNAi strategy. © 2016 Society of Chemical Industry.

  16. Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    PubMed

    Takabatake, Reona; Koiwa, Tomohiro; Kasahara, Masaki; Takashima, Kaori; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Oguchi, Taichi; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

    2011-01-01

    To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

  17. A Quantitative Toxicogenomics Assay for High-throughput and Mechanistic Genotoxicity Assessment and Screening of Environmental Pollutants.

    PubMed

    Lan, Jiaqi; Gou, Na; Rahman, Sheikh Mokhles; Gao, Ce; He, Miao; Gu, April Z

    2016-03-15

    The ecological and health concern of mutagenicity and carcinogenicity potentially associated with an overwhelmingly large and ever-increasing number of chemicals demands for cost-effective and feasible method for genotoxicity screening and risk assessment. This study proposed a genotoxicity assay using GFP-tagged yeast reporter strains, covering 38 selected protein biomarkers indicative of all the seven known DNA damage repair pathways. The assay was applied to assess four model genotoxic chemicals, eight environmental pollutants and four negative controls across six concentrations. Quantitative molecular genotoxicity end points were derived based on dose response modeling of a newly developed integrated molecular effect quantifier, Protein Effect Level Index (PELI). The molecular genotoxicity end points were consistent with multiple conventional in vitro genotoxicity assays, as well as with in vivo carcinogenicity assay results. Further more, the proposed genotoxicity end point PELI values quantitatively correlated with both comet assay in human cell and carcinogenicity potency assay in mice, providing promising evidence for linking the molecular disturbance measurements to adverse outcomes at a biological relevant level. In addition, the high-resolution DNA damaging repair pathway alternated protein expression profiles allowed for chemical clustering and classification. This toxicogenomics-based assay presents a promising alternative for fast, efficient and mechanistic genotoxicity screening and assessment of drugs, foods, and environmental contaminants.

  18. A Shorter Route to Antibody Binders via Quantitative in vitro Bead-Display Screening and Consensus Analysis

    PubMed Central

    Mankowska, Sylwia A.; Gatti-Lafranconi, Pietro; Chodorge, Matthieu; Sridharan, Sudharsan; Minter, Ralph R.; Hollfelder, Florian

    2016-01-01

    Affinity panning of large libraries is a powerful tool to identify protein binders. However, panning rounds are followed by the tedious re-screening of the clones obtained to evaluate binders precisely. In a first application of Bead Surface Display (BeSD) we show successful in vitro affinity selections based on flow cytometric analysis that allows fine quantitative discrimination between binders. Subsequent consensus analysis of the resulting sequences enables identification of clones that bind tighter than those arising directly from the experimental selection output. This is demonstrated by evolution of an anti-Fas receptor single-chain variable fragment (scFv) that was improved 98-fold vs the parental clone. Four rounds of quantitative screening by fluorescence-activated cell sorting of an error-prone library based on fine discrimination between binders in BeSD were followed by analysis of 200 full-length output sequences that suggested a new consensus design with a Kd ∼140 pM. This approach shortens the time and effort to obtain high affinity reagents and its cell-free nature transcends limitations inherent in previous in vivo display systems. PMID:27819305

  19. A qualitative screening and quantitative measurement of organic contaminants on different types of marine plastic debris.

    PubMed

    Gauquie, Johanna; Devriese, Lisa; Robbens, Johan; De Witte, Bavo

    2015-11-01

    Chemical compounds present on plastic were characterised on different types of plastic litter and beached pellets, using a general GC-MS screening method. A variety of plastic related compounds, such as building blocks, antioxidants, additives and degradation products, were identified next to diverse environmental pollutants and biofilm compounds. A validated method for the analysis of PAHs and PCBs on beached pellets at the Belgian Coast, showed concentrations of ∑ 16 EPA-PAHs of 1076-3007 ng g(-1) plastic, while the concentrations of ∑ 7 OSPAR-PCBs ranged from 31 to 236 ng g(-1) plastic. The wide variety of plastic compounds retrieved in the general screening showed the importance of plastic as a potential source of contaminants and their degradation products.

  20. Quantitative assessment of smoking-induced emphysema progression in longitudinal CT screening for lung cancer

    NASA Astrophysics Data System (ADS)

    Suzuki, H.; Mizuguchi, R.; Matsuhiro, M.; Kawata, Y.; Niki, N.; Nakano, Y.; Ohmatsu, H.; Kusumoto, M.; Tsuchida, T.; Eguchi, K.; Kaneko, M.; Moriyama, N.

    2015-03-01

    Computed tomography has been used for assessing structural abnormalities associated with emphysema. It is important to develop a robust CT based imaging biomarker that would allow quantification of emphysema progression in early stage. This paper presents effect of smoking on emphysema progression using annual changes of low attenuation volume (LAV) by each lung lobe acquired from low-dose CT images in longitudinal screening for lung cancer. The percentage of LAV (LAV%) was measured after applying CT value threshold method and small noise reduction. Progression of emphysema was assessed by statistical analysis of the annual changes represented by linear regression of LAV%. This method was applied to 215 participants in lung cancer CT screening for five years (18 nonsmokers, 85 past smokers, and 112 current smokers). The results showed that LAV% is useful to classify current smokers with rapid progression of emphysema (0.2%/year, p<0.05). This paper demonstrates effectiveness of the proposed method in diagnosis and prognosis of early emphysema in CT screening for lung cancer.

  1. Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease

    PubMed Central

    Zheng, Wei; Padia, Janak; Urban, Daniel J.; Jadhav, Ajit; Goker-Alpan, Ozlem; Simeonov, Anton; Goldin, Ehud; Auld, Douglas; LaMarca, Mary E.; Inglese, James; Austin, Christopher P.; Sidransky, Ellen

    2007-01-01

    Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene. Missense mutations result in reduced enzyme activity that may be due to misfolding, raising the possibility of small-molecule chaperone correction of the defect. Screening large compound libraries by quantitative high-throughput screening (qHTS) provides comprehensive information on the potency, efficacy, and structure–activity relationships (SAR) of active compounds directly from the primary screen, facilitating identification of leads for medicinal chemistry optimization. We used qHTS to rapidly identify three structural series of potent, selective, nonsugar glucocerebrosidase inhibitors. The three structural classes had excellent potencies and efficacies and, importantly, high selectivity against closely related hydrolases. Preliminary SAR data were used to select compounds with high activity in both enzyme and cell-based assays. Compounds from two of these structural series increased N370S mutant glucocerebrosidase activity by 40–90% in patient cell lines and enhanced lysosomal colocalization, indicating chaperone activity. These small molecules have potential as leads for chaperone therapy for Gaucher disease, and this paradigm promises to accelerate the development of leads for other rare genetic disorders. PMID:17670938

  2. Quantitative High Throughput Screening Using a Live Cell cAMP Assay Identifies Small Molecule Agonists of the TSH Receptor

    PubMed Central

    Titus, Steve; Neumann, Susanne; Zheng, Wei; Southall, Noel; Michael, Sam; Klumpp, Carleen; Yasgar, Adam; Shinn, Paul; Thomas, Craig J.; Inglese, Jim; Gershengorn, Marvin C.; Austin, Christopher P.

    2009-01-01

    The thyroid stimulating hormone receptor (TSHR) belongs to the glycoprotein hormone receptor subfamily of seven-transmembrane spanning receptors. TSHR is expressed in thyroid follicular cells and is activated by TSH, which regulates growth and function of these cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small molecule agonist of the TSHR is available. To screen for novel TSHR agonists, we miniaturized a cell-based cAMP assay into 1536-well plate format. This assay uses a HEK293 cell line stably expressing the TSHR and a cyclic nucleotide gated ion channel (CNG), which functions as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal HTRF cAMP-based assay. 49 compounds in several structural classes have been confirmed as small molecule TSHR agonists that will serve as starting compounds for chemical optimization and studies of thyroid physiology in health and disease. PMID:18216391

  3. Development of RNAi Methods for Peregrinus maidis, the Corn Planthopper

    PubMed Central

    Yao, Jianxiu; Rotenberg, Dorith; Afsharifar, Alireza; Barandoc-Alviar, Karen; Whitfield, Anna E.

    2013-01-01

    The corn planthopper, Peregrinus maidis, is a major pest of agronomically-important crops. Peregrinus maidis has a large geographical distribution and transmits Maize mosaic rhabdovirus (MMV) and Maize stripe tenuivirus (MSpV). The objective of this study was to develop effective RNAi methods for P. maidis. Vacuolar-ATPase (V-ATPase) is an essential enzyme for hydrolysis of ATP and for transport of protons out of cells thereby maintaining membrane ion balance, and it has been demonstrated to be an efficacious target for RNAi in other insects. In this study, two genes encoding subunits of P. maidis V-ATPase (V-ATPase B and V-ATPase D) were chosen as RNAi target genes. The open reading frames of V-ATPase B and D were generated and used for constructing dsRNA fragments. Experiments were conducted using oral delivery and microinjection of V-ATPase B and V-ATPase D dsRNA to investigate the effectiveness of RNAi in P. maidis. Real-time quantitative reverse transcriptase-PCR (qRT-PCR) analysis indicated that microinjection of V-ATPase dsRNA led to a minimum reduction of 27-fold in the normalized abundance of V-ATPase transcripts two days post injection, while ingestion of dsRNA resulted in a two-fold reduction after six days of feeding. While both methods of dsRNA delivery resulted in knockdown of target transcripts, the injection method was more rapid and effective. The reduction in V-ATPase transcript abundance resulted in observable phenotypes. Specifically, the development of nymphs injected with 200 ng of either V-ATPase B or D dsRNA was impaired, resulting in higher mortality and lower fecundity than control insects injected with GFP dsRNA. Microscopic examination of these insects revealed that female reproductive organs did not develop normally. The successful development of RNAi in P. maidis to target specific genes will enable the development of new insect control strategies and functional analysis of vital genes and genes associated with interactions between P

  4. Development of RNAi methods for Peregrinus maidis, the corn planthopper.

    PubMed

    Yao, Jianxiu; Rotenberg, Dorith; Afsharifar, Alireza; Barandoc-Alviar, Karen; Whitfield, Anna E

    2013-01-01

    The corn planthopper, Peregrinus maidis, is a major pest of agronomically-important crops. Peregrinus maidis has a large geographical distribution and transmits Maize mosaic rhabdovirus (MMV) and Maize stripe tenuivirus (MSpV). The objective of this study was to develop effective RNAi methods for P. maidis. Vacuolar-ATPase (V-ATPase) is an essential enzyme for hydrolysis of ATP and for transport of protons out of cells thereby maintaining membrane ion balance, and it has been demonstrated to be an efficacious target for RNAi in other insects. In this study, two genes encoding subunits of P. maidis V-ATPase (V-ATPase B and V-ATPase D) were chosen as RNAi target genes. The open reading frames of V-ATPase B and D were generated and used for constructing dsRNA fragments. Experiments were conducted using oral delivery and microinjection of V-ATPase B and V-ATPase D dsRNA to investigate the effectiveness of RNAi in P. maidis. Real-time quantitative reverse transcriptase-PCR (qRT-PCR) analysis indicated that microinjection of V-ATPase dsRNA led to a minimum reduction of 27-fold in the normalized abundance of V-ATPase transcripts two days post injection, while ingestion of dsRNA resulted in a two-fold reduction after six days of feeding. While both methods of dsRNA delivery resulted in knockdown of target transcripts, the injection method was more rapid and effective. The reduction in V-ATPase transcript abundance resulted in observable phenotypes. Specifically, the development of nymphs injected with 200 ng of either V-ATPase B or D dsRNA was impaired, resulting in higher mortality and lower fecundity than control insects injected with GFP dsRNA. Microscopic examination of these insects revealed that female reproductive organs did not develop normally. The successful development of RNAi in P. maidis to target specific genes will enable the development of new insect control strategies and functional analysis of vital genes and genes associated with interactions between P

  5. Preliminary Phytochemical Screening, Quantitative Analysis of Alkaloids, and Antioxidant Activity of Crude Plant Extracts from Ephedra intermedia Indigenous to Balochistan.

    PubMed

    Gul, Rahman; Jan, Syed Umer; Faridullah, Syed; Sherani, Samiullah; Jahan, Nusrat

    2017-01-01

    The aim of this study was to evaluate the antioxidant activity, screening the phytogenic chemical compounds, and to assess the alkaloids present in the E. intermedia to prove its uses in Pakistani folk medicines for the treatment of asthma and bronchitis. Antioxidant activity was analyzed by using 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay. Standard methods were used for the identification of cardiac glycosides, phenolic compounds, flavonoids, anthraquinones, and alkaloids. High performance liquid chromatography (HPLC) was used for quantitative purpose of ephedrine alkaloids in E. intermedia. The quantitative separation was confirmed on Shimadzu 10AVP column (Shampack) of internal diameter (id) 3.0 mm and 50 mm in length. The extract of the solute in flow rate of 1 ml/min at the wavelength 210 nm and methanolic extract showed the antioxidant activity and powerful oxygen free radicals scavenging activities and the IC50 for the E. intermedia plant was near to the reference standard ascorbic acid. The HPLC method was useful for the quantitative purpose of ephedrine (E) and pseudoephedrine (PE) used for 45 samples of one species collected from central habitat in three districts (Ziarat, Shairani, and Kalat) of Balochistan. Results showed that average alkaloid substance in E. intermedia was as follows: PE (0.209%, 0.238%, and 0.22%) and E (0.0538%, 0.0666%, and 0.0514%).

  6. Development, validation and quantitative assessment of an enzymatic assay suitable for small molecule screening and profiling: A case-study.

    PubMed

    Sancenon, Vicente; Goh, Wei Hau; Sundaram, Aishwarya; Er, Kai Shih; Johal, Nidhi; Mukhina, Svetlana; Carr, Grant; Dhakshinamoorthy, Saravanakumar

    2015-06-01

    The successful discovery and subsequent development of small molecule inhibitors of drug targets relies on the establishment of robust, cost-effective, quantitative, and physiologically relevant in vitro assays that can support prolonged screening and optimization campaigns. The current study illustrates the process of developing and validating an enzymatic assay for the discovery of small molecule inhibitors using alkaline phosphatase from bovine intestine as model target. The assay development workflow includes an initial phase of optimization of assay materials, reagents, and conditions, continues with a process of miniaturization and automation, and concludes with validation by quantitative measurement of assay performance and signal variability. The assay is further evaluated for dose-response and mechanism-of-action studies required to support structure-activity-relationship studies. Emphasis is placed on the most critical aspects of assay optimization and other relevant considerations, including the technology, assay materials, buffer constituents, reaction conditions, liquid handling equipment, analytical instrumentation, and quantitative assessments. Examples of bottlenecks encountered during assay development and strategies to address them are provided.

  7. Preliminary Phytochemical Screening, Quantitative Analysis of Alkaloids, and Antioxidant Activity of Crude Plant Extracts from Ephedra intermedia Indigenous to Balochistan

    PubMed Central

    Jan, Syed Umer; Faridullah, Syed; Sherani, Samiullah; Jahan, Nusrat

    2017-01-01

    The aim of this study was to evaluate the antioxidant activity, screening the phytogenic chemical compounds, and to assess the alkaloids present in the E. intermedia to prove its uses in Pakistani folk medicines for the treatment of asthma and bronchitis. Antioxidant activity was analyzed by using 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay. Standard methods were used for the identification of cardiac glycosides, phenolic compounds, flavonoids, anthraquinones, and alkaloids. High performance liquid chromatography (HPLC) was used for quantitative purpose of ephedrine alkaloids in E. intermedia. The quantitative separation was confirmed on Shimadzu 10AVP column (Shampack) of internal diameter (id) 3.0 mm and 50 mm in length. The extract of the solute in flow rate of 1 ml/min at the wavelength 210 nm and methanolic extract showed the antioxidant activity and powerful oxygen free radicals scavenging activities and the IC50 for the E. intermedia plant was near to the reference standard ascorbic acid. The HPLC method was useful for the quantitative purpose of ephedrine (E) and pseudoephedrine (PE) used for 45 samples of one species collected from central habitat in three districts (Ziarat, Shairani, and Kalat) of Balochistan. Results showed that average alkaloid substance in E. intermedia was as follows: PE (0.209%, 0.238%, and 0.22%) and E (0.0538%, 0.0666%, and 0.0514%). PMID:28386582

  8. Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies.

    PubMed

    Capalbo, Antonio; Treff, Nathan R; Cimadomo, Danilo; Tao, Xin; Upham, Kathleen; Ubaldi, Filippo Maria; Rienzi, Laura; Scott, Richard T

    2015-07-01

    Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6%; P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS.

  9. Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries

    PubMed Central

    Inglese, James; Auld, Douglas S.; Jadhav, Ajit; Johnson, Ronald L.; Simeonov, Anton; Yasgar, Adam; Zheng, Wei; Austin, Christopher P.

    2006-01-01

    High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration–response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration–response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure–activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>105 compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery. PMID:16864780

  10. Quantitative High-Throughput Screening Identifies 8-Hydroxyquinolines as Cell-Active Histone Demethylase Inhibitors

    PubMed Central

    Kawamura, Akane; Rose, Nathan R.; Ng, Stanley S.; Quinn, Amy M.; Rai, Ganesha; Mott, Bryan T.; Beswick, Paul; Klose, Robert J.; Oppermann, Udo; Jadhav, Ajit; Heightman, Tom D.; Maloney, David J.; Schofield, Christopher J.; Simeonov, Anton

    2010-01-01

    Background Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. Nε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. Nε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. Principal Findings High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. Conclusions These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation. PMID:21124847

  11. Feasibility, limitation and possible solutions of RNAi-based technology for insect pest control.

    PubMed

    Zhang, Hao; Li, Hai-Chao; Miao, Xue-Xia

    2013-02-01

    Numerous studies indicate that target gene silencing by RNA interference (RNAi) could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi-mediated crop protection. However, there are many limitations using RNAi-based technology for pest control, with the effectiveness target gene selection and reliable double-strand RNA (dsRNA) delivery being two of the major challenges. With respect to target gene selection, at present, the use of homologous genes and genome-scale high-throughput screening are the main strategies adopted by researchers. Once the target gene is identified, dsRNA can be delivered by micro-injection or by feeding as a dietary component. However, micro-injection, which is the most common method, can only be used in laboratory experiments. Expression of dsRNAs directed against insect genes in transgenic plants and spraying dsRNA reagents have been shown to induce RNAi effects on target insects. Hence, RNAi-mediated crop protection has been considered as a potential new-generation technology for pest control, or as a complementary method of existing pest control strategies; however, further development to improve the efficacy of protection and range of species affected is necessary. In this review, we have summarized current research on RNAi-based technology for pest insect management. Current progress has proven that RNAi technology has the potential to be a tool for designing a new generation of insect control measures. To accelerate its practical application in crop protection, further study on dsRNA uptake mechanisms based on the knowledge of insect physiology and biochemistry is needed.

  12. Application of RNAi to Genomic Drug Target Validation in Schistosomes

    PubMed Central

    Guidi, Alessandra; Mansour, Nuha R.; Paveley, Ross A.; Carruthers, Ian M.; Besnard, Jérémy; Hopkins, Andrew L.; Gilbert, Ian H.; Bickle, Quentin D.

    2015-01-01

    Concerns over the possibility of resistance developing to praziquantel (PZQ), has stimulated efforts to develop new drugs for schistosomiasis. In addition to the development of improved whole organism screens, the success of RNA interference (RNAi) in schistosomes offers great promise for the identification of potential drug targets to initiate drug discovery. In this study we set out to contribute to RNAi based validation of putative drug targets. Initially a list of 24 target candidates was compiled based on the identification of putative essential genes in schistosomes orthologous of C. elegans essential genes. Knockdown of Calmodulin (Smp_026560.2) (Sm-Calm), that topped this list, produced a phenotype characterised by waves of contraction in adult worms but no phenotype in schistosomula. Knockdown of the atypical Protein Kinase C (Smp_096310) (Sm-aPKC) resulted in loss of viability in both schistosomula and adults and led us to focus our attention on other kinase genes that were identified in the above list and through whole organism screening of known kinase inhibitor sets followed by chemogenomic evaluation. RNAi knockdown of these kinase genes failed to affect adult worm viability but, like Sm-aPKC, knockdown of Polo-like kinase 1, Sm-PLK1 (Smp_009600) and p38-MAPK, Sm-MAPK p38 (Smp_133020) resulted in an increased mortality of schistosomula after 2-3 weeks, an effect more marked in the presence of human red blood cells (hRBC). For Sm-PLK-1 the same effects were seen with the specific inhibitor, BI2536, which also affected viable egg production in adult worms. For Sm-PLK-1 and Sm-aPKC the in vitro effects were reflected in lower recoveries in vivo. We conclude that the use of RNAi combined with culture with hRBC is a reliable method for evaluating genes important for larval development. However, in view of the slow manifestation of the effects of Sm-aPKC knockdown in adults and the lack of effects of Sm-PLK-1 and Sm-MAPK p38 on adult viability, these

  13. Performance comparison of quantitative semantic features and lung-RADS in the National Lung Screening Trial

    NASA Astrophysics Data System (ADS)

    Li, Qian; Balagurunathan, Yoganand; Liu, Ying; Schabath, Matthew; Gillies, Robert J.

    2016-03-01

    Background: Lung-RADS is the new oncology classification guideline proposed by American College of Radiology (ACR), which provides recommendation for further follow up in lung cancer screening. However, only two features (solidity and size) are included in this system. We hypothesize that additional sematic features can be used to better characterize lung nodules and diagnose cancer. Objective: We propose to develop and characterize a systematic methodology based on semantic image traits to more accurately predict occurrence of cancerous nodules. Methods: 24 radiological image traits were systematically scored on a point scale (up to 5) by a trained radiologist, and lung-RADS was independently scored. A linear discriminant model was used on the semantic features to access their performance in predicting cancer status. The semantic predictors were then compared to lung-RADS classification in 199 patients (60 cancers, 139 normal controls) obtained from the National Lung Screening Trial. Result: There were different combinations of semantic features that were strong predictors of cancer status. Of these, contour, border definition, size, solidity, focal emphysema, focal fibrosis and location emerged as top candidates. The performance of two semantic features (short axial diameter and contour) had an AUC of 0.945, and was comparable to that of lung-RADS (AUC: 0.871). Conclusion: We propose that a semantics-based discrimination approach may act as a complement to the lung-RADS to predict cancer status.

  14. High-throughput RNAi screen in Ewing sarcoma cells identifies leucine rich repeats and WD repeat domain containing 1 (LRWD1) as a regulator of EWS-FLI1 driven cell viability.

    PubMed

    He, Tao; Surdez, Didier; Rantala, Juha K; Haapa-Paananen, Saija; Ban, Jozef; Kauer, Maximilian; Tomazou, Eleni; Fey, Vidal; Alonso, Javier; Kovar, Heinrich; Delattre, Olivier; Iljin, Kristiina

    2017-01-05

    A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.

  15. Identifying targets for topical RNAi therapeutics in psoriasis: assessment of a new in vitro psoriasis model.

    PubMed

    Bracke, S; Desmet, E; Guerrero-Aspizua, S; Tjabringa, S G; Schalkwijk, J; Van Gele, M; Carretero, M; Lambert, J

    2013-08-01

    Diseases of the skin are amenable to RNAi-based therapies and targeting key components in the pathophysiology of psoriasis using RNAi may represent a successful new therapeutic strategy. We aimed to develop a straightforward and highly reproducible in vitro psoriasis model useful to study the effects of gene knockdown by RNAi and to identify new targets for topical RNAi therapeutics. We evaluated the use of keratinocytes derived from psoriatic plaques and normal human keratinocytes (NHKs). To induce a psoriatic phenotype in NHKs, combinations of pro-inflammatory cytokines (IL-1α, IL-17A, IL-6 and TNF-α) were tested. The model based on NHK met our needs of a reliable and predictive preclinical model, and this model was further selected for gene expression analyses, comprising a panel of 55 psoriasis-associated genes and five micro-RNAs (miRNAs). Gene silencing studies were conducted by using small interfering RNAs (siRNAs) and miRNA inhibitors directed against potential target genes such as CAMP and DEFB4 and miRNAs such as miR-203. We describe a robust and highly reproducible in vitro psoriasis model that recapitulates expression of a large panel of genes and miRNAs relevant to the pathogenesis of psoriasis. Furthermore, we show that our model is a powerful first step model system for testing and screening RNAi-based therapeutics.

  16. Quantitative low-energy ion beam characterization by beam profiling and imaging via scintillation screens

    NASA Astrophysics Data System (ADS)

    Germer, S.; Pietag, F.; Polak, J.; Arnold, T.

    2016-11-01

    This study presents the imaging and characterization of low-current ion beams in the neutralized state monitored via single crystal YAG:Ce (Y3Al5O12) scintillators. To validate the presented beam diagnostic tool, Faraday cup measurements and test etchings were performed. Argon ions with a typical energy of 1.0 keV were emitted from an inductively coupled radio-frequency (13.56 MHz) ion beam source with total currents of some mA. Different beam properties, such as, lateral ion current density, beam divergence angle, and current density in pulsed ion beams have been studied to obtain information about the spatial beam profile and the material removal rate distribution. We observed excellent imaging properties with the scintillation screen and achieved a detailed characterization of the neutralized ion beam. A strong correlation between the scintillator light output, the ion current density, and the material removal rate could be observed.

  17. Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens*

    PubMed Central

    Dengjel, Jörn; Høyer-Hansen, Maria; Nielsen, Maria O.; Eisenberg, Tobias; Harder, Lea M.; Schandorff, Søren; Farkas, Thomas; Kirkegaard, Thomas; Becker, Andrea C.; Schroeder, Sabrina; Vanselow, Katja; Lundberg, Emma; Nielsen, Mogens M.; Kristensen, Anders R.; Akimov, Vyacheslav; Bunkenborg, Jakob; Madeo, Frank; Jäättelä, Marja; Andersen, Jens S.

    2012-01-01

    Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection. PMID:22311637

  18. Odorant Screening and Quantitation of Thiols in Carmenere Red Wine by Gas Chromatography-Olfactometry and Stable Isotope Dilution Assays.

    PubMed

    Pavez, Carolina; Agosin, Eduardo; Steinhaus, Martin

    2016-05-04

    The sensory impact of thiols in Vitis vinifera 'Carmenere' red wines was evaluated. For this purpose, aroma extract dilution analysis was applied to the thiols isolated from a Carmenere red wine by affinity chromatography with a mercurated agarose gel. Results revealed the presence of four odorants, identified as 2-furanylmethanethiol, 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, and 2-methyl-3-sulfanyl-1-butanol, with the latter being described here for the first time in Carmenere red wines. Quantitation of the four thiols in the Carmenere wine screened by aroma extract dilution analysis and in three additional Carmenere wines by stable isotope dilution assays resulted in concentrations above the respective orthonasal odor detection threshold values. Triangle tests applied to wine model solutions with and without the addition of the four thiols showed significant differences, thus suggesting that the compounds do have the potential to influence the overall aroma of red wine.

  19. Toxin screening in phytoplankton: detection and quantitation using MALDI triple quadrupole mass spectrometry.

    PubMed

    Sleno, Lekha; Volmer, Dietrich A

    2005-03-01

    The investigation of a MALDI triple quadrupole instrument for the analysis of spirolide toxins in phytoplankton samples is described in this study. A high-frequency (kHz) laser was employed for MALDI, generating a semicontinuous ion beam, thus taking advantage of the high duty cycle obtained in sensitive triple quadrupole MRM experiments. Initially, several experimental parameters such as type of organic matrix and concentration, solvent composition, and matrix-to-analyte ratio were optimized, and their impact on sensitivity and precision of the obtained ion currents for a reference spirolide, 13-desmethyl-C, was studied. In all quantitative experiments, excellent linearities in the concentration range between 0.01 and 1.75 microg/mL were obtained, with R2 values of 0.99 or higher. The average precision of the quantitative MALDI measurements was 7.4+/-2.4% RSD. No systematic errors were apparent with this method as shown by a direct comparison to an electrospray LC/MS/MS method. Most importantly, the MALDI technique was very fast; each sample spot was analyzed in less than 5 s as compared to several minutes with the electrospray assay. To demonstrate the potential of the MALDI triple quadrupole method, its application to quantitative analysis in several different phytoplankton samples was investigated, including crude extracts and samples from mass-triggered fractionation experiments. 13-Desmethyl spirolide C was successfully quantified in these complex samples at concentration levels from 0.05 to 90.4 microg/mL (prior to dilution to have samples fall within the dynamic range of the method) without extensive sample preparation steps. The versatility of the MALDI triple quadrupole method was also exhibited for the identification of unknown spirolide analogues. Through the use of dedicated linked scan functions such as precursor ion and neutral loss scans, several spirolide compounds were tentatively identified directly from the crude extract, without the usual time

  20. Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis).

    PubMed

    Zhao, Chaoyang; Alvarez Gonzales, Miguel A; Poland, Therese M; Mittapalli, Omprakash

    2015-01-01

    The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong RNAi machinery that is capable of degrading target mRNA as a response to exogenous double-stranded RNA (dsRNA) induction, we identified three RNAi pathway core component genes, Dicer-2, Argonaute-2 and R2D2, from the A. planipennis genome sequence. Characterization of these core components revealed that they contain conserved domains essential for the proteins to function in the RNAi pathway. Phylogenetic analyses showed that they are closely related to homologs derived from other coleopteran species. We also delivered the dsRNA fragment of AplaScrB-2, a β-fructofuranosidase-encoding gene horizontally acquired by A. planipennis as we reported previously, into A. planipennis adults through microinjection. Quantitative real-time PCR analysis on the dsRNA-treated beetles demonstrated a significantly decreased gene expression level of AplaScrB-2 appearing on day 2 and lasting until at least day 6. This study is the first record of RNAi applied in A. planipennis.

  1. Rapid and Inexpensive Screening of Genomic Copy Number Variations Using a Novel Quantitative Fluorescent PCR Method

    PubMed Central

    Han, Joan C.; Elsea, Sarah H.; Pena, Heloísa B.; Pena, Sérgio Danilo Junho

    2013-01-01

    Detection of human microdeletion and microduplication syndromes poses significant burden on public healthcare systems in developing countries. With genome-wide diagnostic assays frequently inaccessible, targeted low-cost PCR-based approaches are preferred. However, their reproducibility depends on equally efficient amplification using a number of target and control primers. To address this, the recently described technique called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR) was shown to reliably detect four human syndromes by quantifying DNA amplification in an internally controlled PCR reaction. Here, we confirm its utility in the detection of eight human microdeletion syndromes, including the more common WAGR, Smith-Magenis, and Potocki-Lupski syndromes with 100% sensitivity and 100% specificity. We present selection, design, and performance evaluation of detection primers using variety of approaches. We conclude that MQF-PCR is an easily adaptable method for detection of human pathological chromosomal aberrations. PMID:24288428

  2. Diabetes screening after gestational diabetes in England: a quantitative retrospective cohort study

    PubMed Central

    McGovern, Andrew; Butler, Lucilla; Jones, Simon; van Vlymen, Jeremy; Sadek, Khaled; Munro, Neil; Carr, Helen; de Lusignan, Simon

    2014-01-01

    Background The National Institute for Health and Care Excellence (NICE) recommends postpartum and annual monitoring for diabetes for females who have had a diagnosis of gestational diabetes mellitus (GDM). Aim To describe the current state of follow-up after GDM in primary care, in England. Design and setting A retrospective cohort study in 127 primary care practices. The total population analysed comprised 473 772 females, of whom 2016 had a diagnosis of GDM. Method Two subgroups of females were analysed using electronic general practice records. In the first group of females (n = 788) the quality of postpartum follow-up was assessed during a 6-month period. The quality of long-term annual follow-up was assessed in a second group of females (n = 718), over a 5-year period. The two outcome measures were blood glucose testing performed within 6 months postpartum (first group) and blood glucose testing performed annually (second group). Results Postpartum follow-up was performed in 146 (18.5%) females within 6 months of delivery. Annual rates of long-term follow-up stayed consistently around 20% a year. Publication of the Diabetes in Pregnancy NICE guidelines, in 2008, had no effect on long-term screening rates. Substantial regional differences were identified among rates of follow-up. Conclusion Monitoring of females after GDM is markedly suboptimal despite current recommendations. PMID:24567578

  3. Genome-Wide RNAi High-Throughput Screen Identifies Proteins Necessary for the AHR-Dependent Induction of CYP1A1 by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

    PubMed Central

    Hankinson, Oliver

    2013-01-01

    The aryl hydrocarbon receptor (AHR) has a plethora of physiological roles, and upon dysregulation, carcinogenesis can occur. One target gene of AHR encodes the xenobiotic and drug-metabolizing enzyme CYP1A1, which is inducible by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the AHR. An siRNA library targeted against over 5600 gene candidates in the druggable genome was used to transfect mouse Hepa-1 cells, which were then treated with TCDD, and subsequently assayed for CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) activity. Following redundant siRNA activity (RSA) statistical analysis, we identified 93 hits that reduced EROD activity with a p value ≤ .005 and substantiated 39 of these as positive hits in a secondary screening using endoribonuclease-prepared siRNAs (esiRNAs). Twelve of the corresponding gene products were subsequently confirmed to be necessary for the induction of CYP1A1 messenger RNA by TCDD. None of the candidates were deficient in aryl hydrocarbon nuclear translocator expression. However 6 gene products including UBE2i, RAB40C, CRYGD, DCTN4, RBM5, and RAD50 are required for the expression of AHR as well as for induction of CYP1A1. We also found 2 gene products, ARMC8 and TCF20, to be required for the induction of CYP1A1, but our data are ambiguous as to whether they are required for the expression of AHR. In contrast, SIN3A, PDC, TMEM5, and CD9 are not required for AHR expression but are required for the induction of CYP1A1, implicating a direct role in Cyp1a1 transcription. Our methods, although applied to Cyp1a1, could be modified for identifying proteins that regulate other inducible genes. PMID:23997114

  4. Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons

    PubMed Central

    Straccia, Marco; Garcia-Diaz Barriga, Gerardo; Sanders, Phil; Bombau, Georgina; Carrere, Jordi; Mairal, Pedro Belio; Vinh, Ngoc-Nga; Yung, Sun; Kelly, Claire M; Svendsen, Clive N; Kemp, Paul J; Arjomand, Jamshid; Schoenfeld, Ryan C; Alberch, Jordi; Allen, Nicholas D; Rosser, Anne E; Canals, Josep M

    2015-01-01

    A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening. PMID:26417608

  5. Screening for Suitable Reference Genes for Quantitative Real-Time PCR in Heterosigma akashiwo (Raphidophyceae).

    PubMed

    Ji, Nanjing; Li, Ling; Lin, Lingxiao; Lin, Senjie

    2015-01-01

    The raphidophyte Heterosigma akashiwo is a globally distributed harmful alga that has been associated with fish kills in coastal waters. To understand the mechanisms of H. akashiwo bloom formation, gene expression analysis is often required. To accurately characterize the expression levels of a gene of interest, proper reference genes are essential. In this study, we assessed ten of the previously reported algal candidate genes (rpL17-2, rpL23, cox2, cal, tua, tub, ef1, 18S, gapdh, and mdh) for their suitability as reference genes in this species. We used qRT-PCR to quantify the expression levels of these genes in H. akashiwo grown under different temperatures, light intensities, nutrient concentrations, and time points over a diel cycle. The expression stability of these genes was evaluated using geNorm and NormFinder algorithms. Although none of these genes exhibited invariable expression levels, cal, tub, rpL17-2 and rpL23 expression levels were the most stable across the different conditions tested. For further validation, these selected genes were used to normalize the expression levels of ribulose-1, 5-bisphosphate carboxylase/oxygenase large unite (HrbcL) over a diel cycle. Results showed that the expression of HrbcL normalized against each of these reference genes was the highest at midday and lowest at midnight, similar to the diel patterns typically documented for this gene in algae. While the validated reference genes will be useful for future gene expression studies on H. akashiwo, we expect that the procedure used in this study may be helpful to future efforts to screen reference genes for other algae.

  6. Automated, quantitative cognitive/behavioral screening of mice: for genetics, pharmacology, animal cognition and undergraduate instruction.

    PubMed

    Gallistel, C R; Balci, Fuat; Freestone, David; Kheifets, Aaron; King, Adam

    2014-02-26

    We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be

  7. Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity

    SciTech Connect

    Sirenko, Oksana; Cromwell, Evan F.; Crittenden, Carole; Wignall, Jessica A.; Wright, Fred A.; Rusyn, Ivan

    2013-12-15

    of cardiotoxicity is possible in a high-throughput format. • The assay shows benefits of automated data integration across multiple parameters. • Quantitative assessment of concentration–response is possible using iPSCs. • Multi-parametric screening allows for cardiotoxicity risk assessment.

  8. RNAi induced gene silencing in crop improvement.

    PubMed

    Sinha, Subodh Kumar

    2010-12-01

    The RNA silencing is one of the innovative and efficient molecular biology tools to harness the down-regulation of expression of gene(s) specifically. To accomplish such selective modification of gene expression of a particular trait, homology dependent gene silencing uses a stunning variety of gene silencing viz. co-suppression, post-transcriptional gene silencing, virus-induced gene silencing etc. This family of diverse molecular phenomena has a common exciting feature of gene silencing which is collectively called RNA interference abbreviated to as RNAi. This molecular phenomenon has become a focal point of plant biology and medical research throughout the world. As a result, this technology has turned out to be a powerful tool in understanding the function of individual gene and has ultimately led to the tremendous use in crop improvement. This review article illustrates the application of RNAi in a broad area of crop improvement where this technology has been successfully used. It also provides historical perspective of RNAi discovery and its contemporary phenomena, mechanism of RNAi pathway.

  9. Establishing RNAi in a Non-Model Organism: The Antarctic Nematode Panagrolaimus sp. DAW1

    PubMed Central

    Seybold, Anna C.; Wharton, David A.; Thorne, Michael A. S.

    2016-01-01

    The Antarctic nematode Panagrolaimus sp. DAW1 is one of the only organisms known to survive extensive intracellular freezing throughout its tissues. Although the physiological mechanisms of this extreme adaptation are partly understood, the molecular mechanisms remain largely unknown. RNAi is a method that allows the examination of gene function in a direct, targeted manner, by knocking out specific mRNAs and revealing the effects on the phenotype. In this study we have explored the viability of RNAi in Panagrolaimus sp. DAW1. In the first trial, nematodes were fed E. coli expressing Panagrolaimus sp. DAW1 dsRNA of the embryonic lethal genes rps-2 and dhc, and the blister gene duox. Pd-rps-2(RNAi)-treated nematodes showed a significant decrease in larval hatching. However, qPCR showed no significant decrease in the amount of rps-2 mRNA in Pd-rps-2(RNAi)-treated animals. Several soaking protocols for dsRNA uptake were investigated using the fluorescent dye FITC. Desiccation-enhanced soaking showed the strongest uptake of FITC and resulted in a significant and consistent decrease of mRNA levels of two of the four tested genes (rps-2 and tps-2a), suggesting effective uptake of dsRNA-containing solution by the nematode. These findings suggest that RNAi by desiccation-enhanced soaking is viable in Panagrolaimus sp. DAW1 and provide the first functional genomic approach to investigate freezing tolerance in this non-model organism. RNAi, in conjunction with qPCR, can be used to screen for candidate genes involved in intracellular freezing tolerance in Panagrolaimus sp. DAW1. PMID:27832164

  10. RNAi strategies to suppress insects of fruit and tree crops

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Use of ribonucleic acid interference, RNAi, to reduce plant feeding Hemiptera in fruit tree and grapevines. The successful use of RNAi strategies to reduce insect pests, psyllids and leafhoppers was demonstrated. An RNAi bioassay which absorbs dsRNA into plant tissues provided up to 40 days of act...

  11. Coupling desorption electrospray ionization with solid-phase microextraction for screening and quantitative analysis of drugs in urine.

    PubMed

    Kennedy, Joseph H; Aurand, Craig; Shirey, Robert; Laughlin, Brian C; Wiseman, Justin M

    2010-09-01

    Direct analysis of silica C(18)-coated solid-phase microextraction (SPME) fibers using desorption electrospray ionization mass spectrometry (DESI-MS) for the purpose of analyzing drugs from raw urine is presented. The method combines a simple, inexpensive, and solvent-less sample preparation technique with the specificity and speed of DESI-MS and MS/MS. Extraction of seven drugs from raw urine is performed using specially designed SPME fibers coated uniformly with silica-C(18) stationary phase. Each SPME device is inserted into unprocessed urine under gentle agitation and, then, removed, rinsed, and analyzed directly by DESI-MS (MS/MS). Rapid screening over a wide mass range is afforded by coupling the method with a time of flight (TOF) mass spectrometer while quantitative analysis is performed using selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer. The performance of the SPME DESI-MS/MS method was evaluated by preparing calibration standards and quality control (QC) samples of the seven drug compounds from urine over a range from 20 to 1000 ng/mL, with the exception of meprobamate which was prepared from 200 to 10000 ng/mL. The calibration curves constructed for each analyte had an R(2) > 0.99. The range of precision (%CV) and accuracy values (% bias) for low QC samples was 1-11% and 3-38%, respectively. Precision and accuracy values for high QC samples range from 0.9 to 8% and -31 to -8%. Results from urine specimens of actual exposure to drugs screened using the SPME DESI-MS/MS method showed good agreement with the conventional immunoassays and GC/MS analysis. Liquid desorption of the SPME fiber followed by LC/MS/MS also showed good agreement with the SPME DESI-MS/MS method.

  12. NeuronCyto II: An automatic and quantitative solution for crossover neural cells in high throughput screening

    PubMed Central

    Ong, Kok Haur; De, Jaydeep

    2016-01-01

    Abstract Microscopy is a fundamental technology driving new biological discoveries. Today microscopy allows a large number of images to be acquired using, for example, High Throughput Screening (HTS) and 4D imaging. It is essential to be able to interrogate these images and extract quantitative information in an automated fashion. In the context of neurobiology, it is important to automatically quantify the morphology of neurons in terms of neurite number, length, branching and complexity, etc. One major issue in quantification of neuronal morphology is the “crossover” problem where neurites cross and it is difficult to assign which neurite belongs to which cell body. In the present study, we provide a solution to the “crossover” problem, the software package NeuronCyto II. NeuronCyto II is an interactive and user‐friendly software package for automatic neurite quantification. It has a well‐designed graphical user interface (GUI) with only a few free parameters allowing users to optimize the software by themselves and extract relevant quantitative information routinely. Users are able to interact with the images and the numerical features through the Result Inspector. The processing of neurites without crossover was presented in our previous work. Our solution for the “crossover” problem is developed based on our recently published work with directed graph theory. Both methods are implemented in NeuronCyto II. The results show that our solution is able to significantly improve the reliability and accuracy of the neurons displaying “crossover.” NeuronCyto II is freely available at the website: https://sites.google.com/site/neuroncyto/ , which includes user support and where software upgrades will also be placed in the future. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC. PMID:27233092

  13. 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.

    PubMed

    Birmingham, Amanda; Anderson, Emily M; Reynolds, Angela; Ilsley-Tyree, Diane; Leake, Devin; Fedorov, Yuriy; Baskerville, Scott; Maksimova, Elena; Robinson, Kathryn; Karpilow, Jon; Marshall, William S; Khvorova, Anastasia

    2006-03-01

    Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3' untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.

  14. The Rapid and Sensitive Quantitative Determination of Galactose by Combined Enzymatic and Colorimetric Method: Application in Neonatal Screening.

    PubMed

    Kianmehr, Anvarsadat; Mahrooz, Abdolkarim; Ansari, Javad; Oladnabi, Morteza; Shahbazmohammadi, Hamid

    2016-05-01

    The quantitative measurement of galactose in blood is essential for the early diagnosis, treatment, and dietary monitoring of galactosemia patients. In this communication, we aimed to develop a rapid, sensitive, and cost-effective combined method for galactose determination in dry blood spots. This procedure was based on the combination of enzymatic reactions of galactose dehydrogenase (GalDH), dihydrolipoyl dehydrogenase (DLD), and alkaline phosphates with a colorimetric system. The incubation time and the concentration of enzymes used in new method were also optimized. The analytical performance was studied by the precision, recovery, linearity, and sensitivity parameters. Statistical analysis was applied to method comparison experiment. The regression equation and correlation coefficient (R (2)) were Y = 0.0085x + 0.032 and R (2) = 0.998, respectively. This assay exhibited a recovery in the range of 91.7-114.3 % and had the limit detection of 0.5 mg/dl for galactose. The between-run coefficient of variation (CV) was between 2.6 and 11.1 %. The within-run CV was between 4.9 and 9.2 %. Our results indicated that the new and reference methods were in agreement because no significant biases exist between them. Briefly, a quick and reliable combined enzymatic and colorimetric assay was presented for application in newborn mass screening and monitoring of galactosemia patients.

  15. Rapid screening and quantitative determination of bioactive compounds from fruit extracts of Myristica species and their in vitro antiproliferative activity.

    PubMed

    Pandey, Renu; Mahar, Rohit; Hasanain, Mohammad; Shukla, Sanjeev K; Sarkar, Jayanta; Rameshkumar, K B; Kumar, Brijesh

    2016-11-15

    Efficient and sensitive LC-MS/MS methods have been developed for the rapid screening and determination of bioactive compounds in different fruit parts of four Myristica species, viz., Myristica beddomeii, Myristica fragrans, Myristica fatua and Myristica malabarica. Twenty-one compounds were identified and characterized on the basis of their accurate mass and MS/MS fragmentation pattern using HPLC-QTOF-MS/MS and NMR analysis. Quantitative determination of five major bioactive compounds was performed using multiple-reaction monitoring mode with continuous polarity switching by UHPLC-QqQLIT-MS/MS. Moreover, in vitro antiproliferative activity of these Myristica species was evaluated against five human cancer cell lines A549, DLD-1, DU145, FaDu and MCF-7 using SRB assay. Seventeen phytoconstituents were identified and reported for the first time from M. beddomeii and sixteen from M. fatua. Quantification result showed highest total content of five major bioactive compounds in mace of M. fragrans. Evaluation of in vitro antiproliferative activity revealed potent activity in all investigated species except M. fragrans.

  16. Emerging strategies for RNA interference (RNAi) applications in insects.

    PubMed

    Nandety, Raja Sekhar; Kuo, Yen-Wen; Nouri, Shahideh; Falk, Bryce W

    2015-01-01

    RNA interference (RNAi) in insects is a gene regulatory process that also plays a vital role in the maintenance and in the regulation of host defenses against invading viruses. Small RNAs determine the specificity of the RNAi through precise recognition of their targets. These small RNAs in insects comprise small interfering RNAs (siRNAs), micro RNAs (miRNAs) and Piwi interacting RNAs (piRNAs) of various lengths. In this review, we have explored different forms of the RNAi inducers that are presently in use, and their applications for an effective and efficient fundamental and practical RNAi research with insects. Further, we reviewed trends in next generation sequencing (NGS) technologies and their importance for insect RNAi, including the identification of novel insect targets as well as insect viruses. Here we also describe a rapidly emerging trend of using plant viruses to deliver the RNAi inducer molecules into insects for an efficient RNAi response.

  17. Nanoparticle-Based Delivery of RNAi Therapeutics: Progress and Challenges

    PubMed Central

    Zhou, Jiehua; Shum, Ka-To; Burnett, John C.; Rossi, John J.

    2013-01-01

    RNA interference (RNAi) is an evolutionarily conserved, endogenous process for post-transcriptional regulation of gene expression. Although RNAi therapeutics have recently progressed through the pipeline toward clinical trials, the application of these as ideal, clinical therapeutics requires the development of safe and effective delivery systems. Inspired by the immense progress with nanotechnology in drug delivery, efforts have been dedicated to the development of nanoparticle-based RNAi delivery systems. For example, a precisely engineered, multifunctional nanocarrier with combined passive and active targeting capabilities may address the delivery challenges for the widespread use of RNAi as a therapy. Therefore, in this review, we introduce the major hurdles in achieving efficient RNAi delivery and discuss the current advances in applying nanotechnology-based delivery systems to overcome the delivery hurdles of RNAi therapeutics. In particular, some representative examples of nanoparticle-based delivery formulations for targeted RNAi therapeutics are highlighted. PMID:23667320

  18. Emerging strategies for RNA interference (RNAi) applications in insects

    PubMed Central

    Nandety, Raja Sekhar; Kuo, Yen-Wen; Nouri, Shahideh; Falk, Bryce W

    2015-01-01

    RNA interference (RNAi) in insects is a gene regulatory process that also plays a vital role in the maintenance and in the regulation of host defenses against invading viruses. Small RNAs determine the specificity of the RNAi through precise recognition of their targets. These small RNAs in insects comprise small interfering RNAs (siRNAs), micro RNAs (miRNAs) and Piwi interacting RNAs (piRNAs) of various lengths. In this review, we have explored different forms of the RNAi inducers that are presently in use, and their applications for an effective and efficient fundamental and practical RNAi research with insects. Further, we reviewed trends in next generation sequencing (NGS) technologies and their importance for insect RNAi, including the identification of novel insect targets as well as insect viruses. Here we also describe a rapidly emerging trend of using plant viruses to deliver the RNAi inducer molecules into insects for an efficient RNAi response. PMID:25424593

  19. Clone Mapper: An Online Suite of Tools for RNAi Experiments in Caenorhabditis elegans

    PubMed Central

    Thakur, Nishant; Pujol, Nathalie; Tichit, Laurent; Ewbank, Jonathan J.

    2014-01-01

    RNA interference (RNAi), mediated by the introduction of a specific double-stranded RNA, is a powerful method to investigate gene function. It is widely used in the Caenorhabditis elegans research community. An expanding number of laboratories conduct genome-wide RNAi screens, using standard libraries of bacterial clones each designed to produce a specific double-stranded RNA. Proper interpretation of results from RNAi experiments requires a series of analytical steps, from the verification of the identity of bacterial clones, to the identification of the clones’ potential targets. Despite the popularity of the technique, no user-friendly set of tools allowing these steps to be carried out accurately, automatically, and at a large scale, is currently available. We report here the design and production of Clone Mapper, an online suite of tools specifically adapted to the analysis pipeline typical for RNAi experiments with C. elegans. We show that Clone Mapper overcomes the limitations of existing techniques and provide examples illustrating its potential for the identification of biologically relevant genes. The Clone Mapper tools are freely available via http://www.ciml.univ-mrs.fr/EWBANK_jonathan/software.html. PMID:25187039

  20. Transcriptome analysis and systemic RNAi response in the African sweetpotato weevil (Cylas puncticollis, Coleoptera, Brentidae).

    PubMed

    Prentice, Katterinne; Pertry, Ine; Christiaens, Olivier; Bauters, Lander; Bailey, Ana; Niblett, Chuck; Ghislain, Marc; Gheysen, Godelieve; Smagghe, Guy

    2015-01-01

    The African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptome analysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest.

  1. Simultaneous fingerprint, quantitative analysis and anti-oxidative based screening of components in Rhizoma Smilacis Glabrae using liquid chromatography coupled with Charged Aerosol and Coulometric array Detection.

    PubMed

    Yang, Guang; Zhao, Xin; Wen, Jun; Zhou, Tingting; Fan, Guorong

    2017-04-01

    An analytical approach including fingerprint, quantitative analysis and rapid screening of anti-oxidative components was established and successfully applied for the comprehensive quality control of Rhizoma Smilacis Glabrae (RSG), a well-known Traditional Chinese Medicine with the homology of medicine and food. Thirteen components were tentatively identified based on their retention behavior, UV absorption and MS fragmentation patterns. Chemometric analysis based on coulmetric array data was performed to evaluate the similarity and variation between fifteen batches. Eight discriminating components were quantified using single-compound calibration. The unit responses of those components in coulmetric array detection were calculated and compared with those of several compounds reported to possess antioxidant activity, and four of them were tentatively identified as main contributors to the total anti-oxidative activity. The main advantage of the proposed approach was that it realized simultaneous fingerprint, quantitative analysis and screening of anti-oxidative components, providing comprehensive information for quality assessment of RSG.

  2. Stacking up CRISPR against RNAi for therapeutic gene inhibition.

    PubMed

    Haussecker, Dirk

    2016-09-01

    Both RNA interference (RNAi) and clustered regularly-interspaced short palindromic repeats (CRISPR) technologies allow for the sequence-specific inhibition of gene function and therefore have the potential to be used as therapeutic modalities. By judging the current public and scientific journal interest, it would seem that CRISPR, by enabling clean, durable knockouts, will dominate therapeutic gene inhibition, also at the expense of RNAi. This review aims to look behind prevailing sentiments and to more clearly define the likely scope of the therapeutic applications of the more recently developed CRISPR technology and its relative strengths and weaknesses with regards to RNAi. It is found that largely because of their broadly overlapping delivery constraints, while CRISPR presents formidable competition for DNA-directed RNAi strategies, its impact on RNAi therapeutics triggered by synthetic oligonucleotides will likely be more moderate. Instead, RNAi and genome editing, and in particular CRISPR, are poised to jointly promote a further shift toward sequence-targeted precision medicines.

  3. RNA Interference as a Method for Target-Site Screening in the Western Corn Rootworm, Diabrotica Virgifera Virgifera

    PubMed Central

    Alves, Analiza P.; Lorenzen, Marcé D.; Beeman, Richard W.; Foster, John E.; Siegfried, Blair D.

    2010-01-01

    To test the efficacy of RNA interference (RNAi) as a method for target-site screening in Diabrotica virgifera virgifera LeConte (Coleptera: Chrysomelidae) larvae, genes were identified and tested for which clear RNAi phenotypes had been identified in the Coleopteran model, Tribolium castaneum. Here the cloning of the D. v. vergifera orthologs of laccase 2 (DvvLac2) and chitin synthase 2 (DvvCHS2) is reported. Injection of DvvLac2-specific double-stranded RNA resulted in prevention of post-molt cuticular tanning, while injection of DvvCHS2-specific dsRNA reduced chitin levels in midguts. Silencing of both DvvLac2 and DvvCHS2 was confirmed by RT-PCR and quantitative RT-PCR. As in T. castaneum, RNAi-mediated gene silencing is systemic in Diabrotica. The results indicate that RNAi-induced silencing of D. v. vergifera genes provides a powerful tool for identifying potential insecticide targets. PMID:21067417

  4. RNA interference as a method for target-site screening in the Western corn rootworm, Diabrotica virgifera virgifera.

    PubMed

    Alves, Analiza P; Lorenzen, Marcé D; Beeman, Richard W; Foster, John E; Siegfried, Blair D

    2010-01-01

    To test the efficacy of RNA interference (RNAi) as a method for target-site screening in Diabrotica virgifera virgifera LeConte (Coleptera: Chrysomelidae) larvae, genes were identified and tested for which clear RNAi phenotypes had been identified in the Coleopteran model, Tribolium castaneum. Here the cloning of the D. v. vergifera orthologs of laccase 2 (DvvLac2) and chitin synthase 2 (DvvCHS2) is reported. Injection of DvvLac2-specific double-stranded RNA resulted in prevention of post-molt cuticular tanning, while injection of DvvCHS2-specific dsRNA reduced chitin levels in midguts. Silencing of both DvvLac2 and DvvCHS2 was confirmed by RT-PCR and quantitative RT-PCR. As in T. castaneum, RNAi-mediated gene silencing is systemic in Diabrotica. The results indicate that RNAi-induced silencing of D. v. vergifera genes provides a powerful tool for identifying potential insecticide targets.

  5. RNAi Codex: a portal/database for short-hairpin RNA (shRNA) gene-silencing constructs.

    PubMed

    Olson, A; Sheth, N; Lee, J S; Hannon, G; Sachidanandam, R

    2006-01-01

    Use of RNA interference (RNAi) in forward genetic screens is proliferating. Currently, short-interfering RNAs (siRNAs) and short-hairpin RNAs (shRNAs) are being used to silence genes to tease out functional information. It is becoming easier to harness RNAi to silence specific genes, owing to the development of libraries of readymade shRNA and siRNA gene-silencing constructs by using a variety of sources. RNAi Codex, which consists of a database of shRNA related information and an associated website, has been developed as a portal for publicly available shRNA resources and is accessible at http://codex.cshl.org. RNAi Codex currently holds data from the Hannon-Elledge shRNA library and allows the use of biologist-friendly gene names to access information on shRNA constructs that can silence the gene of interest. It is designed to hold user-contributed annotations and publications for each construct, as and when such data become available. We will describe features of RNAi Codex and explain the use of the tool.

  6. Suppression of intestinal immunity through silencing of TCTP by RNAi in transgenic silkworm, Bombyx mori.

    PubMed

    Hu, Cuimei; Wang, Fei; Ma, Sanyuan; Li, Xianyang; Song, Liang; Hua, Xiaoting; Xia, Qingyou

    2015-12-10

    Intestinal immune response is a front line of host defense. The host factors that participate in intestinal immunity response remain largely unknown. We recently reported that Translationally Controlled Tumor Protein (BmTCTP) was obtained by constructing a phage display cDNA library of the silkworm midgut and carrying out high throughput screening of pathogen binding molecules. To further address the function of BmTCTP in silkworm intestinal immunity, transgenic RNAi silkworms were constructed by microinjection piggBac plasmid to Dazao embryos. The antimicrobial capacity of transgenic silkworm decreased since the expression of gut antimicrobial peptide from transgenic silkworm was not sufficiently induced during oral microbial challenge. Moreover, dynamic ERK phosphorylation from transgenic silkworm midgut was disrupted. Taken together, the innate immunity of intestinal was suppressed through disruption of dynamic ERK phosphorylation after oral microbial infection as a result of RNAi-mediated knockdown of midgut TCTP in transgenic silkworm.

  7. Geographic variation in RNAi sensitivity in the migratory locust.

    PubMed

    Sugahara, Ryohei; Tanaka, Seiji; Jouraku, Akiya; Shiotsuki, Takahiro

    2017-03-20

    The RNA interference (RNAi) technology has been widely used in basic and applied research. It is known that RNAi works in some species but not in others, although the cause for this difference remains unclear. Here, we present inter- and intra-populational variations in RNAi sensitivity in the migratory locust Locusta migratoria, and provide information on the genetic background of such variations. In the four strains analyzed, originating from different Japanese localities, most individuals from two of the strains were sensitive to injections of double-stranded RNA (dsRNA) against the corazonin (CRZ) and ecdysone receptor genes, whereas those from the other two strains were resistant. Selection for individuals sensitive to dsCRZ produced a dramatic increase in the RNAi sensitivity in the following generations, although phenotypes also varied in the selected line, suggesting that several genes might control RNAi sensitivity. Reciprocal crosses between a sensitive and a resistant strain suggested that the resistant phenotype is dominant. The expression levels of nine RNAi-associated genes known for other organisms were not correlated with the variation in RNAi sensitivity observed in L. migratoria. Variations in RNAi sensitivity as the ones observed in this study should be considered when using RNAi in basic and applied research as well as in pest management.

  8. Gene Silencing in Insect Cells Using RNAi.

    PubMed

    Wu, Hsuan-Chen; March, John C; Bentley, William E

    2016-01-01

    A technique is described for synthesizing and transfecting double stranded RNA (dsRNA) for RNA interference (RNAi) in Sf-21 cell culture. Transfection with dsRNA only requires an hour and the cells usually recover within 12 h. Suggestions for designing dsRNA are included in the methods. Furthermore, websites are provided for rapid and effective dsRNA design. Three kits are essential for using the described methods: RNAqueous®-4PCR, Megascript™ T7 kit, and the Superscript™ III kit from Life Technologies, Inc.

  9. MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation

    PubMed Central

    Zubakov, Dmitry; Boersma, Anton W. M.; Choi, Ying; van Kuijk, Patricia F.; Wiemer, Erik A. C.

    2010-01-01

    MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids. Electronic supplementary material The online version of this article (doi:10.1007/s00414-009-0402-3) contains

  10. A High Resolution/Accurate Mass (HRAM) Data-Dependent MS3 Neutral Loss Screening, Classification, and Relative Quantitation Methodology for Carbonyl Compounds in Saliva

    NASA Astrophysics Data System (ADS)

    Dator, Romel; Carrà, Andrea; Maertens, Laura; Guidolin, Valeria; Villalta, Peter W.; Balbo, Silvia

    2016-10-01

    Reactive carbonyl compounds (RCCs) are ubiquitous in the environment and are generated endogenously as a result of various physiological and pathological processes. These compounds can react with biological molecules inducing deleterious processes believed to be at the basis of their toxic effects. Several of these compounds are implicated in neurotoxic processes, aging disorders, and cancer. Therefore, a method characterizing exposures to these chemicals will provide insights into how they may influence overall health and contribute to disease pathogenesis. Here, we have developed a high resolution accurate mass (HRAM) screening strategy allowing simultaneous identification and relative quantitation of DNPH-derivatized carbonyls in human biological fluids. The screening strategy involves the diagnostic neutral loss of hydroxyl radical triggering MS3 fragmentation, which is only observed in positive ionization mode of DNPH-derivatized carbonyls. Unique fragmentation pathways were used to develop a classification scheme for characterizing known and unanticipated/unknown carbonyl compounds present in saliva. Furthermore, a relative quantitation strategy was implemented to assess variations in the levels of carbonyl compounds before and after exposure using deuterated d 3 -DNPH. This relative quantitation method was tested on human samples before and after exposure to specific amounts of alcohol. The nano-electrospray ionization (nano-ESI) in positive mode afforded excellent sensitivity with detection limits on-column in the high-attomole levels. To the best of our knowledge, this is the first report of a method using HRAM neutral loss screening of carbonyl compounds. In addition, the method allows simultaneous characterization and relative quantitation of DNPH-derivatized compounds using nano-ESI in positive mode.

  11. Simultaneous quantitation of hexacosanoyl lysophosphatidylcholine, amino acids, acylcarnitines, and succinylacetone during FIA–ESI–MS/MS analysis of dried blood spot extracts for newborn screening

    PubMed Central

    Haynes, Christopher A.; De Jesús, Víctor R.

    2016-01-01

    Objectives The goal of this study was to include the quantitation of hexacosanoyl lysophosphatidylcholine, a biomarker for X-linked adrenoleukodystrophy and other peroxisomal disorders, in the routine extraction and analysis procedure used to quantitate amino acids, acylcarnitines, and succinylacetone during newborn screening. Criteria for the method included use of a single punch from a dried blood spot, one simple extraction of the punch, no high-performance liquid chromatography, and utilizing tandem mass spectrometry to quantitate the analytes. Design and methods Dried blood spot punches were extracted with a methanolic solution of stable-isotope labeled internal standards, formic acid, and hydrazine, followed by flow injection analysis–electrospray ionization–tandem mass spectrometry. Results Quantitation of amino acids, acylcarnitines, and hexacosanoyl lysophosphatidylcholine using this combined method was similar to results obtained using two separate methods. Conclusions A single dried blood spot punch extracted by a rapid (45 min), simple procedure can be analyzed with high throughput (2 min per sample) to quantitate amino acids, acylcarnitines, succinylacetone, and hexacosanoyl lysophosphatidylcholine. PMID:26432925

  12. Towards the elements of successful insect Ribonucleic acid interference (RNAi)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ribonucleic acid interference (RNAi), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. The accumulating body of literature on insect RNAi has revealed that ...

  13. RNAi control of aflatoxins in peanut plants, a multifactorial system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA-interference (RNAi)-mediated control of aflatoxin contamination in peanut plants is a multifactorial and hyper variable system. The use of RNAi biotechnology to silence single genes in plants has inherently high-variability among transgenic events. Also the level of expression of small interfe...

  14. RNAi mediated, stable resistance to Triticum mosaic virus in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Triticum mosaic virus (TriMV), discovered in 2006, affects wheat production systems in the Great Plains of the United States. There are no available TriMV resistant commercial varieties. RNA interference (RNAi) was evaluated as an alternative strategy to generate resistance to TriMV. An RNAi pANDA...

  15. Bringing RNA Interference (RNAi) into the High School Classroom

    ERIC Educational Resources Information Center

    Sengupta, Sibani

    2013-01-01

    RNA interference (abbreviated RNAi) is a relatively new discovery in the field of mechanisms that serve to regulate gene expression (a.k.a. protein synthesis). Gene expression can be regulated at the transcriptional level (mRNA production, processing, or stability) and at the translational level (protein synthesis). RNAi acts in a gene-specific…

  16. "Caenorhabditis Elegans" as an Undergraduate Educational Tool for Teaching RNAi

    ERIC Educational Resources Information Center

    Andersen, Janet; Krichevsky, Alexander; Leheste, Joerg R.; Moloney, Daniel J.

    2008-01-01

    Discovery of RNA-mediated interference (RNAi) is widely recognized as one of the most significant molecular biology breakthroughs in the past 10 years. There is a need for science educators to develop teaching tools and laboratory activities that demonstrate the power of this new technology and help students to better understand the RNAi process.…

  17. A Quantitative High-Throughput Screen for Modulators of IL-6 Signaling: A Model for Interrogating Biological Networks using Chemical Libraries

    PubMed Central

    Johnson, Ronald L.; Huang, Ruili; Jadhav, Ajit; Southall, Noel; Wichterman, Jennifer; MacArthur, Ryan; Xia, Menghang; Bi, Kun; Printen, John; Austin, Christopher P.; Inglese, James

    2009-01-01

    Small molecule modulators are critical for dissecting and understanding signaling pathways at the molecular level. Interleukin 6 (IL-6) is a cytokine that signals via the JAK/STAT pathway and is implicated in cancer and inflammation. To identify modulators of this pathway, we screened a chemical collection against an IL-6 responsive cell line stably expressing a beta-lactamase reporter gene fused to a sis-inducible element (SIE-bla cells). This assay was optimized for a 1536-well microplate format and screened against 11,693 small molecules using quantitative high-throughput screening (qHTS), a method that assays a chemical library at multiple concentrations to generate titration-response profiles for each compound. The qHTS recovered 564 actives with well-fit curves that clustered into 32 distinct chemical series of 13 activators and 19 inhibitors. A retrospective analysis of the qHTS data indicated that single concentration data at 1.5 and 7.7 uM scored 35 and 71% of qHTS actives, respectively, as inactive and were therefore false negatives. Following counter screens to identify fluorescent and nonselective series, we found four activator and one inhibitor series that modulated SIE-bla cells but did not show similar activity in reporter gene assays induced by EGF and hypoxia. Small molecules within these series will make useful tool compounds to investigate IL-6 signaling mediated by JAK/STAT activation. PMID:19668870

  18. [Inhibiting GDF-8 expression by retrovirus-based RNAi stably].

    PubMed

    Liu, Chaowu; Yang, Zhuo; Zhao, Bin; Liu, Changmei

    2008-02-01

    We cloned human U6 promoter from pAVU6 + 27 vector into pXSN to transcripe small RNA. Meanwhile, a shRNA targeting GDF-8 was cloned down-stream of the hU6 promoter to construct recombinant vector. Then the packing cell GP-293 was co-transfected the recombinant with pVSV-G to gernarate virus particle. Resistant C2C12 cell pools were screened using G418. Levels of mRNA and protein of GDF-8 were tested by Real-Time PCR and western blotting. Cell proliferation and cell cycle were analyzed using MTT and FACS. The expression of GDF-8 was dramatically decreased by the retrovirus-based system in C2C12 cells. Cells proliferated effectively after integrating the recombinant. The cells in G0/G1 phase decreased by 13.7%, while cells in S phase increased by 14.9%. In conclusion, the retrovirus-based RNAi could be used to stably silence GDF-8. It can be a powerful tool in curing muscle atrophy.

  19. RNAi-dependent and RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa.

    PubMed

    Chicas, Agustin; Cogoni, Carlo; Macino, Giuseppe

    2004-01-01

    RNA interference (RNAi) can silence genes at the transcriptional level by targeting locus-specific Lys9H3 methylation or at the post-transcriptional level by targeting mRNA degradation. Here we have cloned and sequenced genomic regions methylated in Lys9H3 in Neurospora crassa to test the requirements for components of the RNAi pathway in this modification. We find that 90% of clones map to repeated sequences and relics of transposons that have undergone repeat-induced point mutations (RIP). We find siRNAs derived from transposon relics indicating that the RNAi machinery targets these regions. This is confirmed by the fact that the presence of these siRNAs depends on components of the RNAi pathway such as the RdRP (QDE-1), the putative RecQ helicase (QDE-3) and the two Dicer enzymes. We show that Lys9H3 methylation of RIP sequences is not affected in mutants of the RNAi pathway indicating that the RNAi machinery is not involved in transcriptional gene silencing in Neurospora. We find that RIP regions are transcribed and that the transcript level increases in the mutants of the RNAi pathway. These data suggest that the biological function of the Neurospora RNAi machinery is to control transposon relics and repeated sequences by targeting degradation of transcripts derived from these regions.

  20. RNAi-dependent and RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa

    PubMed Central

    Chicas, Agustin; Cogoni, Carlo; Macino, Giuseppe

    2004-01-01

    RNA interference (RNAi) can silence genes at the transcriptional level by targeting locus-specific Lys9H3 methylation or at the post-transcriptional level by targeting mRNA degradation. Here we have cloned and sequenced genomic regions methylated in Lys9H3 in Neurospora crassa to test the requirements for components of the RNAi pathway in this modification. We find that 90% of clones map to repeated sequences and relics of transposons that have undergone repeat-induced point mutations (RIP). We find siRNAs derived from transposon relics indicating that the RNAi machinery targets these regions. This is confirmed by the fact that the presence of these siRNAs depends on components of the RNAi pathway such as the RdRP (QDE-1), the putative RecQ helicase (QDE-3) and the two Dicer enzymes. We show that Lys9H3 methylation of RIP sequences is not affected in mutants of the RNAi pathway indicating that the RNAi machinery is not involved in transcriptional gene silencing in Neurospora. We find that RIP regions are transcribed and that the transcript level increases in the mutants of the RNAi pathway. These data suggest that the biological function of the Neurospora RNAi machinery is to control transposon relics and repeated sequences by targeting degradation of transcripts derived from these regions. PMID:15302921

  1. RNAi therapeutics for brain cancer: current advancements in RNAi delivery strategies.

    PubMed

    Malhotra, Meenakshi; Toulouse, André; Godinho, Bruno M D C; Mc Carthy, David John; Cryan, John F; O'Driscoll, Caitriona M

    2015-10-01

    Malignant primary brain tumors are aggressive cancerous cells that invade the surrounding tissues of the central nervous system. The current treatment options for malignant brain tumors are limited due to the inability to cross the blood-brain barrier. The advancements in current research has identified and characterized certain molecular markers that are essential for tumor survival, progression, metastasis and angiogenesis. These molecular markers have served as therapeutic targets for the RNAi based therapies, which enable site-specific silencing of the gene responsible for tumor proliferation. However, to bring about therapeutic success, an efficient delivery carrier that can cross the blood-brain barrier and reach the targeted site is essential. The current review focuses on the potential of targeted, non-viral and viral particles containing RNAi therapeutic molecules as delivery strategies specifically for brain tumors.

  2. RNAi-mediated crop protection against insects.

    PubMed

    Price, Daniel R G; Gatehouse, John A

    2008-07-01

    Downregulation of the expression of specific genes through RNA interference (RNAi), has been widely used for genetic research in insects. The method has relied on the injection of double-stranded RNA (dsRNA), which is not possible for practical applications in crop protection. By contrast, specific suppression of gene expression in nematodes is possible through feeding with dsRNA. This approach was thought to be unfeasible in insects, but recent results have shown that dsRNA fed as a diet component can be effective in downregulating targeted genes. More significantly, expression of dsRNA directed against suitable insect target genes in transgenic plants has been shown to give protection against pests, opening the way for a new generation of insect-resistant crops.

  3. Current issues of RNAi therapeutics delivery and development.

    PubMed

    Haussecker, D

    2014-12-10

    12 years following the discovery of the RNAi mechanism in Man, a number of RNAi therapeutics development candidates have emerged with profiles suggesting that they could become drugs of significant medical importance for diseases like TTR amyloidosis, HBV, solid cancers, and hemophilia. Despite this robust progress, the perception of RNAi therapeutics has been on a roller-coaster ride driven not only by science, but also regulatory trends, the stock markets, and Big Pharma business development decisions [1]. This presentation provides an update on the current state of RNAi therapeutics development with a particular focus on what RNAi delivery can achieve today and key challenges to be overcome to expand therapeutic opportunities. The delivery of RNAi triggers to disease-relevant cell types clearly represents the rate-limiting factor in broadly expanding the applicability of RNAi therapeutics. Today, with at least 3 delivery options (lipid nanoparticles/LNPs, GalNAc-siRNA conjugates, Dynamic PolyConjugates/DPCs) for which profound gene knockdowns have been demonstrated in non-human primates and in the clinic, RNAi therapeutics should in principle be able to address most diseases related to gene expression in the liver. Given the central importance of the liver in systemic physiology, this already represents a significant therapeutic and commercial opportunity rivaling that of e.g. monoclonal antibodies. Beyond the liver, there is a reason to believe that current RNAi therapeutics technologies can address a number of solid tumors (e.g. LNPs), diseases of the eye (e.g. self-delivering RNAi triggers) as well as diseases involving the respiratory epithelium (e.g. aerosolized LNPs), certain phagocytic cells (LNPs), hematopoietic stem cells and their progeny (lentiviral DNA-directed RNAi), vascular endothelial cells (cationic lipoplexes), and certain cell types in the kidney (self-delivering RNAi triggers, DPCs; Table 1). Despite this success, there has been a sense that

  4. Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

    PubMed

    Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

    2009-08-01

    An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

  5. Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing).

    PubMed

    Hajeri, Subhas; Killiny, Nabil; El-Mohtar, Choaa; Dawson, William O; Gowda, Siddarame

    2014-04-20

    A transient expression vector based on Citrus tristeza virus (CTV) is unusually stable. Because of its stability it is being considered for use in the field to control Huanglongbing (HLB), which is caused by Candidatus Liberibacter asiaticus (CLas) and vectored by Asian citrus psyllid, Diaphorina citri. In the absence of effective control strategies for CLas, emphasis has been on control of D. citri. Coincident cohabitation in phloem tissue by CLas, D. citri and CTV was exploited to develop a novel method to mitigate HLB through RNA interference (RNAi). Since CTV has three RNA silencing suppressors, it was not known if CTV-based vector could induce RNAi in citrus. Yet, expression of sequences targeting citrus phytoene desaturase gene by CTV-RNAi resulted in photo-bleaching phenotype. CTV-RNAi vector, engineered with truncated abnormal wing disc (Awd) gene of D. citri, induced altered Awd expression when silencing triggers ingested by feeding D. citri nymphs. Decreased Awd in nymphs resulted in malformed-wing phenotype in adults and increased adult mortality. This impaired ability of D. citri to fly would potentially limit the successful vectoring of CLas bacteria between citrus trees in the grove. CTV-RNAi vector would be relevant for fast-track screening of candidate sequences for RNAi-mediated pest control.

  6. Profiling Environmental Chemicals in the Antioxidant Response Element Pathway using Quantitative High Throughput Screening (qHTS)

    EPA Science Inventory

    The antioxidant response element (ARE) signaling pathway plays an important role in the amelioration of oxidative stress, which can contribute to a number of diseases, including cancer. We screened 1408 NTP-provided substances in 1536-well qHTS format at concentrations ranging fr...

  7. First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass.

    PubMed

    Wang, Guangliang; Rajpurohit, Surendra K; Delaspre, Fabien; Walker, Steven L; White, David T; Ceasrine, Alexis; Kuruvilla, Rejji; Li, Ruo-Jing; Shim, Joong S; Liu, Jun O; Parsons, Michael J; Mumm, Jeff S

    2015-07-28

    Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes.

  8. First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass

    PubMed Central

    Wang, Guangliang; Rajpurohit, Surendra K; Delaspre, Fabien; Walker, Steven L; White, David T; Ceasrine, Alexis; Kuruvilla, Rejji; Li, Ruo-jing; Shim, Joong S; Liu, Jun O; Parsons, Michael J; Mumm, Jeff S

    2015-01-01

    Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes. DOI: http://dx.doi.org/10.7554/eLife.08261.001 PMID:26218223

  9. iBeetle-Base: a database for RNAi phenotypes in the red flour beetle Tribolium castaneum.

    PubMed

    Dönitz, Jürgen; Schmitt-Engel, Christian; Grossmann, Daniela; Gerischer, Lizzy; Tech, Maike; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor

    2015-01-01

    The iBeetle-Base (http://ibeetle-base.uni-goettingen.de) makes available annotations of RNAi phenotypes, which were gathered in a large scale RNAi screen in the red flour beetle Tribolium castaneum (iBeetle screen). In addition, it provides access to sequence information and links for all Tribolium castaneum genes. The iBeetle-Base contains the annotations of phenotypes of several thousands of genes knocked down during embryonic and metamorphic epidermis and muscle development in addition to phenotypes linked to oogenesis and stink gland biology. The phenotypes are described according to the EQM (entity, quality, modifier) system using controlled vocabularies and the Tribolium morphological ontology (TrOn). Furthermore, images linked to the respective annotations are provided. The data are searchable either for specific phenotypes using a complex 'search for morphological defects' or a 'quick search' for gene names and IDs. The red flour beetle Tribolium castaneum has become an important model system for insect functional genetics and is a representative of the most species rich taxon, the Coleoptera, which comprise several devastating pests. It is used for studying insect typical development, the evolution of development and for research on metabolism and pest control. Besides Drosophila, Tribolium is the first insect model organism where large scale unbiased screens have been performed.

  10. Quantitative comparison of functional screening by measuring intracellular Ca2+ with radioligand binding at recombinant human dopamine receptors.

    PubMed

    Kassack, Matthias U

    2002-01-01

    The purpose of this study was to test whether screening at dopamine receptors performed with a recently described functional assay for G-protein coupled receptors (GPCRs) provides data that correlate significantly with radioligand binding data in the literature, thus possibly allowing researchers to replace radioligand binding with nonradioactive functional screening. Human dopamine receptors hD1 and hD2L (representing Gs [hD1] or Gi [hD2L] coupled GPCRs) were recombinantly expressed in human embryonic kidney (HEK293) cells. Cells were loaded with Oregon Green 488 BAPTA-1/AM and evenly distributed in 384 well plates. Seventeen test compounds were screened for agonistic activity by injection into the cell suspension and monitoringH of intracellular Ca2+ with a fluorescence microplate reader. Then, standard agonists (100nM SKF38393 for hD1, 30nM quinpirole for hD2L) were injected into wells preincubated with test compounds (screening for antagonism). Injection of various agonists resulted in a concentration-dependent increase in fluorescence. Further, preincubation of antagonists with dopamine receptor expressing cells inhibits concentration-dependent the agonist-induced increase in fluorescence. Calculated apparent functional Ki values correlate with radioligand binding data in the literature (r2 = 0.7796 for D1, r2 = 0.7743 for D2). The correlation between apparent functional Ki values and radioligand binding data for the 17 tested compounds suggests that screening of test compounds at dopamine receptors with the functional Ca2+ assay can replace radioligand binding studies. Furthermore, besides apparent Ki values, information about agonistic or antagonistic properties of a test compound can be obtained with the functional Ca2+ assay.

  11. RNAi applications in therapy development for neurodegenerative disease.

    PubMed

    Maxwell, M M

    2009-01-01

    RNA-mediated interference (RNAi) is a powerful tool for experimental manipulation of gene expression and is widely used to investigate gene function both in vitro and in vivo. RNAi refers to an evolutionarily conserved cellular mechanism for sequence-specific post-transcriptional gene silencing, in which double-stranded RNAs promote selective degradation of homologous cellular mRNAs. Because RNAi-based techniques can be employed to reduce expression of specific genes, this approach holds great promise as a therapy for diverse diseases, including devastating neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases and amyotrophic lateral sclerosis (ALS). Importantly, in recent years RNAi has also emerged as a key tool in target identification and validation studies designed to complement traditional (i.e., small molecule-based) drug development strategies. These studies harness the power of RNAi-mediated reverse genetics to probe disease-associated pathways in both cell-based and animal models, and thus may provide critical data needed to focus drug development efforts around disease-relevant targets. This review highlights recent progress in the preclinical development of RNAi-based therapeutics for neurodegenerative disease and discusses the particular challenges that disorders of the central nervous system (CNS) pose for this approach. It further describes current applications of RNAi techniques for target identification and validation studies and underscores the importance of this methodology to developing treatments for neurological diseases.

  12. Cost-effectiveness of quantitative ultrasound as a technique for screening of osteoporotic fracture risk: report on a health technology assessment conducted in 2001

    PubMed Central

    Aidelsburger, Pamela; Hessel, Franz; Wasem, Jürgen

    2004-01-01

    Aim: On behalf of the German Agency for Health Technology Assessment (DAHTA@DIMDI) a rapid economic HTA was conducted. Aim of the HTA was to evaluate the cost-effectiveness of quantitative ultrasound (QUS) for screening of osteoporotic fracture risk. Study population was formed by postmenopausal women. QUS was compared to the dual X-ray absorptiometry (DXA) as the most frequently used method of measurement. Methods: According to the recommendations for rapid economic HTA a comprehensive literature search was conducted. Data of identified and relevant publications have been extracted in form of a qualitative and quantitative information synthesis. The authors calculated incremental cost-effectiveness ratios for different screening procedures: (1) one-step proceeding comparing QUS with DXA, (2) two-step proceeding starting with QUS followed by DXA in pathologic cases. Results: An additional case diagnosed by DXA in a one-step proceeding rises additional costs of about 1,000 EURO. A two-step proceeding with QUS is cost-effective as long as the costs of one QUS examination are lower than 31%-51% of the costs of one DXA examination. Discussion: All considered studies showed methodological limitations. None of them included long term effects like avoided bone fractures. Considering long-term effects probably would change the results. Due to the weakness of data no concluding judgement about the cost-effectiveness of QUS can be given. PMID:19675686

  13. In vitro screening of compounds against laboratory and field isolates of human hookworm reveals quantitative differences in anthelmintic susceptibility.

    PubMed

    Treger, Rebecca S; Otchere, Joseph; Keil, Martin F; Quagraine, Josephine E; Rai, Ganesha; Mott, Bryan T; Humphries, Debbie L; Wilson, Michael; Cappello, Michael; Vermeire, Jon J

    2014-01-01

    A panel of 80 compounds was screened for anthelmintic activity against a laboratory strain of Ancylostoma ceylanicum and field isolates of hookworm obtained from school children in the Kintampo North District of the Brong Ahafo Region of Ghana. Although the laboratory strain of A. ceylanicum was more susceptible to the compounds tested than the field isolates of hookworm, a twofold increase in compound concentration resulted in comparable egg hatch percent inhibition for select compounds. These data provide evidence that the efficacy of anthelmintic compounds may be species-dependent and that field and laboratory strains of hookworm differ in their sensitivities to the anthelmintics tested. These data also suggest that both compound concentration and hookworm species must be considered when screening to identify novel anthelmintic compounds.

  14. Quantitative EEG Magnitudes in Children with and without Attention Deficit Disorder during Neurological Screening and Cognitive Tasks.

    ERIC Educational Resources Information Center

    Crawford, Helen J.; Barabasz, Marianne

    1996-01-01

    Quantitative EEG magnitude data were obtained from children with and without attention deficit disorder (ADD). The data suggest that the right fronto-centro-temporal region is not as "cognitively activated" relative to the left hemisphere in those children with ADD. Neurotherapy training of the right frontal and central regions in ADD…

  15. Core RNAi Machinery and Sid1, a Component for Systemic RNAi, in the Hemipteran Insect, Aphis glycines.

    PubMed

    Bansal, Raman; Michel, Andy P

    2013-02-08

    RNA interference (RNAi) offers a novel tool to manage hemipteran pests. For the success of RNAi based pest control in the field, a robust and systemic RNAi response is a prerequisite. We identified and characterized major genes of the RNAi machinery, Dicer2 (Dcr2), Argonaute2 (Ago2), and R2d2 in Aphis glycines, a serious pest of soybean. The A. glycines genome encodes for at least one copy of Dcr2, R2d2 and Ago2. Comparative and molecular evolution analyses (dN/dS) showed that domain regions of encoded proteins are highly conserved, whereas linker (non-domain) regions are diversified. Sequence homology and phylogenetic analyses suggested that the RNAi machinery of A. glycines is more similar to that of Tribolium casteneum as compared to that of Drosophila melanogaster. We also characterized Sid1, a major gene implicated in the systemic response for RNAi-mediated gene knockdown. Through qPCR, Dcr2, R2d2, Ago2, and Sid1 were found to be expressed at similar levels in various tissues, but higher expression of Dcr2, R2d2, and Ago2 was seen in first and second instars. Characterization of RNAi pathway and Sid1 in A. glycines will provide the foundation of future work for controlling one of the most important insect pests of soybean in North America.

  16. Functional genomic screening to enhance oncolytic virotherapy.

    PubMed

    Mahoney, D J; Stojdl, D F

    2013-02-05

    Functional genomic screening has emerged as a powerful approach for understanding complex biological phenomena. Of the available tools, genome-wide RNA interference (RNAi) technology is unquestionably the most incisive, as it directly probes gene function. Recent applications of RNAi screening have been impressive. Notable amongst these are its use in elucidated mechanism(s) for signal transduction, various aspects of cell biology, tumourigenesis and metastasis, resistance to cancer therapeutics, and the host's response to a pathogen. Herein we discuss how recent RNAi screening efforts have helped turn our attention to the targetability of non-oncogene support pathways for cancer treatment, with a particular focus on a recent study that identified a non-oncogene addiction to the ER stress response as a synergist target for oncolytic virus therapy (OVT). Moreover, we give our thoughts on the future of RNAi screening as a tool to enhance OVT and describe recent technical improvements that are poised to make genome-scale RNAi experiments more sensitive, less noisy, more applicable in vivo, and more easily validated in clinically relevant animal models.

  17. The development of RNA interference (RNAi) in gastrointestinal nematodes.

    PubMed

    Selkirk, Murray E; Huang, Stanley C; Knox, David P; Britton, Collette

    2012-04-01

    Despite the utility of RNAi for defining gene function in Caenorhabditis elegans and early successes reported in parasitic nematodes, RNAi has proven to be stubbornly inconsistent or ineffective in the animal parasitic nematodes examined to date. Here, we summarise some of our experiences with RNAi in parasitic nematodes affecting animals and discuss the available data in the context of our own unpublished work, taking account of mode of delivery, larval activation, site of gene transcription and the presence/absence of essential RNAi pathway genes as defined by comparisons to C. elegans. We discuss future directions briefly including the evaluation of nanoparticles as a means to enhance delivery of interfering RNA to the target worm tissue.

  18. Autonomously folded α-helical lockers promote RNAi*

    PubMed Central

    Guyader, Christian P. E.; Lamarre, Baptiste; De Santis, Emiliana; Noble, James E.; Slater, Nigel K.; Ryadnov, Maxim G.

    2016-01-01

    RNAi is an indispensable research tool with a substantial therapeutic potential. However, the complete transition of the approach to an applied capability remains hampered due to poorly understood relationships between siRNA delivery and gene suppression. Here we propose that interfacial tertiary contacts between α-helices can regulate siRNA cytoplasmic delivery and RNAi. We introduce a rationale of helical amphipathic lockers that differentiates autonomously folded helices, which promote gene silencing, from helices folded with siRNA, which do not. Each of the helical designs can deliver siRNA into cells via energy-dependent endocytosis, while only autonomously folded helices with pre-locked hydrophobic interfaces were able to promote statistically appreciable gene silencing. We propose that it is the amphipathic locking of interfacing helices prior to binding to siRNA that enables RNAi. The rationale offers structurally balanced amphipathic scaffolds to advance the exploitation of functional RNAi. PMID:27721465

  19. Autonomously folded α-helical lockers promote RNAi.

    PubMed

    Guyader, Christian P E; Lamarre, Baptiste; De Santis, Emiliana; Noble, James E; Slater, Nigel K; Ryadnov, Maxim G

    2016-10-10

    RNAi is an indispensable research tool with a substantial therapeutic potential. However, the complete transition of the approach to an applied capability remains hampered due to poorly understood relationships between siRNA delivery and gene suppression. Here we propose that interfacial tertiary contacts between α-helices can regulate siRNA cytoplasmic delivery and RNAi. We introduce a rationale of helical amphipathic lockers that differentiates autonomously folded helices, which promote gene silencing, from helices folded with siRNA, which do not. Each of the helical designs can deliver siRNA into cells via energy-dependent endocytosis, while only autonomously folded helices with pre-locked hydrophobic interfaces were able to promote statistically appreciable gene silencing. We propose that it is the amphipathic locking of interfacing helices prior to binding to siRNA that enables RNAi. The rationale offers structurally balanced amphipathic scaffolds to advance the exploitation of functional RNAi.

  20. Beyond Drosophila: RNAi in vivo and functional genomics in insects.

    PubMed

    Bellés, Xavier

    2010-01-01

    The increasing availability of insect genomes has revealed a large number of genes with unknown functions and the resulting problem of how to discover these functions. The RNA interference (RNAi) technique, which generates loss-of-function phenotypes by depletion of a chosen transcript, can help to overcome this challenge. RNAi can unveil the functions of new genes, lead to the discovery of new functions for old genes, and find the genes for old functions. Moreover, the possibility of studying the functions of homologous genes in different species can allow comparisons of the genetic networks regulating a given function in different insect groups, thereby facilitating an evolutionary insight into developmental processes. RNAi also has drawbacks and obscure points, however, such as those related to differences in species sensitivity. Disentangling these differences is one of the main challenges in the RNAi field.

  1. Autonomously folded α-helical lockers promote RNAi*

    NASA Astrophysics Data System (ADS)

    Guyader, Christian P. E.; Lamarre, Baptiste; de Santis, Emiliana; Noble, James E.; Slater, Nigel K.; Ryadnov, Maxim G.

    2016-10-01

    RNAi is an indispensable research tool with a substantial therapeutic potential. However, the complete transition of the approach to an applied capability remains hampered due to poorly understood relationships between siRNA delivery and gene suppression. Here we propose that interfacial tertiary contacts between α-helices can regulate siRNA cytoplasmic delivery and RNAi. We introduce a rationale of helical amphipathic lockers that differentiates autonomously folded helices, which promote gene silencing, from helices folded with siRNA, which do not. Each of the helical designs can deliver siRNA into cells via energy-dependent endocytosis, while only autonomously folded helices with pre-locked hydrophobic interfaces were able to promote statistically appreciable gene silencing. We propose that it is the amphipathic locking of interfacing helices prior to binding to siRNA that enables RNAi. The rationale offers structurally balanced amphipathic scaffolds to advance the exploitation of functional RNAi.

  2. RNAi as a Route Toward Breast Cancer Therapy

    DTIC Science & Technology

    2011-09-01

    CONTRACTING ORGANIZATION: Cold Spring Harbor Laboratory... Cold Spring Harbor , NY 11724... Cold Spring Harbor Laboratory Cold Spring Harbor , NY 11724 RNAi, sequencing No abstract provided. 8 1 SEP 2010-31 AUG 2011Annual01-09-2011 hannon

  3. Quantitation of bacteria through adsorption of intracellular biomolecules on carbon paste and screen-printed carbon electrodes and voltammetry of redox-active probes.

    PubMed

    Obuchowska, Agnes

    2008-03-01

    A new electrochemical method for the quantitation of bacteria that is rapid, inexpensive, and amenable to miniaturization is reported. Cyclic voltammetry was used to quantitate M. luteus, C. sporogenes, and E. coli JM105 in exponential and stationary phases, following exposure of screen-printed carbon working electrodes (SPCEs) to lysed culture samples. Ferricyanide was used as a probe. The detection limits (3s) were calculated and the dynamic ranges for E. coli (exponential and stationary phases), M. luteus (exponential and stationary phases), and C. sporogenes (exponential phase) lysed by lysozyme were 3 x 10(4) to 5 x 10(6) colony-forming units (CFU) mL(-1), 5 x 10(6) to 2 x 10(8) CFU mL(-1) and 3 x 10(3) to 3 x 10(5) CFU mL(-1), respectively. Good overlap was obtained between the calibration curves when the electrochemical signal was plotted against the dry bacterial weight, or between the protein concentration in the bacterial lysate. In contrast, unlysed bacteria did not change the electrochemical signal of ferricyanide. The results indicate that the reduction of the electrochemical signal in the presence of the lysate is mainly due to the fouling of the electrode by proteins. Similar results were obtained with carbon-paste electrodes although detection limits were better with SPCEs. The method described herein was applied to quantitation of bacteria in a cooling tower water sample.

  4. Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high throughput screening platform

    PubMed Central

    Nishihara, Kana; Huang, Ruili; Zhao, Jinghua; Shahane, Sampada A.; Witt, Kristine L.; Smith-Roe, Stephanie L.; Tice, Raymond R.; Takeda, Shunichi; Xia, Menghang

    2016-01-01

    DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70 −/−/RAD54 −/− and REV3 −/−) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many well-known genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity—2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether—were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA. PMID:26243743

  5. RNAi Trigger Delivery into Anopheles gambiae Pupae.

    PubMed

    Regna, Kimberly; Harrison, Rachel M; Heyse, Shannon A; Chiles, Thomas C; Michel, Kristin; Muskavitch, Marc A T

    2016-03-08

    RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

  6. Biotechnological uses of RNAi in plants: risk assessment considerations.

    PubMed

    Casacuberta, Josep M; Devos, Yann; du Jardin, Patrick; Ramon, Matthew; Vaucheret, Hervé; Nogué, Fabien

    2015-03-01

    RNAi offers opportunities to generate new traits in genetically modified (GM) plants. Instead of expressing novel proteins, RNAi-based GM plants reduce target gene expression. Silencing of off-target genes may trigger unintended effects, and identifying these genes would facilitate risk assessment. However, using bioinformatics alone is not reliable, due to the lack of genomic data and insufficient knowledge of mechanisms governing mRNA-small (s)RNA interactions.

  7. Phylogenetic Origin and Diversification of RNAi Pathway Genes in Insects.

    PubMed

    Dowling, Daniel; Pauli, Thomas; Donath, Alexander; Meusemann, Karen; Podsiadlowski, Lars; Petersen, Malte; Peters, Ralph S; Mayer, Christoph; Liu, Shanlin; Zhou, Xin; Misof, Bernhard; Niehuis, Oliver

    2017-01-06

    RNA interference (RNAi) refers to the set of molecular processes found in eukaryotic organisms in which small RNA molecules mediate the silencing or down-regulation of target genes. In insects, RNAi serves a number of functions, including regulation of endogenous genes, anti-viral defense, and defense against transposable elements. Despite being well studied in model organisms, such as Drosophila, the distribution of core RNAi pathway genes and their evolution in insects is not well understood. Here we present the most comprehensive overview of the distribution and diversity of core RNAi pathway genes across 100 insect species, encompassing all currently recognized insect orders. We inferred the phylogenetic origin of insect-specific RNAi pathway genes and also identified several hitherto unrecorded gene expansions using whole-body transcriptome data from the international 1KITE (1000 Insect Transcriptome Evolution) project as well as other resources such as i5K (5000 Insect Genome Project). Specifically, we traced the origin of the double stranded RNA binding protein R2D2 to the last common ancestor of winged insects (Pterygota), the loss of Sid-1/Tag-130 orthologs in Antliophora (fleas, flies and relatives, and scorpionflies in a broad sense), and confirm previous evidence for the splitting of the Argonaute proteins Aubergine and Piwi in Brachyceran flies (Diptera, Brachycera). Our study offers new reference points for future experimental research on RNAi-related pathway genes in insects.

  8. Large scale RNAi reveals the requirement of nuclear envelope breakdown for nuclear import of human papillomaviruses.

    PubMed

    Aydin, Inci; Weber, Susanne; Snijder, Berend; Samperio Ventayol, Pilar; Kühbacher, Andreas; Becker, Miriam; Day, Patricia M; Schiller, John T; Kann, Michael; Pelkmans, Lucas; Helenius, Ari; Schelhaas, Mario

    2014-05-01

    A two-step, high-throughput RNAi silencing screen was used to identify host cell factors required during human papillomavirus type 16 (HPV16) infection. Analysis of validated hits implicated a cluster of mitotic genes and revealed a previously undetermined mechanism for import of the viral DNA (vDNA) into the nucleus. In interphase cells, viruses were endocytosed, routed to the perinuclear area, and uncoated, but the vDNA failed to be imported into the nucleus. Upon nuclear envelope perforation in interphase cells HPV16 infection occured. During mitosis, the vDNA and L2 associated with host cell chromatin on the metaphase plate. Hence, we propose that HPV16 requires nuclear envelope breakdown during mitosis for access of the vDNA to the nucleoplasm. The results accentuate the value of genes found by RNAi screens for investigation of viral infections. The list of cell functions required during HPV16 infection will, moreover, provide a resource for future virus-host cell interaction studies.

  9. Quantitative phase imaging of cellular and subcellular structures for non-invasive screening diagnostics of socially significant diseases

    NASA Astrophysics Data System (ADS)

    Vasilenko, Irina; Metelin, Vladislav; Nasyrov, Marat; Belyakov, Vladimir; Kuznetsov, Alexander; Sukhenko, Evgeniy

    2015-03-01

    The objective of the present study is to increase the quality of the early diagnosis using cytological differential-diagnostic criteria for reactive changes in the nuclear structures of the immunocompetent cells. The morphofunctional status of living cells were estimated in the real time using new technologic platform of the hardware-software complex for phase cell imaging. The level of functional activity for lymphocyte subpopulations was determined on the base of modification of nuclear structures and decreasing of nuclear phase thickness. The dynamics of nuclear parameters was used as the quantitative measuring for cell activating level and increasing of proliferative potential.

  10. The RootScope: a simple high-throughput screening system for quantitating gene expression dynamics in plant roots

    PubMed Central

    2013-01-01

    Background High temperature stress responses are vital for plant survival. The mechanisms that plants use to sense high temperatures are only partially understood and involve multiple sensing and signaling pathways. Here we describe the development of the RootScope, an automated microscopy system for quantitating heat shock responses in plant roots. Results The promoter of Hsp17.6 was used to build a Hsp17.6p:GFP transcriptional reporter that is induced by heat shock in Arabidopsis. An automated fluorescence microscopy system which enables multiple roots to be imaged in rapid succession was used to quantitate Hsp17.6p:GFP response dynamics. Hsp17.6p:GFP signal increased with temperature increases from 28°C to 37°C. At 40°C the kinetics and localization of the response are markedly different from those at 37°C. This suggests that different mechanisms mediate heat shock responses above and below 37°C. Finally, we demonstrate that Hsp17.6p:GFP expression exhibits wave like dynamics in growing roots. Conclusions The RootScope system is a simple and powerful platform for investigating the heat shock response in plants. PMID:24119322

  11. A quantitative screening-level approach to incorporate chemical exposure and risk/safety into alternative assessment evaluations.

    PubMed

    Arnold, Scott M; Greggs, Bill; Goyak, Katy O; Landenberger, Bryce D; Mason, Ann M; Howard, Brett; Zaleski, Rosemary; Howard, Brett; Zaleski, Rosemary T

    2017-03-10

    As the general public and retailers ask for disclosure of chemical ingredients in the marketplace, a number of hazard screening tools were developed to evaluate the so called "greenness" of individual chemical ingredients and/or formulations. The majority of these tools focus only on hazard, often using chemical lists, ignoring the other part of the risk equation: exposure. Using a hazard-only focus can result in regrettable substitutions, changing one chemical ingredient for another that turns out to be more hazardous or shifts the toxicity burden to others. To minimize the incidents of regrettable substitutions, BizNGO describes 'Common Principles' to frame a process for informed substitution. Two of the six principles state reduce hazard and minimize exposure. A number of frameworks have emerged to evaluate and assess alternatives. One framework developed by leading experts under the auspices of the U.S. National Academy of Sciences recommended that hazard and exposure be specifically addressed in the same step when assessing candidate alternatives. For the alternative assessment community, this paper serves as an informational resource for considering exposure in an alternatives assessment using elements of problem formulation; product identity, use, and composition; hazard analysis; exposure analysis; and risk characterization. These conceptual elements build upon practices from government, academia, and industry and are exemplified through two hypothetical case studies demonstrating the questions asked and decisions faced in new product development. These two case studies - inhalation exposure to a generic paint product and environmental exposure to a shampoo rinsed down the drain - demonstrate the criteria, considerations, and methods required to combine exposure models addressing human health and environmental impacts to provide a screening level hazard/exposure (risk) analysis. This paper informs practices for these elements within a comparative risk

  12. Development of a screening method for genetically modified soybean by plasmid-based quantitative competitive polymerase chain reaction.

    PubMed

    Shimizu, Eri; Kato, Hisashi; Nakagawa, Yuki; Kodama, Takashi; Futo, Satoshi; Minegishi, Yasutaka; Watanabe, Takahiro; Akiyama, Hiroshi; Teshima, Reiko; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2008-07-23

    A novel type of quantitative competitive polymerase chain reaction (QC-PCR) system for the detection and quantification of the Roundup Ready soybean (RRS) was developed. This system was designed based on the advantage of a fully validated real-time PCR method used for the quantification of RRS in Japan. A plasmid was constructed as a competitor plasmid for the detection and quantification of genetically modified soy, RRS. The plasmid contained the construct-specific sequence of RRS and the taxon-specific sequence of lectin1 (Le1), and both had 21 bp oligonucleotide insertion in the sequences. The plasmid DNA was used as a reference molecule instead of ground seeds, which enabled us to precisely and stably adjust the copy number of targets. The present study demonstrated that the novel plasmid-based QC-PCR method could be a simple and feasible alternative to the real-time PCR method used for the quantification of genetically modified organism contents.

  13. The Discovery of Geranylgeranyltransferase-I Inhibitors with Novel Scaffolds by the Means of Quantitative Structure-Activity Relationship Modeling, Virtual Screening, and Experimental Validation

    PubMed Central

    Peterson, Yuri K.; Wang, Xiang S.; Casey, Patrick J.; Tropsha, Alexander

    2009-01-01

    Geranylgeranylation is critical to the function of several proteins including Rho, Rap1, Rac, Cdc42, and G-protein gamma subunits. Geranylgeranyltransferase type I (GGTase-I) inhibitors (GGTIs) have therapeutic potential to treat inflammation, multiple sclerosis, atherosclerosis, and many other diseases. Following our standard QSAR modeling workflow, we have developed and rigorously validated Quantitative Structure Activity Relationship (QSAR) models for 48 GGTIs using variable selection k nearest neighbor (kNN), automated lazy learning (ALL), and partial least square (PLS) methods. The QSAR models were employed for virtual screening of 9.5 million commercially available chemicals yielding 47 diverse computational hits. Seven of these compounds with novel scaffolds and high predicted GGTase-I inhibitory activities were tested in vitro, and all were found to be bona fide and selective micromolar inhibitors. Notably, these novel hits could not be identified using traditional similarity search. These data demonstrate that rigorously developed QSAR models can serve as reliable virtual screening tools. PMID:19537691

  14. A novel high-throughput and quantitative method based on visible color shifts for screening Bacillus subtilis THY-15 for surfactin production.

    PubMed

    Yang, Huan; Yu, Huimin; Shen, Zhongyao

    2015-08-01

    A novel chromatic visible screening method using bromothymol blue (BTB) as a color indicator and cetylpyridinium chloride (CPC) as a mediator was constructed to obtain the high titer surfactin-producing strains. The reliability and quantification accuracy of color shift were also confirmed. Regular chromatic responses from faint yellow-green to dark green and bright blue reflected the different ranges of surfactin concentrations. Moreover, the quantitative accuracy of surfactin quantification in the range of 100-500 mg/L was verified by reverse-phase high-performance liquid chromatography (RP-HPLC) using different fermentation supernatant samples. Using this CPC-BTB method, a superior surfactin producer, Bacillus subtilis THY-15, was successfully screened. The producer's surfactin (Srf) titer reached 1240 mg/L. RP-HPLC analysis of THY-15 revealed four surfactin isoforms. As identified by amino acid analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, the isoforms of surfactin in fraction 1, 2 and 4 had the same circular peptide sequence of Glu-Leu-Leu-Val-Asp-Leu-Leu but different iso-C13, C14 and C15 fatty acid chains, but the isoform in fraction 3 possessed a special peptide sequence of Glu-Val-Leu-Leu-Asp-Leu-Val.

  15. Screening biological stains with qPCR versus lateral flow immunochromatographic test strips: a quantitative comparison using analytical figures of merit.

    PubMed

    Oechsle, Crystal Simson; Haddad, Sandra; Sgueglia, Joanne B; Grgicak, Catherine M

    2014-01-01

    Biological fluid identification is an important facet of evidence examination in forensic laboratories worldwide. While identifying bodily fluids may provide insight into which downstream DNA methods to employ, these screening techniques consume a vital portion of the available evidence, are usually qualitative, and rely on visual interpretation. In contrast, qPCR yields information regarding the amount and proportion of amplifiable genetic material. In this study, dilution series of either semen or male saliva were prepared in either buffer or female blood. The samples were subjected to both lateral flow immunochromatographic test strips and qPCR analysis. Analytical figures of merit-including sensitivity, minimum distinguishable signal (MDS) and limit of detection (LOD)-were calculated and compared between methods. By applying the theory of the propagation of random errors, LODs were determined to be 0.05 μL of saliva for the RSID™ Saliva cards, 0.03 μL of saliva for Quantifiler(®) Duo, and 0.001 μL of semen for Quantifiler(®) Duo. In conclusion, quantitative PCR was deemed a viable and effective screening method for subsequent DNA profiling due to its stability in different matrices, sensitivity, and low limits of detection.

  16. Application of Quantitative Structure–Activity Relationship Models of 5-HT1A Receptor Binding to Virtual Screening Identifies Novel and Potent 5-HT1A Ligands

    PubMed Central

    2015-01-01

    The 5-hydroxytryptamine 1A (5-HT1A) serotonin receptor has been an attractive target for treating mood and anxiety disorders such as schizophrenia. We have developed binary classification quantitative structure–activity relationship (QSAR) models of 5-HT1A receptor binding activity using data retrieved from the PDSP Ki database. The prediction accuracy of these models was estimated by external 5-fold cross-validation as well as using an additional validation set comprising 66 structurally distinct compounds from the World of Molecular Bioactivity database. These validated models were then used to mine three major types of chemical screening libraries, i.e., drug-like libraries, GPCR targeted libraries, and diversity libraries, to identify novel computational hits. The five best hits from each class of libraries were chosen for further experimental testing in radioligand binding assays, and nine of the 15 hits were confirmed to be active experimentally with binding affinity better than 10 μM. The most active compound, Lysergol, from the diversity library showed very high binding affinity (Ki) of 2.3 nM against 5-HT1A receptor. The novel 5-HT1A actives identified with the QSAR-based virtual screening approach could be potentially developed as novel anxiolytics or potential antischizophrenic drugs. PMID:24410373

  17. RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways

    PubMed Central

    Wu, Jun; Pei, Rongjuan; Xu, Yang; Yang, Dongliang; Roggendorf, Michael; Lu, Mengji

    2013-01-01

    Background & Aims Our previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi. Methods Primary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with defined siRNAs. The mRNA levels of target genes and ISGs were detected by real-time RT-PCR. The involvement of the signaling pathways including RIG-I/MDA5, PKR, and TLR3/7/8/9 was examined by specific inhibition and the analysis of their activation by Western blotting. Results In PMHs from WHV Tg mice, specific siRNAs targeting WHV, mouse β-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-β, MxA, and IP-10. The enhanced ISG expression by siRNA transfection were abolished by siRNA-specific 2′-O-methyl antisense RNA and the inhibitors 2-AP and chloroquine blocking PKR and other TLR-mediated signaling pathways. Furthermore, Western blotting revealed that RNAi results in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-êB, indicating the possible role of IRF3 in the RNAi-directed induction of ISGs. In contrast, silencing of RIG-I and MDA5 failed to block RNAi-mediated MxA induction. Conclusions RNAi is capable of enhancing innate immune responses through the PKR- and TLR-dependent signaling pathways in primary hepatocytes. The immune stimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo. PMID:23700487

  18. Clinical validation of a type-specific real-time quantitative human papillomavirus PCR against the performance of hybrid capture 2 for the purpose of cervical cancer screening.

    PubMed

    Depuydt, C E; Benoy, I H; Beert, J F A; Criel, A M; Bogers, J J; Arbyn, M

    2012-12-01

    To be acceptable for use in cervical cancer screening, a new assay that detects DNA of high-risk human papillomavirus (hrHPV) types must demonstrate high reproducibility and performance not inferior to that of a clinically validated HPV test. In the present study, a real-time quantitative PCR (qPCR) assay targeting the E6 and E7 genes of hrHPV was compared with Hybrid Capture 2 (hc2) in a Belgian cervical cancer screening setting. In women >30 years old, the sensitivity and specificity for intraepithelial neoplasias of grade 2 or worse (93 cases of cervical intraepithelial neoplasias of grade 2 or worse (CIN2+) and 1,207 cases of no CIN or CIN1) were 93.6% and 95.6%, respectively, and those of hc2 were 83.9% and 94.5%, respectively {relative sensitivity of qPCR/hc2 = 1.12 [95% confidence interval (CI), 1.01 to 1.23]; relative specificity = 1.01 [95% CI, 0.99 to 1.03]}. A score test showed that the sensitivity (P < 0.0001) and specificity (P < 0.0001) of the qPCR assay were not inferior to those of hc2 at the required thresholds of 90% and 98%, respectively. The overall agreement of hrHPV positivity between the two runs of the qPCR tests was 98.7% (95% CI, 97.5 to 99.4%), with a kappa value of 0.96 (95% CI, 0.83 to 1.00). The qPCR assay used in this study can be considered a reliable HPV assay that fulfills the clinical validation criteria defined for use in cervical cancer screening.

  19. Interspecies quantitative structure-activity relationships (QSARs) for eco-toxicity screening of chemicals: the role of physicochemical properties.

    PubMed

    Furuhama, A; Hasunuma, K; Aoki, Y

    2015-01-01

    In addition to molecular structure profiles, descriptors based on physicochemical properties are useful for explaining the eco-toxicities of chemicals. In a previous study we reported that a criterion based on the difference between the partition coefficient (log POW) and distribution coefficient (log D) values of chemicals enabled us to identify aromatic amines and phenols for which interspecies relationships with strong correlations could be developed for fish-daphnid and algal-daphnid toxicities. The chemicals that met the log D-based criterion were expected to have similar toxicity mechanisms (related to membrane penetration). Here, we investigated the applicability of log D-based criteria to the eco-toxicity of other kinds of chemicals, including aliphatic compounds. At pH 10, use of a log POW - log D > 0 criterion and omission of outliers resulted in the selection of more than 100 chemicals whose acute fish toxicities or algal growth inhibition toxicities were almost equal to their acute daphnid toxicities. The advantage of log D-based criteria is that they allow for simple, rapid screening and prioritizing of chemicals. However, inorganic molecules and chemicals containing certain structural elements cannot be evaluated, because calculated log D values are unavailable.

  20. A Rapid, Semi-Quantitative Assay to Screen for Modulators of Alpha-Synuclein Oligomerization Ex vivo

    PubMed Central

    Delenclos, Marion; Trendafilova, Teodora; Jones, Daryl R.; Moussaud, Simon; Baine, Ann-Marie; Yue, Mei; Hirst, Warren D.; McLean, Pamela J.

    2016-01-01

    Alpha synuclein (αsyn) aggregates are associated with the pathogenesis of Parkinson's disease and others related disorders. Although modulation of αsyn aggregation is an attractive therapeutic target, new powerful methodologies are desperately needed to facilitate in vivo screening of novel therapeutics. Here, we describe an in vivo rodent model with the unique ability to rapidly track αsyn-αsyn interactions and thus oligomerization using a bioluminescent protein complementation strategy that monitors spatial and temporal αsyn oligomerization ex vivo. We find that αsyn forms oligomers in vivo as early as 1 week after stereotactic AAV injection into rat substantia nigra. Strikingly, although abundant αsyn expression is also detected in striatum at 1 week, no αsyn oligomers are detected at this time point. By 4 weeks, oligomerization of αsyn is detected in both striatum and substantia nigra homogenates. Moreover, in a proof-of-principle experiment, the effect of a previously described Hsp90 inhibitor known to prevent αsyn oligomer formation, demonstrates the utility of this rapid and sensitive animal model to monitor αsyn oligomerization status in the rat brain. PMID:26834539

  1. A Rapid, Semi-Quantitative Assay to Screen for Modulators of Alpha-Synuclein Oligomerization Ex vivo.

    PubMed

    Delenclos, Marion; Trendafilova, Teodora; Jones, Daryl R; Moussaud, Simon; Baine, Ann-Marie; Yue, Mei; Hirst, Warren D; McLean, Pamela J

    2015-01-01

    Alpha synuclein (αsyn) aggregates are associated with the pathogenesis of Parkinson's disease and others related disorders. Although modulation of αsyn aggregation is an attractive therapeutic target, new powerful methodologies are desperately needed to facilitate in vivo screening of novel therapeutics. Here, we describe an in vivo rodent model with the unique ability to rapidly track αsyn-αsyn interactions and thus oligomerization using a bioluminescent protein complementation strategy that monitors spatial and temporal αsyn oligomerization ex vivo. We find that αsyn forms oligomers in vivo as early as 1 week after stereotactic AAV injection into rat substantia nigra. Strikingly, although abundant αsyn expression is also detected in striatum at 1 week, no αsyn oligomers are detected at this time point. By 4 weeks, oligomerization of αsyn is detected in both striatum and substantia nigra homogenates. Moreover, in a proof-of-principle experiment, the effect of a previously described Hsp90 inhibitor known to prevent αsyn oligomer formation, demonstrates the utility of this rapid and sensitive animal model to monitor αsyn oligomerization status in the rat brain.

  2. High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

    PubMed

    Guo, Baoshan; Lei, Cheng; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

    2017-04-11

    The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry.

  3. Silencing of odorant binding protein gene AlinOBP4 by RNAi induces declining electrophysiological responses of Adelphocoris lineolatus to six semiochemicals.

    PubMed

    Zhang, Xue-Ying; Zhu, Xiao-Qiang; Gu, Shao-Hua; Zhou, Yan-Le; Wang, Song-Ying; Zhang, Yong-Jun; Guo, Yu-Yuan

    2016-06-06

    Odorant binding proteins (OBPs) are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. Here, we injected AlinOBP4-siRNA into the conjunctivum between prothorax and mesothorax of the lucerne plant bug, Adelphocoris lineolatus and evaluated the silencing of AlinOBP4 by reverse transcription polymerase chain reaction (RT-PCR) analysis, quantitative real-time PCR (qPCR) test and electroantennogram (EAG) assay. The combination of RT-PCR and qPCR analyses revealed that the levels of messenger RNA transcript were significantly reduced ∼95% in AlinOBP4-siRNA-treated A. lineolatus males and ∼75% in RNAi-treated females within 48 hours. It was found that there are different EAG responses between male and female bugs when the AlinOBP4 gene was silenced by RNAi. The EAGs of A. lineolatus to two plant volatiles, tridecanal and hexyl alcohol, were reduced 9.09% and 79.45% in RNAi-treated males, 62.08% and 62.08% in RNAi-treated females compared to the controls, separately. Antennae of RNAi-treated bugs showed significantly lower electrophysiological responses to four sex pheromone analogs, butyl butanoate, 1-hexyl butyrate, (E)-2-hexenyl butyrate and hexyl hexanoate. The EAG recordings were reduced 35.43%, 35.24%, 39.96% and 78.47% in RNAi-treated males and 64.52%, 18.13%, 36.88% and 49.52% in RNAi-treated females, respectively. The results suggested that AlinOBP4 might play dual-roles in the identification of plant volatiles and sex pheromones. It was suspected that AlinOBP4 may have different functions in odor perception between male and female A. lineolatus.

  4. RNAi pathway participates in chromosome segregation in mammalian cells.

    PubMed

    Huang, Chuan; Wang, Xiaolin; Liu, Xu; Cao, Shuhuan; Shan, Ge

    2015-01-01

    The RNAi machinery is a mighty regulator in a myriad of life events. Despite lines of evidence that small RNAs and components of the RNAi pathway may be associated with structure and behavior of mitotic chromosomes in diverse organisms, a direct role of the RNAi pathway in mammalian mitotic chromosome segregation remains elusive. Here we report that Dicer and AGO2, two central components of the mammalian RNAi pathway, participate in the chromosome segregation. Knockdown of Dicer or AGO2 results in a higher incidence of chromosome lagging, and this effect is independent from microRNAs as examined with DGCR8 knockout cells. Further investigation has revealed that α-satellite RNA, a noncoding RNA derived from centromeric repeat region, is managed by AGO2 under the guidance of endogenous small interference RNAs (ASAT siRNAs) generated by Dicer. Furthermore, the slicer activity of AGO2 is essential for the chromosome segregation. Level and distribution of chromosome-associated α-satellite RNA have crucial regulatory effect on the localization of centromeric proteins such as centromere protein C1 (CENPC1). With these results, we also provide a paradigm in which the RNAi pathway participates in vital cellular events through the maintenance of level and distribution of noncoding RNAs in cells.

  5. Intravenous, non-viral RNAi gene therapy of brain cancer.

    PubMed

    Pardridge, William M

    2004-07-01

    RNA interference (RNAi) has the potential to knock down oncogenes in cancer, including brain cancer. However, the therapeutic potential of RNAi will not be realised until the rate-limiting step of delivery is solved. The development of RNA-based therapeutics is not practical, due to the instability of RNA in vivo. However, plasmid DNA can be engineered to express short hairpin RNA (shRNA), similar to endogenous microRNAs. Intravenous, non-viral RNAi-based gene therapy is enabled with a new gene-targeting technology, which encapsulates the plasmid DNA inside receptor-specific pegylated immunoliposomes (PILs). The feasibility of this RNAi approach was evaluated by showing it was possible to achieve a 90% knockdown of brain tumour-specific gene expression with a single intravenous injection in adult rats or mice with intracranial brain cancer. The survival of mice with intracranial human brain cancer was extended by nearly 90% with weekly intravenous injections of PILs carrying plasmid DNA expressing a shRNA directed against the human epidermal growth factor receptor. RNAi-based gene therapy can be coupled with gene therapy that replaces mutated tumour suppressor genes to build a polygenic approach to the gene therapy of cancer.

  6. RNAi-based targeted gene knockdown in the model oleaginous microalgae Nannochloropsis oceanica.

    PubMed

    Wei, Li; Xin, Yi; Wang, Qintao; Yang, Juan; Hu, Hanhua; Xu, Jian

    2017-03-01

    Microalgae are promising feedstock for renewable fuels such as biodiesel, yet development of industrial oleaginous strains has been hindered by the paucity and inefficiency of reverse genetics tools. Here we established an efficient RNAi-based targeted gene-knockdown method for Nannochloropsis spp., which are emerging model organisms for industrial microalgal oil production. The method achieved a 40-80% success rate in Nannochloropsis oceanica strain IMET1. When transcript level of one carbonic anhydrase (CA) was inhibited by 62-83% via RNAi, mutant cells exhibited photosynthetic oxygen evolution (POE) rates that were 68-100% higher than wild-type (WT) at pH 6.0, equivalent to WT at pH 8.2, yet 39-45% lower than WT at pH 9.0. Moreover, the mutant POE rates were negatively correlated with the increase of culture pH, an exact opposite of WT. Thus, a dynamic carbon concentration mechanism (CCM) that is highly sensitive to pH homeostasis was revealed, where the CA inhibition likely partially abrogated the mechanism that normally deactivates CCM under a high level of dissolved CO2 . Extension of the method to another sequenced N. oceanica strain of CCMP 1779 demonstrated comparable performance. Finally, McrBC-PCR followed by bisulfite sequencing revealed that the gene knockdown is mediated by the CG, CHG and CHH types of DNA methylation at the coding region of the targeted gene. The efficiency, robustness and general applicability of this reverse genetics approach suggested the possibility of large-scale RNAi-based gene function screening in industrial microalgae.

  7. RNA Viruses and RNAi: Quasispecies Implications for Viral Escape.

    PubMed

    Presloid, John B; Novella, Isabel S

    2015-06-19

    Due to high mutation rates, populations of RNA viruses exist as a collection of closely related mutants known as a quasispecies. A consequence of error-prone replication is the potential for rapid adaptation of RNA viruses when a selective pressure is applied, including host immune systems and antiviral drugs. RNA interference (RNAi) acts to inhibit protein synthesis by targeting specific mRNAs for degradation and this process has been developed to target RNA viruses, exhibiting their potential as a therapeutic against infections. However, viruses containing mutations conferring resistance to RNAi were isolated in nearly all cases, underlining the problems of rapid viral evolution. Thus, while promising, the use of RNAi in treating or preventing viral diseases remains fraught with the typical complications that result from high specificity of the target, as seen in other antiviral regimens.

  8. Antitumor therapeutic application of self-assembled RNAi-AuNP nanoconstructs: Combination of VEGF-RNAi and photothermal ablation

    PubMed Central

    Son, Sejin; Kim, Namho; You, Dong Gil; Yoon, Hong Yeol; Yhee, Ji Young; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, Sun Hwa

    2017-01-01

    Nucleic acid-directed self-assembly provides an attractive method to fabricate prerequisite nanoscale structures for a wide range of technological applications due to the remarkable programmability of DNA/RNA molecules. In this study, exquisite RNAi-AuNP nanoconstructs with various geometries were developed by utilizing anti-VEGF siRNA molecules as RNAi-based therapeutics in addition to their role as building blocks for programmed self-assembly. In particular, the anti-VEGF siRNA-functionalized AuNP nanoconstructs can take additional advantage of gold-nanoclusters for photothermal cancer therapeutic agent. A noticeable technical aspect of self-assembled RNAi-AuNP nanoconstructs in this study is the precise conjugation and separation of designated numbers of therapeutic siRNA onto AuNP to develop highly sophisticated RNA-based building blocks capable of creating various geometries of RNAi-AuNP nano-assemblies. The therapeutic potential of RNAi-AuNP nanoconstructs was validated in vivo as well as in vitro by combining heat generation capability of AuNP and anti-angiogenesis mechanism of siRNA. This strategy of combining anti-VEGF mechanism for depleting angiogenesis process at initial tumor progression and complete ablation of residual tumors with photothermal activity of AuNP at later tumor stage showed effective tumor growth inhibition and tumor ablation with PC-3 tumor bearing mice. PMID:28042312

  9. Development of RNAi technology for targeted therapy--a track of siRNA based agents to RNAi therapeutics.

    PubMed

    Zhou, Yinjian; Zhang, Chunling; Liang, Wei

    2014-11-10

    RNA interference (RNAi) was intensively studied in the past decades due to its potential in therapy of diseases. The target specificity and universal treatment spectrum endowed siRNA advantages over traditional small molecules and protein drugs. However, barriers exist in the blood circulation system and the diseased tissues blocked the actualization of RNAi effect, which raised function versatility requirements to siRNA therapeutic agents. Appropriate functionalization of siRNAs is necessary to break through these barriers and target diseased tissues in local or systemic targeted application. In this review, we summarized that barriers exist in the delivery process and popular functionalized technologies for siRNA such as chemical modification and physical encapsulation. Preclinical targeted siRNA delivery and the current status of siRNA based RNAi therapeutic agents in clinical trial were reviewed and finally the future of siRNA delivery was proposed. The valuable experience from the siRNA agent delivery study and the RNAi therapeutic agents in clinical trial paved ways for practical RNAi therapeutics to emerge early.

  10. Enhancement of larval RNAi efficiency by over-expressing Argonaute2 in Bombyx mori.

    PubMed

    Li, Zhiqian; Zeng, Baosheng; Ling, Lin; Xu, Jun; You, Lang; Aslam, Abu F M; Tan, Anjiang; Huang, Yongping

    2015-01-01

    RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.

  11. ElectroTaxis-on-a-Chip (ETC): an integrated quantitative high-throughput screening platform for electrical field-directed cell migration.

    PubMed

    Zhao, Siwei; Zhu, Kan; Zhang, Yan; Zhu, Zijie; Xu, Zhengping; Zhao, Min; Pan, Tingrui

    2014-11-21

    Both endogenous and externally applied electrical stimulation can affect a wide range of cellular functions, including growth, migration, differentiation and division. Among those effects, the electrical field (EF)-directed cell migration, also known as electrotaxis, has received broad attention because it holds great potential in facilitating clinical wound healing. Electrotaxis experiment is conventionally conducted in centimetre-sized flow chambers built in Petri dishes. Despite the recent efforts to adapt microfluidics for electrotaxis studies, the current electrotaxis experimental setup is still cumbersome due to the needs of an external power supply and EF controlling/monitoring systems. There is also a lack of parallel experimental systems for high-throughput electrotaxis studies. In this paper, we present a first independently operable microfluidic platform for high-throughput electrotaxis studies, integrating all functional components for cell migration under EF stimulation (except microscopy) on a compact footprint (the same as a credit card), referred to as ElectroTaxis-on-a-Chip (ETC). Inspired by the R-2R resistor ladder topology in digital signal processing, we develop a systematic approach to design an infinitely expandable microfluidic generator of EF gradients for high-throughput and quantitative studies of EF-directed cell migration. Furthermore, a vacuum-assisted assembly method is utilized to allow direct and reversible attachment of our device to existing cell culture media on biological surfaces, which separates the cell culture and device preparation/fabrication steps. We have demonstrated that our ETC platform is capable of screening human cornea epithelial cell migration under the stimulation of an EF gradient spanning over three orders of magnitude. The screening results lead to the identification of the EF-sensitive range of that cell type, which can provide valuable guidance to the clinical application of EF-facilitated wound healing.

  12. Quantitative High-Throughput Drug Screening Identifies Novel Classes of Drugs with Anticancer Activity in Thyroid Cancer Cells: Opportunities for Repurposing

    PubMed Central

    Zhang, Lisa; He, Mei; Zhang, Yaqin; Nilubol, Naris; Shen, Min

    2012-01-01

    Context: Despite increased understanding of the pathogenesis and targets for thyroid cancer and other cancers, developing a new anticancer chemical agent remains an expensive and long process. An alternative approach is the exploitation of clinically used and/or bioactive compounds. Objective: Our objective was to identify agents with an anticancer effect in thyroid cancer cell lines using quantitative high-throughput screening (qHTS). Design: We used the newly assembled National Institutes of Health Chemical Genomic Center's pharmaceutical collection, which contains 2816 clinically approved drugs and bioactive compounds to perform qHTS. Results: Multiple agents, across a variety of therapeutic categories and with different modes of action, were found to have an antiproliferative effect. We found the following therapeutic categories were the most enriched categories with antiproliferative activity: cardiotonic and antiobesity agents. Sixteen agents had an efficacy of greater than 60% and a 50% inhibitory concentration (IC50) in the nanomolar range. We validated the results of the qHTS using two agents (bortezomib and ouabain) in additional cell lines representing different histological subtypes of thyroid cancer and with different mutations (BRAF V600E, RET/PTC1, p53, PTEN). Both agents induced apoptosis, and ouabain also caused cell cycle arrest. Conclusions: To our knowledge, this is the first study to use qHTS of a large drug library to identify candidate drugs for anticancer therapy. Our results indicate such a screening approach can lead to the discovery of novel agents in different therapeutic categories and drugs with nonclassic chemotherapy mode of action. Our approach could lead to drug repurposing and accelerate clinical trials of compounds with well-established pharmacokinetics and toxicity profiles. PMID:22170715

  13. ElectroTaxis-on-a-Chip (ETC): an integrated quantitative high-throughput screening platform for electrical field-directed cell migration†

    PubMed Central

    Zhao, Siwei; Zhu, Kan; Zhang, Yan; Zhu, Zijie; Xu, Zhengping

    2015-01-01

    Both endogenous and externally applied electrical stimulation can affect a wide range of cellular functions, including growth, migration, differentiation and division. Among those effects, the electrical field (EF)-directed cell migration, also known as electrotaxis, has received broad attention because it holds great potential in facilitating clinical wound healing. Electrotaxis experiment is conventionally conducted in centimetre-sized flow chambers built in Petri dishes. Despite the recent efforts to adapt microfluidics for electrotaxis studies, the current electrotaxis experimental setup is still cumbersome due to the needs of an external power supply and EF controlling/monitoring systems. There is also a lack of parallel experimental systems for high-throughput electrotaxis studies. In this paper, we present a first independently operable microfluidic platform for high-throughput electrotaxis studies, integrating all functional components for cell migration under EF stimulation (except microscopy) on a compact footprint (the same as a credit card), referred to as ElectroTaxis-on-a-Chip (ETC). Inspired by the R–2R resistor ladder topology in digital signal processing, we develop a systematic approach to design an infinitely expandable microfluidic generator of EF gradients for high-throughput and quantitative studies of EF-directed cell migration. Furthermore, a vacuum-assisted assembly method is utilized to allow direct and reversible attachment of our device to existing cell culture media on biological surfaces, which separates the cell culture and device preparation/fabrication steps. We have demonstrated that our ETC platform is capable of screening human cornea epithelial cell migration under the stimulation of an EF gradient spanning over three orders of magnitude. The screening results lead to the identification of the EF-sensitive range of that cell type, which can provide valuable guidance to the clinical application of EF-facilitated wound healing

  14. A quantitative method for acylcarnitines and amino acids using high resolution chromatography and tandem mass spectrometry in newborn screening dried blood spot analysis.

    PubMed

    Miller, John H; Poston, Philip A; Karnes, H Thomas

    2012-08-15

    We have developed a high resolution liquid chromatographic separation with electrospray ionization (ESI) mass spectrometry detection for the combined analysis of twelve acylcarnitines and seven amino acids commonly measured in newborn screening heritable metabolic disorders. Samples were prepared by punching 3.2 mm disks out of dried blood spots and extracting with a mixture of methanol and 0.1% formic acid containing stable isotopically labeled internal standards. Analysis was performed on an UHPLC system using a HILIC amide, 2.1 mm × 50 mm, 1.7 μm column. A normal phase gradient, employing 10mM ammonium acetate in 90:10 acetonitrile/water for mobile phase B and 0.1% formic acid in water for mobile phase A, was used. Optimized multiple reaction monitoring (MRM) was used for detection of amino acids and acylcarnitines on a Waters Premier mass spectrometer. Quantification of analytes was performed using internal calibration by fortification of sodium heparin whole blood with analytes at appropriate levels to encompass the range around the reported cut-off values. The method was fully validated with respect to precision, accuracy, recovery, linearity, matrix suppression and extraction robustness. Precision and accuracy were evaluated over 3 days and determined to be acceptable with an overall precision within 10% and accuracy within 15% of theoretical for all analytes except for acetylcarnitne at one fortified level, which quantitated 21.8% lower than the expected value. This method is suitable as a second-tier test for newborn screening of specific disorders associated with abnormal levels of acylcarnitines and amino acids, potentially reducing false positive cases and shortening the time to diagnosis.

  15. Maximizing the quantitative accuracy and reproducibility of Förster resonance energy transfer measurement for screening by high throughput widefield microscopy

    PubMed Central

    Schaufele, Fred

    2013-01-01

    Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) provides insights into the proximities and orientations of FPs as surrogates of the biochemical interactions and structures of the factors to which the FPs are genetically fused. As powerful as FRET methods are, technical issues have impeded their broad adoption in the biologic sciences. One hurdle to accurate and reproducible FRET microscopy measurement stems from variable fluorescence backgrounds both within a field and between different fields. Those variations introduce errors into the precise quantification of fluorescence levels on which the quantitative accuracy of FRET measurement is highly dependent. This measurement error is particularly problematic for screening campaigns since minimal well-to-well variation is necessary to faithfully identify wells with altered values. High content screening depends also upon maximizing the numbers of cells imaged, which is best achieved by low magnification high throughput microscopy. But, low magnification introduces flat-field correction issues that degrade the accuracy of background correction to cause poor reproducibility in FRET measurement. For live cell imaging, fluorescence of cell culture media in the fluorescence collection channels for the FPs commonly used for FRET analysis is a high source of background error. These signal-to-noise problems are compounded by the desire to express proteins at biologically meaningful levels that may only be marginally above the strong fluorescence background. Here, techniques are presented that correct for background fluctuations. Accurate calculation of FRET is realized even from images in which a non-flat background is 10-fold higher than the signal. PMID:23927839

  16. Quantitative and Qualitative Analysis of Circulating Cell-Free DNA Can Be Used as an Adjuvant Tool for Prostate Cancer Screening: A Meta-Analysis.

    PubMed

    Yin, Changqing; Luo, Changliang; Hu, Wei; Ding, Xu; Yuan, Chunhui; Wang, Fubing

    2016-01-01

    As part of "liquid biopsy," lots of literature indicated the potential diagnostic value of circulating cell-free DNA (cfDNA) in the management of prostate cancer (PCa). However, the literature on the accuracy of cfDNA detection in PCa has been inconsistent. Hence, we performed this meta-analysis to assess the diagnostic value of cfDNA in PCa. A total of 19 articles were included in this analysis according to the inclusion and exclusion criteria. We then investigated two main subgroups in this meta-analysis, including qualitative analysis of abnormal level of cfDNA and qualitative analysis of single-gene methylation alterations. Overall, the results of quantitative analysis showed sensitivity of 0.73 (95% CI, 0.62-0.82) and specificity of 0.80 (95% CI, 0.70-0.87), with an area under the curve (AUC) of 0.83 (95% CI, 0.80-0.86). For qualitative assessment, the values were 0.34 (95% CI, 0.22-0.48), 0.99 (95% CI, 0.97-1.00), and 0.91 (95% CI, 0.88-0.93), respectively. Our results suggest the pooled specificity of each subgroup is much higher than the specificity of prostate-specific antigen (PSA). However, they are not recommended for PCa screening alone, because their sensitivities are not higher than the conventional serum biomarkers PSA. We conclude that analysis of cfDNA can be used as an adjuvant tool for PCa screening.

  17. Quantitative and Qualitative Analysis of Circulating Cell-Free DNA Can Be Used as an Adjuvant Tool for Prostate Cancer Screening: A Meta-Analysis

    PubMed Central

    Luo, Changliang; Hu, Wei; Ding, Xu; Yuan, Chunhui

    2016-01-01

    As part of “liquid biopsy,” lots of literature indicated the potential diagnostic value of circulating cell-free DNA (cfDNA) in the management of prostate cancer (PCa). However, the literature on the accuracy of cfDNA detection in PCa has been inconsistent. Hence, we performed this meta-analysis to assess the diagnostic value of cfDNA in PCa. A total of 19 articles were included in this analysis according to the inclusion and exclusion criteria. We then investigated two main subgroups in this meta-analysis, including qualitative analysis of abnormal level of cfDNA and qualitative analysis of single-gene methylation alterations. Overall, the results of quantitative analysis showed sensitivity of 0.73 (95% CI, 0.62–0.82) and specificity of 0.80 (95% CI, 0.70–0.87), with an area under the curve (AUC) of 0.83 (95% CI, 0.80–0.86). For qualitative assessment, the values were 0.34 (95% CI, 0.22–0.48), 0.99 (95% CI, 0.97–1.00), and 0.91 (95% CI, 0.88–0.93), respectively. Our results suggest the pooled specificity of each subgroup is much higher than the specificity of prostate-specific antigen (PSA). However, they are not recommended for PCa screening alone, because their sensitivities are not higher than the conventional serum biomarkers PSA. We conclude that analysis of cfDNA can be used as an adjuvant tool for PCa screening. PMID:27766004

  18. Screening for seemingly healthy newborns with congenital cytomegalovirus infection by quantitative real-time polymerase chain reaction using newborn urine: an observational study

    PubMed Central

    Yamaguchi, Akira; Oh-ishi, Tsutomu; Arai, Takashi; Sakata, Hideaki; Adachi, Nodoka; Asanuma, Satoshi; Oguma, Eiji; Kimoto, Hirofumi; Matsumoto, Jiro; Fujita, Hidetoshi; Uesato, Tadashi; Fujita, Jutaro; Shirato, Ken; Ohno, Hideki; Kizaki, Takako

    2017-01-01

    Objective Approximately 8–10% of newborns with asymptomatic congenital cytomegalovirus (cCMV) infection develop sensorineural hearing loss (SNHL). However, the relationship between CMV load, SNHL and central nervous system (CNS) damage in cCMV infection remains unclear. This study aimed to examine the relationship between urinary CMV load, SNHL and CNS damage in newborns with cCMV infection. Study design The study included 23 368 newborns from two maternity hospitals in Saitama Prefecture, Japan. Urine screening for cCMV infection (quantitative real-time PCR) and newborn hearing screening (automated auditory brainstem response (AABR) testing) were conducted within 5 days of birth to examine the incidence of cCMV infection and SNHL, respectively. CNS damage was assessed by MRI of cCMV-infected newborns. Results The incidence of cCMV infection was 60/23 368 (0.257%; 95% CI 0.192% to 0.322%). The geometric mean urinary CMV DNA copy number in newborns with cCMV was 1.79×106 copies/mL (95% CI 7.97×105 to 4.02×106). AABR testing revealed abnormalities in 171 of the 22 229 (0.769%) newborns whose parents approved hearing screening. Of these 171 newborns, 22 had SNHL (12.9%), and 5 of these 22 were infected with cCMV (22.7%). Newborns with both cCMV and SNHL had a higher urinary CMV DNA copy number than newborns with cCMV without SNHL (p=0.036). MRI revealed CNS damage, including white matter abnormalities, in 83.0% of newborns with cCMV. Moreover, newborns with CNS damage had a significantly greater urinary CMV load than newborns without CNS damage (p=0.013). Conclusions We determined the incidence of cCMV infection and urinary CMV DNA copy number in seemingly healthy newborns from two hospitals in Saitama Prefecture. SNHL and CNS damage were associated with urinary CMV DNA copy number. Quantification of urinary CMV load may effectively predict the incidence of late-onset SNHL and neurodevelopmental disorders. PMID:28110288

  19. RNAi in fission yeast finds new targets and new ways of targeting at the nuclear periphery.

    PubMed

    Holoch, Daniel; Moazed, Danesh

    2012-04-15

    RNAi in Schizosaccharomyces pombe is critical for centromeric heterochromatin formation. It has remained unclear, however, whether RNAi also regulates the expression of protein-coding loci. In the April 1, 2012, issue of Genes & Development, Woolcock and colleagues (pp. 683-667) reported an elegant mechanism for the conditional RNAi-mediated repression of stress response genes involving association with Dcr1 at the nuclear pore. Unexpectedly, the initial targeting of RNAi components to these genes does not require small RNA guides.

  20. Efficiency of gene silencing in Arabidopsis: direct inverted repeats vs. transitive RNAi vectors.

    SciTech Connect

    Filichkin, Sergei A; DiFazio, Steven P; Brunner, Amy M; Davis, John M; Yang, Zamin Koo; Kalluri, Udaya C; Arias, Renee S; Etherington, Elizabeth; Tuskan, Gerald A; Strauss, S

    2007-01-01

    We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1, ETTIN and TTG1). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required.

  1. Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".

    PubMed

    Vaghi, Valentina; Polacchini, Alessio; Baj, Gabriele; Pinheiro, Vera L M; Vicario, Annalisa; Tongiorgi, Enrico

    2014-10-03

    The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5'- and 3'UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5'- and 3'UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5'UTR or a 3'UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5'UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3'UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent "a quantitative code" for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests.

  2. Rapid quantitative detection of Brucella melitensis by a label-free impedance immunosensor based on a gold nanoparticle-modified screen-printed carbon electrode.

    PubMed

    Wu, Haiyun; Zuo, Yueming; Cui, Chuanjin; Yang, Wei; Ma, Haili; Wang, Xiaowen

    2013-07-04

    A rapid and simple method for quantitative monitoring of Brucella melitensis using electrochemical impedance spectroscopy (EIS) is reported for the first time. The label-free immunosensors were fabricated by immobilizing Brucella melitensis antibody on the surface of gold nanoparticle-modified screen-printed carbon electrodes (GNP-SPCEs). Cyclic voltammetry (CV) and EIS were used to characterize the Brucella melitensis antigen interaction on the surface of GNP-SPCEs with antibody. A general electronic equivalent model of an electrochemical cell was introduced for interpretation of the impedance components of the system. The results showed that the change in electron-transfer resistance (Rct) was significantly different due to the binding of Brucella melitensis cells. A linear relationship between the Rct variation and logarithmic value of the cell concentration was found from 4 × 10(4) to 4 × 10(6) CFU/mL in pure culture. The label-free impedance biosensor was able to detect as low as 1 × 10(4) and 4 × 10(5) CFU/mL of Brucella melitensis in pure culture and milk samples, respectively, in less than 1.5 h. Moreover, a good selectivity versus Escherichia coli O157:H7 and Staphylococcus aureus cells was obtained for our developed immunosensor demonstrating its specificity towards only Brucella melitensis.

  3. Non-transgenic RNAi technology to control insects on citrus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This research demonstrated a non-transgenic delivery method for ribonucleic acid interference, RNAi, that reduced fitness as measured in increased mortality over time, of two insect pests of citrus, ie. psyllids and leafhoppers. The Asian citrus psyllid transmits a deadly plant-infecting bacterium o...

  4. RNAi at work: Targeting invertebrate pests and beneficial organisms' diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Invertebrates present two types of large scale RNAi application opportunities: pest control and beneficial insect health. The former involves the introduction of sustainable applications to keep pest populations low, and the latter represents the challenge of keeping beneficial organisms healthy. RN...

  5. Mosquito and Drosophila entomobirnaviruses suppress dsRNA- and siRNA-induced RNAi.

    PubMed

    van Cleef, Koen W R; van Mierlo, Joël T; Miesen, Pascal; Overheul, Gijs J; Fros, Jelke J; Schuster, Susan; Marklewitz, Marco; Pijlman, Gorben P; Junglen, Sandra; van Rij, Ronald P

    2014-07-01

    RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthermore, we show that RNAi is suppressed in CYV-infected cells and that the viral VP3 protein is responsible for RNAi antagonism. We demonstrate that VP3 can functionally replace B2, the well-characterized RNAi suppressor of Flock House virus. VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs. Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3. Finally, we show that the RNAi-suppressive activity of VP3 is conserved in Drosophila X virus, a birnavirus that persistently infects Drosophila cell cultures. Together, our data indicate that mosquito-specific viruses may encode RNAi antagonists to suppress antiviral RNAi.

  6. Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi.

    PubMed

    Zuber, Johannes; McJunkin, Katherine; Fellmann, Christof; Dow, Lukas E; Taylor, Meredith J; Hannon, Gregory J; Lowe, Scott W

    2011-01-01

    Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown. We demonstrated that this system improves the study of essential genes and was sufficiently robust to eradicate aggressive cancer in mice by suppressing a single gene. Further, we applied this system for in vivo negative-selection screening with pooled shRNAs and propose a streamlined, inexpensive workflow that will facilitate the use of RNA interference (RNAi) for the identification and evaluation of essential therapeutic targets.

  7. Antifungal drug resistance evoked via RNAi-dependent epimutations.

    PubMed

    Calo, Silvia; Shertz-Wall, Cecelia; Lee, Soo Chan; Bastidas, Robert J; Nicolás, Francisco E; Granek, Joshua A; Mieczkowski, Piotr; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M; Cardenas, Maria E; Heitman, Joseph

    2014-09-25

    Microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, Hsp90 and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidylprolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild-type phenotype when grown without exposure to the drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNAs and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves the generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

  8. Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium

    PubMed Central

    Tomoyasu, Yoshinori; Miller, Sherry C; Tomita, Shuichiro; Schoppmeier, Michael; Grossmann, Daniela; Bucher, Gregor

    2008-01-01

    Background RNA interference (RNAi) is a highly conserved cellular mechanism. In some organisms, such as Caenorhabditis elegans, the RNAi response can be transmitted systemically. Some insects also exhibit a systemic RNAi response. However, Drosophila, the leading insect model organism, does not show a robust systemic RNAi response, necessitating another model system to study the molecular mechanism of systemic RNAi in insects. Results We used Tribolium, which exhibits robust systemic RNAi, as an alternative model system. We have identified the core RNAi genes, as well as genes potentially involved in systemic RNAi, from the Tribolium genome. Both phylogenetic and functional analyses suggest that Tribolium has a somewhat larger inventory of core component genes than Drosophila, perhaps allowing a more sensitive response to double-stranded RNA (dsRNA). We also identified three Tribolium homologs of C. elegans sid-1, which encodes a possible dsRNA channel. However, detailed sequence analysis has revealed that these Tribolium homologs share more identity with another C. elegans gene, tag-130. We analyzed tag-130 mutants, and found that this gene does not have a function in systemic RNAi in C. elegans. Likewise, the Tribolium sid-like genes do not seem to be required for systemic RNAi. These results suggest that insect sid-1-like genes have a different function than dsRNA uptake. Moreover, Tribolium lacks homologs of several genes important for RNAi in C. elegans. Conclusion Although both Tribolium and C. elegans show a robust systemic RNAi response, our genome-wide survey reveals significant differences between the RNAi mechanisms of these organisms. Thus, insects may use an alternative mechanism for the systemic RNAi response. Understanding this process would assist with rendering other insects amenable to systemic RNAi, and may influence pest control approaches. PMID:18201385

  9. Defense and counterdefense in the RNAi-based antiviral immune system in insects.

    PubMed

    van Mierlo, Joël T; van Cleef, Koen W R; van Rij, Ronald P

    2011-01-01

    RNA interference (RNAi) is an important pathway to combat virus infections in insects and plants. Hallmarks of antiviral RNAi in these organisms are: (1) an increase in virus replication after inactivation of major actors in the RNAi pathway, (2) production of virus-derived small interfering RNAs (v-siRNAs), and (3) suppression of RNAi by dedicated viral proteins. In this chapter, we will review the mechanism of RNAi in insects, its function as an antiviral immune system, viral small RNA profiles, and viral counterdefense strategies. We will also consider alternative, inducible antiviral immune responses.

  10. Application of Screening Experimental Designs to Assess Chromatographic Isotope Effect upon Isotope-Coded Derivatization for Quantitative Liquid Chromatography–Mass Spectrometry

    PubMed Central

    2015-01-01

    Isotope effect may cause partial chromatographic separation of labeled (heavy) and unlabeled (light) isotopologue pairs. Together with a simultaneous matrix effect, this could lead to unacceptable accuracy in quantitative liquid chromatography–mass spectrometry assays, especially when electrospray ionization is used. Four biologically relevant reactive aldehydes (acrolein, malondialdehyde, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal) were derivatized with light or heavy (d3-, 13C6-, 15N2-, or 15N4-labeled) 2,4-dinitrophenylhydrazine and used as model compounds to evaluate chromatographic isotope effects. For comprehensive assessment of retention time differences between light/heavy pairs under various gradient reversed-phase liquid chromatography conditions, major chromatographic parameters (stationary phase, mobile phase pH, temperature, organic solvent, and gradient slope) and different isotope labelings were addressed by multiple-factor screening using experimental designs that included both asymmetrical (Addelman) and Plackett–Burman schemes followed by statistical evaluations. Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of 15N or 13C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus 15N labeling gave unacceptable differences (>15%) upon quantifying some of the model aldehydes from biological matrixes. On the basis of our results, we recommend the modification of the AIDA protocol by replacing d3-2,4-dinitrophenylhydrazine with 15N- or 13C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects. PMID:24922593

  11. Correlating Quantitative Fecal Immunochemical Test Results with Neoplastic Findings on Colonoscopy in a Population-Based Colorectal Cancer Screening Program: A Prospective Study

    PubMed Central

    McGahan, Colleen E.

    2016-01-01

    Background and Aims. The Canadian Partnership Against Cancer (CPAC) recommends a fecal immunochemical test- (FIT-) positive predictive value (PPV) for all adenomas of ≥50%. We sought to assess FIT performance among average-risk participants of the British Columbia Colon Screening Program (BCCSP). Methods. From Nov-2013 to Dec-2014 consecutive participants of the BCCSP were assessed. Data was obtained from a prospectively collected database. A single quantitative FIT (NS-Plus, Alfresa Pharma Corporation, Japan) with a cut-off of ≥10 μg/g (≥50 ng/mL) was used. Results. 20,322 FIT-positive participants underwent CSPY. At a FIT cut-off of ≥10 μg/g (≥50 ng/mL) the PPV for all adenomas was 52.0%. Increasing the FIT cut-off to ≥20 μg/g (≥100 ng/mL) would increase the PPV for colorectal cancer (CRC) by 1.5% and for high-risk adenomas (HRAs) by 6.5% at a cost of missing 13.6% of CRCs and 32.4% of HRAs. Conclusions. As the NS-Plus FIT cut-off rises, the PPV for CRC and HRAs increases but at the cost of missed lesions. A cut-off of ≥10 μg/g (≥50 ng/mL) produces a PPV for all adenomas exceeding national recommendations. Health authorities need to take into consideration endoscopic resources when selecting a FIT positivity threshold. PMID:28116286

  12. Argonaute2 is the catalytic engine of mammalian RNAi.

    PubMed

    Liu, Jidong; Carmell, Michelle A; Rivas, Fabiola V; Marsden, Carolyn G; Thomson, J Michael; Song, Ji-Joon; Hammond, Scott M; Joshua-Tor, Leemor; Hannon, Gregory J

    2004-09-03

    Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.

  13. Optical imaging of RNAi-mediated silencing of cancer

    NASA Astrophysics Data System (ADS)

    Ochiya, Takahiro; Honma, Kimi; Takeshita, Fumitaka; Nagahara, Shunji

    2008-02-01

    RNAi has rapidly become a powerful tool for drug target discovery and validation in an in vitro culture system and, consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Cancer is one obvious application for RNAi therapeutics, because abnormal gene expression is thought to contribute to the pathogenesis and maintenance of the malignant phenotype of cancer and thereby many oncogenes and cell-signaling molecules present enticing drug target possibilities. RNAi, potent and specific, could silence tumor-related genes and would appear to be a rational approach to inhibit tumor growth. In subsequent in vivo studies, the appropriate cancer model must be developed for an evaluation of siRNA effects on tumors. How to evaluate the effect of siRNA in an in vivo therapeutic model is also important. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide cancer inhibition in real time and are sensitive to subtle changes, are crucial for rapid advancement of these approaches. Bioluminescent imaging is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity.

  14. Lentiviral vector engineering for anti-HIV RNAi gene therapy.

    PubMed

    ter Brake, Olivier; Westerink, Jan-Tinus; Berkhout, Ben

    2010-01-01

    RNA interference or RNAi-based gene therapy for the treatment of HIV-1 infection has recently emerged as a highly effective antiviral approach. The lentiviral vector system is a good candidate for the expression of antiviral short hairpin RNAs (shRNA) in HIV-susceptible cells. However, this strategy can give rise to vector problems because the anti-HIV shRNAs can also target the HIV-based lentiviral vector system. In addition, there may be self-targeting of the shRNA-encoding sequences within the vector RNA genome in the producer cell. The insertion of microRNA (miRNA) cassettes in the vector may introduce Drosha cleavage sites that will also result in the destruction of the vector genome during the production and/or the transduction process. Here, we describe possible solutions to these lentiviral-RNAi problems. We also describe a strategy for multiple shRNA expression to establish a combinatorial RNAi therapy.

  15. RNAi for insect control: current perspective and future challenges.

    PubMed

    Katoch, Rajan; Sethi, Amit; Thakur, Neelam; Murdock, Larry L

    2013-10-01

    The research on the RNA interference (RNAi) for the control of insect pests has made significant growth in recent years. The availability of the genomic sequences of insects has further widened the horizons for the testing of this technology to various insect groups. Different modes of application of double-stranded RNA (dsRNA) have been tested; however, the practicability of delivery of dsRNA in insects still remains the biggest challenge. Till date, the oral delivery of dsRNA in insects is one of the efficient approaches for the practical application of this technique. The uptake of dsRNA from the insect gut is mediated either by SID-1/SID-2 transmembrane proteins or by endocytosis; however, the systemic RNAi machinery still remains to be revealed in insect species. The RNAi-mediated gene knockdown has shown striking results in different insect groups, pointing it to be the upcoming technique for insect control. However, before the successful application of this technique for insect control, some potential issues need to be resolved. This review presents the account of prospects and challenges for the use of this technology for insect control.

  16. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented.

  17. RNAi construct of a cytochrome P450 gene CYP82D109 blocks an early step in the biosynthesis of hemigossypolone and gossypol in transgenic cotton plants.

    PubMed

    Wagner, Tanya A; Liu, Jinggao; Puckhaber, Lorraine S; Bell, Alois A; Williams, Howard; Stipanovic, Robert D

    2015-07-01

    Naturally occurring terpenoid aldehydes from cotton, such as hemigossypol, gossypol, hemigossypolone, and the heliocides, are important components of disease and herbivory resistance in cotton. These terpenoids are predominantly found in the glands. Differential screening identified a cytochrome P450 cDNA clone (CYP82D109) from a Gossypium hirsutum cultivar that hybridized to mRNA from glanded cotton but not glandless cotton. Both the D genome cotton Gossypium raimondii and A genome cotton Gossypium arboreum possessed three additional paralogs of the gene. G. hirsutum was transformed with a RNAi construct specific to this gene family and eight transgenic plants were generated stemming from at least five independent transformation events. HPLC analysis showed that RNAi plants, when compared to wild-type Coker 312 (WT) plants, had a 90% reduction in hemigossypolone and heliocides levels, and a 70% reduction in gossypol levels in the terminal leaves, respectively. Analysis of volatile terpenes by GC-MS established presence of an additional terpene (MW: 218) from the RNAi leaf extracts. The (1)H and (13)C NMR spectroscopic analyses showed this compound was δ-cadinen-2-one. Double bond rearrangement of this compound gives 7-hydroxycalamenene, a lacinilene C pathway intermediate. δ-Cadinen-2-one could be derived from δ-cadinene via a yet to be identified intermediate, δ-cadinen-2-ol. The RNAi construct of CYP82D109 blocks the synthesis of desoxyhemigossypol and increases the induction of lacinilene C pathway, showing that these pathways are interconnected. Lacinilene C precursors are not constitutively expressed in cotton leaves, and blocking the gossypol pathway by the RNAi construct resulted in a greater induction of the lacinilene C pathway compounds when challenged by pathogens.

  18. Integrated siRNA design based on surveying of features associated with high RNAi effectiveness

    PubMed Central

    Gong, Wuming; Ren, Yongliang; Xu, Qiqi; Wang, Yejun; Lin, Dong; Zhou, Haiyan; Li, Tongbin

    2006-01-01

    Background Short interfering RNAs have allowed the development of clean and easily regulated methods for disruption of gene expression. However, while these methods continue to grow in popularity, designing effective siRNA experiments can be challenging. The various existing siRNA design guidelines suffer from two problems: they differ considerably from each other, and they produce high levels of false-positive predictions when tested on data of independent origins. Results Using a distinctly large set of siRNA efficacy data assembled from a vast diversity of origins (the siRecords data, containing records of 3,277 siRNA experiments targeting 1,518 genes, derived from 1,417 independent studies), we conducted extensive analyses of all known features that have been implicated in increasing RNAi effectiveness. A number of features having positive impacts on siRNA efficacy were identified. By performing quantitative analyses on cooperative effects among these features, then applying a disjunctive rule merging (DRM) algorithm, we developed a bundle of siRNA design rule sets with the false positive problem well curbed. A comparison with 15 online siRNA design tools indicated that some of the rule sets we developed surpassed all of these design tools commonly used in siRNA design practice in positive predictive values (PPVs). Conclusion The availability of the large and diverse siRNA dataset from siRecords and the approach we describe in this report have allowed the development of highly effective and generally applicable siRNA design rule sets. Together with ever improving RNAi lab techniques, these design rule sets are expected to make siRNAs a more useful tool for molecular genetics, functional genomics, and drug discovery studies. PMID:17129386

  19. UP-TORR: online tool for accurate and Up-to-Date annotation of RNAi Reagents.

    PubMed

    Hu, Yanhui; Roesel, Charles; Flockhart, Ian; Perkins, Lizabeth; Perrimon, Norbert; Mohr, Stephanie E

    2013-09-01

    RNA interference (RNAi) is a widely adopted tool for loss-of-function studies but RNAi results only have biological relevance if the reagents are appropriately mapped to genes. Several groups have designed and generated RNAi reagent libraries for studies in cells or in vivo for Drosophila and other species. At first glance, matching RNAi reagents to genes appears to be a simple problem, as each reagent is typically designed to target a single gene. In practice, however, the reagent-gene relationship is complex. Although the sequences of oligonucleotides used to generate most types of RNAi reagents are static, the reference genome and gene annotations are regularly updated. Thus, at the time a researcher chooses an RNAi reagent or analyzes RNAi data, the most current interpretation of the RNAi reagent-gene relationship, as well as related information regarding specificity (e.g., predicted off-target effects), can be different from the original interpretation. Here, we describe a set of strategies and an accompanying online tool, UP-TORR (for Updated Targets of RNAi Reagents; www.flyrnai.org/up-torr), useful for accurate and up-to-date annotation of cell-based and in vivo RNAi reagents. Importantly, UP-TORR automatically synchronizes with gene annotations daily, retrieving the most current information available, and for Drosophila, also synchronizes with the major reagent collections. Thus, UP-TORR allows users to choose the most appropriate RNAi reagents at the onset of a study, as well as to perform the most appropriate analyses of results of RNAi-based studies.

  20. RNAi technologies in agricultural biotechnology: The Toxicology Forum 40th Annual Summer Meeting.

    PubMed

    Sherman, James H; Munyikwa, Tichafa; Chan, Stephen Y; Petrick, Jay S; Witwer, Kenneth W; Choudhuri, Supratim

    2015-11-01

    During the 40th Annual Meeting of The Toxicology Forum, the current and potential future science, regulations, and politics of agricultural biotechnology were presented and discussed. The meeting session described herein focused on the technology of RNA interference (RNAi) in agriculture. The general process by which RNAi works, currently registered RNAi-based plant traits, example RNAi-based traits in development, potential use of double stranded RNA (dsRNA) as topically applied pesticide active ingredients, research related to the safety of RNAi, biological barriers to ingested dsRNA, recent regulatory RNAi science reviews, and regulatory considerations related to the use of RNAi in agriculture were discussed. Participants generally agreed that the current regulatory framework is robust and appropriate for evaluating the safety of RNAi employed in agricultural biotechnology and were also supportive of the use of RNAi to develop improved crop traits. However, as with any emerging technology, the potential range of future products, potential future regulatory frameworks, and public acceptance of the technology will continue to evolve. As such, continuing dialogue was encouraged to promote education of consumers and science-based regulations.

  1. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Smialowska, Agata; Djupedal, Ingela; Wang, Jingwen; Kylsten, Per; Swoboda, Peter; Ekwall, Karl

    2014-02-07

    Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.

  2. Multiple sensors ensure guide strand selection in human RNAi pathways.

    PubMed

    Noland, Cameron L; Doudna, Jennifer A

    2013-05-01

    Small RNAs guide RNA-induced silencing complexes (RISCs) to bind to cognate mRNA transcripts and trigger silencing of protein expression during RNA interference (RNAi) in eukaryotes. A fundamental aspect of this process is the asymmetric loading of one strand of a short interfering RNA (siRNA) or microRNA (miRNA) duplex onto RISCs for correct target recognition. Here, we use a reconstituted system to determine the extent to which the core components of the human RNAi machinery contribute to RNA guide strand selection. We show that Argonaute2 (Ago2), the endonuclease that binds directly to siRNAs and miRNAs within RISC, has intrinsic but substrate-dependent RNA strand selection capability. This activity can be enhanced substantially when Ago2 is in complex with the endonuclease Dicer and the double-stranded RNA-binding proteins (dsRBPs)-trans-activation response (TAR) RNA-binding protein (TRBP) or protein activator of PKR (PACT). The extent to which human Dicer/dsRBP complexes contribute to strand selection is dictated by specific duplex parameters such as thermodynamics, 5' nucleotide identity, and structure. Surprisingly, our results also suggest that strand selection for some miRNAs is enhanced by PACT-containing complexes but not by those containing TRBP. Furthermore, overall mRNA targeting by miRNAs is disfavored for complexes containing TRBP but not PACT. These findings demonstrate that multiple proteins collaborate to ensure optimal strand selection in humans and reveal the possibility of delineating RNAi pathways based on the presence of TRBP or PACT.

  3. Modulation of the transcriptional response of innate immune and RNAi genes upon exposure to dsRNA and LPS in silkmoth-derived Bm5 cells overexpressing BmToll9-1 receptor.

    PubMed

    Liu, Jisheng; Kolliopoulou, Anna; Smagghe, Guy; Swevers, Luc

    2014-07-01

    Injection or feeding of dsRNA is commonly used to induce specific gene silencing by RNAi in insects but very little research has been carried out to investigate non-specific effects on gene expression of dsRNA as pathogen-associated molecular pattern (PAMP). This study focuses on the potential role of the BmToll9-1 receptor to modulate the transcriptional response of innate immune and RNAi genes to dsRNA and lipopolysaccharide (LPS), which was used for comparison. To study this role, we took advantage of the silkmoth-derived Bm5 cell line, which does not express BmToll9-1 endogenously, and engineered a transformed cell line that permanently expresses BmToll9-1. Quantitative mRNA expression studies showed that BmToll9-1 can significantly alter the transcriptional response to dsRNA and LPS: (1) BmToll9-1 promotes the transcriptional response of Dicer2, encoding a key component of the RNAi machinery, and, to a lesser extent, that of transcription factors in the Jak-STAT and Toll pathways; and (2) BmToll9-1 represses the transcriptional induction of the IMD and Jak-STAT pathway genes, as well as the antimicrobial peptide (AMP) effector genes, by LPS. Thus, BmToll9-1 was identified as a modulator of innate immune and RNAi machinery gene expression that could be related to its preferential expression in the larval gut, the major barrier of pathogen entry. While BmToll9-1 was found to modulate RNAi-related gene expression, a reporter-based RNAi assay established no evidence for a direct interaction of BmToll9-1 with the intracellular RNAi machinery.

  4. Baculovirus-mediated Gene Delivery and RNAi Applications

    PubMed Central

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-01-01

    Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism. PMID:25912715

  5. ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in Caenorhabditis elegans

    PubMed Central

    Andralojc, Karolina M.; Kelly, Ashley L.; Tanner, Paige C.

    2017-01-01

    Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3). The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules). ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline. PMID:28182654

  6. A Combination of Screening and Computational Approaches for the Identification of Novel Compounds That Decrease Mast Cell Degranulation

    PubMed Central

    McShane, Marisa P.; Friedrichson, Tim; Giner, Angelika; Meyenhofer, Felix; Barsacchi, Rico; Bickle, Marc

    2015-01-01

    High-content screening of compound libraries poses various challenges in the early steps in drug discovery such as gaining insights into the mode of action of the selected compounds. Here, we addressed these challenges by integrating two biological screens through bioinformatics and computational analysis. We screened a small-molecule library enriched in amphiphilic compounds in a degranulation assay in rat basophilic leukemia 2H3 (RBL-2H3) cells. The same library was rescreened in a high-content image-based endocytosis assay in HeLa cells. This assay was previously applied to a genome-wide RNAi screen that produced quantitative multiparametric phenotypic profiles for genes that directly or indirectly affect endocytosis. By correlating the endocytic profiles of the compounds with the genome-wide siRNA profiles, we identified candidate pathways that may be inhibited by the compounds. Among these, we focused on the Akt pathway and validated its inhibition in HeLa and RBL-2H3 cells. We further showed that the compounds inhibited the translocation of the Akt-PH domain to the plasma membrane. The approach performed here can be used to integrate chemical and functional genomics screens for investigating the mechanism of action of compounds. PMID:25838434

  7. Differential effects of RNAi treatments on field populations of the western corn rootworm.

    PubMed

    Chu, Chia-Ching; Sun, Weilin; Spencer, Joseph L; Pittendrigh, Barry R; Seufferheld, Manfredo J

    2014-03-01

    RNA interference (RNAi) mediated crop protection against insect pests is a technology that is greatly anticipated by the academic and industrial pest control communities. Prior to commercialization, factors influencing the potential for evolution of insect resistance to RNAi should be evaluated. While mutations in genes encoding the RNAi machinery or the sequences targeted for interference may serve as a prominent mechanism of resistance evolution, differential effects of RNAi on target pests may also facilitate such evolution. However, to date, little is known about how variation of field insect populations could influence the effectiveness of RNAi treatments. To approach this question, we evaluated the effects of RNAi treatments on adults of three western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) populations exhibiting different levels of gut cysteine protease activity, tolerance of soybean herbivory, and immune gene expression; two populations were collected from crop rotation-resistant (RR) problem areas and one from a location where RR was not observed (wild type; WT). Our results demonstrated that RNAi targeting DvRS5 (a highly expressed cysteine protease gene) reduced gut cysteine protease activity in all three WCR populations. However, the proportion of the cysteine protease activity that was inhibited varied across populations. When WCR adults were treated with double-stranded RNA of an immune gene att1, different changes in survival among WT and RR populations on soybean diets occurred. Notably, for both genes, the sequences targeted for RNAi were the same across all populations examined. These findings indicate that the effectiveness of RNAi treatments could vary among field populations depending on their physiological and genetic backgrounds and that the consistency of an RNAi trait's effectiveness on phenotypically different populations should be considered or tested prior to wide deployment. Also, genes that are potentially subjected

  8. Aptamer-targeted RNAi for HIV-1 therapy.

    PubMed

    Zhou, Jiehua; Rossi, John J

    2011-01-01

    The highly specific mechanism of RNA (RNAi) that inhibits the expression of disease genes is increasingly being harnessed to develop a new class of therapeutics for a wide variety of human maladies. The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. Herein, we demonstrate novel cell type-specific dual inhibitory function anti-gp120 aptamer-siRNA delivery systems for HIV-1 therapy, in which both the aptamer and the siRNA portions have potent anti-HIV activities. The envelope glycoprotein is expressed on the surface of HIV-1 infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. The Dicer substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells. Our results provide a set of novel aptamer-targeted RNAi therapeutics to combat HIV and further validate the use of anti-gp120 aptamers for delivery of Dicer substrate siRNAs.

  9. RNAi and Antiviral Defense in the Honey Bee.

    PubMed

    Brutscher, Laura M; Flenniken, Michelle L

    2015-01-01

    Honey bees play an important agricultural and ecological role as pollinators of numerous agricultural crops and other plant species. Therefore, investigating the factors associated with high annual losses of honey bee colonies in the US is an important and active area of research. Pathogen incidence and abundance correlate with Colony Collapse Disorder- (CCD-) affected colonies in the US and colony losses in the US and in some European countries. Honey bees are readily infected by single-stranded positive sense RNA viruses. Largely dependent on the host immune response, virus infections can either remain asymptomatic or result in deformities, paralysis, or death of adults or larvae. RNA interference (RNAi) is an important antiviral defense mechanism in insects, including honey bees. Herein, we review the role of RNAi in honey bee antiviral defense and highlight some parallels between insect and mammalian immune systems. A more thorough understanding of the role of pathogens on honey bee health and the immune mechanisms bees utilize to combat infectious agents may lead to the development of strategies that enhance honey bee health and result in the discovery of additional mechanisms of immunity in metazoans.

  10. RNAi and Antiviral Defense in the Honey Bee

    PubMed Central

    Brutscher, Laura M.; Flenniken, Michelle L.

    2015-01-01

    Honey bees play an important agricultural and ecological role as pollinators of numerous agricultural crops and other plant species. Therefore, investigating the factors associated with high annual losses of honey bee colonies in the US is an important and active area of research. Pathogen incidence and abundance correlate with Colony Collapse Disorder- (CCD-) affected colonies in the US and colony losses in the US and in some European countries. Honey bees are readily infected by single-stranded positive sense RNA viruses. Largely dependent on the host immune response, virus infections can either remain asymptomatic or result in deformities, paralysis, or death of adults or larvae. RNA interference (RNAi) is an important antiviral defense mechanism in insects, including honey bees. Herein, we review the role of RNAi in honey bee antiviral defense and highlight some parallels between insect and mammalian immune systems. A more thorough understanding of the role of pathogens on honey bee health and the immune mechanisms bees utilize to combat infectious agents may lead to the development of strategies that enhance honey bee health and result in the discovery of additional mechanisms of immunity in metazoans. PMID:26798663

  11. RNAi-mediated down-regulation of DCL1 and AGO1 induces developmental changes in resynthesized Arabidopsis allotetraploids

    PubMed Central

    Lackey, Erika; Ng, Danny W-K.; Chen, Z. Jeffrey

    2010-01-01

    Summary Both natural and newly synthesized allopolyploids display nonadditive gene expression changes through genetic and epigenetic mechanisms. The nonadditively expressed genes include many microRNA (miRNA) targets, suggesting a role for miRNAs and their targets in morphological variation in the allopolyploids and their progenitors. We produced dominant-negative transgenic allotetraploid plants in Arabidopsis using RNA interference (RNAi) that downregulates the expression of miRNA biogenesis genes, including DCL1 and AGO1. RNAi of DCL1 and AGO1 led to dominant negative phenotypes and decreased accumulation of several miRNAs and a tasiRNA tested in the transgenic resynthesized allotetraploids. The results demonstrated that miRNA biogenesis genes are effectively downregulated in the resynthesized allotetraploids containing redundant homoeologous genes that are difficult to be manipulated by conventional mutation screens. These lines will be useful for studying the effects of miRNA biogenesis genes on growth and developmental variation in the allopolyploids. PMID:20409179

  12. The lin-35/Rb and RNAi pathways cooperate to regulate a key cell cycle transition in C. elegans

    PubMed Central

    Ouellet, Jimmy; Roy, Richard

    2007-01-01

    Background The Retinoblastoma gene product (Rb) has been shown to regulate the transcription of key genes involved in cell growth and proliferation. Consistent with this, mutations in Rb are associated with numerous types of cancer making it a critical tumour suppressor gene. Its function is conferred through a large multiprotein complex that exhibits a dual function in both activation and repression of gene targets. In C. elegans, the Rb orthologue lin-35 functions redundantly with other transcriptional regulators to appropriately specify both vulval and pharyngeal cell fates. Results In C. elegans the intestinal cells must alter their cell cycle from the mitotic cell divisions typical of embryogenesis to karyokinesis and then endoreplication, which facilitates growth during larval development. While screening for genes that affect the ability of the intestinal cells to appropriately make this cell cycle transition during post-embryonic development, we isolated mutants that either compromise this switch and remain mononucleate, or cause these cells to undergo multiple rounds of nuclear division. Among these mutants we identified a novel allele of lin-35/Rb, while we also found that the components of the synMuv B complex, which are involved in vulval specification, are also required to properly regulate the developmentally-controlled cell cycle transition typical of these intestinal cells during larval development. More importantly, our work uncovered a role for certain members of the pathways involved in RNAi in mediating the efficient transition between these cell cycle programs, suggesting that lin-35/Rb cooperates with these RNAi components. Furthermore, our findings suggest that met-2, a methyltransferase as well as hpl-1 and hpl-2, two C. elegans homologues of the heterochromatin protein HP1 are also required for this transition. Conclusion Our findings are consistent with lin-35/Rb, synMuv and RNAi components cooperating, probably through their additive

  13. RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?

    PubMed Central

    Unniyampurath, Unnikrishnan; Pilankatta, Rajendra; Krishnan, Manoj N.

    2016-01-01

    The recent emergence of multiple technologies for modifying gene structure has revolutionized mammalian biomedical research and enhanced the promises of gene therapy. Over the past decade, RNA interference (RNAi) based technologies widely dominated various research applications involving experimental modulation of gene expression at the post-transcriptional level. Recently, a new gene editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system, has received unprecedented acceptance in the scientific community for a variety of genetic applications. Unlike RNAi, the CRISPR/Cas9 system is bestowed with the ability to introduce heritable precision insertions and deletions in the eukaryotic genome. The combination of popularity and superior capabilities of CRISPR/Cas9 system raises the possibility that this technology may occupy the roles currently served by RNAi and may even make RNAi obsolete. We performed a comparative analysis of the technical aspects and applications of the CRISPR/Cas9 system and RNAi in mammalian systems, with the purpose of charting out a predictive picture on whether the CRISPR/Cas9 system will eclipse the existence and future of RNAi. The conclusion drawn from this analysis is that RNAi will still occupy specific domains of biomedical research and clinical applications, under the current state of development of these technologies. However, further improvements in CRISPR/Cas9 based technology may ultimately enable it to dominate RNAi in the long term. PMID:26927085

  14. Delivery of dsRNA for RNAi in insects: an overview and future directions.

    PubMed

    Yu, Na; Christiaens, Olivier; Liu, Jisheng; Niu, Jinzhi; Cappelle, Kaat; Caccia, Silvia; Huvenne, Hanneke; Smagghe, Guy

    2013-02-01

    RNA interference (RNAi) refers to the process of exogenous double-stranded RNA (dsRNA) silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi-based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.

  15. Using RNAi in C. "elegans" to Demonstrate Gene Knockdown Phenotypes in the Undergraduate Biology Lab Setting

    ERIC Educational Resources Information Center

    Roy, Nicole M.

    2013-01-01

    RNA interference (RNAi) is a powerful technology used to knock down genes in basic research and medicine. In 2006 RNAi technology using "Caenorhabditis elegans" ("C. elegans") was awarded the Nobel Prize in medicine and thus students graduating in the biological sciences should have experience with this technology. However,…

  16. RNA-seq validation of RNAi identifies additional gene connectivity in Tribolium castaneum (Coleoptera: Tenebrionidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) is a functional genomics tool to validate phenotypes by delivering targeted, gene-specific, and complementary dsRNA into a host via injection, feeding, or other means in order to reduce gene expression. RNAi in the red flour beetle, Tribolium castaneum, has been successful du...

  17. Development of a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR).

    PubMed

    Sigl, Reinhard; Ploner, Christian; Shivalingaiah, Giridhar; Kofler, Reinhard; Geley, Stephan

    2014-01-01

    RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success, however, depends on the effective expression of RNAi-inducing small double-stranded interfering RNA molecules (siRNAs) in target cells. In many cell types, RNAi can be achieved by transfection of chemically synthesised siRNAs, which results in transient knockdown of protein expression. Expression of double-stranded short hairpin RNA (shRNA) provides another means to induce RNAi in cells that are hard to transfect. To facilitate the generation of stable, conditional RNAi cell lines, we have developed novel one- and two-component vector GATEWAY-compatible lentiviral tetracycline-regulated RNAi (GLTR) systems. The combination of a modified RNA-polymerase-III-dependent H1 RNA promoter (designated 'THT') for conditional shRNA expression with different lentiviral delivery vectors allows (1) the use of fluorescent proteins for colour-coded combinatorial RNAi or for monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one vector system (pGLTR-X). All three systems were found to be suitable for the analysis of essential genes, such as CDC27, a component of the mitotic ubiquitin ligase APC/C, in cell lines and primary human cells.

  18. Analysis of Nuclear RNA Interference (RNAi) in Human Cells by Subcellular Fractionation and Argonaute Loading

    PubMed Central

    Gagnon, Keith T.; Li, Liande; Janowski, Bethany A.; Corey, David R.

    2014-01-01

    RNA interference (RNAi) is well known for its ability to regulate gene expression in the cytoplasm of mammalian cells. In mammalian cell nuclei, however, the impact of RNAi has remained more controversial. A key technical hurdle has been a lack of optimized protocols for the isolation and analysis of cell nuclei. Here we describe a simplified protocol for nuclei isolation from cultured cells that incorporates a method for obtaining nucleoplasmic and chromatin fractions and removing cytoplasmic contamination. Cell fractions can then be used to detect the presence and activity of RNAi factors in the nucleus. We present a protocol for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. These protocols facilitate characterization of nuclear RNAi and can be applied to the analysis of other nuclear proteins and pathways. From cellular fractionation to analysis of Argonaute loading results, this protocol takes 4–6 d to complete. PMID:25079428

  19. RNAi effects on actin mRNAs in Homalodisca vitripennis cells

    PubMed Central

    Rosa, Cristina; Kamita, Shizuo G; Dequine, Haley; Wuriyanghan, Ha; Lindbo, John A; Falk, Bryce W

    2010-01-01

    The xylem feeding leafhopper Homalodisaca vitripennis (H. vitripennis) is an unusually robust and efficient vector of Xylella fastidiosa, a Gram-negative bacterium which causes several very important plant diseases. Here we investigated RNA interference (RNAi) to target actin, a key component of insect cells and whole bodies, in H. vitripennis cells. RNAi effectors were delivered via lipid based transfection and real-time RT-PCR, RNA hybridization, and microscopic analyses were employed to verify RNAi effects. When actin dsRNAs were used, a 10-fold decrease in the target H. vitripennis actin mRNA level was seen in cells. Altered phenotypic effects also were evident in transfected cells, as were small interfering RNAs, hallmarks of RNAi. The use of H. vitripennis cells and RNAi offers new opportunities to research hemipterans, the most important insect vectors of plant pathogens. PMID:20628496

  20. Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery.

    PubMed

    Swevers, Luc; Ioannidis, Konstantinos; Kolovou, Marianna; Zografidis, Aris; Labropoulou, Vassiliki; Santos, Dulce; Wynant, Niels; Broeck, Jozef Vanden; Wang, Luoluo; Cappelle, Kaat; Smagghe, Guy

    RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.

  1. Amelioration of psoriasis by anti-TNF-alpha RNAi in the xenograft transplantation model.

    PubMed

    Jakobsen, Maria; Stenderup, Karin; Rosada, Cecilia; Moldt, Brian; Kamp, Søren; Dam, Tomas N; Jensen, Thomas G; Mikkelsen, Jacob Giehm

    2009-10-01

    Tumor necrosis factor-alpha (TNF-alpha) is upregulated in psoriatic skin and represents a prominent target in psoriasis treatment. The level of TNF-alpha-encoding mRNA, however, is not increased in psoriatic skin, and it remains unclear whether intervention strategies based on RNA interference (RNAi) are therapeutically relevant. To test this hypothesis the present study describes first the in vitro functional screening of a panel of short hairpin RNAs (shRNAs) targeting human TNF-alpha mRNA and, next, the transfer of the most potent TNF-alpha shRNA variant, as assessed in vitro, to human skin in the psoriasis xenograft transplantation model by the use of lentiviral vectors. TNF-alpha shRNA treatment leads to amelioration of the psoriasis phentotype in the model, as documented by reduced epidermal thickness, normalization of the skin morphology, and reduced levels of TNF-alpha mRNA as detected in skin biopsies 3 weeks after a single vector injection of lentiviral vectors encoding TNF-alpha shRNA. Our data show efficient lentiviral gene delivery to psoriatic skin and therapeutic applicability of anti-TNF-alpha shRNAs in human skin. These findings validate TNF-alpha mRNA as a target molecule for a potential persistent RNA-based treatment of psoriasis and establish the use of small RNA effectors as a novel platform for target validation in psoriasis and other skin disorders.

  2. Endogenous RNAi and adaptation to environment in C. elegans

    PubMed Central

    Grishok, Alla

    2012-01-01

    The contributions of short RNAs to the control of repetitive elements are well documented in animals and plants. Here, the role of endogenous RNAi and AF10 homolog ZFP-1 in the adaptation of C. elegans to the environment is discussed. First, modulation of insulin signaling through regulation of transcription of the PDK-1 kinase (Mansisidor et al., PLoS Genetics, 2011) is reviewed. Second, an siRNA-based natural selection model is proposed in which variation in endogenous siRNA pools between individuals is subject to natural selection similarly to DNA-based genetic variation. The value of C. elegans for the research of siRNA-based epigenetic variation and adaptation is highlighted. PMID:24058837

  3. RITS- connecting transcription, RNAi and heterochromatin assembly in Fission Yeast

    PubMed Central

    Creamer, Kevin M.; Partridge, Janet F.

    2011-01-01

    In recent years a bevy of evidence has been unearthed indicating that ‘silent’ heterochromatin is not as transcriptionally inert as once thought. In the unicellular yeast Schizosaccharomyces pombe, processing of transcripts derived from centromeric repeats into homologous small interfering RNA (siRNA) is essential for the formation of centromeric heterochromatin. Deletion of genes required for siRNA biogenesis revealed that core components of the canonical RNAi pathway are essential for centromeric heterochromatin assembly as well as for centromere function. Subsequent purification of the RITS (RNA-induced initiation of transcriptional gene silencing) complex provided the critical link between siRNAs and heterochromatin assembly, with RITS acting as a physical bridge between non-coding RNA scaffolds and chromatin. Here, we review current understanding of how RITS promotes heterochromatin formation and how it participates in transcription coupled silencing. PMID:21823226

  4. RNAi-mediated gene function analysis in skin.

    PubMed

    Beronja, Slobodan; Fuchs, Elaine

    2013-01-01

    We have recently developed a method for RNAi-mediated gene function analysis in skin (Beronja et al., Nat Med 16:821-827, 2010). It employs ultrasound-guided in utero microinjections of lentivirus into the amniotic cavity of embryonic day 9 mice, which result in rapid, efficient, and stable transduction into mouse skin. Our technique greatly extends the available molecular and genetic toolbox for comprehensive functional examination of outstanding problems in epidermal biology. In its simplest form, as a single-gene function analysis via shRNA-mediated gene knockdown, our technique requires no animal mating and may need as little as only a few days between manipulation and phenotypic analysis.

  5. Transgenic, inducible RNAi in megakaryocytes and platelets in mice

    PubMed Central

    TAKIGUCHI, M.; JAMES, C.; JOSEFSSON, E. C.; CARMICHAEL, C. L.; PREMSRIRUT, P. K.; LOWE, S. W.; HAMILTON, J. R.; HUANG, D. C. S.; KILE, B. T.; DICKINS, R. A.

    2012-01-01

    Summary Background RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)-regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. Objective We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. Methods and results Using a tet-regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet-on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV-rtTA mice) display inducible reporter gene expression with unusual and near-complete efficiency in megakaryocytes and platelets. To test whether the CMV-rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet-regulated short hairpin RNA (shRNA) targeting Bcl-xL, a pro-survival Bcl-2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl-xL in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl-xL levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl-xL, clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. Conclusions We have established a novel transgenic strategy for inducible gene knockdown inmegakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice. PMID:21138522

  6. Identification of benzodiazepine Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput screen against RUNX1-CBFβ interaction.

    PubMed

    Cunningham, Lea; Finckbeiner, Steven; Hyde, R Katherine; Southall, Noel; Marugan, Juan; Yedavalli, Venkat R K; Dehdashti, Seameen Jean; Reinhold, William C; Alemu, Lemlem; Zhao, Ling; Yeh, Jing-Ruey Joanna; Sood, Raman; Pommier, Yves; Austin, Christopher P; Jeang, Kuan-Teh; Zheng, Wei; Liu, Paul

    2012-09-04

    Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for ~24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia (ALL). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-y survival rate of ~50%. We hypothesize that the interaction between RUNX1 and CBFβ is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput AlphaScreen and time-resolved fluorescence resonance energy transfer (TR-FRET) methods to quantify the RUNX1-CBFβ interaction and screen a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFβ directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued preleukemic phenotype in a RUNX1-ETO transgenic zebrafish, and reduced leukemia burden in a mouse CBFB-MYH11 leukemia model. Our data thus confirmed that RUNX1-CBFβ interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose.

  7. A high-throughput screening system for barley/powdery mildew interactions based on automated analysis of light micrographs

    PubMed Central

    Ihlow, Alexander; Schweizer, Patrick; Seiffert, Udo

    2008-01-01

    Background To find candidate genes that potentially influence the susceptibility or resistance of crop plants to powdery mildew fungi, an assay system based on transient-induced gene silencing (TIGS) as well as transient over-expression in single epidermal cells of barley has been developed. However, this system relies on quantitative microscopic analysis of the barley/powdery mildew interaction and will only become a high-throughput tool of phenomics upon automation of the most time-consuming steps. Results We have developed a high-throughput screening system based on a motorized microscope which evaluates the specimens fully automatically. A large-scale double-blind verification of the system showed an excellent agreement of manual and automated analysis and proved the system to work dependably. Furthermore, in a series of bombardment experiments an RNAi construct targeting the Mlo gene was included, which is expected to phenocopy resistance mediated by recessive loss-of-function alleles such as mlo5. In most cases, the automated analysis system recorded a shift towards resistance upon RNAi of Mlo, thus providing proof of concept for its usefulness in detecting gene-target effects. Conclusion Besides saving labor and enabling a screening of thousands of candidate genes, this system offers continuous operation of expensive laboratory equipment and provides a less subjective analysis as well as a complete and enduring documentation of the experimental raw data in terms of digital images. In general, it proves the concept of enabling available microscope hardware to handle challenging screening tasks fully automatically. PMID:18215267

  8. Lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of the Epstein-Barr-associated gastric carcinoma cell line GT38 in vitro

    PubMed Central

    Wang, Fangjun; Chen, Weichang; Liu, Pengfei; Zhou, Jundong; Liu, Bingtuan; Ye, Wu; Wang, Wenping; Shen, Xiuyun

    2017-01-01

    In this study, lentivirus-mediated RNA interference (RNAi) was applied to inhibit latent membrane protein 2A (LMP2A) gene expression, in order to explore the effects of LMP2A silencing on the growth of an Epstein-Barr virus-associated gastric carcinoma (EBVaGC) cell line in vitro. Lentivirus-mediated RNAi technology was employed to specifically knock down the LMP2A gene in the EBV-positive gastric carcinoma cell line GT38. After infection, reverse transcription-quantitative polymerase chain reaction, western blotting, flow cytometry and colony formation assays were conducted to evaluate the expression of LMP2A and the biological behavior of the GT38 cell line in vitro. The results showed that the expression of the LMP2A gene was clearly downregulated in the infected cells, which indicated that a highly efficient and stable lentivirus vector was successfully constructed. In the GT38 cells in which the expression of LMP2A was downregulated, the proliferation and colony formation of the cells was significantly inhibited. In addition, it was found that the cell cycle of the GT38 cells was arrested in the G0/G1 phase and the apoptosis rate was increased. These results indicate that lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of the EBVaGC cell line GT38 in vitro, and suggests that LMP2A is a potential target for gene therapy in the treatment of EBVaGC. PMID:28123488

  9. Theophylline controllable RNAi-based genetic switches regulate expression of lncRNA TINCR and malignant phenotypes in bladder cancer cells

    PubMed Central

    Chen, Zhicong; Liu, Yuchen; He, Anbang; Li, Jianfa; Chen, Mingwei; Zhan, Yonghao; Lin, Junhao; Zhuang, Chengle; Liu, Li; Zhao, Guoping; Huang, Weiren; Cai, Zhiming

    2016-01-01

    TINCR is a well-known lncRNA which acts as a master regulator in somatic differentiation development. However, it is still unclear whether TINCR is also involved in caner occurrence and progression. In this study, we observed that TINCR was up-regulated in bladder cancer tissues and cells and contributed to oncogenesis and cancer progression. Silencing TINCR expression inhibited cell proliferation and promoted apoptosis in vitro, indicating that TINCR may be the potential therapeutic target for treating bladder urothelial carcinoma. Thus we used the synthetic biology approach to create theophylline controllable RNAi-based genetic switches which silenced TINCR in a dosage-dependent manner. Both RNAi-OFF and ON switches can be used to quantitatively control the expression of TINCR in bladder cancer to suppress the progression of bladder cancer. These findings suggest that lncRNA-TINCR could promote bladder cancer development and progression and artificial control of its expression through inducible RNAi may represent a new kind of therapeutic strategy for treating human bladder cancer. PMID:27586866

  10. RNAi in Arthropods: Insight into the Machinery and Applications for Understanding the Pathogen-Vector Interface

    PubMed Central

    Barnard, Annette-Christi; Nijhof, Ard M.; Fick, Wilma; Stutzer, Christian; Maritz-Olivier, Christine

    2012-01-01

    The availability of genome sequencing data in combination with knowledge of expressed genes via transcriptome and proteome data has greatly advanced our understanding of arthropod vectors of disease. Not only have we gained insight into vector biology, but also into their respective vector-pathogen interactions. By combining the strengths of postgenomic databases and reverse genetic approaches such as RNAi, the numbers of available drug and vaccine targets, as well as number of transgenes for subsequent transgenic or paratransgenic approaches, have expanded. These are now paving the way for in-field control strategies of vectors and their pathogens. Basic scientific questions, such as understanding the basic components of the vector RNAi machinery, is vital, as this allows for the transfer of basic RNAi machinery components into RNAi-deficient vectors, thereby expanding the genetic toolbox of these RNAi-deficient vectors and pathogens. In this review, we focus on the current knowledge of arthropod vector RNAi machinery and the impact of RNAi on understanding vector biology and vector-pathogen interactions for which vector genomic data is available on VectorBase. PMID:24705082

  11. Delivery of RNAi Therapeutics to the Airways-From Bench to Bedside.

    PubMed

    Qiu, Yingshan; Lam, Jenny K W; Leung, Susan W S; Liang, Wanling

    2016-09-20

    RNA interference (RNAi) is a potent and specific post-transcriptional gene silencing process. Since its discovery, tremendous efforts have been made to translate RNAi technology into therapeutic applications for the treatment of different human diseases including respiratory diseases, by manipulating the expression of disease-associated gene(s). Similar to other nucleic acid-based therapeutics, the major hurdle of RNAi therapy is delivery. Pulmonary delivery is a promising approach of delivering RNAi therapeutics directly to the airways for treating local conditions and minimizing systemic side effects. It is a non-invasive route of administration that is generally well accepted by patients. However, pulmonary drug delivery is a challenge as the lungs pose a series of anatomical, physiological and immunological barriers to drug delivery. Understanding these barriers is essential for the development an effective RNA delivery system. In this review, the different barriers to pulmonary drug delivery are introduced. The potential of RNAi molecules as new class of therapeutics, and the latest preclinical and clinical studies of using RNAi therapeutics in different respiratory conditions are discussed in details. We hope this review can provide some useful insights for moving inhaled RNAi therapeutics from bench to bedside.

  12. In vivo RNAi Library Screen to Identify Mediators of Disease Progression and Drug Resistance in CML

    DTIC Science & Technology

    2006-09-01

    O. N., Ozawa, K., Ishikawa, T., Yazaki, Y., and Hirai, H. Development of acute lymphoblastic leukemia and myeloproliferative disorder in...of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood 1998;92:3780-92. 7...Consistent with previous reports (27), these animals developed a CML-like myeloproliferative disease and, occasionally, an acute leukemia. Although p53

  13. RNAi screen for kinases and phosphatases that play a role in antigen presentation by dendritic cells.

    PubMed

    Moita, Catarina F; Chora, Ângelo; Hacohen, Nir; Moita, Luis F

    2012-07-01

    Effective CD8(+) T-cell responses against tumor or microbial antigens that are not directly expressed in antigen-presenting cells (APCs) depend on the cross-presentation of these antigens on MHC class I in APCs. To identify signaling molecules that regulate cross-presentation, we used lentiviral-based RNA interference to test the roles of hundreds of kinases and phosphatases in this process. Our study uncovered eight previously unknown genes, consisting of one positive and seven negative regulators of antigen cross-presentation. Depletion of Acvr1c, a type I receptor for TGF-β family of signaling molecules, led to an increase in CD80 and CD86 co-stimulator surface expression and secreted IL-12 in mouse bone marrow-derived DCs, as well as antigen-specific T-cell proliferation.

  14. An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians

    PubMed Central

    Forsthoefel, David J.; James, Noelle P.; Escobar, David J.; Stary, Joel M.; Vieira, Ana P.; Waters, Forrest A.; Newmark, Phillip A.

    2012-01-01

    SUMMARY Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of post-mitotic tissues. Understanding how these processes are orchestrated requires characterizing cell type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis, and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling post-embryonic organogenesis. PMID:23079596

  15. An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians.

    PubMed

    Forsthoefel, David J; James, Noëlle P; Escobar, David J; Stary, Joel M; Vieira, Ana P; Waters, Forrest A; Newmark, Phillip A

    2012-10-16

    Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of postmitotic tissues. Understanding how these processes are orchestrated requires characterizing cell-type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling postembryonic organogenesis.

  16. Quantitative assessment of touch-screen panel by nondestructive inspection with three-dimensional real-time display optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Cho, Nam Hyun; Park, Kibeom; Kim, Jae-Young; Jung, Yeongri; Kim, Jeehyun

    2015-05-01

    We investigated the use of optical coherence tomography (OCT) to measure several materials immersed in optical adhesives. The effects of variations in the concentration, physical characteristics, and thickness of the materials were studied, and these parameters were found to significantly affect the OCT measurement. The materials were selected for their distinct spectral properties in the infrared region. To ensure reliability, we acquired images using a scanning electron microscope after performing the semiconductor production process. We verified the feasibility of the application of OCT for defect inspection and product verification of touch-screen panels.

  17. Potential and development of inhaled RNAi therapeutics for the treatment of pulmonary tuberculosis.

    PubMed

    Man, Dede K W; Chow, Michael Y T; Casettari, Luca; Gonzalez-Juarrero, Mercedes; Lam, Jenny K W

    2016-07-01

    Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (Mtb), continues to pose a serious threat to public health, and the situation is worsening with the rapid emergence of multidrug resistant (MDR) TB. Current TB regimens require long duration of treatment, and their toxic side effects often lead to poor adherence and low success rates. There is an urgent need for shorter and more effective treatment for TB. In recent years, RNA interference (RNAi) has become a powerful tool for studying gene function by silencing the target genes. The survival of Mtb in host macrophages involves the attenuation of the antimicrobial responses mounted by the host cells. RNAi technology has helped to improve our understanding of how these bacilli interferes with the bactericidal effect and host immunity during TB infection. It has been suggested that the host-directed intervention by modulation of host pathways can be employed as a novel and effective therapy against TB. This therapeutic approach could be achieved by RNAi, which holds enormous potential beyond a laboratory to the clinic. RNAi therapy targeting TB is being investigated for enhancing host antibacterial capacity or improving drug efficacy on drug resistance strains while minimizing the associated adverse effects. One of the key challenges of RNAi therapeutics arises from the delivery of the RNAi molecules into the target cells, and inhalation could serve as a direct administration route for the treatment of pulmonary TB in a non-invasive manner. However, there are still major obstacles that need to be overcome. This review focuses on the RNAi candidates that are currently explored for the treatment of TB and discusses the major barriers of pulmonary RNAi delivery. From this, we hope to stimulate further studies of local RNAi therapeutics for pulmonary TB treatment.

  18. Newborn Screening

    MedlinePlus

    ... Activities Importance of Newborn Screening Newborn Screening and Molecular Biology Branch Pulse Oximetry Screening for CCHDs Sickle Cell Disease Laboratory SCID Quality Assurance Training and Resources ...

  19. Diet-delivered RNAi in Helicoverpa armigera--Progresses and challenges.

    PubMed

    Lim, Zhi Xian; Robinson, Karl E; Jain, Ritesh G; Chandra, G Sharath; Asokan, R; Asgari, Sassan; Mitter, Neena

    2016-02-01

    Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.

  20. Screensaver: an open source lab information management system (LIMS) for high throughput screening facilities

    PubMed Central

    2010-01-01

    Background Shared-usage high throughput screening (HTS) facilities are becoming more common in academe as large-scale small molecule and genome-scale RNAi screening strategies are adopted for basic research purposes. These shared facilities require a unique informatics infrastructure that must not only provide access to and analysis of screening data, but must also manage the administrative and technical challenges associated with conducting numerous, interleaved screening efforts run by multiple independent research groups. Results We have developed Screensaver, a free, open source, web-based lab information management system (LIMS), to address the informatics needs of our small molecule and RNAi screening facility. Screensaver supports the storage and comparison of screening data sets, as well as the management of information about screens, screeners, libraries, and laboratory work requests. To our knowledge, Screensaver is one of the first applications to support the storage and analysis of data from both genome-scale RNAi screening projects and small molecule screening projects. Conclusions The informatics and administrative needs of an HTS facility may be best managed by a single, integrated, web-accessible application such as Screensaver. Screensaver has proven useful in meeting the requirements of the ICCB-Longwood/NSRB Screening Facility at Harvard Medical School, and has provided similar benefits to other HTS facilities. PMID:20482787

  1. [Near-infrared spectra combining with CARS and SPA algorithms to screen the variables and samples for quantitatively determining the soluble solids content in strawberry].

    PubMed

    Li, Jiang-bo; Guo, Zhi-ming; Huang, Wen-qian; Zhang, Bao-hua; Zhao, Chun-jiang

    2015-02-01

    In using spectroscopy to quantitatively or qualitatively analyze the quality of fruit, how to obtain a simple and effective correction model is very critical for the application and maintenance of the developed model. Strawberry as the research object, this research mainly focused on selecting the key variables and characteristic samples for quantitatively determining the soluble solids content. Competitive adaptive reweighted sampling (CARS) algorithm was firstly proposed to select the spectra variables. Then, Samples of correction set were selected by successive projections algorithm (SPA), and 98 characteristic samples were obtained. Next, based on the selected variables and characteristic samples, the second variable selection was performed by using SPA method. 25 key variables were obtained. In order to verify the performance of the proposed CARS algorithm, variable selection algorithms including Monte Carlo-uninformative variable elimination (MC-UVE) and SPA were used as the comparison algorithms. Results showed that CARS algorithm could eliminate uninformative variables and remove the collinearity information at the same time. Similarly, in order to assess the performance of the proposed SPA algorithm for selecting the characteristic samples, SPA algorithm was compared with classical Kennard-Stone algorithm Results showed that SPA algorithm could be used for selection of the characteristic samples in the calibration set. Finally, PLS and MLR model for quantitatively predicting the SSC (soluble solids content) in the strawberry were proposed based on the variables/samples subset (25/98), respectively. Results show that models built by using the 0.59% and 65.33% information of original variables and samples could obtain better performance than using the ones obtained by using all information of the original variables and samples. MLR model was the best with R(pre)2 = 0.9097, RMSEP=0.3484 and RPD = 3.3278.

  2. Discovery of novel targets with high throughput RNA interference screening.

    PubMed

    Kassner, Paul D

    2008-03-01

    High throughput technologies have the potential to affect all aspects of drug discovery. Considerable attention is paid to high throughput screening (HTS) for small molecule lead compounds. The identification of the targets that enter those HTS campaigns had been driven by basic research until the advent of genomics level data acquisition such as sequencing and gene expression microarrays. Large-scale profiling approaches (e.g., microarrays, protein analysis by mass spectrometry, and metabolite profiling) can yield vast quantities of data and important information. However, these approaches usually require painstaking in silico analysis and low-throughput basic wet-lab research to identify the function of a gene and validate the gene product as a potential therapeutic drug target. Functional genomic screening offers the promise of direct identification of genes involved in phenotypes of interest. In this review, RNA interference (RNAi) mediated loss-of-function screens will be discussed and as well as their utility in target identification. Some of the genes identified in these screens should produce similar phenotypes if their gene products are antagonized with drugs. With a carefully chosen phenotype, an understanding of the biology of RNAi and appreciation of the limitations of RNAi screening, there is great potential for the discovery of new drug targets.

  3. The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides

    DOE PAGES

    Nicolas, Francisco E.; Vila, Ana; Moxon, Simon; ...

    2015-03-25

    Here, RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which theymore » derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway. In conclusion, this work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M

  4. RNAi knockdown of parafusin inhibits the secretory pathway.

    PubMed

    Liu, Li; Wyroba, Elzbieta; Satir, Birgit H

    2011-10-01

    Several glycolytic enzymes and their isoforms have been found to be important in cell signaling unrelated to glycolysis. The involvement of parafusin (PFUS), a member of the phosphoglucomutase (PGM) superfamily with no phosphoglucomutase activity, in Ca(2+)-dependent exocytosis has been controversial. This protein was first described in Paramecium tetraurelia, but is widely found. Earlier work showed that parafusin is a secretory vesicle scaffold component with unusual post-translational modifications (cyclic phosphorylation and phosphoglucosylation) coupled to stages in the exocytic process. Using RNAi, we demonstrate that parafusin synthesis can be reversibly blocked, with minor or no effect on other PGM isoforms. PFUS knockdown produces an inhibition of dense core secretory vesicle (DCSV) synthesis leading to an exo(-) phenotype. Although cell growth is unaffected, vesicle content is not packaged properly and no new DCSVs are formed. We conclude that PFUS and its orthologs are necessary for proper scaffold maturation. Because of this association, parafusin is an important signaling component for regulatory control of the secretory pathway.

  5. The insect ecdysone receptor is a good potential target for RNAi-based pest control.

    PubMed

    Yu, Rong; Xu, Xinping; Liang, Yongkang; Tian, Honggang; Pan, Zhanqing; Jin, Shouheng; Wang, Na; Zhang, Wenqing

    2014-01-01

    RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests.

  6. Long-term effects and parental RNAi in the blood feeder Rhodnius prolixus (Hemiptera; Reduviidae).

    PubMed

    Paim, Rafaela M M; Araujo, Ricardo N; Lehane, Michael J; Gontijo, Nelder F; Pereira, Marcos H

    2013-11-01

    RNA interference (RNAi) has been widely employed as a useful alternative to study gene function in insects, including triatomine bugs. However, several aspects related to the RNAi mechanism and functioning are still unclear. The aim of this study is to investigate the persistence and the occurrence of systemic and parental RNAi in the triatomine bug Rhodnius prolixus. For such, the nitrophorins 1 to 4 (NP1-4), which are salivary hemeproteins, and the rhodniin, an intestinal protein, were used as targets for RNAi. The dsRNA for both molecules were injected separately into 3rd and 5th instar nymphs of R. prolixus and the knockdown (mRNA levels and phenotype) were progressively evaluated along several stages of the insect's life. We observed that the NP1-4 knockdown persisted for more than 7 months after the dsRNA injection, and at least 5 months in rhodniin knockdown, passing through various nymphal stages until the adult stage, without continuous input of dsRNA. The parental RNAi was successful from the dsRNA injection in 5th instar nymphs for both knockdown targets, when the RNAi effects (mRNA levels and phenotype) were observed at least in the 2nd instar nymphs of the F1 generation. However, the parental RNAi did not occur when the dsRNA was injected in the 3rd instars. The confirmation of the long persistence and parental transmission of RNAi in R. prolixus can improve and facilitate the utilization of this tool in insect functional genomic studies.

  7. Vitellogenin RNAi halts ovarian growth and diverts reproductive proteins and lipids in young grasshoppers.

    PubMed

    Tokar, Derek R; Veleta, Katherine A; Canzano, Joseph; Hahn, Daniel A; Hatle, John D

    2014-11-01

    Reduced reproduction extends lifespan of females in many animals. To test the effects of reproduction on storage of macronutrients, we block reproductive output in the lubber grasshopper by injecting RNAi against the precursor to egg-yolk protein, vitellogenin, in early adulthood. Controls were injected with either buffer or RNAi against the major storage protein in the hemolymph, hexamerin-90. Vitellogenin RNAi greatly reduced both levels of mRNA for vitellogenin and ovarian growth, in comparison to both controls. Fat body mass was increased upon vitellogenin RNAi, but concentrations of the three hexameric storage proteins from the hemolymph were not. Surprisingly, hemolymph vitellogenin levels were increased upon vitellogenin RNAi. Total reproductive protein (hemolymph vitellogenin plus ovarian vitellin) was unchanged by vitellogenin RNAi, as reproductive protein was diverted to the hemolymph. Similarly, the increased lipid storage upon vitellogenin RNAi was largely attributable to the reduction in lipid in the ovary, due to decreased ovarian growth. A BLAST search revealed that the 515 bp sequence of vitellogenin used for RNAi had three 11 bp regions identical to the vitellogenin receptor of the cockroach Leucophaea maderae. This suggests that our treatment, in addition to reducing levels of vitellogenin transcript, may have also blocked transport of vitellogenin from the hemolymph to the ovary. This would be consistent with halted ovarian growth simultaneous with high levels of vitellogenin in the hemolymph. Nonetheless, the accumulation of vitellogenin, instead of hexameric storage proteins, is inconsistent with a simple model of the trade-off between reproduction and storage. This was observed in young females; future studies will address whether investment of proteins may shift to the soma as individuals age. Overall, our results suggest that blockage of reproduction in young grasshoppers redirects lipids to storage and reproductive proteins to the hemolymph.

  8. Systemic RNAi in western corn rootworm, Diabrotica virgifera virgifera, does not involve transitive pathways.

    PubMed

    Li, Huarong; Bowling, Andrew J; Gandra, Premchand; Rangasamy, Murugesan; Pence, Heather E; McEwan, Robert E; Khajuria, Chitvan; Siegfried, Blair D; Narva, Kenneth E

    2016-08-13

    Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double-stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA-dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi-mediated knockdown of Dv v-ATPase C mRNA throughout the WCR gut and other tissues using high-sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v-ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.

  9. The status of RNAi-based transgenic research in plant nematology

    PubMed Central

    Dutta, Tushar K.; Banakar, Prakash; Rao, Uma

    2015-01-01

    With the understanding of nematode-plant interactions at the molecular level, new avenues for engineering resistance have opened up, with RNA interference being one of them. Induction of RNAi by delivering double-stranded RNA (dsRNA) has been very successful in the model non-parasitic nematode, Caenorhabditis elegans, while in plant nematodes, dsRNA delivery has been accomplished by soaking nematodes with dsRNA solution mixed with synthetic neurostimulants. The success of in vitro RNAi of target genes has inspired the use of in planta delivery of dsRNA to feeding nematodes. The most convincing success of host-delivered RNAi has been achieved against root-knot nematodes. Plant-mediated RNAi has been shown to lead to the specific down-regulation of target genes in invading nematodes, which had a profound effect on nematode development. RNAi-based transgenics are advantageous as they do not produce any functional foreign proteins and target organisms in a sequence-specific manner. Although the development of RNAi-based transgenics against plant nematodes is still in the preliminary stage, they offer novel management strategy for the future. PMID:25628609

  10. RNAi and antiviral defense in Drosophila: setting up a systemic immune response.

    PubMed

    Karlikow, Margot; Goic, Bertsy; Saleh, Maria-Carla

    2014-01-01

    RNA interference (RNAi) controls gene expression in eukaryotic cells and thus, cellular homeostasis. In addition, in plants, nematodes and arthropods it is a central antiviral effector mechanism. Antiviral RNAi has been well described as a cell autonomous response, which is triggered by double-stranded RNA (dsRNA) molecules. This dsRNA is the precursor for the silencing of viral RNA in a sequence-specific manner. In plants, systemic antiviral immunity has been demonstrated, however much less is known in animals. Recently, some evidence for a systemic antiviral response in arthropods has come to light. Cell autonomous RNAi may not be sufficient to reach an efficient antiviral response, and the organism might rely on the spread and uptake of an RNAi signal of unknown origin. In this review, we offer a perspective on how RNAi-mediated antiviral immunity could confer systemic protection in insects and we propose directions for future research to understand the mechanism of RNAi-immune signal sorting, spreading and amplification.

  11. Preclinical evaluation of RNAi as a treatment for transthyretin-mediated amyloidosis

    PubMed Central

    Butler, James S.; Chan, Amy; Costelha, Susete; Fishman, Shannon; Willoughby, Jennifer L. S.; Borland, Todd D.; Milstein, Stuart; Foster, Donald J.; Gonçalves, Paula; Chen, Qingmin; Qin, June; Bettencourt, Brian R.; Sah, Dinah W.; Alvarez, Rene; Rajeev, Kallanthottathil G.; Manoharan, Muthiah; Fitzgerald, Kevin; Meyers, Rachel E.; Nochur, Saraswathy V.; Saraiva, Maria J.; Zimmermann, Tracy S.

    2016-01-01

    Abstract ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis. PMID:27033334

  12. RNAi--a tool for target finding in new drug development.

    PubMed

    Gomase, Virendra S; Tagore, Somnath

    2008-03-01

    RNAi (RNA interference) refers to the introduction of homologous double stranded RNA (dsRNA) to specifically target a gene's product, resulting in null or hypomorphic phenotypes. Long double-stranded RNAs (dsRNAs; typically >200 nt) can be used to silence the expression of target genes in a variety of organisms and cell types (e.g., worms, fruit flies, and plants). The long dsRNAs enter a cellular pathway that is commonly referred to as the RNA interference (RNAi) pathway. RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes. RNAi plays a very important role in endogenous cellular processes, such as heterochromatin formation, developmental control and serves as an antiviral defense mechanism. RNAi has shown great potential for use as a tool for target finding in new drug development, molecular biological discovery, analysis and therapeutics. RNAi pathway is involved in post-transcription silencing, transcriptional silencing and epigenetic silencing as well as its use as a tool for forward genetics and therapeutics.

  13. Canine toys and training devices as sources of exposure to phthalates and bisphenol A: quantitation of chemicals in leachate and in vitro screening for endocrine activity.

    PubMed

    Wooten, Kimberly J; Smith, Philip N

    2013-11-01

    Chewing and mouthing behaviors exhibited by pet dogs are likely to lead to oral exposures to a variety of environmental chemicals. Products intended for chewing and mouthing uses include toys and training devices that are often made of plastics. The goal of the current study was to determine if a subset of phthalates and bisphenol A (BPA), endocrine disrupting chemicals commonly found in plastics, leach out of dog toys and training devices (bumpers) into synthetic canine saliva. In vitro assays were used to screen leachates for endocrine activity. Bumper leachates were dominated by di-2-ethylhexyl phthalate (DEHP) and BPA, with concentrations reaching low μg mL(-1) following short immersions in synthetic saliva. Simulated chewing of bumpers during immersion in synthetic saliva increased concentrations of phthalates and BPA as compared to new bumpers, while outdoor storage had variable effects on concentrations (increased DEHP; decreased BPA). Toys leached substantially lower concentrations of phthalates and BPA, with the exception of one toy which leached considerable amounts of diethyl phthalate. In vitro assays indicated anti-androgenic activity of bumper leachates, and estrogenic activity of both bumper and toy leachates. These results confirm that toys and training devices are potential sources of exposure to endocrine disrupting chemicals in pet dogs.

  14. Parameters for Successful Parental RNAi as An Insect Pest Management Tool in Western Corn Rootworm, Diabrotica virgifera virgifera

    PubMed Central

    Vélez, Ana M.; Fishilevich, Elane; Matz, Natalie; Storer, Nicholas P.; Narva, Kenneth E.; Siegfried, Blair D.

    2016-01-01

    Parental RNAi (pRNAi) is an RNA interference response where the gene knockdown phenotype is observed in the progeny of the treated organism. pRNAi has been demonstrated in female western corn rootworms (WCR) via diet applications and has been described as a potential approach for rootworm pest management. However, it is not clear if plant-expressed pRNAi can provide effective control of next generation WCR larvae in the field. In this study, we evaluated parameters required to generate a successful pRNAi response in WCR for the genes brahma and hunchback. The parameters tested included a concentration response, duration of the dsRNA exposure, timing of the dsRNA exposure with respect to the mating status in WCR females, and the effects of pRNAi on males. Results indicate that all of the above parameters affect the strength of pRNAi phenotype in females. Results are interpreted in terms of how this technology will perform in the field and the potential role for pRNAi in pest and resistance management strategies. More broadly, the described approaches enable examination of the dynamics of RNAi response in insects beyond pRNAi and crop pests. PMID:28029123

  15. Parameters for Successful Parental RNAi as An Insect Pest Management Tool in Western Corn Rootworm, Diabrotica virgifera virgifera.

    PubMed

    Vélez, Ana M; Fishilevich, Elane; Matz, Natalie; Storer, Nicholas P; Narva, Kenneth E; Siegfried, Blair D

    2016-12-24

    Parental RNAi (pRNAi) is an RNA interference response where the gene knockdown phenotype is observed in the progeny of the treated organism. pRNAi has been demonstrated in female western corn rootworms (WCR) via diet applications and has been described as a potential approach for rootworm pest management. However, it is not clear if plant-expressed pRNAi can provide effective control of next generation WCR larvae in the field. In this study, we evaluated parameters required to generate a successful pRNAi response in WCR for the genes brahma and hunchback. The parameters tested included a concentration response, duration of the dsRNA exposure, timing of the dsRNA exposure with respect to the mating status in WCR females, and the effects of pRNAi on males. Results indicate that all of the above parameters affect the strength of pRNAi phenotype in females. Results are interpreted in terms of how this technology will perform in the field and the potential role for pRNAi in pest and resistance management strategies. More broadly, the described approaches enable examination of the dynamics of RNAi response in insects beyond pRNAi and crop pests.

  16. Screening and Validation of Housekeeping Genes of the Root and Cotyledon of Cunninghamia lanceolata under Abiotic Stresses by Using Quantitative Real-Time PCR

    PubMed Central

    Bao, Wenlong; Qu, Yanli; Shan, Xiaoyi; Wan, Yinglang

    2016-01-01

    Cunninghamia lanceolata (Chinese fir) is a fast-growing and commercially important conifer of the Cupressaceae family. Due to the unavailability of complete genome sequences and relatively poor genetic background information of the Chinese fir, it is necessary to identify and analyze the expression levels of suitable housekeeping genes (HKGs) as internal reference for precise analysis. Based on the results of database analysis and transcriptome sequencing, we have chosen five candidate HKGs (Actin, GAPDH, EF1a, 18S rRNA, and UBQ) with conservative sequences in the Chinese fir and related species for quantitative analysis. The expression levels of these HKGs in roots and cotyledons under five different abiotic stresses in different time intervals were measured by qRT-PCR. The data were statistically analyzed using the following algorithms: NormFinder, BestKeeper, and geNorm. Finally, RankAggreg was applied to merge the sequences generated from three programs and rank these according to consensus sequences. The expression levels of these HKGs showed variable stabilities under different abiotic stresses. Among these, Actin was the most stable internal control in root, and GAPDH was the most stable housekeeping gene in cotyledon. We have also described an experimental procedure for selecting HKGs based on the de novo sequencing database of other non-model plants. PMID:27483238

  17. ScreenBEAM: a novel meta-analysis algorithm for functional genomics screens via Bayesian hierarchical modeling | Office of Cancer Genomics

    Cancer.gov

    Functional genomics (FG) screens, using RNAi or CRISPR technology, have become a standard tool for systematic, genome-wide loss-of-function studies for therapeutic target discovery. As in many large-scale assays, however, off-target effects, variable reagents' potency and experimental noise must be accounted for appropriately control for false positives.

  18. Comparison of RNAi sequences in insect-resistant plants to expressed sequences of a beneficial lady beetle: a closer look at RNAi off-target considerations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sequences obtained from transcriptomes of the lady beetle Coleomegilla maculata were compared to those designed for incorporation into crops. Searches of the transcriptomes identified sequences as the most likely to be closely similar to the sequences described in RNAi plant incorporated products. S...

  19. Colorectal polyp model established by transplacental BMP4 RNAi

    PubMed Central

    JIN, XIN; CHEN, ZHONGMEI; XIANG, LI; LUO, QING; GUO, ZHENGHUA; DING, XIONGHUI; JIN, XIANQING

    2014-01-01

    Previous studies have shown that disruption of the bone morphogenetic protein (BMP) signaling pathway is an important cause of intestinal cancer in human and animal models. Thus, the purpose of this study was to construct a Balb/C model of colorectal polyps. Pregnant mice at 9.5 days gestation were injected via the tail vein with the pSES-Si BMP4 plasmid bearing a fluorochrome (DsRed) reporter, in order to silence the BMP4 gene in the first generation (F1); this group of mice was named the pSES-BMP4 group Intestinal fluorescence was detected at 1-, 4- and 8-week-old F1 mice, and reverse transcription-polymerase chain reaction (RT-PCR) and western-blotting assays were used to determine changes in the expression of BMP4. A dissecting microscope and hematoxylin and eosin (H&E) staining were used to observe the cell morphology and appearance of the polyps. DsRed fluorescence was observed in the intestines of 1-week-old F1 mice of the pSES-BMP4 group. BMP4 expression at the mRNA and protein level was reduced in 1-, 4- and 8-week-old F1 mice (P<0.05). However, the level of Smad4 mRNA was only reduced in 8-week-old F1 mice (P<0.05). Multiple hyperplasic polyps emerged in the colon and rectum of the intestines of 4-week-old F1 mice in the pSES-BMP4 group. The size of colorectal polyps increased at 8 weeks, when vessels and polyp pedicles became apparent. In conclusion, silencing of the BMP4 gene using transplacental RNAi injection can induce formation of colorectal polyps in mice. PMID:24806485

  20. Structure-based approach to pharmacophore identification, in silico screening, and three-dimensional quantitative structure-activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function

    SciTech Connect

    Schormann, N.; Senkovich, O.; Walker, K.; Wright, D.L.; Anderson, A.C.; Rosowsky, A.; Ananthan, S.; Shinkre, B.; Velu, S.; Chattopadhyay, D.

    2009-07-10

    We have employed a structure-based three-dimensional quantitative structure-activity relationship (3D-QSAR) approach to predict the biochemical activity for inhibitors of T. cruzi dihydrofolate reductase-thymidylate synthase (DHFR-TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure-based alignment used as input for the QSAR study. Contrary to indirect ligand-based approaches the strategy described here employs a direct receptor-based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D-QSAR models were obtained for T. cruzi DHFR-TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between experimental and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave-one-out method. The derived 3D-QSAR models were tested against a few selected compounds (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D-QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of additional activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability.

  1. Structure-based approach to pharmacophore identification, in silico screening, and three-dimensional quantitative structure-activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function.

    PubMed

    Schormann, N; Senkovich, O; Walker, K; Wright, D L; Anderson, A C; Rosowsky, A; Ananthan, S; Shinkre, B; Velu, S; Chattopadhyay, D

    2008-12-01

    We have employed a structure-based three-dimensional quantitative structure-activity relationship (3D-QSAR) approach to predict the biochemical activity for inhibitors of T. cruzi dihydrofolate reductase-thymidylate synthase (DHFR-TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure-based alignment used as input for the QSAR study. Contrary to indirect ligand-based approaches the strategy described here employs a direct receptor-based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D-QSAR models were obtained for T. cruzi DHFR-TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between experimental and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave-one-out method. The derived 3D-QSAR models were tested against a few selected compounds (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D-QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of additional activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability.

  2. Screening of candidate genes and fine mapping of drought tolerance quantitative trait loci on chromosome 4 in rice (Oryza sativa L.) under drought stress.

    PubMed

    Nie, Yuan-Yuan; Zhang, Lin; Wu, Yun-Hua; Liu, Hao-Jie; Mao, Wei-Wei; Du, Juan; Xiu, Hai-Lin; Wu, Xiao-Yu; Li, Xia; Yan, Yu-Wei; Liu, Guo-Lan; Liu, Hong-Yan; Hu, Song-Ping

    2015-11-01

    Due to severe water resource shortage, genetics of and breeding for DT (drought tolerance) in rice (Oryza sativa L.) have become one of the hot research topics. Identification of grain yield QTLs (quantitative trait loci) directly related to the DT trait of rice can provide useful information for breeding new drought-resistant and water-saving rice varieties via marker-assisted selection. A population of 105 advanced BILs (backcross introgression lines) derived from a cross between Zhenshan97B and IRAT109 in Zhenshan97B background were grown under drought stress in a field experiment and phenotypic traits were investigated. The results showed that in the target interval of RM273-RM255 on chromosome 4, three main-effect QTLs related to panicle length, panicle number, and spikelet number per panicle were identified (LOD [logarithm of the odds] > 2.0). The panicle length-related QTL had two loci located in the neighboring intervals of RM17308-RM17305 and RM17349-RM17190, which explained 18.80% and 20.42%, respectively, of the phenotypic variation, while the panicle number-related QTL was identified in the interval of RM1354-RM17308, explaining 11.47% of the phenotypic variation. As far as the spikelet number per panicle-related QTL was concerned, it was found to be located in the interval of RM17308-RM17305, which explained 28.08% of the phenotypic variation. Using the online Plant-GE query system, a total of 13 matched ESTs (expressed sequence tags) were found in the target region, and of the 13 ESTs, 12 had corresponding predicted genes. For instance, the two ESTs CB096766 and CA765747 were corresponded to the same predicted gene LOC_Os04g46370, while the other four ESTs, CA754286, CB000011, CX056247, and CX056240, were corresponded to the same predicted gene LOC_Os04g46390.

  3. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

    PubMed Central

    Davis, Mark E.; Zuckerman, Jonathan E.; Choi, Chung Hang J.; Seligson, David; Tolcher, Anthony; Alabi, Christopher A.; Yen, Yun; Heidel, Jeremy D.; Ribas, Antoni

    2010-01-01

    Therapeutics that are designed to engage RNA interference (RNAi) pathways have the potential to provide new, major ways of imparting therapy to patients.1,2 Fire et al. first demonstrated that long, double stranded RNAs mediate RNAi in Caenorhabditis elegans,3 and Elbashir et al. opened the pathway to the use of RNAi for human therapy by showing that small interfering RNAs (siRNAs: ca. 21 base pair double stranded RNA) can elicit RNAi in mammalian cells without producing an interferon response.4 We are currently conducting the first-in-human Phase I clinical trial involving the systemic administration of siRNA to patients with solid cancers using a targeted, nanoparticle delivery system. Here we provide evidence of inducing an RNAi mechanism of action in a human from the delivered siRNA. Tumor biopsies from melanoma patients obtained after treatment reveal: (i) the presence of intracellularly-localized nanoparticles in amounts that correlate with dose levels of the nanoparticles administered (this is a first for systemically delivered nanoparticles of any kind), and (ii) reduction in both the specific mRNA (M2 subunit of ribonucleotide reductase (RRM2)) and the protein (RRM2) when compared to pre-dosing tissue. Most importantly, we detect the presence of an mRNA fragment that demonstrates siRNA mediated mRNA cleavage occurs specifically at the site predicted for an RNAi mechanism from a patient who received the highest dose of the nanoparticles. These data when taken in total demonstrate that siRNA administered systemically to a human can produce a specific gene inhibition (reduction in mRNA and protein) by an RNAi mechanism of action. PMID:20305636

  4. Functional analysis of the RNAi response in ovary-derived silkmoth Bm5 cells.

    PubMed

    Kolliopoulou, Anna; Swevers, Luc

    2013-08-01

    Experiments of dsRNA-mediated gene silencing in lepidopteran insects in vivo are characterized by high variability although lepidopteran cell cultures have shown an efficient response to RNAi in transfection experiments. In order to identify the core RNAi factors that regulate the RNAi response of Lepidoptera, we employed the silkmoth ovary-derived Bm5 cells as a test system since this cell line is known to respond potently in silencing after dsRNA transfection. Two parallel approaches were used; involving knock-down of the core RNAi genes or over-expression of the main siRNA pathway factors, in order to study possible inhibition or stimulation of the RNAi silencing response, respectively. Components from all three main small RNA pathways (BmAgo-1 for miRNA, BmAgo-2/BmDcr-2 for siRNA, and BmAgo-3 for piRNA) were found to be involved in the RNAi response that is triggered by dsRNA. Since BmAgo-3, a factor in the piRNA pathway that functions independent of Dicer in Drosophila, was identified as a limiting factor in the RNAi response, sense and antisense ssRNA was also tested to induce gene silencing but proved to be ineffective, suggesting a dsRNA-dependent role for BmAgo-3 in Bombyx mori. After efficient over-expression of the main siRNA factors, immunofluorescence staining revealed a predominant cytoplasmic localization in Bm5 cells. This is the first study in Lepidoptera to provide evidence for possible overlapping of all three known small RNA pathways in the regulation of the dsRNA-mediated silencing response using transfected B. mori-derived Bm5 cells as experimental system.

  5. Dissecting Systemic RNA Interference in the Red Flour Beetle Tribolium castaneum: Parameters Affecting the Efficiency of RNAi

    PubMed Central

    Miller, Sherry C.; Miyata, Keita; Brown, Susan J.; Tomoyasu, Yoshinori

    2012-01-01

    The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects. PMID:23133513

  6. The SID-1 double-stranded RNA transporter is not required for systemic RNAi in the migratory locust.

    PubMed

    Luo, Yuan; Wang, Xianhui; Yu, Dan; Kang, Le

    2012-05-01

    Systemic RNAi, the spreading of RNAi effects to cells and tissues that have not initially encountered a dsRNA trigger, is a common phenomenon in many organisms. However, the underlying mechanisms of systemic RNAi remain largely unknown. Here, we studied the characteristics and possible mechanisms of systemic RNAi in Locusta migratoria. We observed that the locust has pronounced sensitive systemic RNAi in response to dsRNA injection for both broadly-expressed as well as tissue-specific genes. Only a 30 pg (dsRNA / mg tissues) dose could induce significant systemic RNAi effects. Only one SID-1 ortholog (LmSID-1) was identified in the locust genome, which exhibited a progressively increasing expression pattern with development and its expression was enriched in the gonad. To test the role of LmSID-1 in systemic RNAi, we performed in vitro and in vivo experiments. The results from in vivo experiments showed that silencing of LmSID-1 gene dose not influence RNAi effects of other genes. The results from in vitro experiments confirmed that the expression of the LmSID-1 protein in Drosophila S2 cells could not enhance dsRNA uptake. Thus, these findings imply the existence of alternative mechanisms underlying insect systemic RNAi, which may be different from Caenorhabditis elegans or mammals.

  7. [Expression analysis of a transformer gene in Daphnia pulex after RNAi].

    PubMed

    Guo, C Y; Chen, P; Zhang, M M; Ning, J J; Wang, С L; Wang, D L; Zhao, Y L

    2016-01-01

    In order to explore the importance of the transformer (tra) gene in reproductive mode switching in Daphnia pulex, we studied the effect of silencing of this gene using RNA interference (RNAi). We obtained Dptra dsRNA by constructing and using a dsRNA expression vector and transcription method in vitro. D. pulex individuals in different reproductive modes were treated by soaking in a solution of Dptra dsRNA. We then assayed the expression of the endogenous Dptra mRNA after RNAi treatment using RT-PCR and obtained the suppression ratio. Expression of the tra gene in the RNAi groups was down-regulated compared with the controls after 16 h (p < 0.05). We also analyzed the effect of RNAi on the expression of the TRA protein using Western blot, which showed that the expression level of the TRA protein was reduced after RNAi treatment. Our experimental results showed that soaking of D. pulex adults in tra-specific dsRNA transcribed in vitro can specifically reduce the level of tra mRNA and also reduce the expression of the TRA protein, demonstrating effective in vivo silencing of the tra gene.

  8. Recent advances in RNAi-based strategies for therapy and prevention of HIV-1/AIDS.

    PubMed

    Swamy, Manjunath N; Wu, Haoquan; Shankar, Premlata

    2016-08-01

    RNA interference (RNAi) provides a powerful tool to silence specific gene expression and has been widely used to suppress host factors such as CCR5 and/or viral genes involved in HIV-1 replication. Newer nuclease-based gene-editing technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, also provide powerful tools to ablate specific genes. Because of differences in co-receptor usage and the high mutability of the HIV-1 genome, a combination of host factors and viral genes needs to be suppressed for effective prevention and treatment of HIV-1 infection. Whereas the continued presence of small interfering/short hairpin RNA (si/shRNA) mediators is needed for RNAi to be effective, the continued expression of nucleases in the gene-editing systems is undesirable. Thus, RNAi provides the only practical way for expression of multiple silencers in infected and uninfected cells, which is needed for effective prevention/treatment of infection. There have been several advances in the RNAi field in terms of si/shRNA design, targeted delivery to HIV-1 susceptible cells, and testing for efficacy in preclinical humanized mouse models. Here, we comprehensively review the latest advances in RNAi technology towards prevention and treatment of HIV-1.

  9. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation.

    PubMed

    Perkins, Lizabeth A; Holderbaum, Laura; Tao, Rong; Hu, Yanhui; Sopko, Richelle; McCall, Kim; Yang-Zhou, Donghui; Flockhart, Ian; Binari, Richard; Shim, Hye-Seok; Miller, Audrey; Housden, Amy; Foos, Marianna; Randkelv, Sakara; Kelley, Colleen; Namgyal, Pema; Villalta, Christians; Liu, Lu-Ping; Jiang, Xia; Huan-Huan, Qiao; Wang, Xia; Fujiyama, Asao; Toyoda, Atsushi; Ayers, Kathleen; Blum, Allison; Czech, Benjamin; Neumuller, Ralph; Yan, Dong; Cavallaro, Amanda; Hibbard, Karen; Hall, Don; Cooley, Lynn; Hannon, Gregory J; Lehmann, Ruth; Parks, Annette; Mohr, Stephanie E; Ueda, Ryu; Kondo, Shu; Ni, Jian-Quan; Perrimon, Norbert

    2015-11-01

    To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

  10. Key enzymes and proteins of crop insects as candidate for RNAi based gene silencing

    PubMed Central

    Kola, Vijaya Sudhakara Rao; Renuka, P.; Madhav, Maganti Sheshu; Mangrauthia, Satendra K.

    2015-01-01

    RNA interference (RNAi) is a mechanism of homology dependent gene silencing present in plants and animals. It operates through 21–24 nucleotides small RNAs which are processed through a set of core enzymatic machinery that involves Dicer and Argonaute proteins. In recent past, the technology has been well appreciated toward the control of plant pathogens and insects through suppression of key genes/proteins of infecting organisms. The genes encoding key enzymes/proteins with the great potential for developing an effective insect control by RNAi approach are actylcholinesterase, cytochrome P450 enzymes, amino peptidase N, allatostatin, allatotropin, tryptophan oxygenase, arginine kinase, vacuolar ATPase, chitin synthase, glutathione-S-transferase, catalase, trehalose phosphate synthase, vitellogenin, hydroxy-3-methylglutaryl coenzyme A reductase, and hormone receptor genes. Through various studies, it is demonstrated that RNAi is a reliable molecular tool which offers great promises in meeting the challenges imposed by crop insects with careful selection of key enzymes/proteins. Utilization of RNAi tool to target some of these key proteins of crop insects through various approaches is described here. The major challenges of RNAi based insect control such as identifying potential targets, delivery methods of silencing trigger, off target effects, and complexity of insect biology are very well illustrated. Further, required efforts to address these challenges are also discussed. PMID:25954206

  11. Late extraembryonic morphogenesis and its zen(RNAi)-induced failure in the milkweed bug Oncopeltus fasciatus.

    PubMed

    Panfilio, Kristen A

    2009-09-15

    Many insects undergo katatrepsis, essential reorganization by the extraembryonic membranes that repositions the embryo. Knockdown of the zen gene by RNA interference (RNAi) prevents katatrepsis in the milkweed bug Oncopeltus fasciatus. However, the precise morphogenetic defect has been uncertain, and katatrepsis itself has not been characterized in detail. The dynamics of wild type and zen(RNAi) eggs were analyzed from time-lapse movies, supplemented by analysis of fixed specimens. These investigations identify three zen(RNAi) defects. First, a reduced degree of tissue contraction implies a role for zen in baseline compression prior to katatrepsis. Subsequently, a characteristic 'bouncing' activity commences, leading to the initiation of katatrepsis in wild type eggs. The second zen(RNAi) defect is a delay in this activity, suggesting that a temporal window of opportunity is missed after zen knockdown. Ultimately, the extraembryonic membranes fail to rupture in zen(RNAi) eggs: the third defect. Nevertheless, the outer serosal membrane manages to contract, albeit in an aberrant fashion with additional phenotypic consequences for the embryo. These data identify a novel epithelial morphogenetic event - rupture of the 'serosal window' structure - as the ultimate site of defect. Overall, Oncopeltus zen seems to have a role in coordinating a number of pre-katatreptic events during mid embryogenesis.

  12. High-Throughput Construction of Intron-Containing Hairpin RNA Vectors for RNAi in Plants

    PubMed Central

    Yan, Pu; Shen, Wentao; Gao, XinZheng; Li, Xiaoying; Zhou, Peng; Duan, Jun

    2012-01-01

    With the wide use of double-stranded RNA interference (RNAi) for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA) constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA) vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG) cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics. PMID:22675447

  13. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation

    PubMed Central

    Perkins, Lizabeth A.; Holderbaum, Laura; Tao, Rong; Hu, Yanhui; Sopko, Richelle; McCall, Kim; Yang-Zhou, Donghui; Flockhart, Ian; Binari, Richard; Shim, Hye-Seok; Miller, Audrey; Housden, Amy; Foos, Marianna; Randkelv, Sakara; Kelley, Colleen; Namgyal, Pema; Villalta, Christians; Liu, Lu-Ping; Jiang, Xia; Huan-Huan, Qiao; Wang, Xia; Fujiyama, Asao; Toyoda, Atsushi; Ayers, Kathleen; Blum, Allison; Czech, Benjamin; Neumuller, Ralph; Yan, Dong; Cavallaro, Amanda; Hibbard, Karen; Hall, Don; Cooley, Lynn; Hannon, Gregory J.; Lehmann, Ruth; Parks, Annette; Mohr, Stephanie E.; Ueda, Ryu; Kondo, Shu; Ni, Jian-Quan; Perrimon, Norbert

    2015-01-01

    To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT–qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT–qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China). PMID:26320097

  14. Biosafety considerations of RNAi-mediated virus resistance in fruit-tree cultivars and in rootstock.

    PubMed

    Lemgo, Godwin Nana Yaw; Sabbadini, Silvia; Pandolfini, Tiziana; Mezzetti, Bruno

    2013-12-01

    A major application of RNA interference (RNAi) is envisaged for the production of virus-resistant transgenic plants. For fruit trees, this remains the most, if not the only, viable option for the control of plant viral disease outbreaks in cultivated orchards, due to the difficulties associated with the use of traditional and conventional disease-control measures. The use of RNAi might provide an additional benefit for woody crops if silenced rootstock can efficiently transmit the silencing signal to non-transformed scions, as has already been demonstrated in herbaceous plants. This would provide a great opportunity to produce non-transgenic fruit from transgenic rootstock. In this review, we scrutinise some of the concerns that might arise with the use of RNAi for engineering virus-resistant plants, and we speculate that this virus resistance has fewer biosafety concerns. This is mainly because RNAi-eliciting constructs only express small RNA molecules rather than proteins, and because this technology can be applied using plant rootstock that can confer virus resistance to the scion, leaving the scion untransformed. We discuss the main biosafety concerns related to the release of new types of virus-resistant plants and the risk assessment approaches in the application of existing regulatory systems (in particular, those of the European Union, the USA, and Canada) for the evaluation and approval of RNAi-mediated virus-resistant plants, either as transgenic varieties or as plant virus resistance induced by transgenic rootstock.

  15. Effects on RNAi of the tight structure, sequence and position of the targeted region

    PubMed Central

    Yoshinari, Koichi; Miyagishi, Makoto; Taira, Kazunari

    2004-01-01

    RNA interference (RNAi) is a gene-silencing phenomenon that involves the double-stranded RNA-mediated cleavage of mRNA, and small interfering RNAs (siRNAs) can cause RNAi in mammalian cells. There have been many attempts to clarify the mechanism of RNAi, but information about the relationship between the sequence and structure, in particular, a tight structure, of the target RNA and the activities of siRNAs are limited. In the present study, we examined this relationship by introducing the TAR element, which adopts a very stable secondary structure, at different positions within target RNAs. Our results suggested that the activities of siRNAs were affected by the tight stem–loop structure of TAR. In contrast, the position of the target within the mRNA, the binding of the Tat protein to the TAR, and the location of the target within a translated or a noncoding region had only marginal effects on RNAi. When the target sequence was placed in two different orientations, only one orientation had a significant effect on the activities of siRNA, demonstrating that the presence of certain nucleotides at some specific positions was favorable for RNAi. Systematic analysis of 47 different sites within 47 plasmids under identical conditions indicated that it is the target sequence itself, rather than its location, that is the major determinant of siRNA activity. PMID:14762201

  16. The intron in centromeric noncoding RNA facilitates RNAi-mediated formation of heterochromatin

    PubMed Central

    Mutazono, Masatoshi; Morita, Misato; Tsukahara, Chihiro; Chinen, Madoka; Nishioka, Shiori; Yumikake, Tatsuhiro; Dohke, Kohei; Sakamoto, Misuzu; Ideue, Takashi; Nakayama, Jun-ichi; Ishii, Kojiro

    2017-01-01

    In fission yeast, the formation of centromeric heterochromatin is induced through the RNA interference (RNAi)-mediated pathway. Some pre-mRNA splicing mutants (prp) exhibit defective formation of centromeric heterochromatin, suggesting that splicing factors play roles in the formation of heterochromatin, or alternatively that the defect is caused by impaired splicing of pre-mRNAs encoding RNAi factors. Herein, we demonstrate that the splicing factor spPrp16p is enriched at the centromere, and associates with Cid12p (a factor in the RNAi pathway) and the intron-containing dg ncRNA. Interestingly, removal of the dg intron, mutations of its splice sites, or replacement of the dg intron with an euchromatic intron significantly decreased H3K9 dimethylation. We also revealed that splicing of dg ncRNA is repressed in cells and its repression depends on the distance from the transcription start site to the intron. Inefficient splicing was also observed in other intron-containing centromeric ncRNAs, dh and antisense dg, and splicing of antisense dg ncRNA was repressed in the presence of the RNAi factors. Our results suggest that the introns retained in centromeric ncRNAs work as facilitators, co-operating with splicing factors assembled on the intron and serving as a platform for the recruitment of RNAi factors, in the formation of centromeric heterochromatin. PMID:28231281

  17. Key enzymes and proteins of crop insects as candidate for RNAi based gene silencing.

    PubMed

    Kola, Vijaya Sudhakara Rao; Renuka, P; Madhav, Maganti Sheshu; Mangrauthia, Satendra K

    2015-01-01

    RNA interference (RNAi) is a mechanism of homology dependent gene silencing present in plants and animals. It operates through 21-24 nucleotides small RNAs which are processed through a set of core enzymatic machinery that involves Dicer and Argonaute proteins. In recent past, the technology has been well appreciated toward the control of plant pathogens and insects through suppression of key genes/proteins of infecting organisms. The genes encoding key enzymes/proteins with the great potential for developing an effective insect control by RNAi approach are actylcholinesterase, cytochrome P450 enzymes, amino peptidase N, allatostatin, allatotropin, tryptophan oxygenase, arginine kinase, vacuolar ATPase, chitin synthase, glutathione-S-transferase, catalase, trehalose phosphate synthase, vitellogenin, hydroxy-3-methylglutaryl coenzyme A reductase, and hormone receptor genes. Through various studies, it is demonstrated that RNAi is a reliable molecular tool which offers great promises in meeting the challenges imposed by crop insects with careful selection of key enzymes/proteins. Utilization of RNAi tool to target some of these key proteins of crop insects through various approaches is described here. The major challenges of RNAi based insect control such as identifying potential targets, delivery methods of silencing trigger, off target effects, and complexity of insect biology are very well illustrated. Further, required efforts to address these challenges are also discussed.

  18. The intron in centromeric noncoding RNA facilitates RNAi-mediated formation of heterochromatin.

    PubMed

    Mutazono, Masatoshi; Morita, Misato; Tsukahara, Chihiro; Chinen, Madoka; Nishioka, Shiori; Yumikake, Tatsuhiro; Dohke, Kohei; Sakamoto, Misuzu; Ideue, Takashi; Nakayama, Jun-Ichi; Ishii, Kojiro; Tani, Tokio

    2017-02-01

    In fission yeast, the formation of centromeric heterochromatin is induced through the RNA interference (RNAi)-mediated pathway. Some pre-mRNA splicing mutants (prp) exhibit defective formation of centromeric heterochromatin, suggesting that splicing factors play roles in the formation of heterochromatin, or alternatively that the defect is caused by impaired splicing of pre-mRNAs encoding RNAi factors. Herein, we demonstrate that the splicing factor spPrp16p is enriched at the centromere, and associates with Cid12p (a factor in the RNAi pathway) and the intron-containing dg ncRNA. Interestingly, removal of the dg intron, mutations of its splice sites, or replacement of the dg intron with an euchromatic intron significantly decreased H3K9 dimethylation. We also revealed that splicing of dg ncRNA is repressed in cells and its repression depends on the distance from the transcription start site to the intron. Inefficient splicing was also observed in other intron-containing centromeric ncRNAs, dh and antisense dg, and splicing of antisense dg ncRNA was repressed in the presence of the RNAi factors. Our results suggest that the introns retained in centromeric ncRNAs work as facilitators, co-operating with splicing factors assembled on the intron and serving as a platform for the recruitment of RNAi factors, in the formation of centromeric heterochromatin.

  19. Stc1: A Critical Link between RNAi and Chromatin Modification Required for Heterochromatin Integrity

    PubMed Central

    Bayne, Elizabeth H.; White, Sharon A.; Kagansky, Alexander; Bijos, Dominika A.; Sanchez-Pulido, Luis; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Ponting, Chris P.; Rappsilber, Juri; Allshire, Robin C.

    2010-01-01

    Summary In fission yeast, RNAi directs heterochromatin formation at centromeres, telomeres, and the mating type locus. Noncoding RNAs transcribed from repeat elements generate siRNAs that are incorporated into the Argonaute-containing RITS complex and direct it to nascent homologous transcripts. This leads to recruitment of the CLRC complex, including the histone methyltransferase Clr4, promoting H3K9 methylation and heterochromatin formation. A key question is what mediates the recruitment of Clr4/CLRC to transcript-bound RITS. We have identified a LIM domain protein, Stc1, that is required for centromeric heterochromatin integrity. Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex. Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi. We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification. PMID:20211136

  20. Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro.

    PubMed

    Li, Yan; Burclaff, Joseph; Anderson, James T

    2016-01-01

    RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.

  1. siRNA and shRNA screens advance key understanding of host factors required for HIV-1 replication.

    PubMed

    Kok, Kin-Hang; Lei, Ting; Jin, Dong-Yan

    2009-08-27

    A recent RNAi screen used a genome-wide shRNA library to search for cellular factors required for HIV-1 replication. This work complements three other siRNA-based screening studies and potentially opens the door to the discovery of factors that are important for HIV-1 replication in physiological host cells such as T lymphocytes. shRNA screens can be further improved, and they could promise to unravel new pathways and new facets of virus-cell interactions.

  2. Biosafety research for non-target organism risk assessment of RNAi-based GE plants

    PubMed Central

    Roberts, Andrew F.; Devos, Yann; Lemgo, Godwin N. Y.; Zhou, Xuguo

    2015-01-01

    RNA interference, or RNAi, refers to a set of biological processes that make use of conserved cellular machinery to silence genes. Although there are several variations in the source and mechanism, they are all triggered by double stranded RNA (dsRNA) which is processed by a protein complex into small, single stranded RNA, referred to as small interfering RNAs (siRNA) with complementarity to sequences in genes targeted for silencing. The use of the RNAi mechanism to develop new traits in plants has fueled a discussion about the environmental safety of the technology for these applications, and this was the subject of a symposium session at the 13th ISBGMO in Cape Town, South Africa. This paper continues that discussion by proposing research areas that may be beneficial for future environmental risk assessments of RNAi-based genetically modified plants, with a particular focus on non-target organism assessment. PMID:26594220

  3. RNAi-mediated gene knockdown and in vivo diuresis assay in adult female Aedes aegypti mosquitoes.

    PubMed

    Drake, Lisa L; Price, David P; Aguirre, Sarah E; Hansen, Immo A

    2012-07-14

    This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis. The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

  4. Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

    PubMed

    Tetlak, Alicia G; Burnett, Jacob B; Hahn, Daniel A; Hatle, John D

    2015-12-01

    Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction.

  5. RNAi as a Routine Route Toward Breast Cancer Therapy

    DTIC Science & Technology

    2013-09-01

    screens for highly potent shRNAs in a highly parallel and high-throughput fashion. Distribution of the data indicated that approximately 50% of all...from patients that are resistant to trastuzumab therapy. In addition, we will continue to pursue the remaining targets in further validation studies ...CD29hCD49fh cells. Al- though being very elegantly performed and enhancing our un- derstanding of MaSC localization, studies with this mouse model did not

  6. Persistence and transgenerational effect of plant-mediated RNAi in aphids.

    PubMed

    Coleman, A D; Wouters, R H M; Mugford, S T; Hogenhout, S A

    2015-02-01

    Plant-mediated RNA interference (RNAi) has been successfully used as a tool to study gene function in aphids. The persistence and transgenerational effects of plant-mediated RNAi in the green peach aphid (GPA) Myzus persicae were investigated, with a focus on three genes with different functions in the aphid. Rack1 is a key component of various cellular processes inside aphids, while candidate effector genes MpC002 and MpPIntO2 (Mp2) modulate aphid-plant interactions. The gene sequences and functions did not affect RNAi-mediated down-regulation and persistence levels in the aphids. Maximal reduction of gene expression was ~70% and this was achieved at between 4 d and 8 d of exposure of the aphids to double-stranded RNA (dsRNA)-producing transgenic Arabidopsis thaliana. Moreover, gene expression levels returned to wild-type levels within ~6 d after removal of the aphids from the transgenic plants, indicating that a continuous supply of dsRNA is required to maintain the RNAi effect. Target genes were also down-regulated in nymphs born from mothers exposed to dsRNA-producing transgenic plants, and the RNAi effect lasted twice as long (12-14 d) in these nymphs. Investigations of the impact of RNAi over three generations of aphids revealed that aphids reared on dsMpC002 transgenic plants experienced a 60% decline in aphid reproduction levels compared with a 40% decline of aphids reared on dsRack1 and dsMpPIntO2 plants. In a field setting, a reduction of the aphid reproduction by 40-60% would dramatically decrease aphid population growth, contributing to a substantial reduction in agricultural losses.

  7. RNAiFold2T: Constraint Programming design of thermo-IRES switches

    PubMed Central

    Garcia-Martin, Juan Antonio; Dotu, Ivan; Fernandez-Chamorro, Javier; Lozano, Gloria; Ramajo, Jorge; Martinez-Salas, Encarnacion; Clote, Peter

    2016-01-01

    Motivation: RNA thermometers (RNATs) are cis-regulatory elements that change secondary structure upon temperature shift. Often involved in the regulation of heat shock, cold shock and virulence genes, RNATs constitute an interesting potential resource in synthetic biology, where engineered RNATs could prove to be useful tools in biosensors and conditional gene regulation. Results: Solving the 2-temperature inverse folding problem is critical for RNAT engineering. Here we introduce RNAiFold2T, the first Constraint Programming (CP) and Large Neighborhood Search (LNS) algorithms to solve this problem. Benchmarking tests of RNAiFold2T against existent programs (adaptive walk and genetic algorithm) inverse folding show that our software generates two orders of magnitude more solutions, thus allowing ample exploration of the space of solutions. Subsequently, solutions can be prioritized by computing various measures, including probability of target structure in the ensemble, melting temperature, etc. Using this strategy, we rationally designed two thermosensor internal ribosome entry site (thermo-IRES) elements, whose normalized cap-independent translation efficiency is approximately 50% greater at 42 °C than 30 °C, when tested in reticulocyte lysates. Translation efficiency is lower than that of the wild-type IRES element, which on the other hand is fully resistant to temperature shift-up. This appears to be the first purely computational design of functional RNA thermoswitches, and certainly the first purely computational design of functional thermo-IRES elements. Availability: RNAiFold2T is publicly available as part of the new release RNAiFold3.0 at https://github.com/clotelab/RNAiFold and http://bioinformatics.bc.edu/clotelab/RNAiFold, which latter has a web server as well. The software is written in C ++ and uses OR-Tools CP search engine. Contact: clote@bc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID

  8. Next-generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance.

    PubMed

    Ni, Mi; Ma, Wei; Wang, Xiaofang; Gao, Meijing; Dai, Yan; Wei, Xiaoli; Zhang, Lei; Peng, Yonggang; Chen, Shuyuan; Ding, Lingyun; Tian, Yue; Li, Jie; Wang, Haiping; Wang, Xiaolin; Xu, Guowang; Guo, Wangzhen; Yang, Yihua; Wu, Yidong; Heuberger, Shannon; Tabashnik, Bruce E; Zhang, Tianzhen; Zhu, Zhen

    2017-02-15

    Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant 'pyramids' producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross-resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double-stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH-binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt-resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt-resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non-transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.

  9. Current progress in the development of RNAi-based therapeutics for HIV-1.

    PubMed

    Zhou, J; Rossi, J J

    2011-12-01

    Highly active antiretroviral therapy (HAART) treatment for HIV has changed the course of AIDS in societies in which the drugs are readily available. Despite the great success of HAART, drug resistance and toxicity issues still remain a concern for some individuals. Thus, a number of investigators have been exploring other approaches for inhibiting HIV-1 replication. One of the most potent of these is the use of RNA interference (RNAi). This review will focus solely on the use of RNAi for the treatment of HIV-1 infection, including the problems, progress and future prospects.

  10. Gene targeting by RNAi-mediated knockdown of potent DNA ligase IV homologue in the cellulase-producing fungus Talaromyces cellulolyticus.

    PubMed

    Hayata, Koutarou; Asada, Seiya; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Sawayama, Shigeki

    2014-11-01

    The genome of the cellulase-producing fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) was screened for a potent DNA ligase IV gene (ligD homologue). Homologous recombination efficiency in T. cellulolyticus is very low. Therefore, suppression of a non-homologous end-joining system was attempted to enable specific gene knockouts for molecular breeding. The transcript levels of ligD homologue were 0.037 of those of the parental YP-4 strain in the Li20 transformant carrying the RNAi construct targeting the ligD homologue. Transformation of the hairpin-type RNAi vector into T. cellulolyticus could be useful in fungal gene knockdown experiments. Cellulase production and protein secretion were similar in the parental YP-4 strain and the Li20 transformant. Knockout transformation of ligD homologue using the Li20 transformant led to 23.1 % double crossover gene targeting. Our results suggest that the potent DNA ligase IV gene of T. cellulolyticus is related to non-homologous end joining and that the knockdown of the ligD homologue is useful in gene targeting.

  11. Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants

    PubMed Central

    Matilainen, Olli; Kallijärvi, Jukka; Cuellar, Wilmer J.; Lu, Rui; Saarma, Mart; Holmberg, Carina I.; Jäntti, Jussi; Valkonen, Jari P. T.

    2015-01-01

    Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)—mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in

  12. RNAi Efficiency, Systemic Properties, and Novel Delivery Methods for Pest Insect Control: What We Know So Far

    PubMed Central

    Joga, Mallikarjuna R.; Zotti, Moises J.; Smagghe, Guy; Christiaens, Olivier

    2016-01-01

    In recent years, the research on the potential of using RNA interference (RNAi) to suppress crop pests has made an outstanding growth. However, given the variability of RNAi efficiency that is observed in many insects, the development of novel approaches toward insect pest management using RNAi requires first to unravel factors behind the efficiency of dsRNA-mediated gene silencing. In this review, we explore essential implications and possibilities to increase RNAi efficiency by delivery of dsRNA through non-transformative methods. We discuss factors influencing the RNAi mechanism in insects and systemic properties of dsRNA. Finally, novel strategies to deliver dsRNA are discussed, including delivery by symbionts, plant viruses, trunk injections, root soaking, and transplastomic plants. PMID:27909411

  13. RNAi Efficiency, Systemic Properties, and Novel Delivery Methods for Pest Insect Control: What We Know So Far.

    PubMed

    Joga, Mallikarjuna R; Zotti, Moises J; Smagghe, Guy; Christiaens, Olivier

    2016-01-01

    In recent years, the research on the potential of using RNA interference (RNAi) to suppress crop pests has made an outstanding growth. However, given the variability of RNAi efficiency that is observed in many insects, the development of novel approaches toward insect pest management using RNAi requires first to unravel factors behind the efficiency of dsRNA-mediated gene silencing. In this review, we explore essential implications and possibilities to increase RNAi efficiency by delivery of dsRNA through non-transformative methods. We discuss factors influencing the RNAi mechanism in insects and systemic properties of dsRNA. Finally, novel strategies to deliver dsRNA are discussed, including delivery by symbionts, plant viruses, trunk injections, root soaking, and transplastomic plants.

  14. RdRP-synthesized antisense ribosomal siRNAs silence pre-rRNA via the nuclear RNAi pathway.

    PubMed

    Zhou, Xufei; Feng, Xuezhu; Mao, Hui; Li, Mu; Xu, Fei; Hu, Kai; Guang, Shouhong

    2017-03-01

    Expression of rRNA affects cell growth and proliferation, but mechanisms that modulate rRNA levels are poorly understood. We conducted a genetic screen for factors that negatively regulate generation of endogenous short interfering RNA (endo-siRNA) in Caenorhabditis elegans and identified a suppressor of siRNA (susi-1) and antisense ribosomal siRNAs (risiRNAs). risiRNAs show sequence complementary to 18S and 26S rRNAs and require RNA-dependent RNA polymerases (RdRPs) for their production. They act through the nuclear RNA interference (RNAi) pathway to downregulate pre-rRNA. Stress stimuli, including low temperature and UV irradiation, induced the accumulation of risiRNAs. SUSI-1 is a homolog of the human DIS3L2 exonuclease involved in 3'-5' degradation of oligouridylated RNAs. In susi-1 mutant and in low temperature-treated animals, 3'-tail oligouridylated 26S rRNA accumulated. The injection of oligouridylated rRNA elicited nuclear accumulation of NRDE-3. Our findings identify a new subset of 22G-RNAs that regulate pre-rRNA expression and a mechanism to maintain rRNA homeostasis.

  15. Selection of Reference Genes for RT-qPCR Analysis in Coccinella septempunctata to Assess Un-intended Effects of RNAi Transgenic Plants

    PubMed Central

    Yang, Chunxiao; Preisser, Evan L.; Zhang, Hongjun; Liu, Yong; Dai, Liangying; Pan, Huipeng; Zhou, Xuguo

    2016-01-01

    The development of genetically engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is reverse transcriptase-quantitative polymerase chain reaction, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata, a commonly used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper, the ΔCt method, and RefFinder, were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended, respectively, for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata, our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants. PMID:27877186

  16. RNAi of ace1 and ace2 in Blattella germanica reveals their differential contribution to acetylcholinesterase activity and sensitivity to insecticides.

    PubMed

    Revuelta, L; Piulachs, M D; Bellés, X; Castañera, P; Ortego, F; Díaz-Ruíz, J R; Hernández-Crespo, P; Tenllado, F

    2009-12-01

    Cyclorrhapha insect genomes contain a single acetylcholinesterase (AChE) gene while other insects contain at least two ace genes (ace1 and ace2). In this study we tested the hypothesis that the two ace paralogous from Blattella germanica have different contributions to AChE activity, using RNA interference (RNAi) to knockdown each one individually. Paralogous-specific depletion of Bgace transcripts was evident in ganglia of injected cockroaches, although the effects at the protein level were less pronounced. Using spectrophotometric and zymogram measurements, we obtained evidence that BgAChE1 represents 65-75% of the total AChE activity in nerve tissue demonstrating that ace1 encodes a predominant AChE. A significant increase in sensitivity of Bgace1-interfered cockroaches was observed after 48 h of exposure to chlorpyrifos. In contrast, Bgace2 knockdown had a negligible effect on mortality to this organophosphate. These results point out a key role, qualitative and/or quantitative, of AChE1 as target of organophosphate insecticides in this species. Silencing the expression of Bgace1 but not Bgace2 also produced an increased mortality in insects when synergized with lambda-cyhalothrin, a situation which resembles the synergistic effects observed between organophosphates and pyrethroids. Gene silencing of ace genes by RNAi offers an exciting approach for examining a possible functional differentiation in ace paralogous.

  17. Selection of Reference Genes for RT-qPCR Analysis in Coccinella septempunctata to Assess Un-intended Effects of RNAi Transgenic Plants.

    PubMed

    Yang, Chunxiao; Preisser, Evan L; Zhang, Hongjun; Liu, Yong; Dai, Liangying; Pan, Huipeng; Zhou, Xuguo

    2016-01-01

    The development of genetically engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is reverse transcriptase-quantitative polymerase chain reaction, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata, a commonly used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper, the ΔCt method, and RefFinder, were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended, respectively, for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata, our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.

  18. RNAi-mediated mortality of the whitefly through transgenic expression of double-stranded RNA homologous to acetylcholinesterase and ecdysone receptor in tobacco plants

    PubMed Central

    Malik, Hassan Jamil; Raza, Amir; Amin, Imran; Scheffler, Jodi A.; Scheffler, Brian E.; Brown, Judith K.; Mansoor, Shahid

    2016-01-01

    The whitefly Bemisia tabaci (Genn.) is a pest and vector of plant viruses to crop and ornamental plants worldwide. Using RNA interference (RNAi) to down regulate whitefly genes by expressing their homologous double stranded RNAs in plants has great potential for management of whiteflies to reduce plant virus disease spread. Using a Tobacco rattle virus-derived plasmid for in planta transient expression of double stranded RNA (dsRNA) homologous to the acetylcholinesterase (AChE) and ecdysone receptor (EcR) genes of B. tabaci, resulted in significant adult whitefly mortality. Nicotiana tabacum L. plants expressing dsRNA homologous to B. tabaci AChE and EcR were constructed by fusing sequences derived from both genes. Mortality of adult whiteflies exposed to dsRNA by feeding on N. tabacum plants, compared to non-dsRNA expressing plants, recorded at 24-hr intervals post-ingestion for three days, was >90% and 10%, respectively. Analysis of gene expression by real time quantitative PCR indicated that whitefly mortality was attributable to the down-regulation of both target genes by RNAi. Results indicated that knock down of whitefly genes involved in neuronal transmission and transcriptional activation of developmental genes, has potential as a bio-pesticide to reduce whitefly population size and thereby decrease virus spread. PMID:27929123

  19. Human Papillomavirus: Current and Future RNAi Therapeutic Strategies for Cervical Cancer

    PubMed Central

    Jung, Hun Soon; Rajasekaran, Nirmal; Ju, Woong; Shin, Young Kee

    2015-01-01

    Human papillomaviruses (HPVs) are small DNA viruses; some oncogenic ones can cause different types of cancer, in particular cervical cancer. HPV-associated carcinogenesis provides a classical model system for RNA interference (RNAi) based cancer therapies, because the viral oncogenes E6 and E7 that cause cervical cancer are expressed only in cancerous cells. Previous studies on the development of therapeutic RNAi facilitated the advancement of therapeutic siRNAs and demonstrated its versatility by siRNA-mediated depletion of single or multiple cellular/viral targets. Sequence-specific gene silencing using RNAi shows promise as a novel therapeutic approach for the treatment of a variety of diseases that currently lack effective treatments. However, siRNA-based targeting requires further validation of its efficacy in vitro and in vivo, for its potential off-target effects, and of the design of conventional therapies to be used in combination with siRNAs and their drug delivery vehicles. In this review we discuss what is currently known about HPV-associated carcinogenesis and the potential for combining siRNA with other treatment strategies for the development of future therapies. Finally, we present our assessment of the most promising path to the development of RNAi therapeutic strategies for clinical settings. PMID:26239469

  20. Logic integration of mRNA signals by an RNAi-based molecular computer

    PubMed Central

    Xie, Zhen; Liu, Siyuan John; Bleris, Leonidas; Benenson, Yaakov

    2010-01-01

    Synthetic in vivo molecular ‘computers’ could rewire biological processes by establishing programmable, non-native pathways between molecular signals and biological responses. Multiple molecular computer prototypes have been shown to work in simple buffered solutions. Many of those prototypes were made of DNA strands and performed computations using cycles of annealing-digestion or strand displacement. We have previously introduced RNA interference (RNAi)-based computing as a way of implementing complex molecular logic in vivo. Because it also relies on nucleic acids for its operation, RNAi computing could benefit from the tools developed for DNA systems. However, these tools must be harnessed to produce bioactive components and be adapted for harsh operating environments that reflect in vivo conditions. In a step toward this goal, we report the construction and implementation of biosensors that ‘transduce’ mRNA levels into bioactive, small interfering RNA molecules via RNA strand exchange in a cell-free Drosophila embryo lysate, a step beyond simple buffered environments. We further integrate the sensors with our RNAi ‘computational’ module to evaluate two-input logic functions on mRNA concentrations. Our results show how RNA strand exchange can expand the utility of RNAi computing and point toward the possibility of using strand exchange in a native biological setting. PMID:20194121

  1. Logic integration of mRNA signals by an RNAi-based molecular computer.

    PubMed

    Xie, Zhen; Liu, Siyuan John; Bleris, Leonidas; Benenson, Yaakov

    2010-05-01

    Synthetic in vivo molecular 'computers' could rewire biological processes by establishing programmable, non-native pathways between molecular signals and biological responses. Multiple molecular computer prototypes have been shown to work in simple buffered solutions. Many of those prototypes were made of DNA strands and performed computations using cycles of annealing-digestion or strand displacement. We have previously introduced RNA interference (RNAi)-based computing as a way of implementing complex molecular logic in vivo. Because it also relies on nucleic acids for its operation, RNAi computing could benefit from the tools developed for DNA systems. However, these tools must be harnessed to produce bioactive components and be adapted for harsh operating environments that reflect in vivo conditions. In a step toward this goal, we report the construction and implementation of biosensors that 'transduce' mRNA levels into bioactive, small interfering RNA molecules via RNA strand exchange in a cell-free Drosophila embryo lysate, a step beyond simple buffered environments. We further integrate the sensors with our RNAi 'computational' module to evaluate two-input logic functions on mRNA concentrations. Our results show how RNA strand exchange can expand the utility of RNAi computing and point toward the possibility of using strand exchange in a native biological setting.

  2. Suppressors of RNAi from plant viruses are subject to episodic positive selection

    PubMed Central

    Murray, Gemma G. R.; Kosakovsky Pond, Sergei L.; Obbard, Darren J.

    2013-01-01

    Viral suppressors of RNAi (VSRs) are proteins that actively inhibit the antiviral RNA interference (RNAi) immune response, providing an immune evasion route for viruses. It has been hypothesized that VSRs are engaged in a molecular ‘arms race’ with RNAi pathway genes. Two lines of evidence support this. First, VSRs from plant viruses display high sequence diversity, and are frequently gained and lost over evolutionary time scales. Second, Drosophila antiviral RNAi genes show high rates of adaptive evolution. Here, we investigate whether VSRs diversify faster than other genes and, if so, whether this is a result of positive selection, as might be expected in an arms race. By analysis of 12 plant RNA viruses, we show that the relative rate of protein evolution is higher for VSRs than for other genes, but that this is not attributable to pervasive positive selection. We argue that, because evolutionary time scales are extremely different for viruses and eukaryotes, it is improbable that viral adaptation (as measured by the ratio of non-synonymous to synonymous change) will be dominated by one-to-one coevolution with eukaryotes. Instead, for plant virus VSRs, we find strong evidence of episodic selection—diversifying selection that acts on a subset of lineages—which might be attributable to frequent shifts between different host genotypes or species. PMID:23804618

  3. Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

    PubMed Central

    Börner, Kathleen; Niopek, Dominik; Cotugno, Gabriella; Kaldenbach, Michaela; Pankert, Teresa; Willemsen, Joschka; Zhang, Xian; Schürmann, Nina; Mockenhaupt, Stefan; Serva, Andrius; Hiet, Marie-Sophie; Wiedtke, Ellen; Castoldi, Mirco; Starkuviene, Vytaute; Erfle, Holger; Gilbert, Daniel F.; Bartenschlager, Ralf; Boutros, Michael; Binder, Marco; Streetz, Konrad; Kräusslich, Hans-Georg; Grimm, Dirk

    2013-01-01

    As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems. PMID:24049077

  4. RNAi-based insecticidal crops: potential effects on non-target species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNAi is a sequence specific mechanism that silences protein production when particular mRNAs are bound and enzymatically cleaved. Genetically modified crops that silence critical gene function in insect pests have been developed, and are a likely future direction for commercial pest management. Pote...

  5. Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications

    PubMed Central

    Meade, Bryan R; Gogoi, Khirud; Hamil, Alexander S; Palm-Apergi, Caroline; van den Berg, Arjen; Hagopian, Jonathan C; Springer, Aaron D; Eguchi, Akiko; Kacsinta, Apollo D; Dowdy, Connor F; Presente, Asaf; Lönn, Peter; Kaulich, Manuel; Yoshioka, Naohisa; Gros, Edwige; Cui, Xian-Shu; Dowdy, Steven F

    2015-01-01

    RNA interference (RNAi) has great potential to treat human disease1–3. However, in vivo delivery of short interfering RNAs (siRNAs), which are negatively charged double-stranded RNA macromolecules, remains a major hurdle4–9. Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined molecular conjugates9. Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technology that will open new avenues for development of RNAi therapeutics. PMID:25402614

  6. An Arabidopsis Tissue-Specific RNAi Method for Studying Genes Essential to Mitosis

    PubMed Central

    Burgos-Rivera, Brunilís; Dawe, R. Kelly

    2012-01-01

    A large fraction of the genes in plants can be considered essential in the sense that when absent the plant fails to develop past the first few cell divisions. The fact that angiosperms pass through a haploid gametophyte stage can make it challenging to propagate such mutants even in the heterozygous condition. Here we describe a tissue-specific RNAi method that allows us to visualize cell division phenotypes in petals, which are large dispensable organs. Portions of the APETALA (AP3) and PISTILLATA (PI) promoters confer early petal-specific expression. We show that when either promoter is used to drive the expression of a beta-glucuronidase (GUS) RNAi transgene in plants uniformly expressing GUS, GUS expression is knocked down specifically in petals. We further tested the system by targeting the essential kinetochore protein CENPC and two different components of the Spindle Assembly Checkpoint (MAD2 and BUBR1). Plant lines expressing petal-specific RNAi hairpins targeting these genes exhibited an array of petal phenotypes. Cytological analyses of the affected flower buds confirmed that CENPC knockdown causes cell cycle arrest but provided no evidence that either MAD2 or BUBR1 are required for mitosis (although both genes are required for petal growth by this assay). A key benefit of the petal-specific RNAi method is that the phenotypes are not expressed in the lineages leading to germ cells, and the phenotypes are faithfully transmitted for at least four generations despite their pronounced effects on growth. PMID:23236491

  7. RNAi: a potential new class of therapeutic for human genetic disease.

    PubMed

    Seyhan, Attila A

    2011-11-01

    Dominant negative genetic disorders, in which a mutant allele of a gene causes disease in the presence of a second, normal copy, have been challenging since there is no cure and treatments are only to alleviate the symptoms. Current therapies involving pharmacological and biological drugs are not suitable to target mutant genes selectively due to structural indifference of the normal variant of their targets from the disease-causing mutant ones. In instances when the target contains single nucleotide polymorphism (SNP), whether it is an enzyme or structural or receptor protein are not ideal for treatment using conventional drugs due to their lack of selectivity. Therefore, there is a need to develop new approaches to accelerate targeting these previously inaccessible targets by classical therapeutics. Although there is a cooling trend by the pharmaceutical industry for the potential of RNA interference (RNAi), RNAi and other RNA targeting drugs (antisense, ribozyme, etc.) still hold their promise as the only drugs that provide an opportunity to target genes with SNP mutations found in dominant negative disorders, genes specific to pathogenic tumor cells, and genes that are critical for mediating the pathology of various other diseases. Because of its exquisite specificity and potency, RNAi has attracted a considerable interest as a new class of therapeutic for genetic diseases including amyotrophic lateral sclerosis, Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), spinocerebellar ataxia, dominant muscular dystrophies, and cancer. In this review, progress and challenges in developing RNAi therapeutics for genetic diseases will be discussed.

  8. VSG switching in Trypanosoma brucei: antigenic variation analysed using RNAi in the absence of immune selection.

    PubMed

    Aitcheson, Niall; Talbot, Suzanne; Shapiro, Jesse; Hughes, Katie; Adkin, Carl; Butt, Thomas; Sheader, Karen; Rudenko, Gloria

    2005-09-01

    Trypanosoma brucei relies on antigenic variation of its variant surface glycoprotein (VSG) coat for survival. We show that VSG switching can be efficiently studied in vitro using VSG RNAi in place of an immune system to select for switch variants. Contrary to models predicting an instant switch after inhibition of VSG synthesis, switching was not induced by VSG RNAi and occurred at a rate of 10(-4) per division. We find a highly reproducible hierarchy of VSG activation, which appears to be capable of resetting, whereby more than half of the switch events over 12 experiments were to one of two VSGs. We characterized switched clones according to switch mechanism using marker genes in the active VSG expression site (ES). Transcriptional switches between ESs were the preferred switching mechanism, whereby at least 10 of the 17 ESs identified in T. brucei 427 can be functionally active in vitro. We could specifically select for switches mediated by DNA rearrangements by inducing VSG RNAi in the presence of drug selection for the active ES. Most of the preferentially activated VSGs could be activated by multiple mechanisms. This VSG RNAi-based procedure provides a rapid and powerful means for analysing VSG switching in African trypanosomes entirely in vitro.

  9. 'Smart' non-viral delivery systems for targeted delivery of RNAi to the lungs.

    PubMed

    Ramsey, Joanne M; Hibbitts, Alan; Barlow, James; Kelly, Ciara; Sivadas, Neeraj; Cryan, Sally-Ann

    2013-01-01

    The emergence of RNAi offers a potentially exciting new therapeutic paradigm for respiratory diseases. However, effective delivery remains a key requirement for their translation into the clinic and has been a major factor in the limited clinical success seen to date. Inhalation offers tissue-specific targeting of the RNAi to treat respiratory diseases and a diminished risk of off-target effects. In order to deliver RNAi directly to the respiratory tract via inhalation, 'smart' non-viral carriers are required to protect the RNAi during delivery/aerosolization and enhance cell-specific uptake to target cells. Here, we review the state-of-the-art in therapeutic aerosol bioengineering, and specifically non-viral siRNA delivery platforms, for delivery via inhalation. This includes developments in inhaler device engineering and particle engineering, including manufacturing methods and excipients used in therapeutic aerosol bioengineering that underpin the development of smart, cell type-specific delivery systems to target siRNA to respiratory epithelial cells and/or alveolar macrophages.

  10. RNAiFold: a web server for RNA inverse folding and molecular design.

    PubMed

    Garcia-Martin, Juan Antonio; Clote, Peter; Dotu, Ivan

    2013-07-01

    Synthetic biology and nanotechnology are poised to make revolutionary contributions to the 21st century. In this article, we describe a new web server to support in silico RNA molecular design. Given an input target RNA secondary structure, together with optional constraints, such as requiring GC-content to lie within a certain range, requiring the number of strong (GC), weak (AU) and wobble (GU) base pairs to lie in a certain range, the RNAiFold web server determines one or more RNA sequences, whose minimum free-energy secondary structure is the target structure. RNAiFold provides access to two servers: RNA-CPdesign, which applies constraint programming, and RNA-LNSdesign, which applies the large neighborhood search heuristic; hence, it is suitable for larger input structures. Both servers can also solve the RNA inverse hybridization problem, i.e. given a representation of the desired hybridization structure, RNAiFold returns two sequences, whose minimum free-energy hybridization is the input target structure. The web server is publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold, which provides access to two specialized servers: RNA-CPdesign and RNA-LNSdesign. Source code for the underlying algorithms, implemented in COMET and supported on linux, can be downloaded at the server website.

  11. Fruitless RNAi knockdown in the desert locust, Schistocerca gregaria, influences male fertility.

    PubMed

    Boerjan, Bart; Tobback, Julie; Vandersmissen, Hans Peter; Huybrechts, Roger; Schoofs, Liliane

    2012-02-01

    In Drosophila melanogaster, the male-specific splice isoform of the fruitless gene (Fru(M)) encodes a set of transcription factors that are involved in the regulation of male courtship and copulation. Recent insights from non-drosophilid insects suggest a conserved evolutionary role for the transcription factor Fruitless. In the desert locust, Schistocerca gregaria and the German cockroach, Blatella germanica, both orthopteran insects, a conserved functional role for fruitless has been proposed. Fru specific RNAi knockdown in the third nymphal stage of male Schistocera gregaria delays copulation initiation and results in reduced progeny. In order to identify the origin of the observed phenotypic effects following a fruitless RNAi treatment in the male, we show that the fru knockdown has no detectable effect on spermio- or spermatogenesis and on the transfer of spermatozoa during copulation. Nevertheless, it is clear that the male seminal vesicles contain significantly less spermatozoa after fru RNAi as compared to gfp RNAi controls. We conclude that a lowered male fertility, caused by the fru knockdown in male desert locusts may be the direct cause for the reduction of the progeny numbers in their naïve female copulation partners.

  12. RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding

    PubMed Central

    Younis, Adnan; Siddique, Muhammad Irfan; Kim, Chang-Kil; Lim, Ki-Byung

    2014-01-01

    RNA interference (RNAi) is a promising gene regulatory approach in functional genomics that has significant impact on crop improvement which permits down-regulation in gene expression with greater precise manner without affecting the expression of other genes. RNAi mechanism is expedited by small molecules of interfering RNA to suppress a gene of interest effectively. RNAi has also been exploited in plants for resistance against pathogens, insect/pest, nematodes, and virus that cause significant economic losses. Keeping beside the significance in the genome integrity maintenance as well as growth and development, RNAi induced gene syntheses are vital in plant stress management. Modifying the genes by the interference of small RNAs is one of the ways through which plants react to the environmental stresses. Hence, investigating the role of small RNAs in regulating gene expression assists the researchers to explore the potentiality of small RNAs in abiotic and biotic stress management. This novel approach opens new avenues for crop improvement by developing disease resistant, abiotic or biotic stress tolerant, and high yielding elite varieties. PMID:25332689

  13. RNAi as a management tool for the western corn rootworm, Diabrotica virgifera virgifera.

    PubMed

    Fishilevich, Elane; Vélez, Ana M; Storer, Nicholas P; Li, Huarong; Bowling, Andrew J; Rangasamy, Murugesan; Worden, Sarah E; Narva, Kenneth E; Siegfried, Blair D

    2016-09-01

    The western corn rootworm (WCR), Diabrotica virgifera virgifera, is the most important pest of corn in the US Corn Belt. Economic estimates indicate that costs of control and yield loss associated with WCR damage exceed $US 1 billion annually. Historically, corn rootworm management has been extremely difficult because of its ability to evolve resistance to both chemical insecticides and cultural control practices. Since 2003, the only novel commercialized developments in rootworm management have been transgenic plants expressing Bt insecticidal proteins. Four transgenic insecticidal proteins are currently registered for rootworm management, and field resistance to proteins from the Cry3 family highlights the importance of developing traits with new modes of action. One of the newest approaches for controlling rootworm pests involves RNA interference (RNAi). This review describes the current understanding of the RNAi mechanisms in WCR and the use of this technology for WCR management. Further, the review addresses ecological risk assessment of RNAi and insect resistance management of RNAi for corn rootworm. © 2016 Society of Chemical Industry.

  14. An RNAi-Enhanced Logic Circuit for Cancer Specific Detection and Destruction

    DTIC Science & Technology

    2013-02-01

    cancer specific detection and destruction. PRINCIPAL INVESTIGATOR: Ron Weiss...2013 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER An RNAi-enhanced logic circuit for cancer specific detection and destruction. 5b. GRANT NUMBER...ABSTRACT Modern breast cancer therapies utilize non-specific approaches to kill or remove cancerous cells, inflicting significant collateral damage to

  15. [Mutation frequencies in HIV-1 subtype-A genome in regions containing efficient RNAi targets].

    PubMed

    Kravatsky, Y V; Chechetkin, V R; Fedoseeva, D M; Gorbacheva, M A; Kretova, O V; Tchurikov, N A

    2016-01-01

    The development of gene-therapy technology using RNAi for AIDS/HIV-1 treatment is a prospective alternative to traditional anti-retroviral therapy. RNAi targets could be selected in HIV-1 transcripts and in CCR5 mRNA. Previously, we experimentally selected a number of efficient siRNAs that target HIV-1 RNAs. The viral genome mutates frequently, and RNAi strength is very sensitive, even for a single mismatches. That is why it is important to study nucleotide sequences of targets in clinical isolates of HIV-1. In the present study, we analyzed mutations in 6 of about 300-bp regions containing RNAi targets from HIV-1 subtype A isolates in Russia. Estimates of the mean frequencies of mutations in the targets were obtained and the frequencies of mutations in the different codon positions were compared. The frequencies of mutations in the vicinity of the targets and directly within the targets were also compared and have been shown to be approximately the same. The frequencies of indels in the chosen regions have been assessed. Their frequencies have proved to be two to three orders of magnitude less compared to that for mutations.

  16. Health Screening

    MedlinePlus

    Screenings are tests that look for diseases before you have symptoms. Screening tests can find diseases early, when they're easier ... Overweight and obesity Prostate cancer in men Which tests you need depends on your age, your sex, ...

  17. Depression Screening

    MedlinePlus

    ... Centers Diseases + Condition Centers Mental Health Medical Library Depression Screening (PHQ-9) - Instructions The following questions are ... this tool, there is also text-only version . Depression Screening - Manual Instructions The following questions are a ...

  18. Caenorhabditis elegans, a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance.

    PubMed

    Addo, Matthew Glover; Cossard, Raynald; Pichard, Damien; Obiri-Danso, Kwasi; Rötig, Agnès; Delahodde, Agnès

    2010-09-01

    The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown. Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C.elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans. This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.

  19. Quantitative Property-Property Relationship for Screening-Level Prediction of Intrinsic Clearance of Volatile Organic Chemicals in Rats and Its Integration within PBPK Models to Predict Inhalation Pharmacokinetics in Humans

    PubMed Central

    Peyret, Thomas; Krishnan, Kannan

    2012-01-01

    The objectives of this study were (i) to develop a screening-level Quantitative property-property relationship (QPPR) for intrinsic clearance (CLint) obtained from in vivo animal studies and (ii) to incorporate it with human physiology in a PBPK model for predicting the inhalation pharmacokinetics of VOCs. CLint, calculated as the ratio of the in vivo Vmax (μmol/h/kg bw rat) to the Km (μM), was obtained for 26 VOCs from the literature. The QPPR model resulting from stepwise linear regression analysis passed the validation step (R2 = 0.8; leave-one-out cross-validation Q2 = 0.75) for CLint normalized to the phospholipid (PL) affinity of the VOCs. The QPPR facilitated the calculation of CLint (L PL/h/kg bw rat) from the input data on log Pow, log blood: water PC and ionization potential. The predictions of the QPPR as lower and upper bounds of the 95% mean confidence intervals (LMCI and UMCI, resp.) were then integrated within a human PBPK model. The ratio of the maximum (using LMCI for CLint) to minimum (using UMCI for CLint) AUC predicted by the QPPR-PBPK model was 1.36 ± 0.4 and ranged from 1.06 (1,1-dichloroethylene) to 2.8 (isoprene). Overall, the integrated QPPR-PBPK modeling method developed in this study is a pragmatic way of characterizing the impact of the lack of knowledge of CLint in predicting human pharmacokinetics of VOCs, as well as the impact of prediction uncertainty of CLint on human pharmacokinetics of VOCs. PMID:22685458

  20. Orthologs of Human Disease Associated Genes and RNAi Analysis of Silencing Insulin Receptor Gene in Bombyx mori

    PubMed Central

    Zhang, Zan; Teng, Xiaolu; Chen, Maohua; Li, Fei

    2014-01-01

    The silkworm, Bombyx mori L., is an important economic insect that has been domesticated for thousands of years to produce silk. It is our great interest to investigate the possibility of developing the B. mori as human disease model. We searched the orthologs of human disease associated genes in the B. mori by bi-directional best hits of BLAST and confirmed by searching the OrthoDB. In total, 5006 genes corresponding to 1612 kinds of human diseases had orthologs in the B. mori, among which, there are 25 genes associated with diabetes mellitus. Of these, we selected the insulin receptor gene of the B. mori (Bm-INSR) to study its expression in different tissues and at different developmental stages and tissues. Quantitative PCR showed that Bm-INSR was highly expressed in the Malpighian tubules but expressed at low levels in the testis. It was highly expressed in the 3rd and 4th instar larvae, and adult. We knocked down Bm-INSR expression using RNA interference. The abundance of Bm-INSR transcripts were dramatically reduced to ~4% of the control level at 6 days after dsRNA injection and the RNAi-treated B. mori individuals showed apparent growth inhibition and malformation such as abnormal body color in black, which is the typical symptom of diabetic patients. Our results demonstrate that B. mori has potential use as an animal model for diabetic mellitus research. PMID:25302617

  1. Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis.

    PubMed

    Wagner, Daniel E; Ho, Jaclyn J; Reddien, Peter W

    2012-03-02

    Pluripotency is a central, well-studied feature of embryonic development, but the role of pluripotent cell regulation in somatic tissue regeneration remains poorly understood. In planarians, regeneration of entire animals from tissue fragments is promoted by the activity of adult pluripotent stem cells (cNeoblasts). We utilized transcriptional profiling to identify planarian genes expressed in adult proliferating, regenerative cells (neoblasts). We also developed quantitative clonal analysis methods for expansion and differentiation of cNeoblast descendants that, together with RNAi, revealed gene roles in stem cell biology. Genes encoding two zinc finger proteins, Vasa, a LIM domain protein, Sox and Jun-like transcription factors, two candidate RNA-binding proteins, a Setd8-like protein, and PRC2 (Polycomb) were required for proliferative expansion and/or differentiation of cNeoblast-derived clones. These findings suggest that planarian stem cells utilize molecular mechanisms found in germ cells and other pluripotent cell types and identify genetic regulators of the planarian stem cell system.

  2. RNAi Technology for Insect Management and Protection of Beneficial Insects from Diseases: Lessons, Challenges and Risk Assessments.

    PubMed

    Zotti, M J; Smagghe, G

    2015-06-01

    The time has passed for us to wonder whether RNA interference (RNAi) effectively controls pest insects or protects beneficial insects from diseases. The RNAi era in insect science began with studies of gene function and genetics that paved the way for the development of novel and highly specific approaches for the management of pest insects and, more recently, for the treatment and prevention of diseases in beneficial insects. The slight differences in components of RNAi pathways are sufficient to provide a high degree of variation in responsiveness among insects. The current framework to assess the negative effects of genetically modified (GM) plants on human health is adequate for RNAi-based GM plants. Because of the mode of action of RNAi and the lack of genomic data for most exposed non-target organisms, it becomes difficult to determine the environmental risks posed by RNAi-based technologies and the benefits provided for the protection of crops. A better understanding of the mechanisms that determine the variability in the sensitivity of insects would accelerate the worldwide release of commercial RNAi-based approaches.

  3. Double screening

    SciTech Connect

    Gratia, Pierre; Hu, Wayne; Joyce, Austin; Ribeiro, Raquel H.

    2016-06-15

    Attempts to modify gravity in the infrared typically require a screening mechanism to ensure consistency with local tests of gravity. These screening mechanisms fit into three broad classes; we investigate theories which are capable of exhibiting more than one type of screening. Specifically, we focus on a simple model which exhibits both Vainshtein and kinetic screening. We point out that due to the two characteristic length scales in the problem, the type of screening that dominates depends on the mass of the sourcing object, allowing for different phenomenology at different scales. We consider embedding this double screening phenomenology in a broader cosmological scenario and show that the simplest examples that exhibit double screening are radiatively stable.

  4. 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

  5. Gene Network Polymorphism Illuminates Loss and Retention of Novel RNAi Silencing Components in the Cryptococcus Pathogenic Species Complex

    PubMed Central

    Clancey, Shelly Applen; Wang, Xuying; Heitman, Joseph

    2016-01-01

    RNAi is a ubiquitous pathway that serves central functions throughout eukaryotes, including maintenance of genome stability and repression of transposon expression and movement. However, a number of organisms have lost their RNAi pathways, including the model yeast Saccharomyces cerevisiae, the maize pathogen Ustilago maydis, the human pathogen Cryptococcus deuterogattii, and some human parasite pathogens, suggesting there may be adaptive benefits associated with both retention and loss of RNAi. By comparing the RNAi-deficient genome of the Pacific Northwest Outbreak C. deuterogattii strain R265 with the RNAi-proficient genomes of the Cryptococcus pathogenic species complex, we identified a set of conserved genes that were lost in R265 and all other C. deuterogattii isolates examined. Genetic and molecular analyses reveal several of these lost genes play roles in RNAi pathways. Four novel components were examined further. Znf3 (a zinc finger protein) and Qip1 (a homolog of N. crassa Qip) were found to be essential for RNAi, while Cpr2 (a constitutive pheromone receptor) and Fzc28 (a transcription factor) are involved in sex-induced but not mitosis-induced silencing. Our results demonstrate that the mitotic and sex-induced RNAi pathways rely on the same core components, but sex-induced silencing may be a more specific, highly induced variant that involves additional specialized or regulatory components. Our studies further illustrate how gene network polymorphisms involving known components of key cellular pathways can inform identification of novel elements and suggest that RNAi loss may have been a core event in the speciation of C. deuterogattii and possibly contributed to its pathogenic trajectory. PMID:26943821

  6. Colon cancer screening

    MedlinePlus

    Screening for colon cancer; Colonoscopy - screening; Sigmoidoscopy - screening; Virtual colonoscopy - screening; Fecal immunochemical test; Stool DNA test; sDNA test; Colorectal cancer - screening; Rectal ...

  7. siRNA Genome Screening Approaches to Therapeutic Drug Repositioning

    PubMed Central

    Perwitasari, Olivia; Bakre, Abhijeet; Tompkins, S. Mark; Tripp, Ralph A.

    2013-01-01

    Bridging high-throughput screening (HTS) with RNA interference (RNAi) has allowed for rapid discovery of the molecular basis of many diseases, and identification of potential pathways for developing safe and effective treatments. These features have identified new host gene targets for existing drugs paving the pathway for therapeutic drug repositioning. Using RNAi to discover and help validate new drug targets has also provided a means to filter and prioritize promising therapeutics. This review summarizes these approaches across a spectrum of methods and targets in the host response to pathogens. Particular attention is given to the utility of drug repurposing utilizing the promiscuous nature of some drugs that affect multiple molecules or pathways, and how these biological pathways can be targeted to regulate disease outcome. PMID:24275945

  8. Quantitative proteomic analysis of wheat grain proteins reveals differential effects of silencing of omega-5 gliadin genes in transgenic lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel wheat lines with altered flour compositions can be used to decipher the roles of specific gluten proteins in flour quality. Grain proteins from transgenic wheat lines in which genes encoding the omega-5 gliadins were silenced by RNA interference (RNAi) were analyzed in detail by quantitative 2...

  9. Mode of bacterial pathogenesis determines phenotype in elt-2 and elt-7 RNAi Caenorhabditis elegans.

    PubMed

    Elliott, Samantha L; Sturgeon, Craig R; Travers, Deborah M; Montgomery, Madeline C

    2011-05-01

    Caenorhabditis elegans has become a useful model for studying innate immunity. ELT-2, which is homologous to human GATA-4, -5 and -6, is considered the primary GATA transcription factor controlling intestinal immunity in C. elegans. In this study, we characterize the timeline of intestinal distension in nematodes where ELT-2 and another intestinal GATA transcription factor, ELT-7, are abrogated by RNAi using two different models: colonization and toxin-based infections by Pseudomonas aeruginosa. We show that both ELT-2 and ELT-7 are important for survival of C. elegans exposed to P. aeruginosa. Intestinal distension is accelerated in elt-2 RNAi nematodes, and is observed in colonization but not toxin-based Pseudomonas infection. Upon onset of intestinal distension, nematodes die within 24 h, regardless of experimental treatment. These data provide new insight into the role of ELT-2 and ELT-7 in protecting C. elegans against P. aeruginosa infection.

  10. Engineering cherry rootstocks with resistance to Prunus necrotic ring spot virus through RNAi-mediated silencing.

    PubMed

    Song, Guo-qing; Sink, Kenneth C; Walworth, Aaron E; Cook, Meridith A; Allison, Richard F; Lang, Gregory A

    2013-08-01

    Prunus necrotic ringspot virus (PNRSV) is a major pollen-disseminated ilarvirus that adversely affects many Prunus species. In this study, an RNA interference (RNAi) vector pART27-PNRSV containing an inverted repeat (IR) region of PNRSV was transformed into two hybrid (triploid) cherry rootstocks, 'Gisela 6' (GI 148-1) and 'Gisela 7'(GI 148-8)', which are tolerant and sensitive, respectively, to PNRSV infection. One year after inoculation with PNRSV plus Prune Dwarf Virus, nontransgenic 'Gisela 6' exhibited no symptoms but a significant PNRSV titre, while the transgenic 'Gisela 6' had no symptoms and minimal PNRSV titre. The nontransgenic 'Gisela 7' trees died, while the transgenic 'Gisela 7' trees survived. These results demonstrate the RNAi strategy is useful for developing viral resistance in fruit rootstocks, and such transgenic rootstocks may have potential to enhance production of standard, nongenetically modified fruit varieties while avoiding concerns about transgene flow and exogenous protein production that are inherent for transformed fruiting genotypes.

  11. RNAi targeting multiple cell adhesion molecules reduces immune cell recruitment and vascular inflammation after myocardial infarction

    PubMed Central

    Hulsmans, Maarten; Courties, Gabriel; Sun, Yuan; Heidt, Timo; Vinegoni, Claudio; Borodovsky, Anna; Fitzgerald, Kevin; Wojtkiewicz, Gregory R.; Iwamoto, Yoshiko; Tricot, Benoit; Khan, Omar F.; Kauffman, Kevin J.; Xing, Yiping; Shaw, Taylor E.; Libby, Peter; Langer, Robert; Weissleder, Ralph; Swirski, Filip K.

    2016-01-01

    Myocardial infarction (MI) leads to a systemic surge of vascular inflammation in mice and humans, resulting in secondary ischemic complications and high mortality. We show that, in ApoE−/− mice with coronary ligation, increased sympathetic tone up-regulates not only hematopoietic leukocyte production but also plaque endothelial expression of adhesion molecules. To counteract the resulting arterial leukocyte recruitment, we developed nanoparticle-based RNA interference (RNAi) that effectively silences five key adhesion molecules. Simultaneously encapsulating small interfering RNA (siRNA)–targeting intercellular cell adhesion molecules 1 and 2 (Icam1 and Icam2), vascular cell adhesion molecule 1 (Vcam1), and E- and P-selectins (Sele and Selp) into polymeric endothelial-avid nanoparticles reduced post-MI neutrophil and monocyte recruitment into atherosclerotic lesions and decreased matrix-degrading plaque protease activity. Five-gene combination RNAi also curtailed leukocyte recruitment to ischemic myocardium. Therefore, targeted multigene silencing may prevent complications after acute MI. PMID:27280687

  12. RNAi-directed down-regulation of RSV results in increased resistance in rice (Oryza sativa L.).

    PubMed

    Zhou, Yong; Yuan, Yuan; Yuan, Fuhai; Wang, Man; Zhong, Huan; Gu, Minghong; Liang, Guohua

    2012-05-01

    Rice stripe disease (RSD), caused by rice stripe virus (RSV), is a serious disease in temperate rice-growing areas. We have created an RNAi construct containing coat protein gene (CP) and disease specific protein gene (SP) sequences from RSV. The RNAi construct was transformed into two susceptible japonica varieties, Suyunuo and Guanglingxiangjing, to develop resistance against RSD. The homozygous progeny of rice plants in the T(5) and T(7) generations containing RNAi constructs, after self-fertilization were strongly resistant to viral infection. RT-PCR indicated that viral replication of SP and CP in the transgenic plants was significantly inhibited. There were no obvious morphological or developmental differences between the transgenic and wild-type plants from seedling stage to maturity. The excellent agronomic traits of these two varieties, such as high yield and good quality were maintained. Suppression of virus genes using RNAi is therefore a practical and effective strategy for controlling viral infection in crops.

  13. Blood-brain barrier transport of non-viral gene and RNAi therapeutics.

    PubMed

    Boado, Ruben J

    2007-09-01

    The development of gene- and RNA interference (RNAi)-based therapeutics represents a challenge for the drug delivery field. The global brain distribution of DNA genes, as well as the targeting of specific regions of the brain, is even more complicated because conventional delivery systems, i.e. viruses, have poor diffusion in brain when injected in situ and do not cross the blood-brain barrier (BBB), which is only permeable to lipophilic molecules of less than 400 Da. Recent advances in the "Trojan Horse Liposome" (THL) technology applied to the transvascular non-viral gene therapy of brain disorders presents a promising solution to the DNA/RNAi delivery obstacle. The THL is comprised of immunoliposomes carrying either a gene for protein replacement or small hairpin RNA (shRNA) expression plasmids for RNAi effect, respectively. The THL is engineered with known lipids containing polyethyleneglycol (PEG), which stabilizes its structure in vivo in circulation. The tissue target specificity of THL is given by conjugation of approximately 1% of the PEG residues to peptidomimetic monoclonal antibodies (MAb) that bind to specific endogenous receptors (i.e. insulin and transferrin receptors) located on both the BBB and the brain cellular membranes, respectively. These MAbs mediate (a) receptor-mediated transcytosis of the THL complex through the BBB, (b) endocytosis into brain cells and (c) transport to the brain cell nuclear compartment. The present review presents an overview of the THL technology and its current application to gene therapy and RNAi, including experimental models of Parkinson's disease and brain tumors.

  14. RNAi-mediated resistance to rice black-streaked dwarf virus in transgenic rice.

    PubMed

    Ahmed, Mohamed M S; Bian, Shiquan; Wang, Muyue; Zhao, Jing; Zhang, Bingwei; Liu, Qiaoquan; Zhang, Changquan; Tang, Shuzhu; Gu, Minghong; Yu, Hengxiu

    2016-11-30

    Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, causes significant economic losses in rice production in China and many other Asian countries. Development of resistant varieties by using conventional breeding methods is limited, as germplasm with high level of resistance to RBSDV have not yet been found. One of the most promising methods to confer resistance against RBSDV is the use of RNA interference (RNAi) technology. RBSDV non-structural protein P7-2, encoded by S7-2 gene, is a potential F-box protein and involved in the plant-virus interaction through the ubiquitination pathway. P8, encoded by S8 gene, is the minor core protein that possesses potent active transcriptional repression activity. In this study, we transformed rice calli using a mini-twin T-DNA vector harboring RNAi constructs of the RBSDV genes S7-2 or S8, and obtained plants harboring the target gene constructs and the selectable marker gene, hygromycin phosphotransferase (HPT). From the offspring of these transgenic plants, we obtained selectable marker (HPT gene)-free plants. Homozygous T5 transgenic lines which harbored either S7-2-RNAi or S8-RNAi exhibited high level resistance against RBSDV under field infection pressure from indigenous viruliferous small brown planthoppers. Thus, our results showed that RNA interference with the expression of S7-2 or S8 genes seemed an effective way to induce high level resistance in rice against RBSD disease.

  15. funRNA: a fungi-centered genomics platform for genes encoding key components of RNAi

    PubMed Central

    2014-01-01

    Background RNA interference (RNAi) is involved in genome defense as well as diverse cellular, developmental, and physiological processes. Key components of RNAi are Argonaute, Dicer, and RNA-dependent RNA polymerase (RdRP), which have been functionally characterized mainly in model organisms. The key components are believed to exist throughout eukaryotes; however, there is no systematic platform for archiving and dissecting these important gene families. In addition, few fungi have been studied to date, limiting our understanding of RNAi in fungi. Here we present funRNA http://funrna.riceblast.snu.ac.kr/, a fungal kingdom-wide comparative genomics platform for putative genes encoding Argonaute, Dicer, and RdRP. Description To identify and archive genes encoding the abovementioned key components, protein domain profiles were determined from reference sequences obtained from UniProtKB/SwissProt. The domain profiles were searched using fungal, metazoan, and plant genomes, as well as bacterial and archaeal genomes. 1,163, 442, and 678 genes encoding Argonaute, Dicer, and RdRP, respectively, were predicted. Based on the identification results, active site variation of Argonaute, diversification of Dicer, and sequence analysis of RdRP were discussed in a fungus-oriented manner. funRNA provides results from diverse bioinformatics programs and job submission forms for BLAST, BLASTMatrix, and ClustalW. Furthermore, sequence collections created in funRNA are synced with several gene family analysis portals and databases, offering further analysis opportunities. Conclusions funRNA provides identification results from a broad taxonomic range and diverse analysis functions, and could be used in diverse comparative and evolutionary studies. It could serve as a versatile genomics workbench for key components of RNAi. PMID:25522231

  16. Sex-induced silencing defends the genome of Cryptococcus neoformans via RNAi

    PubMed Central

    Wang, Xuying; Hsueh, Yen-Ping; Li, Wenjun; Floyd, Anna; Skalsky, Rebecca; Heitman, Joseph

    2010-01-01

    Cosuppression is a silencing phenomenon triggered by the introduction of homologous DNA sequences into the genomes of organisms as diverse as plants, fungi, flies, and nematodes. Here we report sex-induced silencing (SIS), which is triggered by tandem integration of a transgene array in the human fungal pathogen Cryptococcus neoformans. A SXI2a-URA5 transgene array was found to be post-transcriptionally silenced during sexual reproduction. More than half of the progeny that inherited the SXI2a-URA5 transgene became uracil-auxotrophic due to silencing of the URA5 gene. In vegetative mitotic growth, silencing of this transgene array occurred at an ∼250-fold lower frequency, indicating that silencing is induced during the sexual cycle. Central components of the RNAi pathway—including genes encoding Argonaute, Dicer, and an RNA-dependent RNA polymerase—are all required for both meiotic and mitotic transgene silencing. URA5-derived ∼22-nucleotide (nt) small RNAs accumulated in the silenced isolates, suggesting that SIS is mediated by RNAi via sequence-specific small RNAs. Through deep sequencing of the small RNA population in C. neoformans, we also identified abundant small RNAs mapping to repetitive transposable elements, and these small RNAs were absent in rdp1 mutant strains. Furthermore, a group of retrotransposons was highly expressed during mating of rdp1 mutant strains, and an increased transposition/mutation rate was detected in their progeny, indicating that the RNAi pathway squelches transposon activity during the sexual cycle. Interestingly, Ago1, Dcr1, Dcr2, and Rdp1 are translationally induced in mating cells, and Ago1, Dcr1, and Dcr2 localize to processing bodies (P bodies), whereas Rdp1 appears to be nuclear, providing mechanistic insights into the elevated silencing efficiency during sexual reproduction. We hypothesize that the SIS RNAi pathway operates to defend the genome during sexual development. PMID:21078820

  17. The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides

    SciTech Connect

    Nicolas, Francisco E.; Vila, Ana; Moxon, Simon; Cascales, Maria D.; Torres-Martinez, Santiago; Ruiz-Vazquez, Rosa M.; Garre, Victoriano

    2015-03-25

    Here, RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway. In conclusion, this work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous

  18. Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L.

    PubMed

    Abdurakhmonov, Ibrokhim Y; Buriev, Zabardast T; Saha, Sukumar; Jenkins, Johnie N; Abdukarimov, Abdusattor; Pepper, Alan E

    2014-01-01

    Simultaneous improvement of fibre quality, early-flowering, early-maturity and productivity in Upland cotton (G. hirsutum) is a challenging task for conventional breeding. The influence of red/far-red light ratio on the fibre length prompted us to examine the phenotypic effects of RNA interference (RNAi) of the cotton PHYA1 gene. Here we show a suppression of up to ~70% for the PHYA1 transcript, and compensatory overexpression of up to ~20-fold in the remaining phytochromes in somatically regenerated PHYA1 RNAi cotton plants. Two independent transformants of three generations exhibited vigorous root and vegetative growth, early-flowering, significantly improved upper half mean fibre length and an improvement in other major fibre characteristics. Small decreases in lint traits were observed but seed cotton yield was increased an average 10-17% compared with controls. RNAi-associated phenotypes were heritable and transferable via sexual hybridization. These results should aid in the development of early-maturing and productive Upland cultivars with superior fibre quality.

  19. Attacking HIV-1 RNA versus DNA by sequence-specific approaches: RNAi versus CRISPR-Cas.

    PubMed

    Herrera-Carrillo, Elena; Berkhout, Ben

    2016-10-15

    Human immunodeficiency virus type 1 (HIV-1) infection can be effectively controlled by potent antiviral drugs, but this never results in a cure. The patient should therefore take these drugs for the rest of his/her life, which can cause drug-resistance and adverse effects. Therefore, more durable therapeutic strategies should be considered, such as a stable gene therapy to protect the target T cells against HIV-1 infection. The development of potent therapeutic regimens based on the RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR-Cas) mechanisms will be described, which can be delivered by lentiviral vectors. These mechanisms attack different forms of the viral genome, the RNA and DNA, respectively, but both mechanisms act in a strictly sequence-specific manner. Early RNAi experiments demonstrated profound virus inhibition, but also indicated that viral escape is possible. Such therapy failure can be prevented by the design of a combinatorial RNAi attack on the virus and this gene therapy is currently being tested in a preclinical humanized mouse model. Recent CRISPR-Cas studies also document robust virus inhibition, but suggest a novel viral escape route that is induced by the cellular nonhomologous end joining DNA repair pathway, which is activated by CRISPR-Cas-induced DNA breaks. We will compare these two approaches for durable HIV-1 suppression and discuss the respective advantages and disadvantages. The potential for future clinical applications will be described.

  20. RNAi: Mammalian oocytes do it without RNA-dependent RNA polymerase

    PubMed Central

    STEIN, PAULA; SVOBODA, PETR; ANGER, MARTIN; SCHULTZ, RICHARD M.

    2003-01-01

    Studies in mutant organisms deficient in RNA interference (RNAi) and related post-transcriptional gene silencing implicated a role for a single class of RNA-dependent RNA polymerases (RdRp). Nevertheless, sequence homologs to these RdRps have not been found in coelomate organisms such as Drosophila or mammals. This lack of homologous sequences does not exclude that an RdRp functions in RNAi in these organisms because an RdRp could be acquired by horizontal transfer from an RNA virus. In fact, such a sequence is found in mice (Aquarius) and we observe that it is expressed in mouse oocytes and early embryos, which exhibit RNAi. We report here that cordycepin, an inhibitor of RNA synthesis, does not prevent Mos double-strand RNA (dsRNA) to target endogenous Mos mRNA in mouse oocytes and that targeting a chimeric Mos–EGFP mRNA with dsRNA to EGFP does not reduce the endogenous Mos mRNA, but does target the chimeric mRNA. These results indicate that an RdRp is not involved in dsRNA-mediated mRNA degradation in mammalian oocytes, and possibly in mammals in general, and therefore that only homologous sequences to the dsRNA are targeted for degradation. PMID:12554861

  1. Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies

    PubMed Central

    Weinstein, Shiri; Toker, Itai A.; Emmanuel, Rafi; Ramishetti, Srinivas; Hazan-Halevy, Inbal; Rosenblum, Daniel; Goldsmith, Meir; Abraham, Avigdor; Benjamini, Ohad; Bairey, Osnat; Raanani, Pia; Nagler, Arnon; Lieberman, Judy

    2016-01-01

    Despite progress in systemic small interfering RNA (siRNA) delivery to the liver and to solid tumors, systemic siRNA delivery to leukocytes remains challenging. The ability to silence gene expression in leukocytes has great potential for identifying drug targets and for RNAi-based therapy for leukocyte diseases. However, both normal and malignant leukocytes are among the most difficult targets for siRNA delivery as they are resistant to conventional transfection reagents and are dispersed in the body. We used mantle cell lymphoma (MCL) as a prototypic blood cancer for validating a novel siRNA delivery strategy. MCL is an aggressive B-cell lymphoma that overexpresses cyclin D1 with relatively poor prognosis. Down-regulation of cyclin D1 using RNA interference (RNAi) is a potential therapeutic approach to this malignancy. Here, we designed lipid-based nanoparticles (LNPs) coated with anti-CD38 monoclonal antibodies that are specifically taken up by human MCL cells in the bone marrow of xenografted mice. When loaded with siRNAs against cyclin D1, CD38-targeted LNPs induced gene silencing in MCL cells and prolonged survival of tumor-bearing mice with no observed adverse effects. These results highlight the therapeutic potential of cyclin D1 therapy in MCL and present a novel RNAi delivery system that opens new therapeutic opportunities for treating MCL and other B-cell malignancies. PMID:26699502

  2. The RNAi machinery regulates growth and development in the filamentous fungus Trichoderma atroviride.

    PubMed

    Carreras-Villaseñor, Nohemi; Esquivel-Naranjo, Edgardo U; Villalobos-Escobedo, J Manuel; Abreu-Goodger, Cei; Herrera-Estrella, Alfredo

    2013-07-01

    The RNAi machinery is generally involved in genome protection in filamentous fungi; however, the physiological role of RNAi has been poorly studied in fungal models. Here, we report that in the filamentous fungus Trichoderma atroviride, the products of the dcr2 and rdr3 genes control reproductive development, because mutations in these genes affect conidiation. In addition, Dcr1 together with Dcr2 control vegetative growth since Δdcr1, Δdcr2 and Δdcr1Δdcr2 present morphological alterations. Whole-genome transcriptional analysis of WT, Δdcr1, Δdcr2 and Δdcr1Δdcr2 show that each Dicer controls different biological processes, such as development or metabolism, which could explain the lack of conidiation in the mutants. Finally, we observed sRNAs that are differentially expressed in the WT and Δdcr2. The expression of some of these sRNAs correlates with the expression of differential transcripts, suggesting that these mRNAs may contain the corresponding targets. Together these data show that in T. atroviride, the RNAi machinery plays a central role in endogenous processes such as development and fitness, beyond controlling genome protection against invasive nucleic acids as reported for other fungi.

  3. The Antiviral RNAi Response in Vector and Non-vector Cells against Orthobunyaviruses

    PubMed Central

    Watson, Mick; Blomström, Anne-Lie; Skelton, Jessica K.; Kohl, Alain; Elliott, Richard M.; Schnettler, Esther

    2017-01-01

    Background Vector arthropods control arbovirus replication and spread through antiviral innate immune responses including RNA interference (RNAi) pathways. Arbovirus infections have been shown to induce the exogenous small interfering RNA (siRNA) and Piwi-interacting RNA (piRNA) pathways, but direct antiviral activity by these host responses in mosquito cells has only been demonstrated against a limited number of positive-strand RNA arboviruses. For bunyaviruses in general, the relative contribution of small RNA pathways in antiviral defences is unknown. Methodology/Principal Findings The genus Orthobunyavirus in the Bunyaviridae family harbours a diverse range of mosquito-, midge- and tick-borne arboviruses. We hypothesized that differences in the antiviral RNAi response in vector versus non-vector cells may exist and that could influence viral host range. Using Aedes aegypti-derived mosquito cells, mosquito-borne orthobunyaviruses and midge-borne orthobunyaviruses we showed that bunyavirus infection commonly induced the production of small RNAs and the effects of the small RNA pathways on individual viruses differ in specific vector-arbovirus interactions. Conclusions/Significance These findings have important implications for our understanding of antiviral RNAi pathways and orthobunyavirus-vector interactions and tropism. PMID:28060823

  4. Viral RNA silencing suppressors (RSS): novel strategy of viruses to ablate the host RNA interference (RNAi) defense system.

    PubMed

    Bivalkar-Mehla, Shalmali; Vakharia, Janaki; Mehla, Rajeev; Abreha, Measho; Kanwar, Jagat Rakesh; Tikoo, Akshay; Chauhan, Ashok

    2011-01-01

    Pathogenic viruses have developed a molecular defense arsenal for their survival by counteracting the host anti-viral system known as RNA interference (RNAi). Cellular RNAi, in addition to regulating gene expression through microRNAs, also serves as a barrier against invasive foreign nucleic acids. RNAi is conserved across the biological species, including plants, animals and invertebrates. Viruses in turn, have evolved mechanisms that can counteract this anti-viral defense of the host. Recent studies of mammalian viruses exhibiting RNA silencing suppressor (RSS) activity have further advanced our understanding of RNAi in terms of host-virus interactions. Viral proteins and non-coding viral RNAs can inhibit the RNAi (miRNA/siRNA) pathway through different mechanisms. Mammalian viruses having dsRNA-binding regions and GW/WG motifs appear to have a high chance of conferring RSS activity. Although, RSSs of plant and invertebrate viruses have been well characterized, mammalian viral RSSs still need in-depth investigations to present the concrete evidences supporting their RNAi ablation characteristics. The information presented in this review together with any perspective research should help to predict and identify the RSS activity-endowed new viral proteins that could be the potential targets for designing novel anti-viral therapeutics.

  5. RNAiFold 2.0: a web server and software to design custom and Rfam-based RNA molecules

    PubMed Central

    Garcia-Martin, Juan Antonio; Dotu, Ivan; Clote, Peter

    2015-01-01

    Several algorithms for RNA inverse folding have been used to design synthetic riboswitches, ribozymes and thermoswitches, whose activity has been experimentally validated. The RNAiFold software is unique among approaches for inverse folding in that (exhaustive) constraint programming is used instead of heuristic methods. For that reason, RNAiFold can generate all sequences that fold into the target structure or determine that there is no solution. RNAiFold 2.0 is a complete overhaul of RNAiFold 1.0, rewritten from the now defunct COMET language to C++. The new code properly extends the capabilities of its predecessor by providing a user-friendly pipeline to design synthetic constructs having the functionality of given Rfam families. In addition, the new software supports amino acid constraints, even for proteins translated in different reading frames from overlapping coding sequences; moreover, structure compatibility/incompatibility constraints have been expanded. With these features, RNAiFold 2.0 allows the user to design single RNA molecules as well as hybridization complexes of two RNA molecules. Availability: the web server, source code and linux binaries are publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold2.0. PMID:26019176

  6. Genetic screening

    PubMed Central

    Andermann, Anne; Blancquaert, Ingeborg

    2010-01-01

    Abstract OBJECTIVE To provide a primer for primary care professionals who are increasingly called upon to discuss the growing number of genetic screening services available and to help patients make informed decisions about whether to participate in genetic screening, how to interpret results, and which interventions are most appropriate. QUALITY OF EVIDENCE As part of a larger research program, a wide literature relating to genetic screening was reviewed. PubMed and Internet searches were conducted using broad search terms. Effort was also made to identify the gray literature. MAIN MESSAGE Genetic screening is a type of public health program that is systematically offered to a specified population of asymptomatic individuals with the aim of providing those identified as high risk with prevention, early treatment, or reproductive options. Ensuring an added benefit from screening, as compared with standard clinical care, and preventing unintended harms, such as undue anxiety or stigmatization, depends on the design and implementation of screening programs, including the recruitment methods, education and counseling provided, timing of screening, predictive value of tests, interventions available, and presence of oversight mechanisms and safeguards. There is therefore growing apprehension that economic interests might lead to a market-driven approach to introducing and expanding screening before program effectiveness, acceptability, and feasibility have been demonstrated. As with any medical intervention, there is a moral imperative for genetic screening to do more good than harm, not only from the perspective of individuals and families, but also for the target population and society as a whole. CONCLUSION Primary care professionals have an important role to play in helping their patients navigate the rapidly changing terrain of genetic screening services by informing them about the benefits and risks of new genetic and genomic technologies and empowering them to

  7. Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity.

    PubMed

    Mathur, Kalika; Anand, Abhishek; Dubey, Sunil Kumar; Sanan-Mishra, Neeti; Bhatnagar, Raj K; Sunil, Sujatha

    2016-11-30

    RNAi pathway is an antiviral defence mechanism employed by insects that result in degradation of viral RNA thereby curbing infection. Several viruses including flaviviruses encode viral suppressors of RNAi (VSRs) to counteract the antiviral RNAi pathway. Till date, no VSR has been reported in alphaviruses. The present study was undertaken to evaluate chikungunya virus (CHIKV) proteins for RNAi suppressor activity. We systematically analyzed all nine CHIKV proteins for RNAi suppressor activity using Sf21 RNAi sensor cell line based assay. Two non-structural proteins, namely, nsP2 and nsP3 were found to exhibit RNAi suppressor activity. We further validated the findings in natural hosts, namely in Aedes and in mammalian cell lines and further through EMSA and Agrobacterium infiltration in GFP silenced transgenic tobacco plants. Domains responsible for maximum RNAi suppressor activity were also identified within these proteins. RNA binding motifs in these domains were identified and their participation in RNAi suppression evaluated using site directed mutagenesis. Sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. Further validation of RNAi suppressor activity of these proteins awaits establishment of specific virus infection models.

  8. Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity

    PubMed Central

    Mathur, Kalika; Anand, Abhishek; Dubey, Sunil Kumar; Sanan-Mishra, Neeti; Bhatnagar, Raj K.; Sunil, Sujatha

    2016-01-01

    RNAi pathway is an antiviral defence mechanism employed by insects that result in degradation of viral RNA thereby curbing infection. Several viruses including flaviviruses encode viral suppressors of RNAi (VSRs) to counteract the antiviral RNAi pathway. Till date, no VSR has been reported in alphaviruses. The present study was undertaken to evaluate chikungunya virus (CHIKV) proteins for RNAi suppressor activity. We systematically analyzed all nine CHIKV proteins for RNAi suppressor activity using Sf21 RNAi sensor cell line based assay. Two non-structural proteins, namely, nsP2 and nsP3 were found to exhibit RNAi suppressor activity. We further validated the findings in natural hosts, namely in Aedes and in mammalian cell lines and further through EMSA and Agrobacterium infiltration in GFP silenced transgenic tobacco plants. Domains responsible for maximum RNAi suppressor activity were also identified within these proteins. RNA binding motifs in these domains were identified and their participation in RNAi suppression evaluated using site directed mutagenesis. Sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. Further validation of RNAi suppressor activity of these proteins awaits establishment of specific virus infection models. PMID:27901124

  9. Quadruple screen test

    MedlinePlus

    Quad screen; Multiple marker screening; AFP plus; Triple screen test; AFP maternal; MSAFP; 4-marker screen; Down syndrome - quadruple; Trisomy 21 - quadruple; Turner syndrome - quadruple; Spina bifida - ...

  10. MRSA Screening

    MedlinePlus

    ... Tests Online. AACC is a not-for-profit organization and does not endorse non-AACC products and services. Advertising ... aureus Screening Related tests: Wound Culture All content on Lab Tests Online has been ...

  11. Hypertension screening

    NASA Technical Reports Server (NTRS)

    Foulke, J. M.

    1975-01-01

    An attempt was made to measure the response to an announcement of hypertension screening at the Goddard Space Center, to compare the results to those of previous statistics. Education and patient awareness of the problem were stressed.

  12. Developmental Screening

    MedlinePlus

    Learn More about Your Child’s Development: Developmental Monitoring and Screening Taking a first step, waving “bye-bye,” and pointing to something interesting are all developmental milestones, ...

  13. Get Screened

    MedlinePlus

    ... health. Depending on your age, sex, and medical history, you may need to be screened for things like: Certain types of cancer High blood pressure or high cholesterol Diabetes Osteoporosis (weak bones) ...

  14. Identification of pathogenetically relevant genes in lymphomagenesis by shRNA library screens.

    PubMed

    Ngo, Vu N

    2013-01-01

    RNA interference (RNAi) is a conserved posttranscriptional gene silencing mechanism that has recently emerged as a breakthrough genetic tool in functional genomics and drug target discovery. An increasing number of studies applying RNAi in high-throughput screens have begun to unravel complex signaling networks underlying diverse cellular processes. This chapter describes an approach to construct a conditional small-hairpin (sh)RNA library and its application in human lymphoma cell lines. A library cloning procedure outlines the incorporation of shRNA sequences and random 60-mer "bar code" oligonucleotides, enabling rapid identification of the hairpin by microarrays. Lymphoma cell lines are optimized for efficient retroviral transduction and tetracycline inducibility. The shRNA library is suitable for identifying molecular targets in cancer, but also versatile for various screening strategies.

  15. [HCC screening].

    PubMed

    Albrecht, T

    2008-01-01

    Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed tumour diseases throughout the world. In the vast majority of cases those affected are high-risk patients with chronic viral hepatitis and/or liver cirrhosis, which means there is a clearly identifiable target group for HCC screening. With resection, transplantation, and interventional procedures for local ablation, following early diagnosis curative treatment options are available with which 5-year survival rates of over 60% can be reached. Such early diagnosis is a reality only in a minority of patients, however, and in the majority of cases the disease is already in an advanced stage at diagnosis. One of the objects of HCC screening is diagnosis in an early stage when curative treatment is still possible. Precisely this is achieved by screening, so that the proportion of patients treated with curative intent is decisively higher. There is not yet any clear evidence as to whether this leads to a lowering of the mortality of HCC. As lower mortality is the decisive indicator of success for a screening programme the benefit of HCC screening has so far been neither documented nor refuted. Nonetheless, in large regions of the world it is the practice for high-risk patients to undergo HCC screening in the form of twice-yearly ultrasound examination and determination of AFP.

  16. Cricket body size is altered by systemic RNAi against insulin signaling components and epidermal growth factor receptor.

    PubMed

    Dabour, Noha; Bando, Tetsuya; Nakamura, Taro; Miyawaki, Katsuyuki; Mito, Taro; Ohuchi, Hideyo; Noji, Sumihare

    2011-09-01

    A long-standing problem of developmental biology is how body size is determined. In Drosophila melanogaster, the insulin/insulin-like growth factor (I/IGF) and target of rapamycin (TOR) signaling pathways play important roles in this process. However, the detailed mechanisms by which insect body growth is regulated are not known. Therefore, we have attempted to utilize systemic nymphal RNA interference (nyRNAi) to knockdown expression of insulin signaling components including Insulin receptor (InR), Insulin receptor substrate (chico), Phosphatase and tensin homologue (Pten), Target of rapamycin (Tor), RPS6-p70-protein kinase (S6k), Forkhead box O (FoxO) and Epidermal growth factor receptor (Egfr) and observed the effects on body size in the Gryllus bimaculatus cricket. We found that crickets treated with double-stranded RNA (dsRNA) against Gryllus InR, chico, Tor, S6k and Egfr displayed smaller body sizes, while Gryllus FoxO nyRNAi-ed crickets exhibited larger than normal body sizes. Furthermore, RNAi against Gryllus chico and Tor displayed slow growth and RNAi against Gryllus chico displayed longer lifespan than control crickets. Since no significant difference in ability of food uptake was observed between the Gryllus chico(nyRNAi) nymphs and controls, we conclude that the adult cricket body size can be altered by knockdown of expressions of Gryllus InR, chico, Tor, S6k, FoxO and Egfr by systemic RNAi. Our results suggest that the cricket is a promising model to study mechanisms underlying controls of body size and life span with RNAi methods.

  17. Variation in RNAi efficacy among insect species is attributable to dsRNA degradation in vivo.

    PubMed

    Wang, Kangxu; Peng, Yingchuan; Pu, Jian; Fu, Wenxi; Wang, Jiale; Han, Zhaojun

    2016-10-01

    RNA interference (RNAi) has become an essential technique in entomology research. However, RNAi efficiency appears to vary significantly among insect species. Here, the sensitivity of four insect species from different orders to RNAi was compared to understand the reason for this variation. A previously reported method was modified to monitor trace amounts of double-stranded RNA (dsRNA). After the administration of dsRNA, the dynamics of its content was determined in the hemolymph, in addition to the capability of its degradation in both the hemolymph and the midgut juice. The results showed that injection of dsRNA targeting the homologous chitinase gene in Periplaneta americana, Zophobas atratus, Locusta migratoria, and Spodoptera litura, with doses (1.0, 2.3, 11.5, and 33.0 μg, respectively) resulting in the same initial hemolymph concentration, caused 82%, 78%, 76%, and 20% depletion, respectively, whereas feeding doses based on body weight (24, 24, 36, and 30 μg) accounted for 47%, 28%, 5%, and 1% depletion. The sensitivity of insects to RNAi was observed to be as follows: P. americana > Z. atratus >L. migratoria >S. litura. In vivo monitoring revealed that RNAi effects among these insect species were highly correlated with the hemolymph dsRNA contents. Furthermore, in vitro experiments demonstrated that the hemolymph contents after dsRNA injection were dependent on hemolymph degradation capacities, and on the degradation capabilities in the midgut juice, when dsRNA was fed. In conclusion, the RNAi efficacy in different insect species was observed to depend on the enzymatic degradation of dsRNA, which functions as the key factor determining the inner target exposure dosages. Thus, enzymatic degradation in vivo should be taken into consideration for efficient use of RNAi in insects.

  18. A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells.

    PubMed

    Jolly, Amber L; Luan, Chi-Hao; Dusel, Brendon E; Dunne, Sara F; Winding, Michael; Dixit, Vishrut J; Robins, Chloe; Saluk, Jennifer L; Logan, David J; Carpenter, Anne E; Sharma, Manu; Dean, Deborah; Cohen, Andrew R; Gelfand, Vladimir I

    2016-01-26

    Long-distance intracellular transport of organelles, mRNA, and proteins ("cargo") occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

  19. A genome-wide RNAi screen for microtubule bundle formation and lysosome motility regulation in Drosophila S2 cells

    PubMed Central

    Jolly, Amber L.; Luan, Chi-Hao; Dusel, Brendon E.; Dunne, Sara Fernandez; Winding, Michael; Dixit, Vishrut J.; Robins, Chloe; Saluk, Jennifer L.; Logan, David J.; Carpenter, Anne E.; Sharma, Manu; Dean, Deborah; Cohen, Andrew R.; Gelfand, Vladimir I.

    2016-01-01

    Summary Long-distance intracellular transport of organelles, mRNA, and proteins (“cargo”) occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins; the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naïve Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes, and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels and signaling proteins having a role in lysosome motility regulation, and find an unexpected relationship between the dynein motor, Rab7a and lysosome motility regulation. PMID:26774481

  20. Quantitative research.

    PubMed

    Watson, Roger

    2015-04-01

    This article describes the basic tenets of quantitative research. The concepts of dependent and independent variables are addressed and the concept of measurement and its associated issues, such as error, reliability and validity, are explored. Experiments and surveys – the principal research designs in quantitative research – are described and key features explained. The importance of the double-blind randomised controlled trial is emphasised, alongside the importance of longitudinal surveys, as opposed to cross-sectional surveys. Essential features of data storage are covered, with an emphasis on safe, anonymous storage. Finally, the article explores the analysis of quantitative data, considering what may be analysed and the main uses of statistics in analysis.

  1. Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription.

    PubMed

    Marza, Esther; Taouji, Saïd; Barroso, Kim; Raymond, Anne-Aurélie; Guignard, Léo; Bonneu, Marc; Pallares-Lupon, Néstor; Dupuy, Jean-William; Fernandez-Zapico, Martin E; Rosenbaum, Jean; Palladino, Francesca; Dupuy, Denis; Chevet, Eric

    2015-03-01

    The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. The AAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.

  2. RNAi prevents and reverses phenotypes induced by mutant human ataxin‐1

    PubMed Central

    Keiser, Megan S.; Monteys, Alejandro Mas; Corbau, Romuald; Gonzalez‐Alegre, Pedro

    2016-01-01

    Objective Spinocerebellar ataxia type 1 is an autosomal dominant fatal neurodegenerative disease caused by a polyglutamine expansion in the coding region of ATXN1. We showed previously that partial suppression of mutant ataxin‐1 (ATXN1) expression, using virally expressed RNAi triggers, could prevent disease symptoms in a transgenic mouse model and a knockin mouse model of the disease, using a single dose of virus. Here, we set out to test whether RNAi triggers targeting ATXN1 could not only prevent, but also reverse disease readouts when delivered after symptom onset. Methods We administered recombinant adeno‐associated virus (rAAV) expressing miS1, an artificial miRNA targeting human ATXN1 mRNA (rAAV.miS1), to a mouse model of spinocerebellar ataxia type 1 (SCA1; B05 mice). Viruses were delivered prior to or after symptom onset at multiple doses. Control B05 mice were treated with rAAVs expressing a control artificial miRNA, or with saline. Animal behavior, molecular phenotypes, neuropathology, and magnetic resonance spectroscopy were done on all groups, and data were compared to wild‐type littermates. Results We found that SCA1 phenotypes could be reversed by partial suppression of human mutant ATXN1 mRNA by rAAV.miS1 when delivered after symptom onset. We also identified the therapeutic range of rAAV.miS1 that could prevent or reverse disease readouts. Interpretation SCA1 disease may be reversible by RNAi therapy, and the doses required for advancing this therapy to humans are delineated. Ann Neurol 2016;80:754–765 PMID:27686464

  3. Self-assembling RNA nanorings based on RNAI/II inverse kissing complexes.

    PubMed

    Grabow, Wade W; Zakrevsky, Paul; Afonin, Kirill A; Chworos, Arkadiusz; Shapiro, Bruce A; Jaeger, Luc

    2011-02-09

    RNA is an attractive biopolymer for nanodesign of self-assembling particles for nanobiotechnology and synthetic biology. Here, we experimentally characterize by biochemical and biophysical methods the formation of thermostable and ribonuclease resistant RNA nanorings previously proposed by computational design. High yields of fully programmable nanorings were produced based on several RNAI/IIi kissing complex variants selected for their ability to promote polygon self-assembly. This self-assembly strategy relying on the particular geometry of bended kissing complexes has potential for developing short interfering RNA delivery agents.

  4. RNAi-mediated TCR knockdown prevents autoimmunity in mice caused by mixed TCR dimers following TCR gene transfer.

    PubMed

    Bunse, Mario; Bendle, Gavin M; Linnemann, Carsten; Bies, Laura; Schulz, Stephan; Schumacher, Ton N; Uckert, Wolfgang

    2014-11-01

    Genetically modified T cells that express a transduced T cell receptor (TCR) α/β heterodimer in addition to their endogenous TCR are used in clinical studies to treat cancer. These cells express two TCR-α and two TCR-β chains that do not only compete for CD3 proteins but also form potentially self-reactive mixed TCR dimers, composed of endogenous and transferred chains. To overcome these deficits, we developed an RNAi-TCR replacement vector that simultaneously silences the endogenous TCR and expresses an RNAi-resistant TCR. Transduction of the virus-specific P14 TCR without RNAi resulted in unequal P14 TCR-α and -β chain surface levels, indicating heterodimerization with endogenous TCR chains. Such unequal expression was also observed following TCR gene optimization. Equal surface levels of the introduced TCR chains were however achieved by silencing the endogenous TCR. Importantly, all mice that received cells transduced with the native or optimized P14 TCR developed lethal TCR gene transfer-induced graft-versus-host-disease (TI-GVHD) due to formation of mixed TCR dimers. In contrast, TI-GVHD was almost completely prevented when using the RNAi-TCR replacement vector. Our data demonstrate that RNAi-assisted TCR replacement reduces the formation of mixed TCR dimers, and thereby significantly reduces the risk of TI-GVHD in TCR gene therapy.

  5. Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects

    PubMed Central

    Shukla, Jayendra Nath; Kalsi, Megha; Sethi, Amit; Narva, Kenneth E.; Fishilevich, Elane; Singh, Satnam; Mogilicherla, Kanakachari; Palli, Subba Reddy

    2016-01-01

    ABSTRACT RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects. PMID:27245473

  6. The possible impact of persistent virus infection on the function of the RNAi machinery in insects: a hypothesis.

    PubMed

    Swevers, Luc; Vanden Broeck, Jozef; Smagghe, Guy

    2013-01-01

    RNAi experiments in insects are characterized by great variability in efficiency; for instance beetles and locusts are very amenable to dsRNA-mediated gene silencing, while other insect groups, most notably lepidopterans, are more refractory to RNAi. Several factors can be forwarded that could affect the efficiency of RNAi, such as the composition and function of the intracellular RNAi machinery, the mechanism of dsRNA uptake, the presence of dsRNA- and siRNA-degrading enzymes and non-specific activation of the innate immune response. In this essay, we investigate the evidence whether persistent infection with RNA viruses could be a major factor that affects the response to exogenous dsRNA in insects. The occurrence of RNA viruses in different insect groups will be discussed, as well as several mechanisms by which viruses could interfere with the process of RNAi. Finally, the impact of RNA virus infection on the design of dsRNA-based insect control strategies will be considered.

  7. Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects.

    PubMed

    Shukla, Jayendra Nath; Kalsi, Megha; Sethi, Amit; Narva, Kenneth E; Fishilevich, Elane; Singh, Satnam; Mogilicherla, Kanakachari; Palli, Subba Reddy

    2016-07-02

    RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects.

  8. The possible impact of persistent virus infection on the function of the RNAi machinery in insects: a hypothesis

    PubMed Central

    Swevers, Luc; Vanden Broeck, Jozef; Smagghe, Guy

    2013-01-01

    RNAi experiments in insects are characterized by great variability in efficiency; for instance beetles and locusts are very amenable to dsRNA-mediated gene silencing, while other insect groups, most notably lepidopterans, are more refractory to RNAi. Several factors can be forwarded that could affect the efficiency of RNAi, such as the composition and function of the intracellular RNAi machinery, the mechanism of dsRNA uptake, the presence of dsRNA- and siRNA-degrading enzymes and non-specific activation of the innate immune response. In this essay, we investigate the evidence whether persistent infection with RNA viruses could be a major factor that affects the response to exogenous dsRNA in insects. The occurrence of RNA viruses in different insect groups will be discussed, as well as several mechanisms by which viruses could interfere with the process of RNAi. Finally, the impact of RNA virus infection on the design of dsRNA-based insect control strategies will be considered. PMID:24204347

  9. Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon.

    PubMed

    Song, Beng-Kah; Nadarajah, Kalaivani; Romanov, Michael N; Ratnam, Wickneswari

    2005-01-01

    The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.

  10. Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens

    PubMed Central

    Meliopoulos, Victoria A.; Andersen, Lauren E.; Birrer, Katherine F.; Simpson, Kaylene J.; Lowenthal, John W.; Bean, Andrew G. D.; Stambas, John; Stewart, Cameron R.; Tompkins, S. Mark; van Beusechem, Victor W.; Fraser, Iain; Mhlanga, Musa; Barichievy, Samantha; Smith, Queta; Leake, Devin; Karpilow, Jon; Buck, Amy; Jona, Ghil; Tripp, Ralph A.

    2012-01-01

    Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA