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Sample records for shrna-mediated rna interference

  1. RNA interference in Lepidoptera

    DEFF Research Database (Denmark)

    Terenius, Ole; Papanicolaou, Alexie; Garbutt, Jennie S.

    2011-01-01

    Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive...... is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success...

  2. The RNA interference revolution

    Directory of Open Access Journals (Sweden)

    G. Lenz

    2005-12-01

    Full Text Available The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.

  3. RNA interference against viruses: strike and counterstrike

    NARCIS (Netherlands)

    Haasnoot, Joost; Westerhout, Ellen M.; Berkhout, Ben

    2007-01-01

    RNA interference (RNAi) is a conserved sequence-specific, gene-silencing mechanism that is induced by double-stranded RNA. RNAi holds great promise as a novel nucleic acid-based therapeutic against a wide variety of diseases, including cancer, infectious diseases and genetic disorders. Antiviral

  4. Triggering of RNA interference with RNA-RNA, RNA-DNA, and DNA-RNA nanoparticles.

    Science.gov (United States)

    Afonin, Kirill A; Viard, Mathias; Kagiampakis, Ioannis; Case, Christopher L; Dobrovolskaia, Marina A; Hofmann, Jen; Vrzak, Ashlee; Kireeva, Maria; Kasprzak, Wojciech K; KewalRamani, Vineet N; Shapiro, Bruce A

    2015-01-27

    Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use.

  5. Generation of siRNA Nanosheets for Efficient RNA Interference

    Science.gov (United States)

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-04-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances.

  6. Suppression of RNA interference by adenovirus virus-associated RNA

    NARCIS (Netherlands)

    Andersson, M. Gunnar; Haasnoot, P. C. Joost; Xu, Ning; Berenjian, Saideh; Berkhout, Ben; Akusjärvi, Göran

    2005-01-01

    We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses

  7. RNA interference in plant parasitic nematodes

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... role in regulating development and genome maintenance. RNAi has recently been demonstrated in ... Key words: RNA interference, RNAi, gene expression, plant parasitic nematodes. INTRODUCTION. Plant parasitic .... species to maximize the spectrum of resistance. RNAi effect has been demonstrated ...

  8. Inhibition of virus replication by RNA interference

    NARCIS (Netherlands)

    Haasnoot, P. C. Joost; Cupac, Daniel; Berkhout, Ben

    2003-01-01

    RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene

  9. RNA interference: its use as antiviral therapy

    NARCIS (Netherlands)

    Haasnoot, J.; Berkhout, B.

    2006-01-01

    RNA interference (RNAi) is a sequence-specific gene-silencing mechanism that has been proposed to function as a defence mechanism of eukaryotic cells against viruses and transposons. RNAi was first observed in plants in the form of a mysterious immune response to viral pathogens. But RNAi is more

  10. RNA interference for the control of whiteflies

    Indian Academy of Sciences (India)

    RNA interference (RNAi)-mediated gene silencing was explored for the control of sap-sucking pest Bemisia tabaci, commonly known as whitefly. dsRNAs and siRNAs were synthesized from five different genes – actin ortholog, ADP/ATP translocase, -tubulin, ribosomal protein L9 (RPL9) and V-ATPase A subunit.

  11. Symbiont-mediated RNA interference in insects.

    Science.gov (United States)

    Whitten, Miranda M A; Facey, Paul D; Del Sol, Ricardo; Fernández-Martínez, Lorena T; Evans, Meirwyn C; Mitchell, Jacob J; Bodger, Owen G; Dyson, Paul J

    2016-02-24

    RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects. © 2016 The Authors.

  12. Symbiont-mediated RNA interference in insects

    Science.gov (United States)

    Whitten, Miranda M. A.; Facey, Paul D.; Del Sol, Ricardo; Fernández-Martínez, Lorena T.; Evans, Meirwyn C.; Mitchell, Jacob J.; Bodger, Owen G.

    2016-01-01

    RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects. PMID:26911963

  13. RNA interference and Register Machines (extended abstract

    Directory of Open Access Journals (Sweden)

    Masahiro Hamano

    2012-11-01

    Full Text Available RNA interference (RNAi is a mechanism whereby small RNAs (siRNAs directly control gene expression without assistance from proteins. This mechanism consists of interactions between RNAs and small RNAs both of which may be single or double stranded. The target of the mechanism is mRNA to be degraded or aberrated, while the initiator is double stranded RNA (dsRNA to be cleaved into siRNAs. Observing the digital nature of RNAi, we represent RNAi as a Minsky register machine such that (i The two registers hold single and double stranded RNAs respectively, and (ii Machine's instructions are interpreted by interactions of enzyme (Dicer, siRNA (with RISC com- plex and polymerization (RdRp to the appropriate registers. Interpreting RNAi as a computational structure, we can investigate the computational meaning of RNAi, especially its complexity. Initially, the machine is configured as a Chemical Ground Form (CGF, which generates incorrect jumps. To remedy this problem, the system is remodeled as recursive RNAi, in which siRNA targets not only mRNA but also the machine instructional analogues of Dicer and RISC. Finally, probabilistic termination is investigated in the recursive RNAi system.

  14. RNA interference in plant parasitic nematodes | Karakas | African ...

    African Journals Online (AJOL)

    RNA interference (RNAi, also called RNA-mediated interference) is a mechanism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. Conserved in most eukaryotic organisms, the RNAi pathway is thought to ...

  15. Induction of RNA interference in dendritic cells.

    Science.gov (United States)

    Li, Mu; Qian, Hua; Ichim, Thomas E; Ge, Wei-Wen; Popov, Igor A; Rycerz, Katarzyna; Neu, John; White, David; Zhong, Robert; Min, Wei-Ping

    2004-01-01

    Dendritic cells (DC) reside at the center of the immunological universe, possessing the ability both to stimulate and inhibit various types of responses. Tolerogenic/regulatory DC with therapeutic properties can be generated through various means of manipulations in vitro and in vivo. Here we describe several attractive strategies for manipulation of DC using the novel technique of RNA interference (RNAi). Additionally, we overview some of our data regarding yet undescribed characteristics of RNAi in DC such as specific transfection strategies, persistence of gene silencing, and multi-gene silencing. The advantages of using RNAi for DC genetic manipulation gives rise to the promise of generating tailor-made DC that can be used effectively to treat a variety of immunologically mediated diseases.

  16. RNA interference as an antiviral approach: Targeting HIV-1

    NARCIS (Netherlands)

    Berkhout, Ben

    2004-01-01

    RNA interference (RNAi) is an evolutionary conserved gene-silencing mechanism in which 21- to 23-mer double-stranded short interfering RNA (siRNA) mediates the sequence-specific degradation of mRNA. The recent discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the

  17. Role of RNA interference in plant improvement

    Science.gov (United States)

    Jagtap, Umesh Balkrishna; Gurav, Ranjit Gajanan; Bapat, Vishwas Anant

    2011-06-01

    Research to alter crops for their better performance involving modern technology is underway in numerous plants, and achievements in transgenic plants are impacting crop improvements in unparalleled ways. Striking progress has been made using genetic engineering technology over the past two decades in manipulating genes from diverse and exotic sources, and inserting them into crop plants for inducing desirable characteristics. RNA interference (RNAi) has recently been identified as a natural mechanism for regulation of gene expression in all higher organisms from plants to humans and promises greater accuracy and precision to plant improvement. The expression of any gene can be down-regulated in a highly explicit manner exclusive of affecting the expression of any other gene by using RNAi technologies. Additional research in this field has been focused on a number of other areas including microRNAs, hairpin RNA, and promoter methylation. Manipulating new RNAi pathways, which generate small RNA molecules to amend gene expression in crops, can produce new quality traits and having better potentiality of protection against abiotic and biotic stresses. Nutritional improvement, change in morphology, or enhanced secondary metabolite synthesis are some of the other advantages of RNAi technology. In addition to its roles in regulating gene expression, RNAi is also used as a natural defense mechanism against molecular parasites such as jumping genes and viral genetic elements that affect genome stability. Even though much advancement has been made on the field of RNAi over the preceding few years, the full prospective of RNAi for crop improvement remains to be fully realized. The intricacy of RNAi pathway, the molecular machineries, and how it relates to plant development are still to be explained.

  18. Development of an RNA-interference procedure for gene ...

    African Journals Online (AJOL)

    The results show successful knockdown of HRF target after 16 h of immersion in dsRNA. It is suggested that optimization of delivery methods for dsRNA and targeting more genes could enhance knockdown. Keywords: Dermanyssus gallinae (Acari: Astigmata), RNA interference (RNAi), gene silencing, histamine releasing ...

  19. Triggering of RNA Interference with RNA–RNA, RNA–DNA, and DNA–RNA Nanoparticles

    Science.gov (United States)

    2015-01-01

    Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use. PMID:25521794

  20. Oligonucleotide Antiviral Therapeutics: Antisense and RNA Interference for Highly Pathogenic RNA Viruses

    National Research Council Canada - National Science Library

    Spurgers, Kevin B; Sharkey, C. M; Warfield, Kelly L; Bavari, Sina

    2008-01-01

    .... Important advances in the field include the characterization of RNA interference in mammalian cells and chemical modifications that can dramatically increase the in vivo stability of therapeutic oligonucleotides...

  1. Application of RNA interference methodology to investigate and ...

    Indian Academy of Sciences (India)

    2014-07-31

    Jul 31, 2014 ... SCMV. In planta transformation was performed on maize (Zea mays) inbred line 8112 mediated by Agrobacterium tumefa- ciens. PCR and ... Development of a maize cultivar, i.e. resis- ... Keywords. maize; sugarcane mosaic virus; RNA interference; double-stranded RNA; in planta transformation. Journal of ...

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

    Science.gov (United States)

    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…

  3. Branched RNA: A New Architecture for RNA Interference

    Directory of Open Access Journals (Sweden)

    Anna Aviñó

    2011-01-01

    Full Text Available Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

  4. Effects of silencing PTTG expression by small interference RNA.

    Science.gov (United States)

    Zhang, S-X; Shan, W-X; Yuan, L-P; Liu, Y-L; Sun, L-Z

    2016-07-01

    We investigated the effects of small interference RNA (siRNA) on the cell proliferation inhibition, sensitivity to radiotherapy effects and cell apoptosis. The siRNA used here was specific to the pituitary tumor transforming gene (PTTG). Vectors containing the specific functional siRNAs for PTTG were designed and constructed. Cells were divided into four groups: (I) blank control group; (II) radiotherapy group: cells were exposed to X-ray radiation; (III) Group PTTG siRNA: transfected with PTTG siRNA; (IV) PTTG siRNA+ radiotherapy group: transfected with PTTG siRNA and then were exposed to X-ray radiation. HEC-1A cells were transfected by the specific interfering plasmids using Lipofectamine 2000 transfection reagent. The PTTG protein expression levels were analyzed using Western blot Cell proliferation was examined by MTT assay and the HEC-1A cell line apoptosis was evaluated by flow cytometry. Recombinant small interference RNA (siRNA) expression vectors targeting PTTG were successfully constructed. The results of MTT showed that the growth of the HEC-1A cell was negatively influenced after cells were transfected with PTTG siRNA. Furthermore, PTTG siRNA combined with radiotherapy demonstrated more powerful inhibitory effects. Cell apoptosis rates were significantly increased in the radiotherapy group and the PTTG siRNA transfection group when compared to the control group. A more pronounced cell apoptosis rate was observed in the group that was treated with PTTG siRNA combined with radiotherapy. Recombinant small interference RNA (siRNA) expression vector targeting PTTG successfully inhibited the cell proliferation and induced apoptosis in endometrial carcinoma cells and increased the cancer cells vulnerability to the effects of radiation.

  5. RNA Interference and its therapeutic applications

    Directory of Open Access Journals (Sweden)

    Srinivasa Rao T

    2011-10-01

    Full Text Available RNAi is a potent method, requiring only a few molecules of dsRNA per cell to silence the expression. Long molecules of double stranded RNA (dsRNA trigger the process. The dsRNA comes from virus and transposon activity in natural RNAi process, while it can be injected in the cells in experimental processes. The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is called the sense strand, and the other strand which is complimentary is termed the antisense strand. An enzyme complex called DICER thought to be similar to RNAase III then recognizes dsRNA, and cuts it into roughly 22- nucleotide long fragments. These fragments termed siRNAs for “small interfering RNAs” remain in double stranded duplexes with very short 3' overhangs. However, only one of the two strands, known as the guide strand or antisense strand binds the argonaute protein of RNA-induced silencing complex (RISC and target the complementary mRNA resulting gene silencing. The other anti-guide strand or passenger strand is degraded as a RISC substrate during the process of RISC activation. This form of RNAi is termed as post transcriptional gene silencing (PTGS; other forms are also thought to operate at the genomic or transcriptional level in some organisms. In mammals dsRNA longer than 30 base pairs induces a nonspecific antiviral response. This so-called interferon response results in a nonspecific arrest in translation and induction of apoptosis. This cascade induces a global non-specific suppression of translation, which in turn triggers apoptosis. Interestingly, dsRNAs less than 30 nt in length do not activate the antiviral response and specifically switched off genes in human cells without initiating the acute phase response. Thus these siRNAs are suitable for gene target validation and therapeutic applications in many species, including humans. [Vet. World 2011; 4(5.000: 225-229

  6. The first discovery of RNA interference by RNA restriction enzymes to inhibit protein synthesis.

    Science.gov (United States)

    Inouye, Masayori

    2017-01-15

    In this article, I review how an RNA restriction enzyme, a highly sequence-specific endoribonuclease, was for the first time discovered in 2003 and how the concept of RNA interference using RNA restriction enzymes or mRNA interferases has been developed. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA

    NARCIS (Netherlands)

    Konstantinova, P.; de Vries, W.; Haasnoot, J.; ter Brake, O.; de Haan, P.; Berkhout, B.

    2006-01-01

    Inhibition of virus replication by means of RNA interference has been reported for several important human pathogens, including human immunodeficiency virus type 1 (HIV-1). RNA interference against these pathogens has been accomplished by introduction of virus-specific synthetic small interfering

  8. RNA interference in plant parasitic nematodes

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... with homology to C-type lectins did not affect sexual fate, but 41% fewer nematodes were recovered from the plants (Urwin et al., 2002). As expected, treatment with. dsRNA corresponding to the major sperm protein (MSP) had no effect on nematode development or sexual fate. 14 days after treatment.

  9. RNA Interference-Towards RNA becoming a Medicine

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 8; Issue 12. RNA Inteference – Towards RNA becoming a Medicine. Subhanjan Mondal. General Article Volume 8 Issue 12 December 2003 pp 42-49. Fulltext. Click here to view fulltext PDF. Permanent link:

  10. RNA interference-mediated intrinsic antiviral immunity in invertebrates.

    Science.gov (United States)

    Nayak, Arabinda; Tassetto, Michel; Kunitomi, Mark; Andino, Raul

    2013-01-01

    In invertebrates such as insects and nematodes, RNA interference (RNAi) provides RNA-based protection against viruses. This form of immunity restricts viral replication and dissemination from infected cells and viruses, in turn, have evolved evasion mechanisms or RNAi suppressors to counteract host defenses. Recent advances indicate that, in addition to RNAi, other related small RNA pathways contribute to antiviral functions in invertebrates. This has led to a deeper understanding of fundamental aspects of small RNA-based antiviral immunity in invertebrates and its contribution to viral spread and pathogenesis.

  11. Neuron-specific RNA interference using lentiviral vectors

    DEFF Research Database (Denmark)

    Nielsen, Troels Tolstrup; Marion, Ingrid van; Hasholt, Lis

    2009-01-01

    transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding......BACKGROUND: Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise...... the possible promoter choices and eventually allowing cell type specific down-regulation of target genes. METHODS: In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain. RESULTS: We...

  12. Characterization of RNA interference in rat PC12 cells

    DEFF Research Database (Denmark)

    Thonberg, Håkan; Schéele, Camilla C; Dahlgren, Cecilia

    2004-01-01

    Double-stranded RNA can initiate post transcriptional gene silencing in mammalian cell cultures via a mechanism known as RNA interference (RNAi). The sequence-specific degradation of homologous mRNA is triggered by 21-nucleotide RNA-duplexes termed short interfering RNA (siRNA). The homologous...... strand of the siRNA guides a multi-protein complex, RNA-induced silencing complex (RISC), to cleave target mRNA. Although the exact function and composition of RISC is still unclear, it has been shown to include several proteins of the Argonaute protein family. Here we report of a robust system...... to achieve RNAi in a cultured rat neuronal cell line, PC12. Targeting of neuropeptide Y mRNA by synthetic siRNA results in knock down of the mRNA levels with an IC50 of approximately 0.1 nM. The mRNA knockdown lasts for at least 96 h and is not dependent on protein synthesis. Further, PC12 cells were ablated...

  13. Silencing of HIV-1 with RNA interference: a multiple shRNA approach

    NARCIS (Netherlands)

    ter Brake, Olivier; Konstantinova, Pavlina; Ceylan, Mustafa; Berkhout, Ben

    2006-01-01

    Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). Previously, we have shown that stable expression of a single shRNA targeting the HIV-1 Nef gene strongly inhibits HIV-1 replication. However, this was not sufficient to maintain inhibition. One of the

  14. RNA interference in Lepidoptera: An overview of successful and unsuccessful

    NARCIS (Netherlands)

    Terenius, O.; Papanicolaou, A.; Garbutt, J.S.; Eleftherianos, I.; Huvenne, H.; Kanginakudru, S.; Albrechtsen, M.; An, Chunju; Aymeric, J.L.; Barthel, A.; Bebas, P.; Bitra, K.; Bravo, A.; Chevalier, F.; Collinge, D.P.; Crava, C.M.; Maagd, de R.A.; Duvic, B.; Erlandson, M.; Faye, I.; Felfoldi, G.; Fujiwara, H.; Futahashi, R.; Gandhe, A.S.; Gatehouse, H.S.; Gatehouse, L.N.; Giebultowicz, J.M.; Gomez, I.; Grimmelikhuijzen, C.J.P.; Groot, A.T.; Hauser, F.; Heckel, D.G.; Hegedus, D.D.; Hrycaj, S.; Huang, L.; Hull, J.J.; Iatrou, K.; Iga, M.; Kanost, M.R.; Kotwica, J.; Li, Changyou; Li, Jianghong; Liu, Jisheng; Lundmark, M.; Matsumoto, S.; Meyering-Vos, M.; Millichap, P.J.; Monteiro, A.; Mrinal, N.; Niimi, T.; Nowara, D.; Ohnishi, A.; Oostra, V.; Ozaki, K.; Papakonstantinou, M.; Popadic, A.; Rajam, M.V.; Saenko, S.; Simpson, R.M.; Soberon, M.; Strand, M.R.; Tomita, S.; Toprak, U.; Wang, Ping; Wee, Choon Wei; Whyard, S.; Zhang, Wenqing; Nagaraju, J.; Ffrench-Constant, R.H.; Herrero, S.; Gordon, K.; Swevers, L.; Smagghe, G.

    2011-01-01

    Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive

  15. ESF-EMBO symposium: antiviral applications of RNA interference

    NARCIS (Netherlands)

    ter Brake, Olivier; Haasnoot, Joost; Kurreck, Jens; Berkhout, Ben

    2008-01-01

    ABSTRACT: The first ESF-EMBO symposium on applications of antiviral RNA interference (RNAi) was held in the spring of 2008 in Sant Feliu de Guixols at the Costa Brava in Spain. Some 60 participants from the field of RNAi and virology came together to present their latest findings on RNAi-virus

  16. Exploring Fusarium head blight disease control by RNA interference

    Science.gov (United States)

    RNA interference (RNAi) technology provides a novel tool to study gene function and plant protection strategies. Fusarium graminearum is the causal agent of Fusarium head blight (FHB), which reduces crop yield and quality by producing trichothecene mycotoxins including 3-acetyl deoxynivalenol (3-ADO...

  17. RNA Interference - Towards RNA becoming a Medicine -42 ...

    Indian Academy of Sciences (India)

    becoming a Medicine. Subhanjan Mondal is pursuing MSc biotechnol- ogy at MaduaraiKamaraj. University. His interest lies in cell biology and structural biology. Subhanjan Mondal. The central dogma in molecular biology stipulates that the flow of genetic information is from DNA (i.e. the gene) through RNA to proteins ...

  18. RNA Interference of Human α-Synuclein in Mouse.

    Science.gov (United States)

    Kim, Young-Cho; Miller, Adam; Lins, Livia C R F; Han, Sang-Woo; Keiser, Megan S; Boudreau, Ryan L; Davidson, Beverly L; Narayanan, Nandakumar S

    2017-01-01

    α-Synuclein is postulated to play a key role in the pathogenesis of Parkinson's disease (PD). Aggregates of α-synuclein contribute to neurodegeneration and cell death in humans and in mouse models of PD. Here, we use virally mediated RNA interference to knockdown human α-synuclein in mice. We used an siRNA design algorithm to identify eight siRNA sequences with minimal off-targeting potential. One RNA-interference sequence (miSyn4) showed maximal protein knockdown potential in vitro. We then designed AAV vectors expressing miSyn4 and injected them into the mouse substantia nigra. miSyn4 was robustly expressed and did not detectably change dopamine neurons, glial proliferation, or mouse behavior. We then injected AAV2-miSyn4 into Thy1-hSNCA mice over expressing α-synuclein and found decreased human α-synuclein (hSNCA) in both midbrain and cortex. In separate mice, co-injection of AAV2-hSNCA and AAV2-miSyn4 demonstrated decreased hSNCA expression and rescue of hSNCA-mediated behavioral deficits. These data suggest that virally mediated RNA interference can knockdown hSNCA in vivo, which could be helpful for future therapies targeting human α-synuclein.

  19. RNA Interference Therapies for an HIV-1 Functional Cure.

    Science.gov (United States)

    Scarborough, Robert J; Gatignol, Anne

    2017-12-27

    HIV-1 drug therapies can prevent disease progression but cannot eliminate HIV-1 viruses from an infected individual. While there is hope that elimination of HIV-1 can be achieved, several approaches to reach a functional cure (control of HIV-1 replication in the absence of drug therapy) are also under investigation. One of these approaches is the transplant of HIV-1 resistant cells expressing anti-HIV-1 RNAs, proteins or peptides. Small RNAs that use RNA interference pathways to target HIV-1 replication have emerged as competitive candidates for cell transplant therapy and have been included in all gene combinations that have so far entered clinical trials. Here, we review RNA interference pathways in mammalian cells and the design of therapeutic small RNAs that use these pathways to target pathogenic RNA sequences. Studies that have been performed to identify anti-HIV-1 RNA interference therapeutics are also reviewed and perspectives on their use in combination gene therapy to functionally cure HIV-1 infection are provided.

  20. RNA interference-mediated simultaneous silencing of four genes using cross-shaped RNA.

    Science.gov (United States)

    Lee, Tae Yeon; Chang, Chan Il; Lee, Dooyoung; Hong, Sun Woo; Shin, Chanseok; Li, Chiang J; Kim, Soyoun; Haussecker, Dirk; Lee, Dong-Ki

    2013-04-01

    The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.

  1. RNA interference-based nanosystems for inflammatory bowel disease therapy.

    Science.gov (United States)

    Guo, Jian; Jiang, Xiaojing; Gui, Shuangying

    Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn's disease, is a chronic, recrudescent disease that invades the gastrointestinal tract, and it requires surgery or lifelong medicinal therapy. The conventional medicinal therapies for IBD, such as anti-inflammatories, glucocorticoids, and immunosuppressants, are limited because of their systemic adverse effects and toxicity during long-term treatment. RNA interference (RNAi) precisely regulates susceptibility genes to decrease the expression of proinflammatory cytokines related to IBD, which effectively alleviates IBD progression and promotes intestinal mucosa recovery. RNAi molecules generally include short interfering RNA (siRNA) and microRNA (miRNA). However, naked RNA tends to degrade in vivo as a consequence of endogenous ribonucleases and pH variations. Furthermore, RNAi treatment may cause unintended off-target effects and immunostimulation. Therefore, nanovectors of siRNA and miRNA were introduced to circumvent these obstacles. Herein, we introduce non-viral nanosystems of RNAi molecules and discuss these systems in detail. Additionally, the delivery barriers and challenges associated with RNAi molecules will be discussed from the perspectives of developing efficient delivery systems and potential clinical use.

  2. Domain motions of Argonaute, the catalytic engine of RNA interference

    Directory of Open Access Journals (Sweden)

    Wall Michael E

    2007-11-01

    Full Text Available Abstract Background The Argonaute protein is the core component of the RNA-induced silencing complex, playing the central role of cleaving the mRNA target. Visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of Argonaute that might occur during RNA interference. We have taken the next step by performing an all-atom normal mode analysis of the Pyrococcus furiosus and Aquifex aeolicus Argonaute crystal structures, allowing us to quantitatively assess the feasibility of these conformational changes. To perform the analysis, we begin with the energy-minimized X-ray structures. Normal modes are then calculated using an all-atom molecular mechanics force field. Results The analysis reveals low-frequency vibrations that facilitate the accommodation of RNA duplexes – an essential step in target recognition. The Pyrococcus furiosus and Aquifex aeolicus Argonaute proteins both exhibit low-frequency torsion and hinge motions; however, differences in the overall architecture of the proteins cause the detailed dynamics to be significantly different. Conclusion Overall, low-frequency vibrations of Argonaute are consistent with mechanisms within the current reaction cycle model for RNA interference.

  3. RNA interference in the clinic: challenges and future directions

    Science.gov (United States)

    Pecot, Chad V.; Calin, George A.; Coleman, Robert L.; Lopez-Berestein, Gabriel; Sood, Anil K.

    2011-01-01

    Inherent difficulties with blocking many desirable targets using conventional approaches have prompted many to consider using RNA interference (RNAi) as a therapeutic approach. Although exploitation of RNAi has immense potential as a cancer therapeutic, many physiological obstacles stand in the way of successful and efficient delivery. This Review explores current challenges to the development of synthetic RNAi-based therapies and considers new approaches to circumvent biological barriers, to avoid intolerable side effects and to achieve controlled and sustained release. PMID:21160526

  4. Cardiovascular RNA interference therapy: the broadening tool and target spectrum.

    Science.gov (United States)

    Poller, Wolfgang; Tank, Juliane; Skurk, Carsten; Gast, Martina

    2013-08-16

    Understanding of the roles of noncoding RNAs (ncRNAs) within complex organisms has fundamentally changed. It is increasingly possible to use ncRNAs as diagnostic and therapeutic tools in medicine. Regarding disease pathogenesis, it has become evident that confinement to the analysis of protein-coding regions of the human genome is insufficient because ncRNA variants have been associated with important human diseases. Thus, inclusion of noncoding genomic elements in pathogenetic studies and their consideration as therapeutic targets is warranted. We consider aspects of the evolutionary and discovery history of ncRNAs, as far as they are relevant for the identification and selection of ncRNAs with likely therapeutic potential. Novel therapeutic strategies are based on ncRNAs, and we discuss here RNA interference as a highly versatile tool for gene silencing. RNA interference-mediating RNAs are small, but only parts of a far larger spectrum encompassing ncRNAs up to many kilobasepairs in size. We discuss therapeutic options in cardiovascular medicine offered by ncRNAs and key issues to be solved before clinical translation. Convergence of multiple technical advances is highlighted as a prerequisite for the translational progress achieved in recent years. Regarding safety, we review properties of RNA therapeutics, which may immunologically distinguish them from their endogenous counterparts, all of which underwent sophisticated evolutionary adaptation to specific biological contexts. Although our understanding of the noncoding human genome is only fragmentary to date, it is already feasible to develop RNA interference against a rapidly broadening spectrum of therapeutic targets and to translate this to the clinical setting under certain restrictions.

  5. Inhibition of Monkeypox virus replication by RNA interference

    Directory of Open Access Journals (Sweden)

    Jahrling Peter B

    2009-11-01

    Full Text Available Abstract The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV, Vaccinia (VACV, monkeypox (MPV and Variola (VARV viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA. Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R or an important gene in viral entry (E8L, inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses.

  6. Who Watches the Watchmen: Roles of RNA Modifications in the RNA Interference Pathway.

    Science.gov (United States)

    Shelton, Samantha B; Reinsborough, Calder; Xhemalce, Blerta

    2016-07-01

    RNA levels are widely thought to be predictive of RNA function. However, the existence of more than a hundred chemically distinct modifications of RNA alone is a major indication that these moieties may impart distinct functions to subgroups of RNA molecules that share a primary sequence but display distinct RNA "epigenetic" marks. RNAs can be modified on many sites, including 5' and 3' ends, the sugar phosphate backbone, or internal bases, which collectively provide many opportunities for posttranscriptional regulation through a variety of mechanisms. Here, we will focus on how modifications on messenger and microRNAs may affect the process of RNA interference in mammalian cells. We believe that taking RNA modifications into account will not only advance our understanding of this crucial pathway in disease and cancer but will also open the path to exploiting the enzymes that "write" and "erase" them as targets for therapeutic drug development.

  7. Specific RNA Interference in Caenorhabditis elegans by Ingested dsRNA Expressed in Bacillus subtilis

    NARCIS (Netherlands)

    Lezzerini, M.; van de Ven, K.; Veerman, M.; Brul, S.; Budovskaya, Y.V.

    2015-01-01

    In nematodes, genome-wide RNAi-screening has been widely used as a rapid and efficient method to identify genes involved in the aging processes. By far the easiest way of inducing RNA interference (RNAi) in Caenorhabditis elegans is by feeding Escherichia coli that expresses specific double stranded

  8. Biological mechanisms determining the success of RNA interference in insects.

    Science.gov (United States)

    Wynant, Niels; Santos, Dulce; Vanden Broeck, Jozef

    2014-01-01

    Insects constitute the largest group of animals on this planet, having a huge impact on our environment, as well as on our quality of life. RNA interference (RNAi) is a posttranscriptional gene silencing mechanism triggered by double-stranded (ds)RNA fragments. This process not only forms the basis of a widely used reverse genetics research method in many different eukaryotes but also holds great promise to contribute to the species-specific control of agricultural pests and to combat viral infections in beneficial and disease vectoring insects. However, in many economically important insect species, such as flies, mosquitoes, and caterpillars, systemic delivery of naked dsRNA does not trigger effective gene silencing. Although many components of the RNAi pathway have initially been deciphered in the fruit fly, Drosophila melanogaster, it will be of major importance to investigate this process in a wider variety of species, including dsRNA-sensitive insects such as locusts and beetles, to elucidate the factors responsible for the remarkable variability in RNAi efficiency, as observed in different insects. In this chapter, we review the current knowledge on the RNAi pathway, as well as the most recent insights into the mechanisms that might determine successful RNAi in insects. © 2014 Elsevier Inc. All rights reserved.

  9. RNA interference by feeding in vitro synthesized double-stranded RNA to planarians: methodology and dynamics

    Science.gov (United States)

    Rouhana, Labib; Weiss, Jennifer A.; Forsthoefel, David J.; Lee, Hayoung; King, Ryan S.; Inoue, Takeshi; Shibata, Norito; Agata, Kiyokazu; Newmark, Phillip A.

    2013-01-01

    Background The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced via injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. Results We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time, and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. Conclusions This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. PMID:23441014

  10. RNA interference tools for the western flower thrips, Frankliniella occidentalis.

    Science.gov (United States)

    Badillo-Vargas, Ismael E; Rotenberg, Dorith; Schneweis, Brandi A; Whitfield, Anna E

    2015-05-01

    The insect order Thysanoptera is exclusively comprised of small insects commonly known as thrips. The western flower thrips, Frankliniella occidentalis, is an economically important pest amongst thysanopterans due to extensive feeding damage and tospovirus transmission to hundreds of plant species worldwide. Geographically-distinct populations of F. occidentalis have developed resistance against many types of traditional chemical insecticides, and as such, management of thrips and tospoviruses are a persistent challenge in agriculture. Molecular methods for defining the role(s) of specific genes in thrips-tospovirus interactions and for assessing their potential as gene targets in thrips management strategies is currently lacking. The goal of this work was to develop an RNA interference (RNAi) tool that enables functional genomic assays and to evaluate RNAi for its potential as a biologically-based approach for controlling F. occidentalis. Using a microinjection system, we delivered double-stranded RNA (dsRNA) directly to the hemocoel of female thrips to target the vacuolar ATP synthase subunit B (V-ATPase-B) gene of F. occidentalis. Gene expression analysis using real-time quantitative reverse transcriptase-PCR (qRT-PCR) revealed significant reductions of V-ATPase-B transcripts at 2 and 3 days post-injection (dpi) with dsRNA of V-ATPase-B compared to injection with dsRNA of GFP. Furthermore, the effect of knockdown of the V-ATPase-B gene in females at these two time points was mirrored by the decreased abundance of V-ATPase-B protein as determined by quantitative analysis of Western blots. Reduction in V-ATPase-B expression in thrips resulted in increased female mortality and reduced fertility, i.e., number of viable offspring produced. Survivorship decreased significantly by six dpi compared to the dsRNA-GFP control group, which continued decreasing significantly until the end of the bioassay. Surviving female thrips injected with dsRNA-V-ATPase-B produced

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

    Science.gov (United States)

    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.

  12. Role of RNA interference (RNAi) in the moss Physcomitrella patens

    KAUST Repository

    Arif, Muhammad Asif

    2013-01-14

    RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species. 2013 by the authors; licensee MDPI, Basel, Switzerland.

  13. Advances with RNA interference in Alzheimer’s disease research

    Directory of Open Access Journals (Sweden)

    Chen S

    2013-02-01

    Full Text Available Shun Chen,1,2,* Xuemei Ge,2,* Yinghui Chen,3,* Nan Lv,2 Zhenguo Liu,1 Weien Yuan2 1Department of Neurology, Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine, 2School of Pharmacy, Shanghai Jiaotong University, Shanghai, 3Department of Neurology, Jinshan Hospital, Fudan University, Shanghai, People’s Republic of China *These authors contributed equally to this workAbstract: Alzheimer’s disease (AD is a progressive neurodegenerative disorder characterized clinically by memory and cognitive dysfunction. Unfortunately, there is no effective therapeutic method for AD treatment or ways to halt disease progression. Many mechanisms are involved in the disease, including genes mutation and protein dysfunction. RNA interference (RNAi technology may potentially be able to control AD. It can inhibit the protein expression of specific genes by activating a sequence-specific RNA degradation process. This is a powerful tool with which to study gene function, investigate the mechanism of the disease, and validate drug targets. In this review, we highlight the advances in RNAi technology in the investigation and treatment of AD.Keywords: RNAi, β-amyloid, tau, amyloid precursor protein

  14. Assessing the efficiency of RNA interference for maize functional genomics.

    Science.gov (United States)

    McGinnis, Karen; Murphy, Nick; Carlson, Alvar R; Akula, Anisha; Akula, Chakradhar; Basinger, Heather; Carlson, Michelle; Hermanson, Peter; Kovacevic, Nives; McGill, M Annie; Seshadri, Vishwas; Yoyokie, Jessica; Cone, Karen; Kaeppler, Heidi F; Kaeppler, Shawn M; Springer, Nathan M

    2007-04-01

    A large-scale functional genomics project was initiated to study the function of chromatin-related genes in maize (Zea mays). Transgenic lines containing short gene segments in inverted repeat orientation designed to reduce expression of target genes by RNA interference (RNAi) were isolated, propagated, and analyzed in a variety of assays. Analysis of the selectable marker expression over multiple generations revealed that most transgenes were transmitted faithfully, whereas some displayed reduced transmission or transgene silencing. A range of target-gene silencing efficiencies, from nondetectable silencing to nearly complete silencing, was revealed by semiquantitative reverse transcription-PCR analysis of transcript abundance for the target gene. In some cases, the RNAi construct was able to cause a reduction in the steady-state RNA levels of not only the target gene, but also another closely related gene. Correlation of silencing efficiency with expression level of the target gene and sequence features of the inverted repeat did not reveal any factors capable of predicting the silencing success of a particular RNAi-inducing construct. The frequencies of success of this large-scale project in maize, together with parameters for optimization at various steps, should serve as a useful framework for designing future RNAi-based functional genomics projects in crop plants.

  15. Endogenous RNA interference is driven by copy number

    Science.gov (United States)

    Cruz, Cristina; Houseley, Jonathan

    2014-01-01

    A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome. DOI: http://dx.doi.org/10.7554/eLife.01581.001 PMID:24520161

  16. Validation of RNA interferences as technique to study CNS anti-obesity drug targets

    NARCIS (Netherlands)

    Gestel, M.A. van

    2015-01-01

    RNA interference offers the opportunity to unravel the roles of genes in a specific area of the brain. Genes implicated in energy balance are perfect targets for the application of RNA interference as their phenotypes can be easily monitored and the neural circuits involved in energy homeostasis are

  17. Compressed sensing methods for DNA microarrays, RNA interference, and metagenomics.

    Science.gov (United States)

    Rao, Aditya; P, Deepthi; Renumadhavi, C H; Chandra, M Girish; Srinivasan, Rajgopal

    2015-02-01

    Compressed sensing (CS) is a sparse signal sampling methodology for efficiently acquiring and reconstructing a signal from relatively few measurements. Recent work shows that CS is well-suited to be applied to problems in genomics, including probe design in microarrays, RNA interference (RNAi), and taxonomic assignment in metagenomics. The principle of using different CS recovery methods in these applications has thus been established, but a comprehensive study of using a wide range of CS methods has not been done. For each of these applications, we apply three hitherto unused CS methods, namely, l1-magic, CoSaMP, and l1-homotopy, in conjunction with CS measurement matrices such as randomly generated CS m matrix, Hamming matrix, and projective geometry-based matrix. We find that, in RNAi, the l1-magic (the standard package for l1 minimization) and l1-homotopy methods show significant reduction in reconstruction error compared to the baseline. In metagenomics, we find that l1-homotopy as well as CoSaMP estimate concentration with significantly reduced time when compared to the GPSR and WGSQuikr methods.

  18. Unexpected complexity in the interference activity of a cloned influenza defective interfering RNA.

    Science.gov (United States)

    Meng, Bo; Bentley, Kirsten; Marriott, Anthony C; Scott, Paul D; Dimmock, Nigel J; Easton, Andrew J

    2017-07-24

    Defective interfering (DI) viruses are natural antivirals made by nearly all viruses. They have a highly deleted genome (thus being non-infectious) and interfere with the replication of genetically related infectious viruses. We have produced the first potential therapeutic DI virus for the clinic by cloning an influenza A DI RNA (1/244) which was derived naturally from genome segment 1. This is highly effective in vivo, and has unexpectedly broad-spectrum activity with two different modes of action: inhibiting influenza A viruses through RNA interference, and all other (interferon-sensitive) respiratory viruses through stimulating interferon type I. We have investigated the RNA inhibitory mechanism(s) of DI 1/244 RNA. Ablation of initiation codons does not diminish interference showing that no protein product is required for protection. Further analysis indicated that 1/244 DI RNA interferes by replacing the cognate full-length segment 1 RNA in progeny virions, while interfering with the expression of genome segment 1, its cognate RNA, and genome RNAs 2 and 3, but not genome RNA 6, a representative of the non-polymerase genes. Our data contradict the dogma that a DI RNA only interferes with expression from its cognate full-length segment. There is reciprocity as cloned segment 2 and 3 DI RNAs inhibited expression of RNAs from a segment 1 target. These data demonstrate an unexpected complexity in the mechanism of interference by this cloned therapeutic DI RNA.

  19. RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?

    Directory of Open Access Journals (Sweden)

    Unnikrishnan Unniyampurath

    2016-02-01

    Full Text Available 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.

  20. RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?

    Science.gov (United States)

    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

  1. An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference.

    Science.gov (United States)

    Peng, Wenfang; Feng, Mingxia; Feng, Xu; Liang, Yun Xiang; She, Qunxin

    2015-01-01

    CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-α and Cmr-β) in Sulfolobus islandicus, a genetic assay was developed using plasmids carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis identified a trinucleotide sequence in the 3'-region of crRNA that was crucial for RNA interference. Studying mutants lacking Cmr-α or Cmr-β system showed that each Cmr complex exhibited RNA interference. Strikingly, these analyses further revealed that the two Cmr systems displayed distinctive interference features. Whereas Cmr-β complexes targeted transcripts and could be recycled in RNA cleavage, Cmr-α complexes probably targeted nascent RNA transcripts and remained associated with the substrate. Moreover, Cmr-β exhibited much stronger RNA cleavage activity than Cmr-α. Since we previously showed that S. islandicus Cmr-α mediated transcription-dependent DNA interference, the Cmr-α constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Silencing Huntington's chorea: Is RNA Interference a Potential Cure?

    Directory of Open Access Journals (Sweden)

    Gerlinde A. Metz

    2006-01-01

    Full Text Available In 1872, George Huntington described Huntington's disease as characterized by motor, cognitive and psychiatric impairments. Huntington's disease is a dominant and autosomal mutation on chromosome 4 featuring the insertion of numerous CAG repeats. CAG codes for the amino acid, glutmanine that forms part of the Huntingtin protein (htt. Excess glutamine attachments make htt prone to accumulate in neurons. Three genes can be considered when developing therapies for Huntington's disease. They include targeting the symptoms of the disease, the progression of the disease and the cause of the disease. By using RNA interference (RNAi, the cause of the disease can be targeted. RNAi is a method that could potentially silence the formation of abnormal htt. This paper will discuss how RNAi could potentially cure Huntington's disease, by describing the genetic and proteinomic basis of Huntington's disease, the function of RNAi in Huntington's disease and the problems of benefits of RNAi. Preliminary work using RNAi in transgenic mice has shown a decrease in the behavioural expression of the mutant Huntington gene. There are several limitations associated with using RNAi as a gene therapy. For example, the effects of RNAi are short lived. A transposition system such as Sleeping Beauty can be used to increase the integration of the gene, however, for patients who currently have Huntington's disease, RNAi may potentially be used in combination with drugs or other treatments to target both symptoms and the underlying cause of Huntington's disease. This combination could eventually alleviate many painful symptoms associated with Huntington's disease and could even stop the progressive neurodegeneration of Huntington's disease. This review concludes that a substantial amount of new research is still necessary before RNAi is directly applicable to human patients with Huntington's disease.

  3. Immunoregulation by interference RNA (iRNA – mechanisms, role, perspective

    Directory of Open Access Journals (Sweden)

    Emilia Sikora

    2011-08-01

    Full Text Available The functioning of an organism depends on the precise control mechanisms, constantly adjusted to the actual state. Therefore, there is a need for efficient communication between both adjacent and distant cells, which may be executed by proteins such as hormones, neurotransmitters and cytokines. Recently another means of regulation has emerged – short regulatory RNAs (srRNAs. Although discovered only a couple of years ago, the mechanism of RNA interference has already become a topic of thousands of publications, defining its roles in both physiological and pathological processes, such as cancerogenesis and autoimmunization.RNAs regulating cell function may be coded in its genome (both exons and introns or be introduced from the external environment. In mammals microRNAs (miRNAs cooperate with proteins from the Ago/PIWI family to form effector ribonucleoprotein complexes, and owing to their complementarity to the target mRNA, control genes’ expression at the posttranscriptional level, either through the suppression of mRNA translation or through mRNA degradation.SrRNAs are crucial regulators throughout the development of immune cells, starting from hematopoietic stem cells, up to the effector cells of the adaptive immune response. Moreover, some of the regulatory cells perform their function by releasing miRNAs, which are then transported to the target cells, possibly enclosed in the exosomes.

  4. Agroinfiltration-based expression of hairpin RNA in soybean plants for RNA interference against Tetranychus urticae.

    Science.gov (United States)

    Dubey, Vimal Kumar; Lee, Ung Gyu; Kwon, Deok Ho; Lee, Si Hyeock

    2017-10-01

    The coatomer subunit alpha (COPA) and aquaporin 9 (AQ9) genes from the two-spotted spider mite, Tetranychus urticae, were previously determined to exhibit RNA interference (RNAi)-based lethality when their double-stranded RNAs were systemically delivered via multi-unit chambers (Kwon et al., 2016 [8]). In current study, the hairpin RNAs of the COPA and AQ9 were transiently expressed in soybean plants by agroinfiltration. When T. urticae was fed with the soybean plants agroinfiltrated with the COPA and AQ9 hairpin RNA cassettes, the cumulative mortality increased significantly at 6days post-infestation. Quantitative PCR analysis revealed that the transcript level of both COPA and AQ9 was significantly reduced in T. urticae after 2days post-infestation, thereby confirming that the significant increases in mortality resulted from the knockdown of COPA and AQ9 transcripts. Our findings demonstrate the utility of COPA and AQ9 as potential genes for plant host-mediated RNAi control of T. urticae. In addition, we proved the usefulness of agroinfiltration as a rapid validation tool for confirming the RNAi-based lethality of target genes against arthropod pests before producing transgenic plants as agroinfiltration requires less time and skill to validate transgene function. Furthermore, these findings prove the concept that hairpin RNA expressed in plant hosts can also induce RNAi and eventually kill T. urticae, a sap-sucking pest. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Double strand RNA-mediated RNA interference through feeding in larval gypsy moth, Lymantria dispar (Lepidoptera: Erebidae)

    Science.gov (United States)

    RNA interference (RNAi) has gained popularity in several fields of research, silencing targeted genes by degradation of RNA. The objective of this study was to develop RNAi for use as a molecular tool in the control of the invasive pest Lymantria dispar (Lepidoptera: Erebidae), gypsy moth, which ha...

  6. Establishment and characterization of Prnp knockdown neuroblastoma cells using dual microRNA-mediated RNA interference

    Science.gov (United States)

    Lau, Agnes; Westaway, David; McKenzie, Debbie; Aiken, Judd; Kim, Yong-Sun

    2011-01-01

    Prion diseases are fatal transmissible neurodegenerative disorders. In the pathogenesis of the disease, the cellular prion protein (PrPC) is required for replication of abnormal prion (PrPSc), which results in accumulation of PrPSc. Although there have been extensive studies using Prnp knockout systems, the normal function of PrPC remains ambiguous. Compared with conventional germline knockout technologies and transient naked siRNA-dependent knockdown systems, newly constructed durable chained-miRNA could provide a cell culture model that is closer to the disease status and easier to achieve with no detrimental sequelae. The selective silencing of a target gene by RNA interference (RNAi) is a powerful approach to investigate the unknown function of genes in vitro and in vivo. To reduce PrPC expression, a novel dual targeting-microRNA (miRdual) was constructed. The miRdual, which targets N- and C-termini of Prnp simultaneously, more effectively suppressed PrPC expression compared with conventional single site targeting. Furthermore, to investigate the cellular change following PrPC depletion, gene expression analysis of PrPC interacting and/or associating genes and several assays including proliferation, viability and apoptosis were performed. The transcripts 670460F02Rik and Plk3, Ppp2r2b and Csnk2a1 increase in abundance and are reported to be involved in cell proliferation and mitochondrial-mediated apoptosis. Dual-targeting RNAi with miRdual against Prnp will be useful for analyzing the physiological function of PrPC in neuronal cell lines and may provide a potential therapeutic intervention for prion diseases in the future. PMID:21494092

  7. RNA interference machinery-mediated gene regulation in mouse adult neural stem cells.

    Science.gov (United States)

    Cernilogar, Filippo M; Di Giaimo, Rossella; Rehfeld, Frederick; Cappello, Silvia; Lie, D Chichung

    2015-09-19

    Neurogenesis in the brain of adult mammals occurs throughout life in two locations: the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus in the hippocampus. RNA interference mechanisms have emerged as critical regulators of neuronal differentiation. However, to date, little is known about its function in adult neurogenesis. Here we show that the RNA interference machinery regulates Doublecortin levels and is associated with chromatin in differentiating adult neural progenitors. Deletion of Dicer causes abnormal higher levels of Doublecortin. The microRNA pathway plays an important role in Doublecortin regulation. In particular miRNA-128 overexpression can reduce Doublecortin levels in differentiating adult neural progenitors. We conclude that the RNA interference components play an important role, even through chromatin association, in regulating neuron-specific gene expression programs.

  8. RNA interference technology with emphasis on delivery vehicles-prospects and limitations.

    Science.gov (United States)

    Prabha, Shashi; Vyas, Ruchi; Gupta, Nidhi; Ahmed, Bahar; Chandra, Ramesh; Nimesh, Surendra

    2016-09-01

    RNA interference (RNAi)-based therapeutics rely upon safe and efficient delivery of small interfering RNA (siRNA) molecules. This review explores various dimensions of RNAi with emphasis on the development of nanoparticle-based delivery vectors for safe and efficient siRNA delivery. An exhaustive database search has been done regarding studies done to investigate the potential of siRNA delivery employing nanoparticles has been cited in the present review. With the current challenges, there is a need for collaborative work allowing for the successful development of nanoparticle/siRNA complexes as health-promoting biotherapeutics.

  9. Characterisation of the RNA interference response against the long-wavelength receptor of the honeybee.

    Science.gov (United States)

    Leboulle, Gérard; Niggebrügge, Claudia; Roessler, Reinhard; Briscoe, Adriana D; Menzel, Randolf; Hempel de Ibarra, Natalie

    2013-10-01

    Targeted knock-down is the method of choice to advance the study of sensory and brain functions in the honeybee by using molecular techniques. Here we report the results of a first attempt to interfere with the function of a visual receptor, the long-wavelength-sensitive (L-) photoreceptor. RNA interference to inhibit this receptor led to a reduction of the respective mRNA and protein. The interference effect was limited in time and space, and its induction depended on the time of the day most probably because of natural daily variations in opsin levels. The inhibition did not effectively change the physiological properties of the retina. Possible constraints and implications of this method for the study of the bee's visual system are discussed. Overall this study underpins the usefulness and feasibility of RNA interference as manipulation tool in insect brain research. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. RNA virus interference via CRISPR/Cas13a system in plants.

    Science.gov (United States)

    Aman, Rashid; Ali, Zahir; Butt, Haroon; Mahas, Ahmed; Aljedaani, Fatimah; Khan, Muhammad Zuhaib; Ding, Shouwei; Mahfouz, Magdy

    2018-01-04

    CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

  11. Pharmacogenomics-based RNA interference nanodelivery: focus on solid malignant tumors.

    Science.gov (United States)

    Rosen, Yitzhak; Upadhyay, Urvashi M; Elman, Noel M

    2012-07-01

    RNA interference represents one of the most promising strategies in fighting disease. However, small RNA interference faces substantial challenges for in vivo application due to the inherent instability of the RNA interference molecule. Among the nonviral gene delivery carriers, nanoparticles have attracted interest due to their success in various model systems. Nanomaterials have unique properties compared to conventional bulk materials that may be applicable in this setting. The nanoparticle complex carrying small interference RNA can undergo surface modification to achieve targeted modification for tissue-specific delivery. However, toxicity issues of the delivery systems need to be addressed and they require a pharmacogenomic profile of their own. The authors review pharmacogenomics, toxicogenomics, nanoparticle-based drug delivery, and small interference RNA, with a focus on how logically engineered nanoparticle delivery systems can be used for personalized medicine in malignant tumors. Pharmacogenomics may be helpful in addressing possible individualized drug response for both the gene silencing capability of the delivered siRNA and the nanoparticle drug delivery system as both complete and distinct units. This may be done by assessing variations in gene expressions and single nucleotide polymorphisms. Patient profiling may be key as patient noncompliance due to toxicity plays a major role in treatment failure.

  12. Reversal of pathology in CHMP2B-mediated frontotemporal dementia patient cells using RNA interference

    DEFF Research Database (Denmark)

    Nielsen, Troels Tolstrup; Mizielinska, Sarah; Hasholt, Lis

    2012-01-01

    role in the pathogenesis of the disease. METHODS: In the present study, we used lentiviral vectors to efficiently knockdown CHMP2B by delivering microRNA embedded small hairpin RNAs. RESULTS: We show that CHMP2B can be efficiently knocked down in patient fibroblasts using an RNA interference approach...... and that the knockdown causes reversal of the abnormal endosomal phenotype observed in patient fibroblasts. CONCLUSIONS: This is the first description of a treatment that reverses the cellular pathology caused by mutant CHMP2B and suggests that RNA interference might be a feasible therapeutic strategy. Furthermore...

  13. Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

    OpenAIRE

    Irma Sánchez-Vargas; Scott, Jaclyn C; B Katherine Poole-Smith; Alexander W E Franz; Valérie Barbosa-Solomieu; Jeffrey Wilusz; Olson, Ken E.; Blair, Carol D.

    2009-01-01

    A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), ...

  14. Versatile RNA interference nanoplatform for systemic delivery of RNAs.

    Science.gov (United States)

    Choi, Ki Young; Silvestre, Oscar F; Huang, Xinglu; Min, Kyung Hyun; Howard, Gregory P; Hida, Naoki; Jin, Albert J; Carvajal, Nicole; Lee, Sang Wook; Hong, Jong-In; Chen, Xiaoyuan

    2014-05-27

    Development of nontoxic, tumor-targetable, and potent in vivo RNA delivery systems remains an arduous challenge for clinical application of RNAi therapeutics. Herein, we report a versatile RNAi nanoplatform based on tumor-targeted and pH-responsive nanoformulas (NFs). The NF was engineered by combination of an artificial RNA receptor, Zn(II)-DPA, with a tumor-targetable and drug-loadable hyaluronic acid nanoparticle, which was further modified with a calcium phosphate (CaP) coating by in situ mineralization. The NF can encapsulate small-molecule drugs within its hydrophobic inner core and strongly secure various RNA molecules (siRNAs, miRNAs, and oligonucleotides) by utilizing Zn(II)-DPA and a robust CaP coating. We substantiated the versatility of the RNAi nanoplatform by demonstrating effective delivery of siRNA and miRNA for gene silencing or miRNA replacement into different human types of cancer cells in vitro and into tumor-bearing mice in vivo by intravenous administration. The therapeutic potential of NFs coloaded with an anticancer drug doxorubicin (Dox) and multidrug resistance 1 gene target siRNA (siMDR) was also demonstrated in this study. NFs loaded with Dox and siMDR could successfully sensitize drug-resistant OVCAR8/ADR cells to Dox and suppress OVCAR8/ADR tumor cell proliferation in vitro and tumor growth in vivo. This gene/drug delivery system appears to be a highly effective nonviral method to deliver chemo- and RNAi therapeutics into host cells.

  15. RNA virus interference via CRISPR/Cas13a system in plants

    KAUST Repository

    Aman, Rashid

    2017-11-04

    CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produced interference against green fluorescent protein (GFP) expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. crRNAs targeting the HC-Pro and GFP sequences exhibited better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses, and for other RNA manipulations in plants.

  16. RNA interference: A novel tool for plant disease management ...

    African Journals Online (AJOL)

    Transgenic plants expressing RNAi vectors, as well as, dsRNA containing crop sprays have been successful for efficient control of plant pathogens affecting economically important crop species. The present paper discusses strategies and applications of this novel technology in plant disease management for sustainable ...

  17. Antiviral immunity in Drosophila requires systemic RNA interference spread.

    NARCIS (Netherlands)

    Saleh, M.C.; Tassetto, M.; Rij, R.P. van; Goic, B.; Gausson, V.; Berry, B.; Jacquier, C.; Antoniewski, C.; Andino, R.

    2009-01-01

    Multicellular organisms evolved sophisticated defence systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations. In insects, such as Drosophila melanogaster, RNA

  18. Application of RNA interference for the control of female reproductive functions.

    Science.gov (United States)

    Sirotkin, Alexander V

    2012-01-01

    RNA interference, a recently discovered new mechanism controlling gene expression via small RNAs, was shown to be involved in the characterization and control of basic ovarian cell functions. The main classes of small RNAs, as well as their expression in ovaries have been described. Furthermore, the successful application of RNA interference for the study and control of basic ovarian functions (fertility, proliferation, apoptosis, secretory activity, luteogenesis, oocyte maturation and related ovarian cell malignant transformation) and production of recombinant proteins has been demonstrated. Application of RNA interference in reproductive biology and medicine can be successful in three main areas - (1) characterization and prediction of physiological and pathological state (association between particular small RNA and physiological or pathological processes), (2) application of small RNAs for regulation of reproductive processes and (3) treatment of reproductive disorders or their particular indexes. Problems of improvement of small RNA delivery to target ovarian cells and potent RNA interference-related approaches for the treatment of ovarian disorders (especially of ovarian cancer) have been discussed.

  19. Mutual Interference between Genomic RNA Replication and Subgenomic mRNA Transcription in Brome Mosaic Virus

    OpenAIRE

    Grdzelishvili, Valery Z.; Garcia-Ruiz, Hernan; Watanabe, Tokiko; Ahlquist, Paul

    2005-01-01

    Replication by many positive-strand RNA viruses includes genomic RNA amplification and subgenomic mRNA (sgRNA) transcription. For brome mosaic virus (BMV), both processes occur in virus-induced, membrane-associated compartments, require BMV replication factors 1a and 2a, and use negative-strand RNA3 as a template for genomic RNA3 and sgRNA syntheses. To begin elucidating their relations, we examined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expres...

  20. Inhibition of osteoclastogenesis by RNA interference targeting RANK

    Directory of Open Access Journals (Sweden)

    Ma Ruofan

    2012-08-01

    Full Text Available Abstract Background Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK ligand (RANKL as well as the macrophage colony-stimulating factor (M-CSF. The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption. Methods Three pairs of short hairpin RNAs (shRNA targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs using the optimal shRNA by targeting RANK. Results Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p Conclusions These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.

  1. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea

    Science.gov (United States)

    Marraffini, Luciano A.; Sontheimer, Erik J.

    2010-01-01

    Sequence-directed genetic interference pathways control gene expression and preserve genome integrity in all kingdoms of life. The importance of such pathways is highlighted by the extensive study of RNA interference (RNAi) and related processes in eukaryotes. In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids. CRISPR sequences provide an adaptive, heritable record of past infections and express CRISPR RNAs — small RNAs that target invasive nucleic acids. Here, we review the mechanisms of CRISPR interference and its roles in microbial physiology and evolution. We also discuss potential applications of this novel interference pathway. PMID:20125085

  2. Discovery of Prostate Cancer Tumor Suppressors and Mediators of MDV3100 Resistance Through in Vivo RNA Interference Screen

    Science.gov (United States)

    2015-11-01

    RNA Interference Screen PRINCIPAL INVESTIGATOR: Kamlesh K Yadav CONTRACTING ORGANIZATION: Sloan Kettering Institute for Cancer Research New...Suppressors and Mediators of MDV3100 Resistance through in Vivo RNA Interference Screen 5b. GRANT NUMBER W81XWH-13-1-0084 5c. PROGRAM ELEMENT NUMBER 6... rna interference screens, STARR consortium retreat, CSHL, NY 8 o Website(s) or other Internet site(s) o Technologies or techniques o

  3. An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference

    DEFF Research Database (Denmark)

    Peng, Wenfang; Feng, Mingxia; Feng, Xu

    2015-01-01

    CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-α and Cmr-β) in Sulfolobus islandicus, a genetic assay was developed using plasmids...... carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis....... islandicus Cmr-α mediated transcription-dependent DNA interference, the Cmr-α constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA....

  4. RNA Interference as a Therapeutic Strategy for the Treatment of Liver Diseases.

    Science.gov (United States)

    Gonzalez-Rodriguez, Agueda; Valverde, Angela M

    2015-01-01

    RNA interference has emerged as an innovative technology for gene silencing that degrades mRNAs complementary to the antisense strands of double-stranded, short interfering RNAs (siRNAs). Its therapeutic application has important advantages over small-molecule drugs since offers the possibility of targeting virtually all genes and allows selective silencing of one or several genes. So far, a relative small proportion of cellular proteins can bind and respond to chemical drugs. Based on that, RNA interference-mediated gene silencing is widely considered as a crucial breakthrough in molecular biology with a direct translation to medicine. The liver has been widely chosen as a model system for the development of RNA interference therapy due to the convenience and availability of effective delivery into this tissue. Numerous preclinical models have revealed promising results, but the safety of this technology remains the primary challenge in developing siRNA based treatments. Liver diseases comprise a broad spectrum of genetic and non-genetic pathologies including acute fulminant liver injury that demands urgent medical care, or chronic pathologies such as nonalcoholic fatty liver (NAFLD), alcoholic liver disease, liver cirrhosis, viral hepatitis and hepatocellular carcinoma (HCC). In some cases restoration of liver function is not possible and alternatives to liver transplantation offering novel and efficient therapeutic approaches are urgently needed. In this review, we describe recent insights on the advantages of using RNA interference in preclinical settings as a targeted strategy with potential to markedly improve the treatment of liver diseases.

  5. Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference.

    Science.gov (United States)

    Angart, Phillip A; Carlson, Rebecca J; Adu-Berchie, Kwasi; Walton, S Patrick

    2016-10-01

    Efficient short interfering RNA (siRNA)-mediated gene silencing requires selection of a sequence that is complementary to the intended target and possesses sequence and structural features that encourage favorable functional interactions with the RNA interference (RNAi) pathway proteins. In this study, we investigated how terminal sequence and structural characteristics of siRNAs contribute to siRNA strand loading and silencing activity and how these characteristics ultimately result in a functionally asymmetric duplex in cultured HeLa cells. Our results reiterate that the most important characteristic in determining siRNA activity is the 5' terminal nucleotide identity. Our findings further suggest that siRNA loading is controlled principally by the hybridization stability of the 5' terminus (Nucleotides: 1-2) of each siRNA strand, independent of the opposing terminus. Postloading, RNA-induced silencing complex (RISC)-specific activity was found to be improved by lower hybridization stability in the 5' terminus (Nucleotides: 3-4) of the loaded siRNA strand and greater hybridization stability toward the 3' terminus (Nucleotides: 17-18). Concomitantly, specific recognition of the 5' terminal nucleotide sequence by human Argonaute 2 (Ago2) improves RISC half-life. These findings indicate that careful selection of siRNA sequences can maximize both the loading and the specific activity of the intended guide strand.

  6. Sequence-non-specific effects generated by various types of RNA interference triggers.

    Science.gov (United States)

    Olejniczak, Marta; Urbanek, Martyna O; Jaworska, Edyta; Witucki, Lukasz; Szczesniak, Michal W; Makalowska, Izabela; Krzyzosiak, Wlodzimierz J

    2016-02-01

    RNA interference triggers such as short interfering RNA (siRNA) or genetically encoded short hairpin RNA (shRNA) and artificial miRNA (sh-miR) are widely used to silence the expression of specific genes. In addition to silencing selected targets, RNAi reagents may induce various side effects, including immune responses. To determine the molecular markers of immune response activation when using RNAi reagents, we analyzed the results of experiments gathered in the RNAimmuno (v 2.0) and GEO Profiles databases. To better characterize and compare cellular responses to various RNAi reagents in one experimental system, we designed a reagent series in corresponding siRNA, D-siRNA, shRNA and sh-miR forms. To exclude sequence-specific effects the reagents targeted 3 different transcripts (Luc, ATXN3 and HTT). We demonstrate that RNAi reagents induce a broad variety of sequence-non-specific effects, including the deregulation of cellular miRNA levels. Typical siRNAs are weak stimulators of interferon response but may saturate the miRNA biogenesis pathway, leading to the downregulation of highly expressed miRNAs, whereas plasmid-based reagents induce known markers of immune response and may alter miRNA levels and their isomiR composition. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Application of Carbon Nanomaterials in Gene Delivery for Endogenous RNA Interference In Vitro and In Vivo.

    Science.gov (United States)

    Li, Lanying; Wen, Yanli; Xu, Qin; Xu, Li; Liu, Dong; Liu, Gang; Huang, Qing

    2015-01-01

    Knocking down expression by small interfering RNA (siRNA) has shown high affinity, specificity and potency in silencing target gene sites. For effective endogenous RNA interference (RNAi), proper siRNA delivery vehicles are essential, to take the siRNA inside cells and protect them during the circulation. Carbon nanomaterials (CNMs) have been successfully applied in biomedicine and biosensor based on their ultra-high surface functionalization and nucleic acid molecular loading capacity. Recently, CNMs have drawn considerable research interest and expectation as potential non-viral vectors for siRNA delivery. Here we reviewed the recent application of CNMs in gene delivery for RNAi, mainly about fullerenes, carbon nanotubes (CNTs) and graphene.

  8. Exosomes: Nanoparticulate tools for RNA interference and drug delivery.

    Science.gov (United States)

    Shahabipour, Fahimeh; Barati, Nastaran; Johnston, Thomas P; Derosa, Giuseppe; Maffioli, Pamela; Sahebkar, Amirhossein

    2017-07-01

    Exosomes are naturally occurring extracellular vesicles released by most mammalian cells in all body fluids. Exosomes are known as key mediators in cell-cell communication and facilitate the transfer of genetic and biochemical information between distant cells. Structurally, exosomes are composed of lipids, proteins, and also several types of RNAs which enable these vesicles to serve as important disease biomarkers. Moreover, exosomes have emerged as novel drug and gene delivery tools owing to their multiple advantages over conventional delivery systems. Recently, increasing attention has been focused on exosomes for the delivery of drugs, including therapeutic recombinant proteins, to various target tissues. Exosomes are also promising vehicles for the delivery of microRNAs and small interfering RNAs, which is usually hampered by rapid degradation of these RNAs, as well as inefficient tissue specificity of currently available delivery strategies. This review highlights the most recent accomplishments and trends in the use of exosomes for the delivery of drugs and therapeutic RNA molecules. © 2017 Wiley Periodicals, Inc.

  9. A Simple Laboratory Practical to Illustrate RNA Mediated Gene Interference Using Drosophila Cell Culture

    Science.gov (United States)

    Buluwela, Laki; Kamalati, Tahereh; Photiou, Andy; Heathcote, Dean A.; Jones, Michael D.; Ali, Simak

    2010-01-01

    RNA mediated gene interference (RNAi) is now a key tool in eukaryotic cell and molecular biology research. This article describes a five session laboratory practical, spread over a seven day period, to introduce and illustrate the technique. During the exercise, students working in small groups purify PCR products that encode "in vitro"…

  10. Gene Therapy Strategies to Block HIV-1 Replication by RNA Interference

    NARCIS (Netherlands)

    Herrera-Carrillo, Elena; Berkhout, Ben

    2015-01-01

    The cellular mechanism of RNA interference (RNAi) plays an antiviral role in many organisms and can be used for the development of therapeutic strategies against viral pathogens. Persistent infections like the one caused by the human immunodeficiency virus type 1 (HIV-1) likely require a durable

  11. Toward a durable treatment of HIV-1 infection using RNA interference

    NARCIS (Netherlands)

    Eekels, Julia J. M.; Berkhout, Ben

    2011-01-01

    RNA interference (RNAi) is a cellular mechanism that mediates sequence-specific gene silencing at the posttranscriptional level. RNAi can be used as an antiviral approach against human pathogens. An attractive target for RNAi therapeutics is the human immunodeficiency virus type 1 (HIV-1), and the

  12. How Golden Is Silence? Teaching Undergraduates the Power and Limits of RNA Interference

    Science.gov (United States)

    Kuldell, Natalie H.

    2006-01-01

    It is hard and getting harder to strike a satisfying balance in teaching. Time dedicated to student-generated models or ideas is often sacrificed in an effort to "get through the syllabus." I describe a series of RNA interference (RNAi) experiments for undergraduate students that simultaneously explores fundamental concepts in gene regulation,…

  13. The interplay between virus infection and the cellular RNA interference machinery

    NARCIS (Netherlands)

    Berkhout, Ben; Haasnoot, Joost

    2006-01-01

    RNA interference (RNAi) plays a pivotal role in the regulation of gene expression to control cell development and differentiation. In plants, insects and nematodes RNAi also functions as an innate defence response against viruses. Similarly, there is accumulating evidence that RNAi functions as an

  14. Toward a Durable Anti-HIV Gene Therapy Based on RNA Interference

    NARCIS (Netherlands)

    Berkhout, Ben

    2009-01-01

    Basic research in the field of molecular biology led to the discovery of the mechanism of RNA interference (RNAi) in Caenorhabditis elegans in 1998. RNAi is now widely appreciated as an important gene control mechanism in mammals, and several RNAi-based gene-silencing applications have already been

  15. RNA interference for functional genomics and improvement of cotton (Gossypium species)

    Science.gov (United States)

    RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium ssp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function ...

  16. Effects of RNA interference targeting Smad7 on nerve cells ischemic ...

    African Journals Online (AJOL)

    Effects of RNA interference targeting Smad7 on nerve cells ischemic injury induced in PC12 cells. C Mei, J Mang, J He, G Xu, Z Li, W Liang, Z Xu. Abstract. Ischemic cerebrovascular disease is a global health problem. According to the World Health Organization, ischemic stroke is actually the most common cause of death in ...

  17. RNA interference for functional genomics and improvement of cotton (Gossypium sp.)

    NARCIS (Netherlands)

    Abdurakhmonov, Ibrokhim Y.; Ayubov, Mirzakamol S.; Ubaydullaeva, Khurshida A.; Buriev, Zabardast T.; Shermatov, Shukhrat E.; Ruziboev, Haydarali S.; Shapulatov, Umidjon; Saha, Sukumar; Ulloa, Mauricio; Yu, John Z.; Percy, Richard G.; Devor, Eric J.; Sharma, Govind C.; Sripathi, Venkateswara R.; Kumpatla, Siva P.; Krol, van der Sander; Kater, Hake D.; Khamidov, Khakimdjan; Salikhov, Shavkat I.; Jenkins, Johnie N.; Abdukarimov, Abdusattor; Pepper, Alan E.

    2016-01-01

    RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of

  18. Functional specialization among insect chitinase family genes revealed by RNA interference

    Science.gov (United States)

    The biological functions of individual members of the large family of chitinase-like proteins from the red flour beetle, Tribolium castaneum, were examined using gene-specific RNA interference (RNAi). One chitinase, TcCHT5, was found to be required for pupal-adult molting only. A lethal phenotype ...

  19. Production of high-amylose maize lines using RNA interference in ...

    African Journals Online (AJOL)

    To regulate the biosynthetic process of maize starch and produce high-amylose transgenic maize, RNA interference was used to inhibit the starch branching enzyme gene Sbe2. A construct with a 562 bp segment of Sbe2 (pRSBE2a) was cloned as inverted repeats. Highly efficient RNAi vector pRSBE2a was transferred to ...

  20. Effects of RNA interference targeting Smad7 on nerve cells ischemic ...

    African Journals Online (AJOL)

    DR. NJ TONUKARI

    2012-01-10

    Jan 10, 2012 ... Effects of RNA interference targeting Smad7 on nerve cells ischemic injury induced in PC12 cells. Chunli Mei1,2, Jing Mang1, Jinting He1, Guihua Xu1, Zhongshu Li1, Wenzhao Liang1 and. Zhongxin Xu1*. 1Department of Neurology, China-Japan Union Hospital, Jilin University, Changchun 130012, ...

  1. Accumulation of dsRNA in endosomes contributes to inefficient RNA interference in the fall armyworm, Spodoptera frugiperda.

    Science.gov (United States)

    Yoon, June-Sun; Gurusamy, Dhandapani; Palli, Subba Reddy

    2017-11-01

    RNA interference (RNAi) efficiency varies among insects studied. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. We recently showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into small interference RNAs (siRNAs) because they are trapped in acidic bodies. In the current study, we focused on the identification of acidic bodies in which dsRNAs accumulate in Sf9 cells. Time-lapse imaging studies showed that dsRNAs enter Sf9 cells and accumulate in acidic bodies within 20 min after their addition to the medium. CypHer-5E-labeled dsRNA also accumulated in the midgut and fat body dissected from Spodoptera frugiperda larvae with similar patterns observed in Sf9 cells. Pharmacological inhibitor assays showed that the dsRNAs use clathrin mediated endocytosis pathway for transport into the cells. We investigated the potential dsRNA accumulation sites employing LysoTracker and double labeling experiments using the constructs to express a fusion of green fluorescence protein with early or late endosomal marker proteins and CypHer-5E-labeled dsRNA. Interestingly, CypHer-5E-labeled dsRNA accumulated predominantly in early and late endosomes. These data suggest that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi response in lepidopteran insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1.

    Science.gov (United States)

    Li, Weiqiang; Koutmou, Kristin S; Leahy, Daniel J; Li, Min

    2015-07-31

    During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Functional Analysis of the Isopentenyl Diphosphate Isomerase of Salvia miltiorrhiza via Color Complementation and RNA Interference.

    Science.gov (United States)

    Zhang, Xianan; Guan, Hongyu; Dai, Zhubo; Guo, Juan; Shen, Ye; Cui, Guanghong; Gao, Wei; Huang, Luqi

    2015-11-10

    Isopentenyl diphosphate isomerase (IPI) catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1) was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.

  4. Flavivirus sfRNA suppresses antiviral RNA interference in cultured cells and mosquitoes and directly interacts with the RNAi machinery.

    Science.gov (United States)

    Moon, Stephanie L; Dodd, Benjamin J T; Brackney, Doug E; Wilusz, Carol J; Ebel, Gregory D; Wilusz, Jeffrey

    2015-11-01

    Productive arbovirus infections require mechanisms to suppress or circumvent the cellular RNA interference (RNAi) pathway, a major antiviral response in mosquitoes. In this study, we demonstrate that two flaviviruses, Dengue virus and Kunjin virus, significantly repress siRNA-mediated RNAi in infected human cells as well as during infection of the mosquito vector Culex quinquefasciatus. Arthropod-borne flaviviruses generate a small structured non-coding RNA from the viral 3' UTR referred to as sfRNA. Analysis of infections with a mutant Kunjin virus that is unable to generate appreciable amounts of the major sfRNA species indicated that RNAi suppression was associated with the generation of the non-coding sfRNA. Co-immunoprecipitation of sfRNA with RNAi mediators Dicer and Ago2 suggest a model for RNAi suppression. Collectively, these data help to establish a clear role for sfRNA in RNAi suppression and adds to the emerging impact of viral long non-coding RNAs in modulating aspects of anti-viral immune processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. A simple and efficient method to transfect small interference RNA into bovine SCNT embryos.

    Science.gov (United States)

    Zhang, Hui; Wang, LiJun; Li, WenZhe; Mao, QingFu; Wang, YongSheng; Li, Qian; Hua, Song; Zhang, Yong

    2015-10-01

    RNA interference is an important tool to study the gene function. Microinjection and electroporation are usually used to transfer DNA, small interference RNA (siRNA), morpholinos, and protein into oocytes or embryos. This study used a simple and effective method to transfect siRNA into bovine somatic cell nuclear transfer (SCNT) embryos. In this method, siRNA transfection and electrofusion of SCNT were combined. A pair of platinum microelectrodes was used during SCNT to complete electrofusion. A CY3-labeled siRNA-targeted DNA methyltransferase-1 (DNMT1) was chosen to verify the siRNA transfection efficiency of this approach. First, a suitable concentration of siRNA was mixed with Zimmermann's fusion medium. Reconstructed embryos were then added into the microdrops of the mixed fusion medium to simultaneously transfect the siRNA and electrofuse the SCNT embryos. Our results showed that transfecting DNMT1 siRNA via the proposed method caused obvious CY3 fluorescence and significant downregulation of DNMT1 messenger RNA, DNMT1 protein, and global DNA methylation levels in the SCNT embryos. Meanwhile, the survival rate after electrofusion (90.4% vs. 89.4% vs. 89.1%, P > 0.05) and developmental rates of the SCNT embryos (72.8% vs. 74.9% vs. 72.4%, P > 0.05; 29.7% vs. 31.7% vs. 29.7%, P > 0.05) were not significantly affected. In summary, siRNAs were effectively transfected into the SCNT embryos via the proposed method and exert their functions, and the normal development of preimplantation SCNT embryos was not affected by the method used. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Lingo-1 inhibited by RNA interference promotes functional recovery of experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Wang, Chun-Juan; Qu, Chuan-Qiang; Zhang, Jie; Fu, Pei-Cai; Guo, Shou-Gang; Tang, Rong-Hua

    2014-12-01

    Lingo-1 is a negative regulator of myelination. Repairment of demyelinating diseases, such as multiple sclerosis (MS)/experimental autoimmune encephalomyelitis (EAE), requires activation of the myelination program. In this study, we observed the effect of RNA interference on Lingo-1 expression, and the impact of Lingo-1 suppression on functional recovery and myelination/remyelination in EAE mice. Lentiviral vectors encoding Lingo-1 short hairpin RNA (LV/Lingo-1-shRNA) were constructed to inhibit Lingo-1 expression. LV/Lingo-1-shRNA of different titers were transferred into myelin oligodendrocyte glycoprotein-induced EAE mice by intracerebroventricular (ICV) injection. Meanwhile, lentiviral vectors carrying nonsense gene sequence (LVCON053) were used as negative control. The Lingo-1 expression was detected and locomotor function was evaluated at different time points (on days 1,3,7,14,21, and 30 after ICV injection). Myelination was investigated by luxol fast blue (LFB) staining.LV/Lingo-1-shRNA administration via ICV injection could efficiently down-regulate the Lingo-1 mRNA and protein expression in EAE mice on days 7,14,21, and 30 (P RNA interference is, therefore, a promising approach for the treatment of demyelinating diseases, such as MS/EAE. © 2014 Wiley Periodicals, Inc.

  7. RNA interference and its role in the regulation of eucaryotic gene expression.

    Science.gov (United States)

    Szweykowska-Kulińska, Zofia; Jarmołowski, Artur; Figlerowicz, Marek

    2003-01-01

    Several years ago it was discovered that plant transformation with a transcribed sense transgene could shut down the expression of a homologous endogenous gene. Moreover, it was shown that the introduction into the cell of dsRNA (double-stranded RNA) containing nucleotide sequence complementary to an mRNA sequence causes selective degradation of the latter and thus silencing of a specific gene. This phenomenon, called RNA interference (RNAi) was demonstrated to be present in almost all eukaryotic organisms. RNAi is also capable of silencing transposons in germ line cells and fighting RNA virus infection. Enzymes involved in this process exhibit high homology across species. Some of these enzymes are involved in other cellular processes, for instance developmental timing, suggesting strong interconnections between RNAi and other metabolic pathways. RNAi is probably an ancient mechanism that evolved to protect eukaryotic cells against invasive forms of nucleic acids.

  8. eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference

    Science.gov (United States)

    Yi, Tingfang; Arthanari, Haribabu; Akabayov, Barak; Song, Huaidong; Papadopoulos, Evangelos; Qi, Hank H.; Jedrychowski, Mark; Güttler, Thomas; Guo, Cuicui; Luna, Rafael E.; Gygi, Steven P.; Huang, Stephen A.; Wagner, Gerhard

    2015-05-01

    MicroRNA (miRNA) biogenesis and miRNA-guided RNA interference (RNAi) are essential for gene expression in eukaryotes. Here we report that translation initiation factor eIF1A directly interacts with Ago2 and promotes Ago2 activities in RNAi and miR-451 biogenesis. Biochemical and NMR analyses demonstrate that eIF1A binds to the MID domain of Ago2 and this interaction does not impair translation initiation. Alanine mutation of the Ago2-facing Lys56 in eIF1A impairs RNAi activities in human cells and zebrafish. The eIF1A-Ago2 assembly facilitates Dicer-independent biogenesis of miR-451, which mediates erythrocyte maturation. Human eIF1A (heIF1A), but not heIF1A(K56A), rescues the erythrocyte maturation delay in eif1axb knockdown zebrafish. Consistently, miR-451 partly compensates erythrocyte maturation defects in zebrafish with eif1axb knockdown and eIF1A(K56A) expression, supporting a role of eIF1A in miRNA-451 biogenesis in this model. Our results suggest that eIF1A is a novel component of the Ago2-centred RNA-induced silencing complexes (RISCs) and augments Ago2-dependent RNAi and miRNA biogenesis.

  9. A systematic analysis of the effect of target RNA structure on RNA interference

    NARCIS (Netherlands)

    Westerhout, Ellen M.; Berkhout, Ben

    2007-01-01

    RNAi efficiency is influenced by local RNA structure of the target sequence. We studied this structure-based resistance in detail by targeting a perfect RNA hairpin and subsequently destabilized its tight structure by mutation, thereby gradually exposing the target sequence. Although the tightest

  10. HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome

    NARCIS (Netherlands)

    Westerhout, Ellen M.; Ooms, Marcel; Vink, Monique; Das, Atze T.; Berkhout, Ben

    2005-01-01

    HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed

  11. The Role of RNA Interference (RNAi in Arbovirus-Vector Interactions

    Directory of Open Access Journals (Sweden)

    Carol D. Blair

    2015-02-01

    Full Text Available RNA interference (RNAi was shown over 18 years ago to be a mechanism by which arbovirus replication and transmission could be controlled in arthropod vectors. During the intervening period, research on RNAi has defined many of the components and mechanisms of this antiviral pathway in arthropods, yet a number of unexplored questions remain. RNAi refers to RNA-mediated regulation of gene expression. Originally, the term described silencing of endogenous genes by introduction of exogenous double-stranded (dsRNA with the same sequence as the gene to be silenced. Further research has shown that RNAi comprises three gene regulation pathways that are mediated by small RNAs: the small interfering (siRNA, micro (miRNA, and Piwi-interacting (piRNA pathways. The exogenous (exo-siRNA pathway is now recognized as a major antiviral innate immune response of arthropods. More recent studies suggest that the piRNA and miRNA pathways might also have important roles in arbovirus-vector interactions. This review will focus on current knowledge of the role of the exo-siRNA pathway as an arthropod vector antiviral response and on emerging research into vector piRNA and miRNA pathway modulation of arbovirus-vector interactions. Although it is assumed that arboviruses must evade the vector’s antiviral RNAi response in order to maintain their natural transmission cycles, the strategies by which this is accomplished are not well defined. RNAi is also an important tool for arthropod gene knock-down in functional genomics studies and in development of arbovirus-resistant mosquito populations. Possible arbovirus strategies for evasion of RNAi and applications of RNAi in functional genomics analysis and arbovirus transmission control will also be reviewed.

  12. Gene silencing by RNA interference in Sarcoptes scabiei: a molecular tool to identify novel therapeutic targets.

    Science.gov (United States)

    Fernando, Deepani D; Marr, Edward J; Zakrzewski, Martha; Reynolds, Simone L; Burgess, Stewart T G; Fischer, Katja

    2017-06-10

    Scabies is one of the most common and widespread parasitic skin infections globally, affecting a large range of mammals including humans, yet the molecular biology of Sarcoptes scabiei is astonishingly understudied. Research has been hampered primarily due to the difficulty of sampling or culturing these obligatory parasitic mites. A further and major impediment to identify and functionally analyse potential therapeutic targets from the recently emerging molecular databases is the lack of appropriate molecular tools. We performed standard BLAST based searches of the existing S. scabiei genome databases using sequences of genes described to be involved in RNA interference in Drosophila and the mite model organism Tetranychus urticae. Experimenting with the S. scabiei mu-class glutathione S-transferase (SsGST-mu1) as a candidate gene we explored the feasibility of gene knockdown in S. scabiei by double-stranded RNA-interference (dsRNAi). We provide here an analysis of the existing S. scabiei draft genomes, confirming the presence of a double stranded RNA (dsRNA) - mediated silencing machinery. We report for the first time experimental gene silencing by RNA interference (RNAi) in S. scabiei. Non-invasive immersion of S. scabiei in dsRNA encoding an S. scabiei glutathione S-transferase mu-class 1 enzyme (SsGST-mu1) resulted in a 35% reduction in the transcription of the target gene compared to controls. A series of experiments identified the optimal conditions allowing systemic experimental RNAi without detrimental side effects on mite viability. This technique can now be used to address the key questions on the fundamental aspects of mite biology and pathogenesis, and to assess the potential therapeutic benefits of silencing S. scabiei target genes.

  13. RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei

    NARCIS (Netherlands)

    Vondrusková, Eva; van den Burg, Janny; Zíková, Alena; Ernst, Nancy Lewis; Stuart, Kenneth; Benne, Rob; Lukes, Julius

    2005-01-01

    Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth

  14. Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

    Science.gov (United States)

    Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

    2014-04-01

    Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions.

  15. Therapeutic potentials of gene silencing by RNA interference: principles, challenges, and new strategies.

    Science.gov (United States)

    Deng, Yan; Wang, Chi Chiu; Choy, Kwong Wai; Du, Quan; Chen, Jiao; Wang, Qin; Li, Lu; Chung, Tony Kwok Hung; Tang, Tao

    2014-04-01

    During recent decades there have been remarkable advances in biology, in which one of the most important discoveries is RNA interference (RNAi). RNAi is a specific post-transcriptional regulatory pathway that can result in silencing gene functions. Efforts have been done to translate this new discovery into clinical applications for disease treatment. However, technical difficulties restrict the development of RNAi, including stability, off-target effects, immunostimulation and delivery problems. Researchers have attempted to surmount these barriers and improve the bioavailability and safety of RNAi-based therapeutics by optimizing the chemistry and structure of these molecules. This paper aimed to describe the principles of RNA interference, review the therapeutic potential in various diseases and discuss the new strategies for in vivo delivery of RNAi to overcome the challenges. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Gold nanoparticle interference study during the isolation, quantification, purity and integrity analysis of RNA.

    Science.gov (United States)

    Sanabria, Natasha M; Vetten, Melissa; Andraos, Charlene; Boodhia, Kailen; Gulumian, Mary

    2014-01-01

    Investigations have been conducted regarding the interference of nanoparticles (NPs) with different toxicological assay systems, but there is a lack of validation when conducting routine tests for nucleic acid isolation, quantification, integrity, and purity analyses. The interference of citrate-capped gold nanoparticles (AuNPs) was investigated herein. The AuNPs were added to either BEAS-2B bronchial human cells for 24 h, the isolated pure RNA, or added during the isolation procedure, and the resultant interaction was assessed. Total RNA that was isolated from untreated BEAS-2B cells was spiked with various concentrations (v/v%) of AuNPs and quantified. A decrease in the absorbance spectrum (220-340 nm) was observed in a concentration-dependent manner. The 260 and 280 nm absorbance ratios that traditionally infer RNA purity were also altered. Electrophoresis was performed to determine RNA integrity, but could not differentiate between AuNP-exposed samples. However, the spiked post-isolation samples did produce differences in spectra (190-220 nm), where shifts were observed at a shorter wavelength. These shifts could be due to alterations to chromophores found in nucleic acids. The co-isolation samples, spiked with 100 µL AuNP during the isolation procedure, displayed a peak shift to a longer wavelength and were similar to the results obtained from a 24 h AuNP treatment, under non-cytotoxic test conditions. Moreover, hyperspectral imaging using CytoViva dark field microscopy did not detect AuNP spectral signatures in the RNA isolated from treated cells. However, despite the lack of AuNPs in the final RNA product, structural changes in RNA could still be observed between 190-220 nm. Consequently, full spectral analyses should replace the traditional ratios based on readings at 230, 260, and 280 nm. These are critical points of analyses, validation, and optimization for RNA-based techniques used to assess AuNPs effects.

  17. The promise and progress of RNA-interference-based antiviral therapy for respiratory syncytial virus.

    Directory of Open Access Journals (Sweden)

    V. V. Vysochinskayа

    2012-01-01

    Full Text Available Respiratory syncytial virus (RSV is a major cause of morbidity in infants, young children, and the elderly worldwide. Presently, there are no explicit recommendations for RSV treatment apart from supportive care. Recent progress in studies of the mechanism of RNA interference suggests the formation of a new class of antiviral drugs in the treatment of RSV infection and related respiratory diseases.

  18. New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing

    Science.gov (United States)

    Burroughs, Alexander Maxwell; Ando, Yoshinari; Aravind, L

    2014-01-01

    Our understanding of the pervasive involvement of small RNAs in regulating diverse biological processes has been greatly augmented by recent application of deep-sequencing technologies to small RNA across diverse eukaryotes. We review the currently-known small RNA classes and place them in context of the reconstructed evolutionary history of the RNAi protein machinery. This synthesis indicates the earliest versions of eukaryotic RNAi systems likely utilized small RNA processed from three types of precursors: 1) sense-antisense transcriptional products, 2) genome-encoded, imperfectly-complementary hairpin sequences, and 3) larger non-coding RNA precursor sequences. Structural dissection of PIWI proteins along with recent discovery of novel families (including Med13 of the Mediator complex) suggest that emergence of a distinct architecture with the N-terminal domains (also occurring separately fused to endoDNases in prokaryotes) formed via duplication of an ancestral unit was key to their recruitment as primary RNAi effectors and use of small RNAs of certain preferred lengths. Prokaryotic PIWI proteins are typically components of several RNA-directed DNA restriction or CRISPR/Cas systems. However, eukaryotic versions appear to have emerged from a subset that evolved RNA-directed RNA interference. They were recruited alongside RNaseIII domains and RdRP domains, also from prokaryotic systems, to form the core eukaryotic RNAi system. Like certain regulatory systems, RNAi diversified into two distinct but linked arms concomitant with eukaryotic nucleo-cytoplasmic compartmentalization. Subsequent elaboration of RNAi proceeded via diversification of the core protein machinery through lineage-specific expansions and recruitment of new components from prokaryotes (nucleases and small RNA-modifying enzymes), allowing for diversification of associating small RNAs. PMID:24311560

  19. A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust.

    Science.gov (United States)

    Song, Huifang; Zhang, Jianqin; Li, Daqi; Cooper, Anastasia M W; Silver, Kristopher; Li, Tao; Liu, Xiaojian; Ma, Enbo; Zhu, Kun Yan; Zhang, Jianzhen

    2017-07-01

    Application of RNA interference (RNAi) for insect pest management is limited by variable efficiency of RNAi in different insect species. In Locusta migratoria, RNAi is highly efficient through injection of dsRNA, but oral delivery of dsRNA is much less effective. Efforts to understand this phenomenon have shown that dsRNA is more rapidly degraded in midgut fluid than in hemolymph due to nuclease enzyme activity. In the present study, we identified and characterized two full-length cDNAs of double-stranded RNA degrading enzymes (dsRNase) from midgut of L. migratoria, which were named LmdsRNase2 and LmdsRNase3. Gene expression analysis revealed that LmdsRNase2 and LmdsRNase3 were predominantly expressed in the midgut, relatively lower expression in gastric caeca, and trace expression in other tested tissues. Incubation of dsRNA in midgut fluid from LmdsRNase3-suppressed larvae or control larvae injected with dsGFP resulted in high levels of degradation; however, dsRNA incubated in midgut fluid from LmdsRNase2-suppressed larvae was more stable, indicating LmdsRNase2 is responsible for dsRNA degradation in the midgut. To verify the biological function of LmdsRNase2 in vivo, nymphs were injected with dsGFP, dsLmdsRNase2 or dsLmdsRNase3 and chitinase 10 (LmCht10) or chitin synthase 1 (LmCHS1) dsRNA were orally delivered. Mortality associated with reporter gene knockdown was observed only in locusts injected with dsLmdsRNase2 (48% and 22%, for dsLmCht10 and dsLmCHS1, respectively), implicating LmdsRNase2 in reducing RNAi efficiency. Furthermore, recombinantly expressed LmdsRNase2 fusion proteins degraded dsRNA rapidly, whereas LmdsRNase3 did not. These results suggest that rapid degradation of dsRNA by dsRNase2 in the midgut is an important factor causing low RNAi efficiency when dsRNA is orally delivered in the locust. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. RNA Interference Induced by the Cationic Lipid Delivery of siRNA

    Science.gov (United States)

    Bouxsein, Nathan

    2005-03-01

    Recent discoveries demonstrate that the introduction of synthetically prepared duplexes of 19-21 bp short interfering RNAs (siRNA) into mammalian cells results in the cleavage of target mRNA leading to post transcriptional gene silencing [1]. Our work focuses on the cationic-lipid (CL) mediated delivery of siRNA into mammalian cell lines in an approach similar to CL based gene delivery [2]. Co-transfection of a target and a non-target reporter plasmid followed by the CL delivery of a sequence specific siRNA allows us to probe the silencing efficiency (SE) of the target plasmid relative to non-specific silencing of both plasmids. We have created a phase diagram for SE as a function of the complex membrane charge density and as a function of the CL:siRNA charge ratio. X-ray diffraction was performed to probe the structure of the complexes at points along the phase diagram. Funding provided by NIH AI-12520, AI-20611 and GM-59288. [1] Elbashir et. al., Nature, 411 494-498 (2001) [2] Ewert et. al., Curr. Med. Chem. 11 133-149 (2004)

  1. Reversal of chemoresistance with small interference RNA (siRNA) in etoposide resistant acute myeloid leukemia cells (HL-60).

    Science.gov (United States)

    Kachalaki, Saeed; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi; Shanehbandi, Dariush; Mohammadinejad, Sina; Mansoori, Behzad

    2015-10-01

    Overexpression of ATP-binding cassette (ABC) drug transporters is a major barrier in the success of cancer chemotherapy. One way to overcome overexpression of ABC drug transporter-mediated chemoresistance in acute myeloid leukemia is to suppress ABC drug transporter genes expression by small interference RNA (siRNA). In this study was assessed the involvement of ABCB1 gene in the mechanisms of resistance to etoposide in AML cells. The etoposide-resistant HL-60 cells were generated by stepwise exposure increasing concentrations of etoposide. The etoposide-resistant HL-60 cells were transfected with siRNAs using Transfection Reagent. The ABCB1 mRNA expression were assessed by real-time quantitative PCR. The MDR1/P-gp levels were measured by Western blotting. The sensitivity of resistant HL-60 cells to etoposide after transfection was determined using MTT assay. Apoptosis of resistant HL-60 cells after transfection was detected by flow cytometer. It was found that siRNA effectively inhibited ABCB1 expression at both mRNA and protein levels. Knockdown of the ABCB1 gene correlated with increased sensitivity of the resistant HL-60 cells to etoposide and was observed to lower the cytotoxic index (IC50 etoposide value) after transfection. Our results indicate that product of the ABCB1 gene have effective role in resistance to etoposide in acute myeloid leukemia cells. Copyright © 2015. Published by Elsevier Masson SAS.

  2. Viral RNA silencing suppressors (RSS): novel strategy of viruses to ablate the host RNA interference (RNAi) defense system.

    Science.gov (United States)

    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. Copyright © 2010 Elsevier B.V. All rights reserved.

  3. RNA interference and dietary inhibitors induce a similar compensation response in Tribolium castaneum larvae.

    Science.gov (United States)

    Perkin, L C; Elpidina, E N; Oppert, B

    2017-02-01

    Tribolium castaneum is a major agriculture pest damaging stored grains and cereal products. The T. castaneum genome contains 26 cysteine peptidase genes, mostly cathepsins L and B, and seven have a major role in digestion. We targeted the expression of the most highly expressed cathepsin L gene on chromosome 10, TC011001, by RNA interference (RNAi), using double-stranded RNA (dsRNA) constructs of different regions of the gene (3', middle, 5' and entire coding regions). RNA sequencing and quantitation (RNA-seq) was used to evaluate knockdown and specificity amongst the treatments. Overall, target gene expression decreased in all treatment groups, but was more severe and specific in dsRNA targeting the 3' and entire coding regions, encoding the proteolytic active site in the enzyme. Additional cysteine cathepsin genes also were down-regulated (off-target effects), but some were up-regulated in response to RNAi treatment. Notably, some serine peptidase genes were increased in expression, especially in dsRNA targeting 5' and middle regions, and the response was similar to the effects of dietary cysteine protease inhibitors. We manually annotated these serine peptidase genes to gain insight into function and relevance to the RNAi study. The data indicate that T. castaneum larvae compensate for the loss of digestive peptidase activity in the larval gut, regardless of the mechanism of disruption. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  4. Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito's RNA interference pathway.

    Directory of Open Access Journals (Sweden)

    Irma Sánchez-Vargas

    2009-02-01

    Full Text Available A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi, is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA, which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs. These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2 infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.

  5. [Down-regulation of CT120A by RNA interference suppresses lung cancer cells growth].

    Science.gov (United States)

    Pan, Dong-ning; Wei, Lin; Yao, Ming; Wan, Da-fang; Gu, Jian-ren

    2005-06-22

    To validate our obtained outcomes and clarify the relationship between CT120A, a novel human plasma membrane-associated gene, and proliferation of lung cancer cells. A vector-based small hairpin RNA (shRNA) was transfected into the human lung adenocarcinoma SPC-A-1 cells to specifically target CT120A cDNA. RT-PCR and Western blotting were used to analyze the CT120A expression. The cell proliferation rate was analyzed by BrdU-TdR incorporation assay, the ability of cells to grow in soft agarose and the tumorigenicity in nude mice were measured. Flow cytometry was performed to analyze cell apoptosis. When compared with the scrambled control cell line, CT120A transcripts were reduced by 70% and 50% in two shRNA-H stable transfectants, H2 and H3 clones, respectively. The protein of CT120A was reduced by about 80% in both the H2 and H3 clones. By BrdU incorporation assay, up to the 6th day a dramatic decrease in the cell growth rate (30% to 40%) was observed in the shRNA-H2 and shRNA-H3 cell lines. The colony formation rate in soft agarose of the two cell lines was about one half that of the control cells. In addition, a remarkable reduction of tumorigenicity of the two cell lines was observed as compared with that of the control. The suppression of CT120A expression also sensitized cells to ultraviolet-induced apoptosis. Down-regulation of CT120A by RNA interference suppresses lung cancer cell growth. The successful knockdown of CT120A expression by RNA interference implicates that CT120A may be a new candidate of drug target for treatment of lung cancers.

  6. Combined RNA interference of adenine nucleotide translocase-2 and ganciclovir therapy in hepatocellular carcinoma.

    Science.gov (United States)

    Kim, Jung Eun; Hwang, Mi-Hye; Lee, Ho Won; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

    2013-11-01

    The purpose of this study was to investigate the anticancer effects of combined RNA interference (RNAi) of the adenine nucleotide translocase-2 (ANT2) gene and ganciclovir (GCV) therapy for treatment of hepatocellular carcinoma cells (Huh 7) in an animal model. The Huh 7/NTG stable cell line was established by transfection of a vector with the human sodium iodide symporter (hNIS), HSV1-sr39 thymidine kinase (tk), and enhanced green florescent protein (EGFP) fusion gene into Huh 7 cells. mRNA expressions of these genes were evaluated by RT-PCR analysis. The functions of hNIS and HSV1-sr39tk were verified with (125)I uptake and (3)H-penciclovir (PCV) uptake tests. EGFP and hNIS expression was confirmed with confocal microscopy after immunocytochemical staining. We treated the tumor cells with ANT2 shRNA or GCV or both ANT2 shRNA and GCV and treated the in vivo mouse model with a Huh 7/NTG tumor xenograft. The therapeutic effects of the in vivo study were assessed with caliper measurements and gamma camera imaging using (99m)Tc-pertechnetate. Huh 7/NTG cells showed a cell number-dependent increase in (125)I uptake and a 24-fold higher (3)H-PCV uptake compared to parent Huh 7 cells. Huh 7/NTG cells transfected with ANT2 shRNA had lower ANT2 mRNA expression and more impaired proliferation activity than cells transfected with scramble shRNA. Proliferation of Huh 7/NTG cells was also inhibited by GCV treatment. Combined GCV and ANT2 shRNA therapy further inhibited cell proliferation in the in vitro study. The combined therapy with GCV and ANT2 shRNA showed a further decrease in tumor growth in the mouse model. Our results suggest that the combined RNA interference with ANT2 and GCV therapy inhibited hepatocellular carcinoma cell proliferation more than single GCV therapy or ANT2 shRNA therapy in vitro and in vivo. Therefore it could be applied treating incurable hepatocellular carcinoma. © 2013.

  7. Co-expression of Argonaute2 enhances short hairpin RNA-induced RNA interference in Xenopus CNS neurons in vivo

    Directory of Open Access Journals (Sweden)

    Chih-ming Chen

    2009-07-01

    Full Text Available RNA interference (RNAi is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi and pReporter (pRe constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex (RISC, on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNAmir30 enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.

  8. RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems.

    Science.gov (United States)

    Dietrich, Isabelle; Jansen, Stephanie; Fall, Gamou; Lorenzen, Stephan; Rudolf, Martin; Huber, Katrin; Heitmann, Anna; Schicht, Sabine; Ndiaye, El Hadji; Watson, Mick; Castelli, Ilaria; Brennan, Benjamin; Elliott, Richard M; Diallo, Mawlouth; Sall, Amadou A; Failloux, Anna-Bella; Schnettler, Esther; Kohl, Alain; Becker, Stefanie C

    2017-01-01

    The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila

  9. Interference of hepatitis C virus RNA replication by short interfering RNAs

    Science.gov (United States)

    Kapadia, Sharookh B.; Brideau-Andersen, Amy; Chisari, Francis V.

    2003-02-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.

  10. RNA interference technology in crop protection against arthropod pests, pathogens and nematodes.

    Science.gov (United States)

    Zotti, Moises; Dos Santos, Ericmar Avila; Cagliari, Deise; Christiaens, Olivier; Taning, Clauvis Nji Tizi; Smagghe, Guy

    2017-11-30

    Scientists have made significant progress in understanding and unraveling several aspects of double-stranded RNA (dsRNA)-mediated gene silencing during the last two decades. Now that the RNA interference (RNAi) mechanism is well understood, it is time to consider how to apply the acquired knowledge to agriculture and crop protection. Some RNAi-based products are already available for farmers and more are expected to reach the market soon. Tailor-made dsRNA as an active ingredient for biopesticide formulations is considered a raw material that can be used for diverse purposes, from pest control and bee protection against viruses to pesticide resistance management. The RNAi mechanism works at the messenger RNA (mRNA) level, exploiting a sequence-dependent mode of action, which makes it unique in potency and selectivity compared with conventional agrochemicals. Furthermore, the use of RNAi in crop protection can be achieved by employing plant-incorporated protectants through plant transformation, but also by non-transformative strategies such as the use of formulations of sprayable RNAs as direct control agents, resistance factor repressors or developmental disruptors. In this review, RNAi is presented in an agricultural context (discussing products that have been launched on the market or will soon be available), and we go beyond the classical presentation of successful examples of RNAi in pest-insect control and comprehensively explore its potential for the control of plant pathogens, nematodes and mites, and to fight against diseases and parasites in beneficial insects. Moreover, we also discuss its use as a repressor for the management of pesticide-resistant weeds and insects. Finally, this review reports on the advances in non-transformative dsRNA delivery and the production costs of dsRNA, and discusses environmental considerations. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  11. Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety.

    Science.gov (United States)

    Hatano, Akihiko; Shiraishi, Mitsuya; Terado, Nanae; Tanabe, Atsuhiro; Fukuda, Kenji

    2015-10-15

    Thymidine phosphorylase was used to catalyze the conversion of thymidine (or methyluridine) and uracil incorporating stable isotopes to deoxyuridine (or uridine) with the uracil base incorporating the stable isotope. These base-exchange reactions proceeded with high conversion rates (75-96%), and the isolated yields were also good (64-87%). The masses of all synthetic compounds incorporating stable isotopes were identical to the theoretical molecular weights via EIMS. (13)C NMR spectra showed spin-spin coupling between (13)C and (15)N in the synthetic compounds, and the signals were split, further proving incorporation of the isotopes into the compounds. The RNA interference effects of this siRNA with uridine incorporating stable isotopes were also investigated. A 25mer siRNA had a strong knockdown effect on the MARCKS protein. The insertion position and number of uridine moieties incorporating stable isotopes introduced into the siRNA had no influence on the silencing of the target protein. This incorporation of stable isotopes into RNA and DNA has the potential to function as a chemically benign tracer in cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Evolutionarily conserved roles of the dicer helicase domain in regulating RNA interference processing.

    Science.gov (United States)

    Kidwell, Mary Anne; Chan, Jessica M; Doudna, Jennifer A

    2014-10-10

    The enzyme Dicer generates 21-25 nucleotide RNAs that target specific mRNAs for silencing during RNA interference and related pathways. Although their active sites and RNA binding regions are functionally conserved, the helicase domains have distinct activities in the context of different Dicer enzymes. To examine the evolutionary origins of Dicer helicase functions, we investigated two related Dicer enzymes from the thermophilic fungus Sporotrichum thermophile. RNA cleavage assays showed that S. thermophile Dicer-1 (StDicer-1) can process hairpin precursor microRNAs, whereas StDicer-2 can only cleave linear double-stranded RNAs. Furthermore, only StDicer-2 possesses robust ATP hydrolytic activity in the presence of double-stranded RNA. Deletion of the StDicer-2 helicase domain increases both StDicer-2 cleavage activity and affinity for hairpin RNA. Notably, both StDicer-1 and StDicer-2 could complement the distantly related yeast Schizosaccharomyces pombe lacking its endogenous Dicer gene but only in their full-length forms, underscoring the importance of the helicase domain. These results suggest an in vivo regulatory function for the helicase domain that may be conserved from fungi to humans. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. [Construction and identification of a RNA interference plasmid for rat BNIP3 gene].

    Science.gov (United States)

    Chen, Ming; Zhang, Xing-mei; Li, Bo-xing; Sun, Hong-yu; Li, Xiao-wen; Gao, Tian-ming

    2010-11-01

    To construct a RNA interfering plasmid targeting rat Bcl-2/E1B-19K-interacting protein 3 (BNIP3) and assess its effect on exogenous BNIP3 expression in HEK293 cells. The miRNA sequences were designed using Invitrogen BLOCK-iT RNAi Designer and synthesized into double-strand oligonucleotides, which were cloned into the pcDNATM6.2-GW/EmGFP-miR vector, followed by transformation of the product into competent Top10 E. coli cells. After expansion of the transformed bacteria, the plasmid was extracted and sequenced before its co-transfection with pEGFP-C3- rBNIP3 plasmid into HEK293 cells. The interference effect of the constructed plasmid on BNIP3 mRNA and protein expression were detected by real-time PCR and Western blotting. The sequencing result indicated that the interfering plasmid targeting rat BNIP3 was constructed correctly. After transfection into HEK293 cells, the interfering plasmid significantly inhibited exogenous BNIP3 mRNA and protein expressions. The RNA interfering plasmid targeting rat BNIP3 has been constructed successfully, which provides a useful tool for studying the function of BNIP3.

  14. Down-Regulated Expression of RACK1 Gene by RNA Interference Enhances Drought Tolerance in Rice

    OpenAIRE

    Da-hong LI; Liu, Hui; Yang, Yan-li; Ping-ping ZHEN; Jian-sheng LIANG

    2009-01-01

    The receptor for activated C-kinase 1 (RACK1) is a highly conserved scaffold protein with versatile functions, and plays important roles in the regulation of plant growth and development. Transgenic rice plants, in which the expression of RACK1 gene was inhibited by RNA interference (RNAi), were studied to elucidate the possible functions of RACK1 in responses to drought stress in rice. Real-time PCR analysis showed that the expression of RACK1 in transgenic rice plants was inhibited by more ...

  15. Inactivation of the type I interferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells.

    Science.gov (United States)

    Maillard, Pierre V; Van der Veen, Annemarthe G; Deddouche-Grass, Safia; Rogers, Neil C; Merits, Andres; Reis E Sousa, Caetano

    2016-12-01

    RNA interference (RNAi) elicited by long double-stranded (ds) or base-paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence-specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN-responsive cells with type I IFN Notably, transfection with long dsRNA specifically vaccinates IFN-deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  16. Functions of the Vasa gene in Schistosoma japonicum as assessed by RNA interference.

    Science.gov (United States)

    He, Siyu; Zhu, Lulu; Liu, Fengchun; Liu, Quan; Shao, Yanjing; Hua, Mengqing; Ding, Han; Shao, Wei; Du, Yinan; Hou, Xin; Ren, Cuiping; Liu, Miao; Shen, Jijia

    2018-01-05

    Vasa, an enzyme belonging to the helicase family, contributes to the regulation of reproductive system development in many species. Thus, we hypothesized that the Vasa3 gene may function in the reproductive system of the parasite Schistosoma japonicum (S. japonicum), which is a major causative agent of schistosomiasis. It is a severe disease globally affecting humans and animals. To test this hypothesis, we firstly conducted whole mount in situ hybridization analyses and found that the S. japonicum Vasa3 (SjVasa3) gene was expressed mainly in the reproductive organs. We then explored the reproductive functions of Vasa3 in S. japonicum using RNA interference (RNAi) techniques. Coupled schistosomes collected from mice 28days post infection (dpi) were transfected three times with SjVasa3-specific small interfering RNA (siRNA) and cultured in vitro for up to 10days. As measured by quantitative PCR (qPCR) and Western blot analysis, levels of SjVasa3 mRNA and protein in Vasa siRNA treated worms were significantly reduced compared with untreated and scrambled siRNA treated worms. Confocal laser scanning microscopy (CLSM) images showed markedly siRNA induced changes in the morphology of the reproductive organs, especially in the female ovary, vitellarium and the male testes. SjVasa3 gene silencing also significantly reduced egg production. These data demonstrate that SjVasa3 is essential in reproductive organ development and egg production in S. japonicum, and could be a potential target for developing novel compounds to treat schistosomiasis. Copyright © 2017. Published by Elsevier B.V.

  17. HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

    Directory of Open Access Journals (Sweden)

    Valentina Vongrad

    Full Text Available MiRNAs and other small noncoding RNAs (sncRNAs are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM.The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP, which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

  18. RNA interference of four genes in adult Bactrocera dorsalis by feeding their dsRNAs.

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    Xiaoxue Li

    Full Text Available BACKGROUND: RNA interference (RNAi is a powerful method to inhibit gene expression in a sequence specific manner. Recently silencing the target gene through feeding has been successfully carried out in many insect species. METHODOLOGY/PRINCIPAL FINDINGS: Escherichia coli strain HT115 was genetically engineered to express dsRNA targeting genes that encode ribosomal protein Rpl19, V type ATPase D subunit, the fatty acid elongase Noa and a small GTPase Rab11. qRT-PCR showed that mRNA level of four target genes was reduced compared to ds-egfp control by feeding either engineered bacteria or dsRNAs. The maximum down-regulation of each gene varied from 35% to 100%. Tissue specific examination indicated that RNAi could be observed not only in midgut but also in other tissues like the ovary, nervous system and fat body. Silencing of rab11 through ingestion of dsRNA killed 20% of adult flies. Egg production was affected through feeding ds-noa and ds-rab11 compared to ds-egfp group. Adult flies were continuously fed with dsRNA and bacteria expressing dsRNA for 14 days and up-regulations of target genes were observed during this process. The transcripts of noa showed up-regulation compared to ds-egfp control group in four tissues on day 7 after continuous feeding either dsRNA or engineered bacteria. The maximum over-expression is 21 times compared to ds-egfp control group. Up-regulation of rab11 mRNA level could be observed in testes on day 7 after continuous bacteria treatment and in midgut on day 2 after ds-rab11 treatment. This phenomenon could also be observed in rpl19 groups. CONCLUSIONS: Our results suggested that it is feasible to silence genes by feeding dsRNA and bacteria expressing dsRNA in Bactrocera dorsalis. Additionally the over-expression of the target gene after continuously feeding dsRNA or bacteria was observed.

  19. Gene silencing in non-model insects: Overcoming hurdles using symbiotic bacteria for trauma-free sustainable delivery of RNA interference: Sustained RNA interference in insects mediated by symbiotic bacteria: Applications as a genetic tool and as a biocide.

    Science.gov (United States)

    Whitten, Miranda; Dyson, Paul

    2017-03-01

    Insight into animal biology and development provided by classical genetic analysis of the model organism Drosophila melanogaster was an incentive to develop advanced genetic tools for this insect. But genetic systems for the over one million other known insect species are largely undeveloped. With increasing information about insect genomes resulting from next generation sequencing, RNA interference is now the method of choice for reverse genetics, although it is constrained by the means of delivery of interfering RNA. A recent advance to ensure sustained delivery with minimal experimental intervention or trauma to the insect is to exploit commensal bacteria for symbiont-mediated RNA interference. This technology not only offers an efficient means for RNA interference in insects in laboratory conditions, but also has potential for use in the control of human disease vectors, agricultural pests and pathogens of beneficial insects. © 2017 WILEY Periodicals, Inc.

  20. Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus.

    Science.gov (United States)

    Marr, Edward J; Sargison, Neil D; Nisbet, Alasdair J; Burgess, Stewart T G

    2015-12-01

    This is the first report of gene silencing by RNA interference (RNAi) in the European house dust mite, Dermatophagoides pteronyssinus, Trouessart, 1897. Using a non-invasive immersion method first developed for the honey bee mite, Varroa destructor, a significant reduction in the expression of D. pteronyssinus glutathione-S-transferase mu-class 1 enzyme (DpGST-mu1) was achieved following overnight immersion in double stranded RNA encoding DpGST-mu1. Although no detrimental phenotypic changes were observed following silencing, this technique can now be used to address fundamental physiological questions and assess the potential therapeutic benefit in silencing D. pteronyssinus target genes in selected domestic situations of high human-mite interface. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Virus-derived gene expression and RNA interference vector for grapevine.

    Science.gov (United States)

    Kurth, Elizabeth G; Peremyslov, Valera V; Prokhnevsky, Alexey I; Kasschau, Kristin D; Miller, Marilyn; Carrington, James C; Dolja, Valerian V

    2012-06-01

    The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.

  2. Cmr1 enables efficient RNA and DNA interference of a III-B CRISPR–Cas system by binding to target RNA and crRNA

    Science.gov (United States)

    Li, Yingjun; Zhang, Yan; Lin, Jinzhong; Pan, Saifu; Han, Wenyuan; Peng, Nan; Liang, Yun Xiang

    2017-01-01

    Abstract CRISPR–Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems provide adaptive immunity against invasive nucleic acids guided by CRISPR RNAs (crRNAs) in archaea and bacteria. Type III CRISPR–Cas effector complexes show RNA cleavage and RNA-activated DNA cleavage activity, representing the only known system of dual nucleic acid interference. Here, we investigated the function of Cmr1 by genetic assays of DNA and RNA interference activity in the mutants and biochemical characterization of their mutated Cmr complexes. Three cmr1α mutants were constructed including ΔβΔ1α, Δβ1α-M1 and Δβ1α-M2 among which the last two mutants carried a double and a quadruple mutation in the first α-helix region of Cmr1α. Whereas the double mutation of Cmr1α (W58A and F59A) greatly influenced target RNA capture, the quadruple mutation almost abolished crRNA binding to Cmr1α. We found that Cmr2α-6α formed a stable core complex that is active in both RNA and DNA cleavage and that Cmr1α strongly enhances the basal activity of the core complex upon incorporation into the ribonucleoprotein complex. Therefore, Cmr1 functions as an integral activation module in III-B systems, and the unique occurrence of Cmr1 in III-B systems may reflect the adaptive evolution of type III CRISPR–Cas systems in thermophiles. PMID:28977458

  3. Cmr1 enables efficient RNA and DNA interference of a III-B CRISPR-Cas system by binding to target RNA and crRNA.

    Science.gov (United States)

    Li, Yingjun; Zhang, Yan; Lin, Jinzhong; Pan, Saifu; Han, Wenyuan; Peng, Nan; Liang, Yun Xiang; She, Qunxin

    2017-11-02

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems provide adaptive immunity against invasive nucleic acids guided by CRISPR RNAs (crRNAs) in archaea and bacteria. Type III CRISPR-Cas effector complexes show RNA cleavage and RNA-activated DNA cleavage activity, representing the only known system of dual nucleic acid interference. Here, we investigated the function of Cmr1 by genetic assays of DNA and RNA interference activity in the mutants and biochemical characterization of their mutated Cmr complexes. Three cmr1α mutants were constructed including ΔβΔ1α, Δβ1α-M1 and Δβ1α-M2 among which the last two mutants carried a double and a quadruple mutation in the first α-helix region of Cmr1α. Whereas the double mutation of Cmr1α (W58A and F59A) greatly influenced target RNA capture, the quadruple mutation almost abolished crRNA binding to Cmr1α. We found that Cmr2α-6α formed a stable core complex that is active in both RNA and DNA cleavage and that Cmr1α strongly enhances the basal activity of the core complex upon incorporation into the ribonucleoprotein complex. Therefore, Cmr1 functions as an integral activation module in III-B systems, and the unique occurrence of Cmr1 in III-B systems may reflect the adaptive evolution of type III CRISPR-Cas systems in thermophiles. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. RNA-interference-based gene therapy approaches to HIV type-1 treatment: tackling the hurdles from bench to bedside

    NARCIS (Netherlands)

    Von Eije, Karin J.; Berkhout, Ben

    2009-01-01

    RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs (siRNAs) to mediate sequence-specific gene silencing by cleavage of the targeted messenger RNA. RNAi can be used as an antiviral approach to silence HIV type-1 (HIV-1) through stable expression of

  5. Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi

    Directory of Open Access Journals (Sweden)

    Manev Hari

    2003-08-01

    Full Text Available Abstract Background RNA interference (RNAi is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly gene (corresponding to a putative gene CG5652/GM06434, that we named beltless based on an embryonic loss-of-function phenotype. Results Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts. Conclusions We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should

  6. How golden is silence? Teaching undergraduates the power and limits of RNA interference.

    Science.gov (United States)

    Kuldell, Natalie H

    2006-01-01

    It is hard and getting harder to strike a satisfying balance in teaching. Time dedicated to student-generated models or ideas is often sacrificed in an effort to "get through the syllabus." I describe a series of RNA interference (RNAi) experiments for undergraduate students that simultaneously explores fundamental concepts in gene regulation, develops cutting-edge laboratory skills, and embraces student-directed learning. Students design a small interfering RNA (siRNA) against luciferase, add it to cells expressing this gene, and then quantitatively assess the siRNA's effect on both intended and unintended targets, using a luciferase assay and a DNA microarray. Because both RNAi and microarray technologies are relatively new, with no clear consensus on their analysis or limitations, students are encouraged to explore different approaches to the design of their reagents and interpretations of their data. The ability to creatively formulate a hypothesis-driven experimental approach to a scientific question and to critically evaluate collected data is stressed. Equally important, this experiment emphasizes how modern scientific ideas emerge, are debated, tested, and decided.

  7. Tumor-specific RNA interference targeting Pokemon suppresses tumor growth and induces apoptosis in prostate cancer.

    Science.gov (United States)

    Li, Yining; Xu, Shuxiong; Wang, Xiangwei; Shi, Hua; Sun, Zhaolin; Yang, Zhao

    2013-02-01

    To explore the exact mechanism of Pokemon in prostate cancer. Pokemon is a member of the POK family of transcriptional repressors. Its main function is suppression of the p14ARF (alternate reading frame) tumor suppressor gene. Although Pokemon expression has been found to be increased in various types of lymphoma, the exact mechanism of the gene in prostate cancer is not clear. In the present study, prostate cancer cells were transfected with the specific short hairpin ribonucleic acid (RNA) expression vector targeting Pokemon. The expression of Pokemon messenger RNA and its protein was detected by semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The cell growth and cell apoptosis were also examined using the methyl thiazolyl tetrazolium assay and flow cytometry. The results demonstrated that specific RNA interference (RNAi) could decrease the expression levels of Pokemon gene messenger RNA and protein in prostate cancer cells. In addition, that specific RNAi significantly inhibited the cell proliferation and increased the apoptotic rate. In vivo experiments showed that specific RNAi inhibited the tumorigenicity of prostate cancer cells and significantly suppressed tumor growth. Therefore, an RNAi-targeted Pokemon gene strategy could be a potential approach to prostate cancer therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. RNA interference can be used to disrupt gene function in tardigrades.

    Science.gov (United States)

    Tenlen, Jennifer R; McCaskill, Shaina; Goldstein, Bob

    2013-05-01

    How morphological diversity arises is a key question in evolutionary developmental biology. As a long-term approach to address this question, we are developing the water bear Hypsibius dujardini (Phylum Tardigrada) as a model system. We expect that using a close relative of two well-studied models, Drosophila (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda), will facilitate identifying genetic pathways relevant to understanding the evolution of development. Tardigrades are also valuable research subjects for investigating how organisms and biological materials can survive extreme conditions. Methods to disrupt gene activity are essential to each of these efforts, but no such method yet exists for the Phylum Tardigrada. We developed a protocol to disrupt tardigrade gene functions by double-stranded RNA-mediated RNA interference (RNAi). We showed that targeting tardigrade homologs of essential developmental genes by RNAi produced embryonic lethality, whereas targeting green fluorescent protein did not. Disruption of gene functions appears to be relatively specific by two criteria: targeting distinct genes resulted in distinct phenotypes that were consistent with predicted gene functions and by RT-PCR, RNAi reduced the level of a target mRNA and not a control mRNA. These studies represent the first evidence that gene functions can be disrupted by RNAi in the phylum Tardigrada. Our results form a platform for dissecting tardigrade gene functions for understanding the evolution of developmental mechanisms and survival in extreme environments.

  9. A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies

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    Fatih M. Uckun

    2014-12-01

    Full Text Available The purposes of the present study were to further evaluate the biologic significance of the CD22ΔE12 molecular lesion and determine if it could serve as a molecular target for RNA interference (RNAi therapy. We show that both pediatric and adult B-lineage lymphoid malignancies are characterized by a very high incidence of the CD22ΔE12 genetic defect. We provide unprecedented experimental evidence for a previously unrecognized causal link between CD22ΔE12 and aggressive biology of BPL cells by demonstrating that siRNA-mediated knockdown of CD22ΔE12 in primary BPL cells is associated with a marked inhibition of their clonogenicity. These findings provide the preclinical proof-of-concept that siRNA-mediated depletion of CD22ΔE12 may help develop effective treatments for high-risk and relapsed BPL patients who are in urgent need for therapeutic innovations. We also describe a unique polypeptide-based nanoparticle formulation of CD22ΔE12-siRNA as an RNAi therapeutic candidate targeting CD22ΔE12 that is capable of delivering its siRNA cargo into the cytoplasm of leukemia cells causing effective CD22ΔE12 depletion and marked inhibition of leukemic cell growth. Further development and optimization of this nanoparticle or other nanoformulation platforms for CD22ΔE12-siRNA may facilitate the development of an effective therapeutic RNAi strategy against a paradigm shift in therapy of aggressive or chemotherapy-resistant B-lineage lymphoid malignancies.

  10. Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

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    El Far Mohamed

    2009-05-01

    Full Text Available Abstract Background Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC. While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain. Results We show that the TRBP binding site in Dicer is a 165 amino acid (aa region located between the ATPase and the helicase domains. The binding site in TRBP is a 69 aa domain, called C4, located at the C-terminal end of TRBP. The TRBP1 and TRBP2 isoforms, but not TRBPs lacking the C4 site (TRBPsΔC4, co-immunoprecipitated with Dicer. The C4 domain is therefore necessary to bind Dicer, irrespective of the presence of RNA. Immunofluorescence shows that while full-length TRBPs colocalize with Dicer, TRBPsΔC4 do not. tarbp2-/- cells, which do not express TRBP, do not support RNA interference (RNAi mediated by short hairpin or micro RNAs against EGFP. Both TRBPs, but not TRBPsΔC4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPsΔC4, rescued RNAi function. Conclusion The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPsΔC4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPsΔC4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function.

  11. Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells.

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    Li, Yang; Basavappa, Megha; Lu, Jinfeng; Dong, Shuwei; Cronkite, D Alexander; Prior, John T; Reinecker, Hans-Christian; Hertzog, Paul; Han, Yanhong; Li, Wan-Xiang; Cheloufi, Sihem; Karginov, Fedor V; Ding, Shou-Wei; Jeffrey, Kate L

    2016-12-05

    Influenza A virus (IAV) causes annual epidemics and occasional pandemics, and is one of the best-characterized human RNA viral pathogens1. However, a physiologically relevant role for the RNA interference (RNAi) suppressor activity of the IAV non-structural protein 1 (NS1), reported over a decade ago2, remains unknown3. Plant and insect viruses have evolved diverse virulence proteins to suppress RNAi as their hosts produce virus-derived small interfering RNAs (siRNAs) that direct specific antiviral defence4-7 by an RNAi mechanism dependent on the slicing activity of Argonaute proteins (AGOs)8,9. Recent studies have documented induction and suppression of antiviral RNAi in mouse embryonic stem cells and suckling mice10,11. However, it is still under debate whether infection by IAV or any other RNA virus that infects humans induces and/or suppresses antiviral RNAi in mature mammalian somatic cells12-21. Here, we demonstrate that mature human somatic cells produce abundant virus-derived siRNAs co-immunoprecipitated with AGOs in response to IAV infection. We show that the biogenesis of viral siRNAs from IAV double-stranded RNA (dsRNA) precursors in infected cells is mediated by wild-type human Dicer and potently suppressed by both NS1 of IAV as well as virion protein 35 (VP35) of Ebola and Marburg filoviruses. We further demonstrate that the slicing catalytic activity of AGO2 inhibits IAV and other RNA viruses in mature mammalian cells, in an interferon-independent fashion. Altogether, our work shows that IAV infection induces and suppresses antiviral RNAi in differentiated mammalian somatic cells.

  12. [Inhibition of growth and proliferation of Hep-2 cells by targeting human telomerase reverse transcriptase mRNA using RNA interference technology].

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    Chen, Shi-ming; Tao, Ze-zhang; Xiao, Bo-kui; Pan, Song; Liu, Dan; Chi, Hua-ming

    2005-12-01

    To investigate the effect of RNA interference by targeting human telomerase reverse transcriptase (hTERT) mRNA in the larynx cancer cell line, Hep-2. The primary structures of hTERT cDNA were found in GenBank. Then the structure analysis were done according to RNAi strategy which determined the specific base sequences to design shRNA plasmid. Two types of plasmid, pshRNA1 and pshRNA2, involved in fluorescein gene were synthesized based on the specific base sequences. Control pshRNA3, a random sequence, and control pshRNA4, without additional specific sequence were also constructed. Cells were treated daily with pshRNA1-4 or normal culture medium respectively. The pshRNA1-3 was identified by electrophoresis. After administration of pshRNA1-4, fluorescence expression was detected by confocal microscopy, the expression of hTERT of the transfected cells was determined by Western blotting, telomerase activity was measured by TRAP-PCR ELISA, cell viability was determined by MTT assay, morphological changes and apoptosis were examined by inverted microscope and TUNEL respectively. There was a 400 bp balteum in pshRNA1-3 after cut by SalI, which was identical with the size of the objective gene. Many cells presented green fluorescence after being treated by pshRNA1-4, but there are much more dead green fluorescent cells in the pshRNA1 and pshRNA2 group. hTERT protein and telomerase activity was significantly decreased after treated by pshRNA1 or pshRNA2. It was observed that treatment with pshRNA1 or pshRNA2 in the presence of a valid transfection reagent could reduce cell viability of Hep-2 cells within 96 h (P interference may be a promising strategy for the treatment of laryngeal cancer.

  13. Inhibition of full length Hepatitis C Virus particles of 1a genotype through small interference RNA

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    Rehman Sidra

    2011-05-01

    Full Text Available Abstract Background Hepatitis C virus (HCV, a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Currently, the only treatment available consists of a combination of Pegylated interferon alpha (INF-α and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV infection. Results This study was design to assess or explore the silencing effect of small interference RNAs (siRNAs against full length HCV particles of genotype 1a. In the present study six 21-bp siRNAs were designed against different regions of HCV structural genes (Core, E1 and E2. Selected siRNAs were labeled as Csi 301, Csi 29, E1si 52, E1si 192, E2si 86 and E2si 493. Our results demonstrated that siRNAs directed against HCV core gene showed 70% reduction in viral titer in HCV infected liver cells. Moreover, siRNAs against E1 and E2 envelop genes showed a dramatic reduction in HCV viral RNA, E2si 86 exhibited 93% inhibition, while E1si 192, E2si 493 and E1si 52 showed 87%, 80%, and 66% inhibition respectively. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggested that siRNAs targeted against HCV structural genes efficiently silence full length HCV particles and provide an effective therapeutic option against HCV infection.

  14. C6/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response.

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    Doug E Brackney

    2010-10-01

    Full Text Available Mosquitoes rely on RNA interference (RNAi as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV infection in C6/36 (Aedes albopictus cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses. Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae, Sindbis virus (SINV, Togaviridae and La Crosse virus (LACV, Bunyaviridae and total RNA recovered from cell lysates. Small RNA (sRNA libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26-27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand and distribution (position along viral genome of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level.

  15. Cooler temperatures destabilize RNA interference and increase susceptibility of disease vector mosquitoes to viral infection.

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    Zach N Adelman

    Full Text Available The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus, exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery.We utilized transgenic "sensor" strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor" strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2 or Argonaute-2 (AGO-2. We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses.This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.

  16. RNA interference inhibits herpes simplex virus type 1 isolated from saliva samples and mucocutaneous lesions.

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    Silva, Amanda Perse da; Lopes, Juliana Freitas; Paula, Vanessa Salete de

    2014-01-01

    The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplex virus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three different small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene (sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large subunit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplex virus type-1 was isolated from saliva samples and mucocutaneous lesions from infected patients. All mucocutaneous lesions' samples were positive for herpes simplex virus type-1 by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva samples were positive by real-time PCR and 50% were positive by virus isolation. The levels of herpes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-time PCR, whose results demonstrated that the effect of siRNAs on gene expression depends on siRNA concentration. The three siRNA sequences used were able to inhibit viral replication, assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1 was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replication. The results demonstrate that silencing herpes simplex virus type-1 UL39 expression by siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNA based antiviral strategy may be a potential therapeutic alternative. Copyright © 2014. Published by Elsevier Editora Ltda.

  17. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

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    Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

    2014-05-06

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

  18. RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing.

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    Yoo, Aeri; Park, Hyun-Min; Kang, Soon-Suk; Kim, Eun-Soon; Tchah, Hungwon; Kim, Jae Yong

    2016-09-27

    Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

  19. RNA Interference: A Novel Source of Resistance to Combat Plant Parasitic Nematodes

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    Sagar Banerjee

    2017-05-01

    Full Text Available Plant parasitic nematodes cause severe damage and yield loss in major crops all over the world. Available control strategies include use of insecticides/nematicides but these have proved detrimental to the environment, while other strategies like crop rotation and resistant cultivars have serious limitations. This scenario provides an opportunity for the utilization of technological advances like RNA interference (RNAi to engineer resistance against these devastating parasites. First demonstrated in the model free living nematode, Caenorhabtidis elegans; the phenomenon of RNAi has been successfully used to suppress essential genes of plant parasitic nematodes involved in parasitism, nematode development and mRNA metabolism. Synthetic neurotransmitants mixed with dsRNA solutions are used for in vitro RNAi in plant parasitic nematodes with significant success. However, host delivered in planta RNAi has proved to be a pioneering phenomenon to deliver dsRNAs to feeding nematodes and silence the target genes to achieve resistance. Highly enriched genomic databases are exploited to limit off target effects and ensure sequence specific silencing. Technological advances like gene stacking and use of nematode inducible and tissue specific promoters can further enhance the utility of RNAi based transgenics against plant parasitic nematodes.

  20. Effective inhibition of porcine epidemic diarrhea virus by RNA interference in vitro.

    Science.gov (United States)

    Shen, Haiyan; Zhang, Chunhong; Guo, Pengju; Liu, Zhicheng; Zhang, Jianfeng

    2015-10-01

    Porcine epidemic diarrhea virus (PEDV) is a member of the coronaviridae family, which can cause acute and highly contagious enteric disease of swine characterized by severe entero-pathogenic diarrhea in piglets. Currently, the vaccines of PEDV are only partially effective and there is no specific drug available for treatment of PEDV infection. To exploit the possibility of using RNA interference (RNAi) as a strategy against PEDV infection, five shRNA-expressing plasmids targeting the N, M, and S genes of PEDV were constructed and transfected into Vero cells. The cytopathic effect and MTS assays demonstrated that two shRNAs (pSilencer4.1-M1 and pSilencer4.1-N) were capable of protecting cells against PEDV invasion with very high specificity and efficiency. The two shRNA expression plasmids were also able to inhibit the PEDV replication significantly, as shown by detection of virus titers (TCID50/mL). A real-time quantitative RT-PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with these two plasmids were reduced by 95.0 %. Our results suggest that RNAi might be a promising new strategy against PEDV infection.

  1. Potent inhibition of Hendra virus infection via RNA interference and poly I:C immune activation.

    Science.gov (United States)

    McCaskill, Jana L; Marsh, Glenn A; Monaghan, Paul; Wang, Lin-Fa; Doran, Timothy; McMillan, Nigel A J

    2013-01-01

    Hendra virus (HeV) is a highly pathogenic zoonotic paramyxovirus that causes fatal disease in a wide range of species, including humans. HeV was first described in Australia in 1994, and has continued to re-emerge with increasing frequency. HeV is of significant concern to human health due to its high mortality rate, increasing emergence, absence of vaccines and limited post exposure therapies. Here we investigate the use of RNA interference (RNAi) based therapeutics targeting HeV in conjunction with the TLR3 agonist Poly I:C and show that they are potent inhibitors of HeV infection in vitro. We found that short interfering RNAs (siRNAs) targeting the abundantly expressed N, P and M genes of HeV caused over 95% reduction of HeV virus titre, protein and mRNA. Furthermore, we found that the combination of HeV targeting siRNA and Poly I:C had an additive effect in suppressing HeV infection. Our results demonstrate for the first time that RNAi and type I interferon stimulation are effective inhibitors of HeV replication in vitro and may provide an effective therapy for this highly lethal, zoonotic pathogen.

  2. Potent inhibition of Hendra virus infection via RNA interference and poly I:C immune activation.

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    Jana L McCaskill

    Full Text Available Hendra virus (HeV is a highly pathogenic zoonotic paramyxovirus that causes fatal disease in a wide range of species, including humans. HeV was first described in Australia in 1994, and has continued to re-emerge with increasing frequency. HeV is of significant concern to human health due to its high mortality rate, increasing emergence, absence of vaccines and limited post exposure therapies. Here we investigate the use of RNA interference (RNAi based therapeutics targeting HeV in conjunction with the TLR3 agonist Poly I:C and show that they are potent inhibitors of HeV infection in vitro. We found that short interfering RNAs (siRNAs targeting the abundantly expressed N, P and M genes of HeV caused over 95% reduction of HeV virus titre, protein and mRNA. Furthermore, we found that the combination of HeV targeting siRNA and Poly I:C had an additive effect in suppressing HeV infection. Our results demonstrate for the first time that RNAi and type I interferon stimulation are effective inhibitors of HeV replication in vitro and may provide an effective therapy for this highly lethal, zoonotic pathogen.

  3. [Lentiviral vector-mediated RNA interfere gene Nogo receptor to repair spinal cord injury].

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    Lü, Bi-tao; Yuan, Wen; Xu, Sheng-ming

    2010-10-15

    To evaluate the effects of lentiviral vector-mediated RNA interfere gene Nogo receptor (NgR) of rat cortical neurons in repairing spinal cord injury. The recombinant-lentiviral vector with small inferring RNA siNgR199 which had been constructed was transfected into rat cortical neuron cells in vitro in 3 multiplicity of infection (MOI). The infection rate was determined with fluorescent microscope, and the target gene was detected by PCR analysis. Then, the recombinant was injected into the cortical motor area of the rats with severe spinal cord injury, and the saline was also injected into other rats with severe spinal cord injury as a match control. The functional recovery of the rats' hindlimb was assessed using BBB score and the nerve fiber of the injured region was observed by nerve tracing. The rate of recombinant infecting rat cortical neuron in vitro exceeded 99%. PCR analysis confirmed that the effect of lentiviral vector-mediated RNA interfering gene NgR of rat cortical neurons in vitro was 61%. Although all rats with spinal cord injury were observed to have the hindlimb functional recovery, these rats injected with recombinant had better hindlimb functional recovery than others showing by more BBB score (P vector with siNgR199 which had been constructed is able to promote the growth of nerve fiber and the functional recovery of the rats' hindlimb.

  4. Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes.

    Science.gov (United States)

    Göertz, G P; Fros, J J; Miesen, P; Vogels, C B F; van der Bent, M L; Geertsema, C; Koenraadt, C J M; van Rij, R P; van Oers, M M; Pijlman, G P

    2016-11-15

    Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5'-3' exoribonuclease XRN1/Pacman on conserved RNA structures in the 3' untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo Two reproducible small-RNA hot spots within the 3' UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3' SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production. Understanding the flavivirus transmission

  5. Large-Scale RNA Interference Screening to Identify Transcriptional Regulators of a Tumor Suppressor Gene.

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    Forloni, Matteo; Ho, Thuy; Sun, Lisha; Wajapeyee, Narendra

    2017-01-01

    RNA interference (RNAi) is a powerful research tool that can be used to silence the expression of a specific gene. In the past several years, RNAi has provided the opportunity to identify factors and pathways involved in complex biological processes by performing unbiased loss-of-function screens on a genome-wide scale. Here we describe a genome-wide RNAi screening strategy to identify factors that regulates epigenetic silencing of a specific tumor suppressor gene, using RASSF1A as an example. The approach we describe is a general RNAi screening strategy that can be applied to identify other factors that drive and/or maintain epigenetic modifications on specific genes, including cancer-related genes.

  6. Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene.

    Science.gov (United States)

    Yeganeh, Meghdad; Praz, Viviane; Cousin, Pascal; Hernandez, Nouria

    2017-02-15

    Overlapping gene arrangements can potentially contribute to gene expression regulation. A mammalian interspersed repeat (MIR) nested in antisense orientation within the first intron of the Polr3e gene, encoding an RNA polymerase III (Pol III) subunit, is conserved in mammals and highly occupied by Pol III. Using a fluorescence assay, CRISPR/Cas9-mediated deletion of the MIR in mouse embryonic stem cells, and chromatin immunoprecipitation assays, we show that the MIR affects Polr3e expression through transcriptional interference. Our study reveals a mechanism by which a Pol II gene can be regulated at the transcription elongation level by transcription of an embedded antisense Pol III gene. © 2017 Yeganeh et al.; Published by Cold Spring Harbor Laboratory Press.

  7. Down-Regulated Expression of RACK1 Gene by RNA Interference Enhances Drought Tolerance in Rice

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    Da-hong LI

    2009-03-01

    Full Text Available The receptor for activated C-kinase 1 (RACK1 is a highly conserved scaffold protein with versatile functions, and plays important roles in the regulation of plant growth and development. Transgenic rice plants, in which the expression of RACK1 gene was inhibited by RNA interference (RNAi, were studied to elucidate the possible functions of RACK1 in responses to drought stress in rice. Real-time PCR analysis showed that the expression of RACK1 in transgenic rice plants was inhibited by more than 50%. The tolerance to drought stress of the transgenic rice plants was higher as compared with the non-transgenic rice plants. The peroxidation of membrane and the production of malondialdehyde were significantly lower, and the superoxide dismutase activity in transgenic rice plants was significantly higher than those in non-trangenic rice plants. It is suggested that RACK1 negatively regulated the redox system-related tolerance to drought stress of rice plants.

  8. Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference

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    Barrangou, Rodolphe; Birmingham, Amanda; Wiemann, Stefan; Beijersbergen, Roderick L.; Hornung, Veit; Smith, Anja van Brabant

    2015-01-01

    The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today's challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications. PMID:25800748

  9. Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference.

    Science.gov (United States)

    Barrangou, Rodolphe; Birmingham, Amanda; Wiemann, Stefan; Beijersbergen, Roderick L; Hornung, Veit; Smith, Anja van Brabant

    2015-04-20

    The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today's challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Effects of RNA interference-mediated NRP-1 silencing on the proliferation and apoptosis of breast cancer cells.

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    Han, Zhengxiang; Jiang, Guan; Zhang, Yingying; Xu, Jie; Chen, Chong; Zhang, Lansheng; Xu, Zhenyuan; Du, Xiuping

    2015-07-01

    Lentiviral expression vectors carrying human NRP-1 short hairpin RNA (shRNA) were constructed and selected to present highly efficient NRP-1/shRNA interference sequences, in order to investigate the effects of RNA interference (RNAi)-mediated NRP-1 silencing on the biological activities of breast cancer cells. Three pairs of human NRP-1 targeted specific interference sequences and one pair of non-specific control sequences were designed, synthesized and subcloned into pLB lentiviral vectors, which were further identified by polymerase chain reaction (PCR) and sequencing. Recombinant and lentiviral packaging plasmids were co-transfected into 293FT cell lines in order to produce lentiviral particles and to infect breast cancer cells with high NRP-1 expression. Flow cytometry was used to sort green fluorescent protein-positive cells. Fluorescence quantitative-reverse transcription-PCR and western blot analysis were employed to identify the interference silencing sequence with the most efficient silencing profile. A cell counting kit-8 assay and an Annexin V-propidium iodide method in combination with flow cytometry were used to examine the effects of RNA interference-mediated NRP-1 gene silencing on cell proliferation, apoptosis and sensitivity to chemotherapy. The recombinant lentiviral plasmid pLB-NRP-1/shRNA was constructed successfully, as confirmed by PCR and sequencing. After the infection of recombinant lentiviral plasmids, the expression profiles of NRP-1 mRNA, and proteins of MCF-7 and SK-BR-3 cell-specific interference group (pLB-NRP-1/shRNA3) were significantly lower than that of the control group (Pinterference group (pLB-NRP-1/shRNA3) showed lower optical density values and higher apoptotic rates at 48, 72 and 96 h; these differences were statistically significant (Pinterference groups compared with the control group (Pinterference sequences were selected. Furthermore, RNA interference (RNAi)-mediated NRP-1 silencing may induce proliferation suppression

  11. RNA interference of gonadotropin-inhibitory hormone gene induces arousal in songbirds.

    Directory of Open Access Journals (Sweden)

    Takayoshi Ubuka

    Full Text Available Gonadotropin-inhibitory hormone (GnIH was originally identified in quail as a hypothalamic neuropeptide inhibitor of pituitary gonadotropin synthesis and release. However, GnIH neuronal fibers do not only terminate in the median eminence to control anterior pituitary function but also extend widely in the brain, suggesting it has multiple roles in the regulation of behavior. To identify the role of GnIH neurons in the regulation of behavior, we investigated the effect of RNA interference (RNAi of the GnIH gene on the behavior of white-crowned sparrows, a highly social songbird species. Administration of small interfering RNA against GnIH precursor mRNA into the third ventricle of male and female birds reduced resting time, spontaneous production of complex vocalizations, and stimulated brief agonistic vocalizations. GnIH RNAi further enhanced song production of short duration in male birds when they were challenged by playbacks of novel male songs. These behaviors resembled those of breeding birds during territorial defense. The overall results suggest that GnIH gene silencing induces arousal. In addition, the activities of male and female birds were negatively correlated with GnIH mRNA expression in the paraventricular nucleus. Density of GnIH neuronal fibers in the ventral tegmental area was decreased by GnIH RNAi treatment in female birds, and the number of gonadotropin-releasing hormone neurons that received close appositions of GnIH neuronal fiber terminals was negatively correlated with the activity of male birds. In summary, GnIH may decrease arousal level resulting in the inhibition of specific motivated behavior such as in reproductive contexts.

  12. Heterotopia formation in rat but not mouse neocortex after RNA interference knockdown of DCX.

    Science.gov (United States)

    Ramos, Raddy L; Bai, Jilin; LoTurco, Joseph J

    2006-09-01

    Subcortical band heterotopia (SBH) or double cortex is associated with significant impairments in neocortical function including mental retardation and epilepsy. Mutant alleles of DCX in humans typically cause SBH in females and lissencephaly in males, whereas Dcx null mutations in mice neither disrupt neocortical neuronal migration nor cause SBH formation. In utero RNA interference (RNAi) of Dcx in rats, in contrast, creates an animal model of SBH. Possible explanations for the discrepancies in results following loss of Dcx function include species differences and/or differences between RNAi knockdown and genetic deletion. We have carried out a series of in utero RNAi experiments to investigate possible species differences between rat and mouse to determine the molecular specificity of RNAi against Dcx and to identify the cellular constituents of SBH in the rat model. In utero RNAi in the rat consistently leads to both the formation of SBH and laminar displacement of transfected cells in normotopic cortex, whereas the same treatment in mouse fails to induce SBH but does create laminar displacement. Induction of SBH and impaired radial migration following RNAi against Dcx is rescued by overexpression of Dcx. Thus, both disruptions induced by RNAi are specific to interference of Dcx. SBHs contain transfected pyramidal cells as well as nontransfected cell types, including neocortical interneurons and glia. Together these results indicate that there is a species difference between rat and mouse with respect to RNAi-induced SBH formation and that SBH formation involves the recruitment of several unaltered cell types.

  13. A novel approach for inhibition of HIV-1 by RNA interference: counteracting viral escape with a second generation of siRNAs

    NARCIS (Netherlands)

    ter Brake, Olivier; Berkhout, Ben

    2005-01-01

    RNA interference (RNAi) is an evolutionary conserved gene silencing mechanism in which small interfering RNA (siRNA) mediates the sequence specific degradation of mRNA. The recent discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the possibility to use this

  14. Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae.

    Science.gov (United States)

    Williams, Thomas C; Espinosa, Monica I; Nielsen, Lars K; Vickers, Claudia E

    2015-04-01

    Engineering dynamic, environmentally- and temporally-responsive control of gene expression is one of the principle objectives in the field of synthetic biology. Dynamic regulation is desirable because many engineered functions conflict with endogenous processes which have evolved to facilitate growth and survival, and minimising conflict between growth and production phases can improve product titres in microbial cell factories. There are a limited number of mechanisms that enable dynamic regulation in yeast, and fewer still that are appropriate for application in an industrial setting. To address this problem we have identified promoters that are repressed during growth on glucose, and activated during growth on sucrose. Catabolite repression and preferential glucose utilisation allows active growth on glucose before switching to production on sucrose. Using sucrose as an activator of gene expression circumvents the need for expensive inducer compounds and enables gene expression to be triggered during growth on a fermentable, high energy-yield carbon source. The ability to fine-tune the timing and population density at which gene expression is activated from the SUC2 promoter was demonstrated by varying the ratio of glucose to sucrose in the growth medium. Finally, we demonstrated that the system could also be used to repress gene expression (a process also required for many engineering projects). We used the glucose/sucrose system to control a heterologous RNA interference module and dynamically repress the expression of a constitutively regulated GFP gene. The low noise levels and high dynamic range of the SUC2 promoter make it a promising option for implementing dynamic regulation in yeast. The capacity to repress gene expression using RNA interference makes the system highly versatile, with great potential for metabolic engineering applications.

  15. Engineering host-derived resistance against plant parasites through RNA interference: challenges and opportunities.

    Science.gov (United States)

    Runo, Steven

    2011-01-01

    RNA interference (RNAi) has rapidly advanced to become a powerful genetic tool and holds promise to revolutionizing agriculture by providing a strategy for controlling a wide array of crop pests. Numerous studies document RNAi efficacy in achieving silencing in viruses, insects, nematodes and weeds parasitizing crops. In general, host derived pest resistance through RNAi is achieved by genetically transforming host plants with double stranded RNA constructs targeted at essential parasite genes leading to generation of small interfering RNAs (siRNAs). Small interfering RNAs formed in the host are then delivered to the parasite and transported to target cells. Delivery can be oral - worms and insects, viral infections, viruses - or through a vascular connections - parasitic plants, while delivery to target cells is by cell to cell systemic movement of the silencing signal. Despite the overall optimism in generating pest resistant crops through RNAi-mediated silencing, some hurdles have recently begun to emerge. Presently, the main challenge is delivery of sufficient siRNAs, in the right cells, and at the right time to mount; a strong, durable, and broad-spectrum posttranscriptional gene silencing (PTGS) signal. This review highlights the novel strategies available for improving host derived RNAi resistance in downstream applied agriculture.

  16. From The Cover: Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation

    Science.gov (United States)

    Nollen, Ellen A. A.; Garcia, Susana M.; van Haaften, Gijs; Kim, Soojin; Chavez, Alejandro; Morimoto, Richard I.; Plasterk, Ronald H. A.

    2004-04-01

    Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation. protein misfolding | neurodegenerative diseases

  17. Fluorescence Reporter-Based Genome-Wide RNA Interference Screening to Identify Alternative Splicing Regulators.

    Science.gov (United States)

    Misra, Ashish; Green, Michael R

    2017-01-01

    Alternative splicing is a regulated process that leads to inclusion or exclusion of particular exons in a pre-mRNA transcript, resulting in multiple protein isoforms being encoded by a single gene. With more than 90 % of human genes known to undergo alternative splicing, it represents a major source for biological diversity inside cells. Although in vitro splicing assays have revealed insights into the mechanisms regulating individual alternative splicing events, our global understanding of alternative splicing regulation is still evolving. In recent years, genome-wide RNA interference (RNAi) screening has transformed biological research by enabling genome-scale loss-of-function screens in cultured cells and model organisms. In addition to resulting in the identification of new cellular pathways and potential drug targets, these screens have also uncovered many previously unknown mechanisms regulating alternative splicing. Here, we describe a method for the identification of alternative splicing regulators using genome-wide RNAi screening, as well as assays for further validation of the identified candidates. With modifications, this method can also be adapted to study the splicing regulation of pre-mRNAs that contain two or more splice isoforms.

  18. Engineered Hydrogels for Local and Sustained Delivery of RNA-Interference Therapies.

    Science.gov (United States)

    Wang, Leo L; Burdick, Jason A

    2017-01-01

    It has been nearly two decades since RNA-interference (RNAi) was first reported. While there are no approved clinical uses, several phase II and III clinical trials suggest the great promise of RNAi therapeutics. One challenge for RNAi therapies is the controlled localization and sustained presentation to target tissues, to both overcome systemic toxicity concerns and to enhance in vivo efficacy. One approach that is emerging to address these limitations is the entrapment of RNAi molecules within hydrogels for local and sustained release. In these systems, nucleic acids are either delivered as siRNA conjugates or within nanoparticles. A plethora of hydrogels has been implemented using these approaches, including both traditional hydrogels that have already been developed for other applications and new hydrogels developed specifically for RNAi delivery. These hydrogels have been applied to various applications in vivo, including cancer, bone regeneration, inflammation and cardiac repair. This review will examine the design and implementation of such hydrogel RNAi systems and will cover the most recent applications of these systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Inhibition of cisplatin-resistance by RNA interference targeting metallothionein using reducible oligo-peptoplex.

    Science.gov (United States)

    Lee, Jong-Hwan; Chae, Ji-Won; Kim, Jang Kyoung; Kim, Hyung Jin; Chung, Jee Young; Kim, Yong-Hee

    2015-10-10

    Effective intracellular level of a platinum anti-cancer drug, cisplatin, following repeated injections can be decreased either by the active efflux via ATP pump or by interactions with glutathione and metallothionein. Cisplatin in cytoplasm preferably binds to cysteine-rich proteins such as glutathione and metallothionein (MT). Detoxification of cisplatin by intracellular thiol-containing proteins has been considered to be major hurdles to overcome. The short hairpin RNA targeting MT (shMT) was tested to down-regulate MT and recover cisplatin resistance. A reducible polymer, poly(oligo-d-arginine) (rPOA), formed stable complex with shMT and demonstrated superior transfection efficiency. Efficient transfection of shMT/rPOA oligo-peptoplexes was found to significantly inhibit MT over-expression, resulting in 45% decrease of cell viability compared to the cisplatin alone group. This decrease was mediated by the synergistic effect of shMT/rPOA oligo-peptoplex and cisplatin. Co-administration of shMT/rPOA oligo-peptoplex and cisplatin in in vivo tumor model showed noticeable tumor-suppressing effect by inducing reversal of cisplatin resistance following effective intracellular delivery of shMT by rPOA. Combination therapy through co-administration of shMT/rPOA oligo-peptoplex and cisplatin was found to effectively reverse cisplatin resistance by RNA interference and consequently improve anti-cancer activity of cisplatin. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. RNA interference for glioblastoma therapy: Innovation ladder from the bench to clinical trials.

    Science.gov (United States)

    Lozada-Delgado, Eunice L; Grafals-Ruiz, Nilmary; Vivas-Mejía, Pablo E

    2017-11-01

    Glioblastoma multiforme (GBM) is the most common and deadliest type of primary brain tumor with a prognosis of 14months after diagnosis. Current treatment for GBM patients includes "total" tumor resection, temozolomide-based chemotherapy, radiotherapy or a combination of these options. Although, several targeted therapies, gene therapy, and immunotherapy are currently in the clinic and/or in clinical trials, the overall survival of GBM patients has hardly improved over the last two decades. Therefore, novel multitarget modalities are urgently needed. Recently, RNA interference (RNAi) has emerged as a novel strategy for the treatment of most cancers, including GBM. RNAi-based therapies consist of using small RNA oligonucleotides to regulate protein expression at the post-transcriptional level. Despite the therapeutic potential of RNAi molecules, systemic limitations including short circulatory stability and low release into the tumor tissue have halted their progress to the clinic. The effective delivery of RNAi molecules through the blood-brain barrier (BBB) represents an additional challenge. This review focuses on connecting the translational process of RNAi-based therapies from in vitro evidence to pre-clinical studies. We delineate the effect of RNAi in GBM cell lines, describe their effectiveness in glioma mouse models, and compare the proposed drug carriers for the effective transport of RNAi molecules through the BBB to reach the tumor in the brain. Furthermore, we summarize the most important obstacles to overcome before RNAi-based therapy becomes a reality for GBM treatment. Published by Elsevier Inc.

  1. MIMEAnTo: profiling functional RNA in mutational interference mapping experiments.

    Science.gov (United States)

    Smith, Maureen R; Smyth, Redmond P; Marquet, Roland; von Kleist, Max

    2016-11-01

    The mutational interference mapping experiment (MIME) is a powerful method that, coupled to a bioinformatics analysis pipeline, allows the identification of domains and structures in RNA that are important for its function. In MIME, target RNAs are randomly mutated, selected by function, physically separated and sequenced using next-generation sequencing (NGS). Quantitative effects of each mutation at each position in the RNA can be recovered with statistical certainty using the herein developed user-friendly, cross-platform software MIMEAnTo (MIME Analysis Tool). MIMEAnTo is implemented in C ++ using the boost library as well as Qt for the graphical user interface and is distributed under GPL (http://www.gnu.org/licences/gpl). The libraries are statically linked in a stand alone executable and are not required on the system. The plots are generated with gnuplot. Gnuplot-iostream (https://github.com/dstahlke/gnuplot-iostream) serves as gnuplot interface. Standalone executables including examples and source code can be downloaded from https://github.com/maureensmith/MIMEAnTo CONTACTS: msmith@zedat.fu-berlin.de or vkleist@zedat.fu-berlin.deSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. RNA interference technology used for the study of aquatic virus infections.

    Science.gov (United States)

    Reshi, Mohammad Latif; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2014-09-01

    Aquaculture is one of the most important economic activities in Asia and is presently the fastest growing sector of food production in the world. Explosive increases in global fish farming have been accompanied by an increase in viral diseases. Viral infections are responsible for huge economic losses in fish farming, and control of these viral diseases in aquaculture remains a serious challenge. Recent advances in biotechnology have had a significant impact on disease reduction in aquaculture. RNAi is one of the most important technological breakthroughs in modern biology, allowing us to directly observe the effects of the loss of specific genes in living systems. RNA interference technology has emerged as a powerful tool for manipulating gene expression in the laboratory. This technology represents a new therapeutic approach for treating aquatic diseases, including viral infections. RNAi technology is based on a naturally occurring post-transcriptional gene silencing process mediated by the formation of dsRNA. RNAi has been proven widely effective for gene knockdown in mammalian cultured cells, but its utility in fish remains unexplored. This review aims to highlight the RNAi technology that has made significant contributions toward the improvement of aquatic animal health and will also summarize the current status and future strategies concerning the therapeutic applications of RNAi to combat viral disease in aquacultured organisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. RNA interference: a promising technique for the improvement of traditional crops.

    Science.gov (United States)

    Katoch, Rajan; Thakur, Neelam

    2013-03-01

    RNA interference (RNAi) is a homology-dependent gene-silencing technology that involves double-stranded RNA directed against a target gene. This technique has emerged as powerful tool in understanding the functions of a number of genes in recent years. For the improvement in the nutritional status of the plants and reduction in the level of antinutrients, the conventional breeding methods were not completely successful in achieving the tissue-specific regulation of some genes. RNAi has shown successful results in a number of plant species for nutritional improvement, change in morphology and alteration in metabolite synthesis. This technology has been applied mostly in genetic engineering of important crop plants, and till date there are no reports of its application for the improvement of traditional/underutilized crops. In this study, we discuss current knowledge of RNAi function and concept and strategies for the improvement of traditional crops. Practical application. Although RNAi has been extensively used for the improvement of popular crops, no attention has been given for the use of this technology for the improvement of underutilized crops. This study describes the importance of use of this technology for the improvement of underutilized crops.

  4. RNA interference as a resistance mechanism against crop parasites in Africa: a 'Trojan horse' approach.

    Science.gov (United States)

    Runo, Steven; Alakonya, Amos; Machuka, Jesse; Sinha, Neelima

    2011-02-01

    Biological crop pests cause serious economic losses. In Africa, the most prevalent parasites are insect pests, plant pathogenic root-knot nematodes, viruses and parasitic plants. African smallholder farmers struggle to overcome these parasitic constraints to agricultural production. Crop losses and the host range of these parasites have continued to increase in spite of the use of widely advocated control methods. A sustainable method to overcome biological pests in Africa would be to develop crop germplasm resistant to parasites. This is achievable using either genetic modification (GM) or a non-GM approach. However, there is a paucity of resistant genes available for introduction. Additionally, the biological processes underpinning host parasite resistance are not sufficiently well understood. The authors review a technology platform for using RNA-mediated interference (RNAi) as bioengineered resistance to important crop parasites in Africa. To achieve acquired resistance, a host crop is stably transformed with a transgene that encodes a hairpin RNA targeting essential parasitic genes. The RNAi sequence is chosen in such a way that it shares no homology with the host's genes, so it remains 'inactive' until parasitism. Upon parasitism, the RNAi sequence enters the parasite and post-transcriptional gene silencing (PTGS) mechanisms are activated, leading to the death of the parasite. Copyright © 2010 Society of Chemical Industry.

  5. RNA interference technology to control pest sea lampreys--a proof-of-concept.

    Directory of Open Access Journals (Sweden)

    George Heath

    Full Text Available The parasitic sea lamprey (Petromyzon marinus has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin reduced transcript levels 2.5, 3.6, and 5.0-fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species.

  6. RNA interference technology to control pest sea lampreys--a proof-of-concept.

    Science.gov (United States)

    Heath, George; Childs, Darcy; Docker, Margaret F; McCauley, David W; Whyard, Steven

    2014-01-01

    The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0-fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species.

  7. Design and Construction of Shrimp Antiviral DNA Vaccines Expressing Long and Short Hairpins for Protection by RNA Interference.

    Science.gov (United States)

    Chaudhari, Aparna; Pathakota, Gireesh-Babu; Annam, Pavan-Kumar

    2016-01-01

    DNA vaccines present the aquaculture industry with an effective and economically viable method of controlling viral pathogens that drastically affect productivity. Since specific immune response is rudimentary in invertebrates, the presence of RNA interference (RNAi) pathway in shrimps provides a promising new approach to vaccination. Plasmid DNA vaccines that express short or long double stranded RNA in vivo have shown protection against viral diseases. The design, construction and considerations for preparing such vaccines are discussed.

  8. Small Interference RNA Targeting TLR4 Gene Effectively Attenuates Pulmonary Inflammation in a Rat Model

    Directory of Open Access Journals (Sweden)

    Feixiang Wu

    2012-01-01

    Full Text Available Objective. The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA targeting Toll-like receptor 4 (TLR4 gene in ameliorating lipopolysaccharide- (LPS- induced acute lung injury (ALI. Methods. In vitro, alveolar macrophages (AMs were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group, 300 μL of Ad-EGFP (Ad-EGFP group, or 300 μL of Ad-siTLR4 (Ad-siTLR4 group and then were intravenously treated with LPS (50 mg/kg to induce ALI. Results. Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals. Conclusion. TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

  9. Development of a microinjection system for RNA interference in the water flea Daphnia pulex.

    Science.gov (United States)

    Hiruta, Chizue; Toyota, Kenji; Miyakawa, Hitoshi; Ogino, Yukiko; Miyagawa, Shinichi; Tatarazako, Norihisa; Shaw, Joseph R; Iguchi, Taisen

    2013-11-05

    The ubiquitous, freshwater microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have a well annotated, reference genome assembly that revealed an unusually high gene count highlighted by a large gene orphanage,-i.e., previously uncharacterized genes. Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic manipulation, but the establishment of gene manipulation techniques is needed to accurately define gene functions. Although previous investigations developed an RNA interference (RNAi) system for one congener D. magna, these methods are not appropriate for D. pulex because of the smaller size of their early embryos. In these studies, we develop RNAi techniques for D. pulex by first determining the optimum culture conditions of their isolated embryos and then applying these conditions to the development of microinjection techniques and proof-of-principle RNAi experiments. We found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mM sucrose dissolved in M4 media, providing optimal conditions for microinjections. Then, we injected double-stranded (ds)RNA specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for limb development in invertebrates and vertebrates. Injected embryos presented with defects in the second antenna and appendage development, and dsRNA induced the degradation of Dll mRNAs, indicating that this technique successfully inhibited transcription of the target gene. We developed a microinjection system for RNAi studies in D. pulex. These techniques add to the growing genomic toolbox and enhance the genetic tractability of this important model for environmental, evolutionary, and developmental genomics.

  10. [Effects of RNA interference silencing RhoC gene upon paclitaxel sensitivity in ovarian cancer cell lines].

    Science.gov (United States)

    Pan, Ying; Zhang, Wen-ying; Zou, Ji-yan; Sheng, Min-jia; Xuan, Li-li; Hai, De-yang

    2009-06-02

    To explore the changes of paclitaxel sensitivity by RNA interference of RhoC gene in ovarian cancer cell lines. The microRNA (miRNA) targeting RhoC gene was designed and synthesized by in vitro transcription and transfected into ovarian cancer cell line SKOV3. The mRNA and protein of RhoC were detected by the means of RT-PCR and Western blot. The changes of paclitaxel sensitivity after interference were examined by methyl thiazolyl tetrazolium (MTf) assay. At the same time, the mRNA of apoptosis gene Caspase-3 and survivin were evaluated by RT-PCR. In SKOV3 cell, the mRNA and protein of RhoC dramatically decreased at 24, 48 and 72 hours post-transfection. The sensitivity of SKOV3 cell line to paclitaxel increased significantly after turning off the RhoC gene. The proapoptotic gene Caspase-3 increased significantly and anti-apoptosis gene survivin dramatically decreased after the combination treatment of paclitaxel and RhoC-miRNA in comparison with paclitaxel alone treatment. The sensitivity of ovarian cancer cell lines SKOV3 to paclitaxel can be enhanced by RNA interfering RhoC gene. The synergistic mechanism is possibly correlated with the up-regulation of Caspase-3 gene and the down-regulation of survivin gene.

  11. RNA Interference Based Approach to Down Regulate Osmoregulators of Whitefly (Bemisia tabaci): Potential Technology for the Control of Whitefly

    Science.gov (United States)

    Over the past decade RNA interference (RNAi) technology has emerged as a successful tool not only for functional genomics, but in planta expression of short interfering RNAs (siRNAs) could offer potential for insect pest management. Insects feeding exclusively on plant sap depend on osmotic pressure...

  12. Antiviral RNA Interference against Orsay Virus Is neither Systemic nor Transgenerational in Caenorhabditis elegans.

    Science.gov (United States)

    Ashe, Alyson; Sarkies, Peter; Le Pen, Jérémie; Tanguy, Mélanie; Miska, Eric A

    2015-12-01

    Antiviral RNA-mediated silencing (RNA interference [RNAi]) acts as a powerful innate immunity defense in plants, invertebrates, and mammals. In Caenorhabditis elegans, RNAi is systemic; i.e., RNAi silencing signals can move between cells and tissues. Furthermore, RNAi effects can be inherited transgenerationally and may last for many generations. Neither the biological relevance of systemic RNAi nor transgenerational RNAi is currently understood. Here we examined the role of both pathways in the protection of C. elegans from viral infection. We studied the Orsay virus, a positive-strand RNA virus related to Nodaviridae and the first and only virus known to infect C. elegans. Immunity to Orsay virus infection requires the RNAi pathway. Surprisingly, we found that genes required for systemic or transgenerational RNAi did not have a role in antiviral defense. Furthermore, we found that Orsay virus infection did not elicit a systemic RNAi response even when a target for RNAi was provided by using transgenes. Finally, we show that viral siRNAs, the effectors of RNAi, are not inherited to a level that provides any significant resistance to viral infection in the next generation. We conclude that systemic or transgenerational RNAi does not play a role in the defense against natural Orsay virus infection. Furthermore, our data suggest that there is a qualitative difference between experimental RNAi and antiviral RNAi. Our data are consistent with a model of systemic and transgenerational RNAi that requires a nuclear or germ line component that is lacking in almost all RNA virus infections. Since its discovery in Caenorhabditis elegans, RNAi has proven a valuable scientific tool in many organisms. In C. elegans, exogenous RNAi spreads throughout the organism and can be passed between generations; however, there has been controversy as to the endogenous role(s) that the RNAi pathway plays. One endogenous role for which spreading both within the infected organism and between

  13. [Inhibitory effect of RNA interference targeting GFI-1 on the proliferation of atypical chronic myelogenous leukemia NT1 cells].

    Science.gov (United States)

    Yang, X; Liu, H; Lin, Z H; Qian, J; Xu, X R

    2016-08-01

    To investigate the inhibitory effects of RNA interference targeting GFI-1 on growth and proliferation of atypical chronic myelogenous leukemia (aCML) NT1 cells. NT1 cells were transfected with PBS and liposome complex (vehicle group), scrambled siRNA and liposome complex (negative control, NC group), and GFI-1 siRNA and liposome complex (GFI-1 siRNA group), respectively. Real-time quantitative RT-PCR (qRT-PCR) and Western blot were performed to examine the expression levels of GFI-1 mRNA and protein, respectively. The proliferation abilities of NT1 cells of the three groups were evaluated by MTT assay. The cell cycle in cells of the three groups was analyzed by flow cytometry. Moreover, nude mouse xenograft model was used to detect the tumor formation ability in the three group cells. Quantitative real-time PCR data showed that the expression level of GFI-1 mRNA in GFI-1 siRNA group was significantly lower than those of NC group and vehicle group [(0.367±0.017) vs. (0.918±0.006) and (1.010±0.005), respectively, (PRNA interference targeting GFI-1 inhibits the growth and proliferation of NT1 cells, which may provide a new therapeutic target for atypical chronic myelogenous leukemia.

  14. An in vivo RNA interference screen identifies gene networks controlling Drosophila melanogaster blood cell homeostasis

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    Augé Benoit

    2010-06-01

    Full Text Available Abstract Background In metazoans, the hematopoietic system plays a key role both in normal development and in defense of the organism. In Drosophila, the cellular immune response involves three types of blood cells: plasmatocytes, crystal cells and lamellocytes. This last cell type is barely present in healthy larvae, but its production is strongly induced upon wasp parasitization or in mutant contexts affecting larval blood cell homeostasis. Notably, several zygotic mutations leading to melanotic mass (or "tumor" formation in larvae have been associated to the deregulated differentiation of lamellocytes. To gain further insights into the gene regulatory network and the mechanisms controlling larval blood cell homeostasis, we conducted a tissue-specific loss of function screen using hemocyte-specific Gal4 drivers and UAS-dsRNA transgenic lines. Results By targeting around 10% of the Drosophila genes, this in vivo RNA interference screen allowed us to recover 59 melanotic tumor suppressor genes. In line with previous studies, we show that melanotic tumor formation is associated with the precocious differentiation of stem-cell like blood progenitors in the larval hematopoietic organ (the lymph gland and the spurious differentiation of lamellocytes. We also find that melanotic tumor formation can be elicited by defects either in the fat body, the embryo-derived hemocytes or the lymph gland. In addition, we provide a definitive confirmation that lymph gland is not the only source of lamellocytes as embryo-derived plasmatocytes can differentiate into lamellocytes either upon wasp infection or upon loss of function of the Friend of GATA cofactor U-shaped. Conclusions In this study, we identify 55 genes whose function had not been linked to blood cell development or function before in Drosophila. Moreover our analyses reveal an unanticipated plasticity of embryo-derived plasmatocytes, thereby shedding new light on blood cell lineage relationship, and

  15. Transcriptome analysis in cotton boll weevil (Anthonomus grandis and RNA interference in insect pests.

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    Alexandre Augusto Pereira Firmino

    Full Text Available Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, Anthonomus grandis, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, A. grandis, was analyzed. The A. grandis transcriptome analysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to Tribolium castaneum sequences. A comparison of A. grandis, Drosophila melanogaster and Bombyx mori protein families' data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with T. castaneum SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to A. grandis female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, A. grandis. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects.

  16. Update on the clinical utility of an RNA interference-based treatment: focus on Patisiran

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    Rizk M

    2017-11-01

    Full Text Available Malak Rizk, Şükrü Tüzmen Molecular Biology and Genetics Program, Department of Biological Sciences, Faculty of Arts and Sciences, Eastern Mediterranean University (EMU, Famagusta, North Cyprus, Turkey Abstract: RNA interference (RNAi is a naturally existing endogenous mechanism for posttranscriptional gene regulation, nowadays commonly utilized for functional characterization of genes and development of potential treatment strategies for diseases. RNAi-based studies for therapy, after being examined for over a decade, are finally in the pipeline for developing a potential treatment for the mutated transthyretin (TTR gene, which gives rise to a dysfunctional TTR protein. This dysfunctional protein causes TTR amyloidosis (ATTR, an inherited, progressively incapacitating, and often fatal genetic disorder. TTR is a protein produced in the liver, and functions as a carrier for retinol-binding protein and also thyroxine. This protein facilitates the transport of vitamin A around the human body. A mutation or misprint in the code of this protein results in an abnormal folding of the protein. Therefore, not only does the transportation of the vitamin A become disabled, but also there will be formation of clusters called amyloid deposits, which attack the heart and the nerves causing some patients to be unconditionally bound to bed. ATTR is a hereditary autosomal dominant disease with a 50% chance of inheritance by offspring, even with just one of the parents having a single defective allele of this gene. Alnylam Pharmaceuticals worked on the concept of RNAi therapy for years, which led to the introduction of lipid nanoparticles encircling small interfering RNAs. The drug showed extremely positive results since the first trial, and a great percentage of defective protein reduction. This drug was later named Patisiran. Keywords: RNAi, TTR gene, TTR amyloidosis, siRNA, gene silencing, Patisiran

  17. [Influence of RNA interference targeting against human telomerase reverse transcriptase on expression of C-myc protein].

    Science.gov (United States)

    Chi, Huaming; Tao, Zezhang; Chen, Shiming; Xiao, Bokui; Zhan, Hanzhang

    2005-11-01

    To investigate the effect of inhibiting human telomerase reverse transcriptase (hTERT) on expression of C-myc protein by RNA interference (RNAi) in the larynx cancer cell line, Hep-2. The primary structures of hTERT cDNA were found in GeneBank. Then the structure analyses were done according to the strategy of RNAi, which determined the specific base sequences to design shRNA plasmid. One type of plasmid, pshRNA1, involved in fluorescein gene was synthesized based on the specific base sequence. Control pshRNA2-a random sequence-were also constructed. METAFECTENE was used as the transfect ion reagent. Cells were treated daily with pshRNA1-2 or normal culture medium respectively. After administration of pshRNA1-2, hTERT mRNA was detected by RT-PCR, hTERT protein and C-myc protein were examined by Western Blot. The expression of hTERT mRNA and protein were both significantly decreased after treated by pshRNA1 (P < 0.05). The expression of C-myc protein was significantly increased after treated by pshRNA1 (P < 0.01). The inhibition of hTERT expression could increase the expression of C-myc protein in Hep-2 cells.

  18. LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages.

    Science.gov (United States)

    Tsai, Jin-Wu; Chen, Yu; Kriegstein, Arnold R; Vallee, Richard B

    2005-09-12

    Mutations in the human LIS1 gene cause the smooth brain disease classical lissencephaly. To understand the underlying mechanisms, we conducted in situ live cell imaging analysis of LIS1 function throughout the entire radial migration pathway. In utero electroporation of LIS1 small interference RNA and short hairpin dominant negative LIS1 and dynactin cDNAs caused a dramatic accumulation of multipolar progenitor cells within the subventricular zone of embryonic rat brains. This effect resulted from a complete failure in progression from the multipolar to the migratory bipolar state, as revealed by time-lapse analysis of brain slices. Surprisingly, interkinetic nuclear oscillations in the radial glial progenitors were also abolished, as were cell divisions at the ventricular surface. Those few bipolar cells that reached the intermediate zone also exhibited a complete block in somal translocation, although, remarkably, process extension persisted. Finally, axonal growth also ceased. These results identify multiple distinct and novel roles for LIS1 in nucleokinesis and process dynamics and suggest that nuclear position controls neural progenitor cell division.

  19. RNA-Interference Components Are Dispensable for Transcriptional Silencing of the Drosophila Bithorax-Complex

    KAUST Repository

    Cernilogar, Filippo M.

    2013-06-13

    Background:Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.Principal Findings:Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.Conclusions:We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila. © 2013 Cernilogar et al.

  20. Targeting Th17 Cells with Small Molecules and Small Interference RNA

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    Hui Lin

    2015-01-01

    Full Text Available T helper 17 (Th17 cells play a central role in inflammatory and autoimmune diseases via the production of proinflammatory cytokines interleukin- (IL- 17, IL-17F, and IL-22. Anti-IL-17 monoclonal antibodies show potent efficacy in psoriasis but poor effect in rheumatoid arthritis (RA and Crohn’s disease. Alternative agents targeting Th17 cells may be a better way to inhibit the development and function of Th17 cells than antibodies of blocking a single effector cytokine. Retinoic acid-related orphan receptor gamma t (RORγt which acts as the master transcription factor of Th17 differentiation has been an attractive pharmacologic target for the treatment of Th17-mediated autoimmune disease. Recent progress in technology of chemical screen and engineering nucleic acid enable two new classes of therapeutics targeting RORγt. Chemical screen technology identified several small molecule specific inhibitors of RORγt from a small molecule library. Systematic evolution of ligands by exponential enrichment (SELEX technology enabled target specific aptamers to be isolated from a random sequence oligonucleotide library. In this review, we highlight the development and therapeutic potential of small molecules inhibiting Th17 cells by targeting RORγt and aptamer mediated CD4+ T cell specific delivery of small interference RNA against RORγt gene expression to inhibit pathogenic effector functions of Th17 lineage.

  1. Understanding the core of RNA interference: The dynamic aspects of Argonaute-mediated processes

    KAUST Repository

    Zhu, Lizhe

    2016-10-05

    At the core of RNA interference, the Argonaute proteins (Ago) load and utilize small guide nucleic acids to silence mRNAs or cleave foreign nucleic acids in a sequence specific manner. In recent years, based on extensive structural studies of Ago and its interaction with the nucleic acids, considerable progress has been made to reveal the dynamic aspects of various Ago-mediated processes. Here we review these novel insights into the guide-strand loading, duplex unwinding, and effects of seed mismatch, with a focus on two representative Agos, the human Ago 2 (hAgo2) and the bacterial Thermus thermophilus Ago (TtAgo). In particular, comprehensive molecular simulation studies revealed that although sharing similar overall structures, the two Agos have vastly different conformational landscapes and guide-strand loading mechanisms because of the distinct rigidity of their L1-PAZ hinge. Given the central role of the PAZ motions in regulating the exposure of the nucleic acid binding channel, these findings exemplify the importance of protein motions in distinguishing the overlapping, yet distinct, mechanisms of Ago-mediated processes in different organisms.

  2. Ablation of RNA interference and retrotransposons accompany acquisition and evolution of transposases to heterochromatin protein CENPB

    Science.gov (United States)

    Upadhyay, Udita; Srivastava, Suchita; Khatri, Indu; Nanda, Jagpreet Singh; Subramanian, Srikrishna; Arora, Amit; Singh, Jagmohan

    2017-01-01

    Inactivation of retrotransposons is accompanied by the emergence of centromere-binding protein-B (CENPB) in Schizosaccharomyces, as well as in metazoans. The RNA interference (RNAi)-induced transcriptional silencing (RITS) complex, comprising chromodomain protein-1 (Chp1), Tas3 (protein with unknown function), and Argonaute (Ago1), plays an important role in RNAi-mediated heterochromatinization. We find that whereas the Ago1 subunit of the RITS complex is highly conserved, Tas3 is lost and Chp1 is truncated in Schizosaccharomyces cryophilus and Schizosaccharomyces octosporus. We show that truncated Chp1 loses the property of heterochromatin localization and silencing when transformed in Schizosaccharomyces pombe. Furthermore, multiple copies of CENPB, related to Tc1/mariner and Tc5 transposons, occur in all Schizosaccharomyces species, as well as in humans, but with loss of transposase function (except Schizosaccharomyces japonicus). We propose that acquisition of Tc1/mariner and Tc5 elements by horizontal transfer in S. pombe (and humans) is accompanied by alteration of their function from a transposase/endonuclease to a heterochromatin protein, designed to suppress transposon expression and recombination. The resulting redundancy of RITS may have eased the selection pressure, resulting in progressive loss or truncation of tas3 and chp1 genes in S. octosporus and S. cryophilus and triggered similar evolutionary dynamics in the metazoan orthologues. PMID:28228545

  3. Knockdown of E2f1 by RNA interference impairs proliferation of rat cells in vitro

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    Luciana dos Reis Vasques

    2010-01-01

    Full Text Available E2F1 plays a key role in cell-cycle regulation in mammals, since its transcription factor activity controls genes required for DNA synthesis and apoptosis. E2F1 deregulation is a common feature among different tumor types and can be a major cause of cell proliferation. Thus, blocking E2F1 expression by RNA interference represents a promising therapeutic approach. In this study, the introduction of specific short hairpin RNAs (shRNAs reduced E2f1 expression by up to 77%, and impaired rat glioma cell proliferation by approximately 70%, as compared to control cells. Furthermore, we investigated the expression of E2f1 target genes, Cyclin A and Cyclin E. Cyclin A was found to be down-regulated, whereas Cyclin E had similar expression to control cells, indicating that gene(s other than E2f1 control its transcription. Other E2f family members, E2f2 and E2f3, which have been classified in the same subgroup of transcriptional activators, were also analyzed. Expression of both E2f2 and E2f3 was similar to control cells, showing no cross-inactivation or up-regulation to compensate for the absence of E2f1. Nevertheless, their expression was insufficient to maintain the initial proliferation potential. Taken together, our results suggest that shE2f1 is a promising therapy to control tumor cell proliferation.

  4. Harnessing the RNA interference pathway to advance treatment and prevention of hepatocellular carcinoma.

    Science.gov (United States)

    Arbuthnot, Patrick; Thompson, Liam-Jed

    2008-03-21

    Primary liver cancer is the fifth most common malignancy in the world and is a leading cause of cancer-related mortality. Available treatment for hepatocellular carcinoma (HCC), the commonest primary liver cancer, is rarely curative and there is a need to develop therapy that is more effective. Specific and powerful gene silencing that can be achieved by activating RNA interference (RNAi) has generated enthusiasm for exploiting this pathway for HCC therapy. Many studies have been carried out with the aim of silencing HCC-related cellular oncogenes or the hepatocarcinogenic hepatitis B virus (HBV) and hepatitis C virus (HCV). Proof of principle studies have demonstrated promising results, and an early clinical trial assessing RNAi-based HBV therapy is currently in progress. Although the data augur well, there are several significant hurdles that need to be overcome before the goal of RNAi-based therapy for HCC is realized. Particularly important are the efficient and safe delivery of RNAi effecters to target malignant tissue and the limitation of unintended harmful non-specific effects.

  5. A comparative study of transfection methods for RNA interference in bone marrow-derived murine dendritic cells

    DEFF Research Database (Denmark)

    Pedersen, Charlotte Demuth; Fang, J J; Pedersen, Anders Elm

    2009-01-01

    Selective gene silencing using RNA interference (RNAi) has been shown to be an efficient method for manipulation of cellular functions. In this study, we compare three previously established methods for transfection of murine bone marrow-derived DC (BM-DC). We tested the efficacy of electroporation...... with the Mouse Nucleofector kit((R)) from Amaxa Biosystems and lipid-based transfection methods using transfection reagents from Santa Cruz Biotechnology or Genlantis. To analyse the transfection efficacy we used FITC-conjugated siRNA as a positive control together with CD80 and CD86 specific siRNA. We show...... that electroporation using the Mouse Nucleofector kit((R)) from Amaxa Biosystems was not an efficient method to transfect BM-DC with siRNA in our hands. Transfection with Santa Cruz Biotechnology reagents resulted in up to 59% FITC-siRNA positive cells, but did not result in effective silencing of CD80 surface...

  6. Dicer and Argonaute Genes Involved in RNA Interference in the Entomopathogenic Fungus Metarhizium robertsii.

    Science.gov (United States)

    Meng, Huimin; Wang, Zhangxun; Wang, Yulong; Zhu, Hong; Huang, Bo

    2017-04-01

    RNA interference (RNAi) is a gene-silencing mechanism that plays an important role in gene regulation in a number of eukaryotic organisms. Two core components, Dicer and Argonaute, are central in the RNAi machinery. However, the physiological roles of Dicer and Argonaute in the entomopathogenic fungus Metarhizium robertsii have remained unclear. Here, the roles of genes encoding Dicer (M. robertsiidcl1 [Mrdcl1] and Mrdcl2) and Argonaute (Mrago1 and Mrago2) proteins in M. robertsii were investigated. The results showed that the Dicer-like protein MrDCL2 and Argonaute protein MrAGO1 are the major components of the RNAi process occurring in M. robertsii The Dicer and Argonaute genes were not involved in the regulation of growth and diverse abiotic stress response in M. robertsii under the tested conditions. Moreover, our results showed that the Dicer and Argonaute gene mutants demonstrated reduced abilities to produce conidia, compared to the wild type (WT) and the gene-rescued mutant. In particular, the conidial yields in the Δdcl2 and Δago1 mutants were reduced by 55.8% and 59.3%, respectively, compared with those from the control strains. Subsequently, for the WT and Δdcl2 mutant strains, digital gene expression (DGE) profiling analysis of the stage of mycelium growth and conidiogenesis revealed that modest changes occur in development or metabolism processes, which may explain the reduction in conidiation in the Δdcl2 mutant. In addition, we further applied high-throughput sequencing technology to identify small RNAs (sRNAs) that are differentially expressed in the WT and the Δdcl2 mutant and found that 4 known microRNA-like small RNAs (milRNAs) and 8 novel milRNAs were Mrdcl2 dependent in M. robertsiiIMPORTANCE The identification and characterization of components in RNAi have contributed significantly to our understanding of the mechanism and functions of RNAi in eukaryotes. Here, we found that Dicer and Argonaute genes play an important role in regulating

  7. RNA interference in Schistosoma mansoni schistosomula: selectivity, sensitivity and operation for larger-scale screening.

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    Saša Stefanić

    Full Text Available BACKGROUND: The possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3 genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi. METHODOLOGY/PRINCIPAL FINDINGS: We investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (dsRNA (approximately 500 bp designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose-dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days; electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was

  8. Modulating drug resistance by targeting BCRP/ABCG2 using retrovirus-mediated RNA interference.

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    Ni Xie

    Full Text Available The BCRP/ABCG2 transporter, which mediates drug resistance in many types of cells, depends on energy provided by ATP hydrolysis. Here, a retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, thus laying the foundation for further studies and applications.To target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged. The pLenti6/BCRPsi retroviruses (V-BCRPi that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry. The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma. After injecting V-BCRPi-infected JAR tumor cells into the dorsal skin of hairless mice, BCRP/ABCG2 expression in the tumor tissue was determined using immunohistochemistry, fluorescent quantitative RT-PCR and Western blot analyses. After intraperitoneal injection of BCRP/ABCG2-tolerant 5-FU, the tumor volume, weight change, and apoptosis rate of the tumor tissue were determined using in situ hybridization. V-BCRPi increased the sensitivity of the tumor histiocytes to 5-FU and improved the cell apoptosis-promoting effects of 5-FU in the tumor.The goal of the in vivo and in vitro studies was to screen for an RNA interference recombinant retrovirus capable of stably targeting the ATP-binding domain of BCRP/ABCG2 (V-BCRPi to inhibit its function. A new method to improve the chemo-sensitivity of breast cancer and other tumor cells was discovered, and this method could be used for gene therapy and functional studies of malignant tumors.

  9. Modulating drug resistance by targeting BCRP/ABCG2 using retrovirus-mediated RNA interference.

    Science.gov (United States)

    Xie, Ni; Mou, Lisha; Yuan, Jianhui; Liu, Wenlan; Deng, Tingting; Li, Zigang; Jing, Yi; Jin, Yi; Hu, Zhangli

    2014-01-01

    The BCRP/ABCG2 transporter, which mediates drug resistance in many types of cells, depends on energy provided by ATP hydrolysis. Here, a retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, thus laying the foundation for further studies and applications. To target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged. The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry. The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma. After injecting V-BCRPi-infected JAR tumor cells into the dorsal skin of hairless mice, BCRP/ABCG2 expression in the tumor tissue was determined using immunohistochemistry, fluorescent quantitative RT-PCR and Western blot analyses. After intraperitoneal injection of BCRP/ABCG2-tolerant 5-FU, the tumor volume, weight change, and apoptosis rate of the tumor tissue were determined using in situ hybridization. V-BCRPi increased the sensitivity of the tumor histiocytes to 5-FU and improved the cell apoptosis-promoting effects of 5-FU in the tumor. The goal of the in vivo and in vitro studies was to screen for an RNA interference recombinant retrovirus capable of stably targeting the ATP-binding domain of BCRP/ABCG2 (V-BCRPi) to inhibit its function. A new method to improve the chemo-sensitivity of breast cancer and other tumor cells was discovered, and this method could be used for gene therapy and functional studies of malignant tumors.

  10. The efficiency of RNA interference for conferring stable resistance to Plum Pox Virus

    Science.gov (United States)

    Plum transformed with an intron hairpin RNA CP (ihRNA-CP) were resistant to PPV infection through the specific process of RNA silencing involving both small interfering -RNA interfering (siRNA) and a methylated virus transgene. This recognition process specifically targeted the triggered PPV genome...

  11. Ablation of RNA interference and retrotransposons accompany acquisition and evolution of transposases to heterochromatin protein CENPB.

    Science.gov (United States)

    Upadhyay, Udita; Srivastava, Suchita; Khatri, Indu; Nanda, Jagpreet Singh; Subramanian, Srikrishna; Arora, Amit; Singh, Jagmohan

    2017-04-15

    Inactivation of retrotransposons is accompanied by the emergence of centromere-binding protein-B (CENPB) in Schizosaccharomyces, as well as in metazoans. The RNA interference (RNAi)-induced transcriptional silencing (RITS) complex, comprising chromodomain protein-1 (Chp1), Tas3 (protein with unknown function), and Argonaute (Ago1), plays an important role in RNAi-mediated heterochromatinization. We find that whereas the Ago1 subunit of the RITS complex is highly conserved, Tas3 is lost and Chp1 is truncated in Schizosaccharomyces cryophilus and Schizosaccharomyces octosporus We show that truncated Chp1 loses the property of heterochromatin localization and silencing when transformed in Schizosaccharomyces pombe Furthermore, multiple copies of CENPB, related to Tc1/mariner and Tc5 transposons, occur in all Schizosaccharomyces species, as well as in humans, but with loss of transposase function (except Schizosaccharomyces japonicus). We propose that acquisition of Tc1/mariner and Tc5 elements by horizontal transfer in S. pombe (and humans) is accompanied by alteration of their function from a transposase/endonuclease to a heterochromatin protein, designed to suppress transposon expression and recombination. The resulting redundancy of RITS may have eased the selection pressure, resulting in progressive loss or truncation of tas3 and chp1 genes in S. octosporus and S. cryophilus and triggered similar evolutionary dynamics in the metazoan orthologues. © 2017 Upadhyay et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  12. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

    Science.gov (United States)

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang; Singh, Upinder

    2016-04-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Genome-wide exonic small interference RNA-mediated gene silencing regulates sexual reproduction in the homothallic fungus Fusarium graminearum.

    Science.gov (United States)

    Son, Hokyoung; Park, Ae Ran; Lim, Jae Yun; Shin, Chanseok; Lee, Yin-Won

    2017-02-01

    Various ascomycete fungi possess sex-specific molecular mechanisms, such as repeat-induced point mutations, meiotic silencing by unpaired DNA, and unusual adenosine-to-inosine RNA editing, for genome defense or gene regulation. Using a combined analysis of functional genetics and deep sequencing of small noncoding RNA (sRNA), mRNA, and the degradome, we found that the sex-specifically induced exonic small interference RNA (ex-siRNA)-mediated RNA interference (RNAi) mechanism has an important role in fine-tuning the transcriptome during ascospore formation in the head blight fungus Fusarium graminearum. Approximately one-third of the total sRNAs were produced from the gene region, and sRNAs with an antisense direction or 5'-U were involved in post-transcriptional gene regulation by reducing the stability of the corresponding gene transcripts. Although both Dicers and Argonautes partially share their functions, the sex-specific RNAi pathway is primarily mediated by FgDicer1 and FgAgo2, while the constitutively expressed RNAi components FgDicer2 and FgAgo1 are responsible for hairpin-induced RNAi. Based on our results, we concluded that F. graminearum primarily utilizes ex-siRNA-mediated RNAi for ascosporogenesis but not for genome defenses and other developmental stages. Each fungal species appears to have evolved RNAi-based gene regulation for specific developmental stages or stress responses. This study provides new insights into the regulatory role of sRNAs in fungi and other lower eukaryotes.

  14. Knockdown of RNA Interference Pathway Genes in Western Corn Rootworms (Diabrotica virgifera virgifera Le Conte Demonstrates a Possible Mechanism of Resistance to Lethal dsRNA.

    Directory of Open Access Journals (Sweden)

    Ana María Vélez

    Full Text Available RNA interference (RNAi is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2, an endonuclease responsible for formation of small interfering RNA's and Argonaute 2 (Ago2, an essential catalytic component of the RNA-induced silencing complex (RISC have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae. We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2 did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA's. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance.

  15. Knockdown of RNA Interference Pathway Genes in Western Corn Rootworms (Diabrotica virgifera virgifera Le Conte) Demonstrates a Possible Mechanism of Resistance to Lethal dsRNA.

    Science.gov (United States)

    Vélez, Ana María; Khajuria, Chitvan; Wang, Haichuan; Narva, Kenneth E; Siegfried, Blair D

    2016-01-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2), an endonuclease responsible for formation of small interfering RNA's and Argonaute 2 (Ago2), an essential catalytic component of the RNA-induced silencing complex (RISC) have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2) did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA's. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance.

  16. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi)

    National Research Council Canada - National Science Library

    Wilda, Monika; Fuchs, Uta; Wössmann, Wilhelm; Borkhardt, Arndt

    2002-01-01

    .... We used dsRNA targeting the M-BCR/ABL fusion site to kill leukemic cells with such a rearrangement. Transfection of dsRNA specific for the M-BCR/ABL fusion mRNA into K562 cells depleted the corresponding mRNA and the M-BCR/ABL oncoprotein...

  17. Inhibition of targeted platelet-derived growth factor receptor-α RNA interference into vitreous cavity for experimental proliferative vitreoretinopathy

    Directory of Open Access Journals (Sweden)

    Zhu Meng

    2016-01-01

    Full Text Available AIM:To observe the therapeutic effect of platelet-derived growth factor receptor-α RNA interference on inhibiting experimental proliferative vitreoretinopathy(PVR.METHODS:Different concentrations of PDGFR-α shRNA were blended with lip2000,and the final scale of PDGFR-α shRNA/lip2 000 complex was 1:1, 1:2 and 1:3 respectively(the concentration of PDGFR-α shRNA was respectively 2μg, 3μg and 4μg.All the complexes were cultivated in human retinal pigment epithelium(HRPEfor 24h after transfection and then respectively injected 0.1mL to rabbit vitreous cavity.Forty healthy adult rabbits were seleceted in this study and randomly divided into colored balanced salt solution(BSSgroup(N, comprising lipofectamineTM2 000 HRPE cell dilution group(group A. The highest transduction efficiency of 1.0, 1.5 and 2.0μmol/L containing PDGFR-α receptors shRNA, lipofectamineTM2 000 of HRPE cell dilution were selected(respectively group B, C and Dwith 8 eyes each, the right eyes as the experimental eye. The extent of PVR was observed by indirect ophthalmoscope; and slice staining situation was observed by immunohistochemistry.The fundus changes were observed by histopathology.RESULTS:The highest transduction efficiency of PDGFR-αshRNA/lip2 000 ratio was 1:2.The extent of PVR, the histopathology changes and the immunohistochemistry of PDGFR-α in group B,C and group D were significantly lower than that in group A, while group D was much lower than those of group B and C.CONCLUSION:PDGFR-αRNA interference could inhibit the formation of experimental PVR.

  18. Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2'-modified-4'-thionucleosides.

    Science.gov (United States)

    Kikuchi, Yusaku; Yamazaki, Naoshi; Tarashima, Noriko; Furukawa, Kazuhiro; Takiguchi, Yoshiharu; Itoh, Kohji; Minakawa, Noriaki

    2013-09-01

    Gene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a 'target domain' and a 'U1 domain', we prepared adaptor ONs using 2'-modified-4'-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2'-fluoro-4'-thionucleoside and 2'-fluoronucleoside units as well as only 2'-fluoronucleoside units, while those prepared as combination of 2'-OMe nucleoside/2'-OMe-4'-thionucleoside and 2'-fluoronucleoside units did not show significant activity. Measurement of Tm values indicated that a higher hybridization ability of adaptor ONs with complementary RNA is one of the important factors to show potent U1i activity. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleri by ingested dsRNA expressing bacteria

    Science.gov (United States)

    2011-01-01

    Background The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. Results We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene. Conclusion This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals. PMID:21679422

  20. Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

    Science.gov (United States)

    Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. Copyright © 2016

  1. The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2010-04-01

    Full Text Available Abstract Background The RNA interference (RNAi pathway acts as an innate antiviral immune response in Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity. Results As a novel tool for studying arbovirus-mosquito interactions, we engineered a transgenic mosquito line with an impaired RNAi pathway in the midgut of bloodfed females by silencing expression of the Aa-dcr2 gene. In midgut tissue of the transgenic Carb/dcr16 line, Aa-dcr2 expression was reduced ~50% between 1-7 days post-bloodmeal (pbm when compared to the recipient mosquito strain. After infection with SINV-TR339EGFP, Aa-dcr2 expression levels were enhanced in both mosquito strains. In the RNAi pathway impaired mosquito strain SINV titers and midgut infection rates were significantly higher at 7 days pbm. There was also a strong tendency for increased virus dissemination rates among the transgenic mosquitoes. Between 7-14 days pbm, SINV was diminished in midgut tissue of the transgenic mosquitoes. Transgenic impairment of the RNAi pathway and/or SINV infection did not affect longevity of the mosquitoes. Conclusions We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level. Following ingestion by Ae. aegypti, the recombinant SINV-TR339EGFP was confronted with both

  2. [Effect of PC cell-derived growth factor RNA interference on biological behavior of esophageal squamous carcinoma cells].

    Science.gov (United States)

    Zheng, Qing-feng; Liu, Shuo-yan; Wang, Hai-yan; Wang, Feng; Wang, Zhen; Chen, Xiao-feng; Wang, Jian-jian; Ying, Min-gang; Zheng, Xiong-wei; Lin, Xian-dong; Zhou, Zhi-feng; Gong, Fu-sheng; Xie, Yun-qing

    2013-09-01

    To investigate the effect of PC cell-derived growth factor (PCDGF) RNA interference on esophageal squamous carcinoma cells Eca-109 in vitro. The PCDGF-shRNA expression vector was transfected into the Eca-109 cells by liposome. After transfection, the mRNA and protein expressions of PCDGF were detected by RT-PCR and Western-blot respectively. Cell Counting Kit-8 (CCK-8) assay and Boyden chamber method were performed to measure the cell proliferation and invasion ability respectively. The expression levels of PCDGF mRNA and protein were both decreased in Eca-109 cells transfected with PCDGF-shRNA expression vector (transfection group). Twenty-four, 48 and 72 h after transfection, the cells proliferation in the transfection group was inhibited, and the inhibition rate was 20.4%, 21.1% and 20.9% respectively. The cell proliferation activity in the transfection group was significantly lower than that in the non-transfection group, liposome group and negative vector group (all Pgroup,negative vector group, liposome group and transfection group was 118.8±12.0, 100.8±9.0, 114.3±4.7, and 53.5±16.3 respectively. The differences were statistically significant between the transfection group and the other 3 groups (all P<0.05). PCDGF RNA interference can inhibit the proliferation and invasion abilities of esophageal squamous carcinoma cells in vitro. PCDGF gene may be the new target of gene therapy.

  3. Platinum Interference with siRNA Non-seed Regions Fine-Tunes Silencing Capacity

    DEFF Research Database (Denmark)

    Hedman, Hanna K; Kirpekar, Finn; Elmroth, Sofi K C

    2011-01-01

    RNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs......, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized...

  4. Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression

    National Research Council Canada - National Science Library

    Aftab Ahmad; Muhammad Zia-Ur-Rehman; Usman Hameed; Abdul Qayyum Rao; Ammara Ahad; Aneela Yasmeen; Faheem Akram; Kamran Shahzad Bajwa; Jodi Scheffler; Idrees Ahmad Nasir; Ahmad Ali Shahid; Muhammad Javed Iqbal; Tayyab Husnain; Muhammad Saleem Haider; Judith K Brown

    2017-01-01

    Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi...

  5. Gene silencing of hPRLR mRNA by RNA interference in human breast cancer cells.

    Science.gov (United States)

    Wei, Qinjun; Lu, Yajie; Pan, Mei; Shu, Yongquian; Cao, Xin

    2008-01-01

    Up-regulation of the human prolactin receptor (hPRLR) has been implicated in aberrant signaling that may induce abnormal proliferation of breast epithelium. In this study, we evaluated the inhibition of cell proliferation by small-interfering RNA (siRNA) targeting of hPRLR, as well as the anti-tumor efficacy of this treatment on the T-47D breast cancer cell line. The hPRLR-targeted siRNA were chemically synthesized and constructed in a specific siRNA expression vector (pSilencer 2.0), the expression of hPRLR was analyzed by real-time quantitative PCR and Western blot analysis, growth inhibition was measured by MTT assay, and cell cycle analysis was carried out to determine the effect of siRNA treatment on T-47D cells. Our results indicate that the hPRLR siRNA plasmid markedly reduced PRLR gene expression, decreased the growth rate and reduced the frequency of T-47D cells in the G2/M phase, while significantly increasing cells in G0/G1. In summary, RNAi silencing of hPRLR gene expression specifically inhibited the proliferation of T-47D breast cancer cells. This implies that RNAi have therapeutic potential as a treatment for breast cancer by targeting the overexpression of hPRLR, suggesting that this gene product might be a potential therapeutic target.

  6. [Down-regulation of human intercellular adhesion molecule-1 expression in MCF-7 cells infected by lentiviral short hairpin RNA interference vectors].

    Science.gov (United States)

    Di, Dalin; Chen, Lei; Wang, Lina; Wang, Chengdong; Ju, Jiyu

    2015-08-01

    To construct lentiviral interference vectors of human intercellular adhesion molecule-1 (ICAM-1), then infect human breast cancer MCF-7 cells and identify the interference effects. Three short hairpin RNA (shRNA) interference sequences targeting human ICAM-1 gene (ICAM-1 shRNA1, ICAM-1 shRNA2 and ICAM-1 shRNA3) and a negative control sequence (NS) were designed, synthesized and cloned into the pLKO.1-SP6-PGK-GFP vector. After DNA sequencing, three plasmid-based lentiviral packaging system (vector plasmid-psPAX2-pMD2.G) was used to transfect HEK293T cells to package lentiviruses. The supernatants containing viruses were harvested to detect the viral titer. Human MCF-7 breast cancer cells were infected with the lentiviruses and the interference efficiency was detected by real-time quantitative PCR (qRT-PCR) and Western blotting. PCR showed that the designed sequences were successfully inserted into the pLKO.1-SP6-PGK-GFP vector and DNA sequencing results were correct. The qRT-PCR and Western blotting showed that the mRNA and protein expression levels of ICAM-1 in the infected MCF-7 cells decreased significantly in the ICAM-1 shRNA3 group. Lentiviral interference vectors of human ICAM-1 were constructed successfully and the expression of ICAM-1 in MCF-7 cells was down-regulated by ICAM-1 shRNA.

  7. Delivery of dsRNA through topical feeding for RNA interference in the citrus sap piercing-sucking hemipteran, Diaphorina citri.

    Science.gov (United States)

    Killiny, Nabil; Kishk, Abdelaziz

    2017-06-01

    RNA interference (RNAi) is a powerful means to study functional genomics in insects. The delivery of dsRNA is a challenging step in the development of RNAi assay. Here, we describe a new delivery method to increase the effectiveness of RNAi in the Asian citrus psyllid Diaphorina citri. Bromophenol blue droplets were topically applied to fifth instar nymphs and adults on the ventral side of the thorax between the three pairs of legs. In addition to video recordings that showed sucking of the bromophenol blue by the stylets, dissected guts turned blue indicating that the uptake was through feeding. Thus, we called the method topical feeding. We targeted the abnormal wing disc gene (awd), also called nucleoside diphosphate kinase (NDPK), as a reporter gene to prove the uptake of dsRNA via this method of delivery. Our results showed that dsRNA-awd caused reduction of awd expression and nymph mortality. Survival and lifespan of adults emerged from treated nymphs and treated adults were affected. Silencing awd caused wing malformation in the adults emerged from treated nymphs. Topical feeding as a delivery of dsRNA is highly efficient for both nymphs and adults. The described method could be used to increase the efficiency of RNAi in D. citri and other sap piercing-sucking hemipterans. © 2017 Wiley Periodicals, Inc.

  8. Improving model predictions for RNA interference activities that use support vector machine regression by combining and filtering features

    Directory of Open Access Journals (Sweden)

    Peek Andrew S

    2007-06-01

    Full Text Available Abstract Background RNA interference (RNAi is a naturally occurring phenomenon that results in the suppression of a target RNA sequence utilizing a variety of possible methods and pathways. To dissect the factors that result in effective siRNA sequences a regression kernel Support Vector Machine (SVM approach was used to quantitatively model RNA interference activities. Results Eight overall feature mapping methods were compared in their abilities to build SVM regression models that predict published siRNA activities. The primary factors in predictive SVM models are position specific nucleotide compositions. The secondary factors are position independent sequence motifs (N-grams and guide strand to passenger strand sequence thermodynamics. Finally, the factors that are least contributory but are still predictive of efficacy are measures of intramolecular guide strand secondary structure and target strand secondary structure. Of these, the site of the 5' most base of the guide strand is the most informative. Conclusion The capacity of specific feature mapping methods and their ability to build predictive models of RNAi activity suggests a relative biological importance of these features. Some feature mapping methods are more informative in building predictive models and overall t-test filtering provides a method to remove some noisy features or make comparisons among datasets. Together, these features can yield predictive SVM regression models with increased predictive accuracy between predicted and observed activities both within datasets by cross validation, and between independently collected RNAi activity datasets. Feature filtering to remove features should be approached carefully in that it is possible to reduce feature set size without substantially reducing predictive models, but the features retained in the candidate models become increasingly distinct. Software to perform feature prediction and SVM training and testing on nucleic acid

  9. Effect of silencing HOXA5 gene expression using RNA interference on cell cycle and apoptosis in Jurkat cells.

    Science.gov (United States)

    Huang, Hui-Ping; Liu, Wen-Jun; Guo, Qu-Lian; Bai, Yong-Qi

    2016-03-01

    Acute lymphocytic leukemia (ALL) is a common malignant tumor with a high morbidity rate among children, accounting for approximately 80% of leukemia cases. Although there have been improvements in the treatment of patients frequent relapse lead to a poor prognosis. The aim of the present study was to determine whether HOXA5 may be used as a target for gene therapy in leukemia in order to provide a new treatment. Mononuclear cells were extracted from the bone marrow according to the clinical research aims. After testing for ALL in the acute stage, the relative mRNA and protein expression of HOXA5 was detected in the ALL remission groups (n=25 cases per group) and the control group [n=20 cases, immune thrombocytopenia (ITP)]. Gene silencing by RNA interference (RNAi) was used to investigate the effect of silencing HOXA5 after small interfering RNA (siRNA) transfection to Jurkat cells. The HOXA5-specific siRNA was transfected to Jurkat cells using lipofectamine. The experiment was divided into the experimental group (liposomal transfection of HOXA5 targeting siRNA), the negative control group (liposomal transfection of cells with negative control siRNA) and the control group (plus an equal amount of cells and culture media only). Western blotting and quantitative fluorescent polymerase chain reaction (QF‑PCR) were used to detect the relative HOXA5 mRNA expression and protein distribution in each cell group. Cell distribution in the cell cycle and the rate of cells undergoing apoptosis were determined using flow cytometry. The expression of HOXA5 at the mRNA and protein levels in the acute phase of ALL was significantly higher than that in ALL in the remission and control groups. In cells transfected with HOXA5-specific siRNA, the expression of HOXA5 at the mRNA and protein levels decreased significantly (Pcells in the cell cycle was also altered. Specifically, more cells were present in the G0/G1 phase compared to the S phase (Pcells transfected with HOXA5

  10. In silico molecular docking analysis of the human Argonaute 2 PAZ domain reveals insights into RNA interference

    Science.gov (United States)

    Kandeel, Mahmoud; Kitade, Yukio

    2013-07-01

    RNA interference (RNAi) is a critical cellular pathway activated by double stranded RNA and regulates the gene expression of target mRNA. During RNAi, the 3' end of siRNA binds with the PAZ domain, followed by release and rebinding in a cyclic manner, which deemed essential for proper gene silencing. Recently, we provided the forces underlying the recognition of small interfering RNA by PAZ in a computational study based on the structure of Drosophila Argonaute 2 (Ago2) PAZ domain. We have now reanalyzed these data within the view of the new available structures from human Argonauts. While the parameters of weak binding are correlated with higher (RNAi) in the Drosophila model, a different profile is predicted with the human Ago2 PAZ domain. On the basis of the human Ago2 PAZ models, the indicators of stronger binding as the total binding energy and the free energy were associated with better RNAi efficacy. This discrepancy might be attributable to differences in the binding site topology and the difference in the conformation of the bound nucleotides.

  11. Efficacy of RNA interference knockdown using aerosolized short interfering RNAs bound to nanoparticles in three diverse aphid species.

    Science.gov (United States)

    Thairu, M W; Skidmore, I H; Bansal, R; Nováková, E; Hansen, T E; Li-Byarlay, H; Wickline, S A; Hansen, A K

    2017-06-01

    RNA interference (RNAi) has emerged as a promising method for validating gene function; however, its utility in nonmodel insects has proven problematic, with delivery methods being one of the main obstacles. This study investigates a novel method of RNAi delivery in aphids, the aerosolization of short interfering RNA (siRNA)-nanoparticle complexes. By using nanoparticles as a siRNA carrier, the likelihood of cellular uptake is increased, when compared to methods previously used in insects. To determine the efficacy of this RNAi delivery system, siRNAs were aerosolized with and without nanoparticles in three aphid species: Acyrthosiphon pisum, Aphis glycines and Schizaphis graminum. The genes targeted for knockdown were carotene dehydrogenase (tor), which is important for pigmentation in Ac. pisum, and branched chain-amino acid transaminase (bcat), which is essential in the metabolism of branched-chain amino acids in all three aphid species. Overall, we observed modest gene knockdown of tor in Ac. pisum and moderate gene knockdown of bcat in Ap. glycines along with its associated phenotype. We also determined that the nanoparticle emulsion significantly increased the efficacy of gene knockdown. Overall, these results suggest that the aerosolized siRNA-nanoparticle delivery method is a promising new high-throughput and non-invasive RNAi delivery method in some aphid species. © 2017 The Royal Entomological Society.

  12. Targeting MACC1 by RNA interference inhibits proliferation and invasion of bladder urothelial carcinoma in T24 cells.

    Science.gov (United States)

    Xu, Song-Tao; Ding, Xiang; Ni, Qing-Feng; Jin, Shao-Ju

    2015-01-01

    The purpose of this article is to research on whether MACC1 can serve as a potential target for gene therapy of human bladder urothelial carcinoma (BUC). In this study, the expression of MACC1 gene was knocked down by RNA interference (RNAi) in the T24 cell (human BUC cell). The transcription level of MACC1 was detected by RT-PCR. Activities of MACC1, caspase-3, caspase-8, Bax and Met (mesenchymal-epithelial transition factor) protein were measured by Western blot. The cell proliferation and apoptosis were detected by MTT and flow cytometry. The cell's invasion ability was performed on Matrigel transwell assay. We also detect MMP2 (metalloproteinase-2) proteins by ELISA. The results showed that the level of MACC1 mRNA and protein was significantly reduced after RNAi. MTT assay showed that the proliferation of T24 cell was decreased due to RNA interference. Apoptosis studies also showed that MACC1 gene interference in T24 loses its anti-apoptotic effects. The expression of apoptosis proteins (Caspase-3, Caspase-8 and Bax) increased significantly due to the MACC1 RNAi. The level of Met protein was down-regulated obviously due to RNAi. Transwell assay showed that invasion abilities of T24 cells were reduced obviously due to MACC1 RNAi. Further studies showed that the secretion of MMP-2 was reduced by RNAi. It can conclude that the ability of proliferation and invasion in T24 cells can be inhibited by RNAi-targeting MACC1. As a result, MACC1 can serve as a potential target for gene therapy of human bladder urothelial carcinoma.

  13. Small RNA interference-mediated gene silencing of TREK-1 potassium channel in cultured astrocytes.

    Science.gov (United States)

    Wu, Xiao; Tang, Ronghua; Liu, Yang; Song, Jingjiao; Yu, Zhiyuan; Wang, Wei; Xie, Minjie

    2012-12-01

    This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labeled dsRNA oligmers were used to determine the transfection efficiency in cultured astrocytes. TREK-1-specific siRNA duplexes (siT1, siT2, siT3) at the optimal concentration were transfected into cultured astrocytes, and the most efficient siRNA was identified by the method of immunocytochemical staining and Western blotting. The proliferation of astrocytes tranfected with TREK-1-targeting siRNA under hypoxia condition was measured by fluorescence-activated cell sorting (FACS). The results showed that TREK-1 was expressed in cultured astrocytes. The dsRNA oligmers targeting TREK-1 could be transfected efficiently in cultured astrocytes and down-regulate the expression of TREK-1 in astrocytes. Moreover, the down-regulation of TREK-1 in astrocytes contributed to the proliferation of astrocytes under hypoxia condition as determined by cell cycle analysis. It was concluded that siRNA is a powerful technique that can be used to knockdown the expression of TREK-1 in astrocytes, which helps further investigate the function of TREK-1 channel in astrocytes under physicological and pathological condition.

  14. [Inhibition of hepatitis B virus replication and expression by RNA interference in vitro].

    Science.gov (United States)

    Zhu, Cai; Fan, Xue-gong; Li, Ning; Ying, Ruo-su; Tian, Xue-fei

    2005-09-14

    To design pSilencer3.1-H1hygro plasmid expressing short interfering RNAs (siRNA) that target HBV S gene region, and to evaluate inhibitory effect of this siRNA on HBV in vitro. HepG2.2.15 was used as target cell. The plasmid expressing small interfering RNA was transfected into the cultured cells via liposome metafectene, HBsAg and HBeAg were analyzed by time-resolved immunofluorometric assay, HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV S-mRNA was detected by semi-quantitative RT-PCR. The plasmid expressing siRNA was successfully constructed. The S region siRNAs could effectively inhibit both antigens secretion and HBV replication compared with controls, HBsAg levels decreased by 75%, 82%, 89%; HBeAg levels decreased by 32%, 38%, 43%; HBV DNA production decreased by 30%, 43%, 49%; The HBV mRNA species was reduced by 30%, 70%, 90% when transfected with 1 microg, 2 microg, 4 microg HBV S-siRNA, respectively. These results demonstrate that RNAi can substantially inhibit HBV replication and the antigens expression in the infected cells. These inhibitive effect of siRNA on HBV was dose-dependent and sequence-specific.

  15. [Inhibition of hepatitis B virus replication by RNA interference in vitro].

    Science.gov (United States)

    Zhu, Cai; Fan, Xue-Gong; Li, Ning; Ying, Ruo-Su

    2004-09-01

    To design pSilencer3.1-H1hygro plasmid expressing short interfering RNAs (siRNA) that targets HBV core gene region, and to evaluate inhibitory effect of this siRNA on HBV in vitro. HepG2 2.2.15 was used as target cells. The plasmid and liposome metafectene were cotransfected into the cultured cells, HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR. The plasmid expressing siRNA was successfully constructed. The two constructed siRNAs could effectively inhibit HBV replication, and their inhibitive effect on HBV was dose-dependent. These results showed that siRNA could substantially inhibit HBV replication in the infected cells

  16. Decreased expression of RNA interference machinery, Dicer and Drosha, is associated with poor outcome in ovarian cancer patients

    Energy Technology Data Exchange (ETDEWEB)

    Merritt, William M.; Lin, Yvonne G.; Han, Liz Y.; Kamat, Aparna A.; Spannuth, Whitney A.; Schmandt, Rosemarie; Urbauer, Diana; Pennacchio, Len A.; Cheng, Jan-Fang; Zeidan, Alexandra; Wang, Hua; Mueller, Peter; Lenburg, Marc E.; Gray, Joe W.; Mok, Samuel; Birrer, Michael J.; Lopez-Berestein, Gabriel; Coleman, Robert L.; Bar-Eli, Menashe; Sood, Anil K.

    2008-05-06

    The clinical and functional significance of RNA interference (RNAi) machinery, Dicer and Drosha, in ovarian cancer is not known and was examined. Dicer and Drosha expression was measured in ovarian cancer cell lines (n=8) and invasive epithelial ovarian cancer specimens (n=111) and correlated with clinical outcome. Validation was performed with previously published cohorts of ovarian, breast, and lung cancer patients. Anti-Galectin-3 siRNA and shRNA transfections were used for in vitro functional studies. Dicer and Drosha mRNA and protein levels were decreased in 37% to 63% of ovarian cancer cell lines and in 60% and 51% of human ovarian cancer specimens, respectively. Low Dicer was significantly associated with advanced tumor stage (p=0.007), and low Drosha with suboptimal surgical cytoreduction (p=0.02). Tumors with both high Dicer and Drosha were associated with increased median patient survival (>11 years vs. 2.66 years for other groups; p<0.001). In multivariate analysis, high Dicer (HR=0.48; p=0.02), high-grade histology (HR=2.46; p=0.03), and poor chemoresponse (HR=3.95; p<0.001) were identified as independent predictors of disease-specific survival. Findings of poor clinical outcome with low Dicer expression were validated in separate cohorts of cancer patients. Galectin-3 silencing with siRNA transfection was superior to shRNA in cell lines with low Dicer (78-95% vs. 4-8% compared to non-targeting sequences), and similar in cell lines with high Dicer. Our findings demonstrate the clinical and functional impact of RNAi machinery alterations in ovarian carcinoma and support the use of siRNA constructs that do not require endogenous Dicer and Drosha for therapeutic applications.

  17. DICER-ARGONAUTE2 complex in continuous fluorogenic assays of RNA interference enzymes.

    Directory of Open Access Journals (Sweden)

    Mark A Bernard

    Full Text Available Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC have been hindered by lack of methods for continuous monitoring of enzymatic activity. "Quencherless" fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS and hypoxia-inducible factor 1-α subunit (HIF1A. Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (Km=74 nM with substrate inhibition kinetics (Ki=105 nM, demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER bound in the active site of DICER may undergo direct transfer (as AGO2 substrate to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29, suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a

  18. Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

    DEFF Research Database (Denmark)

    Mousaei, Marzieh; Deng, Ling; She, Qunxin

    2016-01-01

    The stringency of crRNA-protospacer DNA base pair matching required for effective CRISPR-Cas interference is relatively low in crenarchaeal Sulfolobus species in contrast to that required in some bacteria. To understand its biological significance we studied crRNA-protospacer interactions...... in Sulfolobus islandicus REY15A which carries multiple, and functionally diverse, interference complexes. A range of mismatches were introduced into a vector-borne protospacer that was identical to spacer 1 of CRISPR locus 2, with a cognate CCN PAM sequence. Two important crRNA annealing regions were identified...... lacking type III-Bα and III-Bβ interference gene cassettes, which showed similar interference levels to those of the wild-type strain. Parallels are drawn to the involvement of two annealing sites for microRNAs on some eukaryal mRNAs which provide enhanced binding capacity and specificity. A biological...

  19. Lentivirus-mediated RNA Interference Targeting LAPTM4B Inhibits Human Ovarian Cancer Cell Invasion In Vitro.

    Science.gov (United States)

    Meng, Fanling; Chen, Xiuwei; Song, Hongtao; Lou, Ge; Fu, Songbin

    2016-01-01

    LAPTM4B (lysosome-associated protein transmembrane 4 beta) play an important role in several human carcinomas. We examines the effects of RNA interference mediated downregulation of human lysosomal-associated protein transmembrane 4 beta expression on the biological behavior of the human serous adenocarcinoma cell line NIH:OVCAR3. This study investigated the expression level of lysosomal-associated protein transmembrane 4 beta in several ovarian cancer cell lines. RNA interference mediated by recombinant lentiviral vectors expressing an artificial lysosomal-associated protein transmembrane 4 beta miRNA was used to induce long-lasting downregulation of lysosomal-associated protein transmembrane 4 beta gene expression in NIH:OVCAR3 cells. Lysosomal-associated protein transmembrane 4 beta expression as well as the motility, migration potential, and proliferation of the tumor cells was measured by flow cytometry, real-time polymerase chain reaction, Western blot analysis, transwell migration assays, wound healing assays, and cell counting kit-8 assays. In addition, the cell cycle analysis utilized fluorescence-activated cell sorting. Four recombinant plasmid expression vectors encoding premiRNAs against lysosomal-associated protein transmembrane 4 beta (pcDNA-LAPTM4B-miR-1, -2, -3, and-4) were constructed and transfected into 293T cells, which overexpress lysosomal-associated protein transmembrane 4 beta. The recombinant lentiviral vector for lysosomal-associated protein transmembrane 4 beta RNA interference was packaged with pcDNA-LAPTM4B-miR-3, which had the highest interfering efficiency, thereby successfully generating stable transfectants. Compared with the control cells, the LAPTM4B-miRNA-transfected NIH:OVCAR3 cells exhibited significant decreases in cell motility and invasion. Furthermore, LAPTM4B depletion resulted in a significant decrease in proliferating cell nuclear antigen, vascular endothelial growth factor, MMP2, MMP9, and CDK12 expression. We propose

  20. Lentivirus mediated shRNA interference targeting MAT2B induces growth-inhibition and apoptosis in hepatocelluar carcinoma.

    Science.gov (United States)

    Wang, Qun; Liu, Quan-Yan; Liu, Zhi-Su; Qian, Qun; Sun, Quan; Pan, Ding-Yu

    2008-08-07

    To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2beta gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-x(L) and bcl-x(S) were detected with western blot. We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved in down-regulating bcl-x(L) and up- regulating bcl-x(S). LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.

  1. Prevention of Chinese sacbrood virus infection in Apis cerana using RNA interference.

    Science.gov (United States)

    Liu, Xuejiao; Zhang, Yi; Yan, Xun; Han, Richou

    2010-11-01

    Chinese sacbrood virus (CSBV) is the pathogen of Chinese sacbrood disease, which poses a serious threat to honeybee Apis cerana, and tends to cause bee colony and even the whole apiary collapse. Here we report on prevention of CSBV infection by feeding second instar larvae of A. cerana with specific sequences of CSBV double-stranded RNA (dsRNA). Protection of the bee larvae from CSBV by ingestion of CSBV-derived dsRNA was further demonstrated by quantitative real-time PCR (qRT-PCR) and northern blot analysis. The result provides a potential method to protect A. cerana from CSBV infection.

  2. Magnetic gold nanoparticle-mediated small interference RNA silencing Bag-1 gene for colon cancer therapy

    National Research Council Canada - National Science Library

    HUANG, WENBAI; LIU, ZHAN'AO; ZHOU, GUANZHOU; TIAN, AILING; SUN, NIANFENG

    ... function of colon cancer. We prepared and evaluated magnetic gold nanoparticle/Bag-1 siRNA recombinant plasmid complex, a gene therapy system, which can transfect cells efficiently, for both therapeutic effect and safety...

  3. Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice

    Science.gov (United States)

    Shih, Yao-Ming; Sun, Cheng-Pu; Chou, Hui-Hsien; Wu, Tzu-Hui; Chen, Chun-Chi; Wu, Ping-Yi; Enya Chen, Yu-Chen; Bissig, Karl-Dimiter; Tao, Mi-Hua

    2015-01-01

    Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients. PMID:26482836

  4. RNA Interference based Approach to Down Regulate Osmoregulators of Whitefly (Bemisia tabaci: Potential Technology for the Control of Whitefly.

    Directory of Open Access Journals (Sweden)

    Amir Raza

    Full Text Available Over the past decade RNA interference (RNAi technology has emerged as a successful tool not only for functional genomics, but in planta expression of short interfering RNAs (siRNAs that could offer great potential for insect pest management. The diet of insects feeding exclusively on phloem sieves contains water and sugars as main components, and the uptake of the liquid food greatly depends on the osmotic pressure within the insect body. Based on this physiological mechanism, transgenic plants of Nicotiana tabacum were generated expressing double stranded RNA (dsRNA against both aquaporin (AQP and a sucrase gene, alpha glucosidase (AGLU. These two genes are involved in osmotic pressure maintenance particularly in sap sucking insects, and the aim was to disrupt osmoregulation within the insect ultimately leading to mortality. Real time quantitative PCR (RT-qPCR was performed to assess the suppression of gene expression in Bemisia tabaci (B. tabaci and mortality was recorded during transgenic tobacco feeding bioassays. Feeding of insects on plants expressing dsRNA significantly reduced the transcript level of the target genes in B. tabaci after six days of feeding and more than 70% mortality was observed in B. tabaci fed on transgenic plants compared to the control plants. Our data shows that down-regulation of genes related to osmoregulation may find practical applications for the control of this important pest in cotton and other crops.

  5. Advances in RNA interference technology and its impact on nutritional improvement, disease and insect control in plants.

    Science.gov (United States)

    Katoch, Rajan; Thakur, Neelam

    2013-03-01

    This review highlights the advances in the knowledge of RNA interference (RNAi) and discusses recent progress on the functionality of different components RNAi machinery operating in the organisms. The silencing of genes by RNA interference has become the technology of choice for investigation of gene functions in different organisms. The refinement in the knowledge of the endogenous RNAi pathways in plants along with the development of new strategies and applications for the improvement of nutritional value of important agricultural crops through suppression of genes in different plants have opened new vistas for nutritional security. The improvement in the nutritional status of the plants and reduction in the level of toxins or antinutrients was desired for long, but the available technology was not completely successful in achieving the tissue specific regulation of some genes. In the recent years, a number of economically important crop plants have been tested successfully for improving plant nutritional value through metabolic engineering using RNAi. The implications of this technology for crop improvement programs, including nutritional enrichment, reduction of antinutrients, disease, and insect control have been successfully tested in variety of crops with commercial considerations. The enhancement of the nutraceutical traits for the desired health benefits in common crop plants through manipulation of gene expression has been elaborated in this article. The tremendous potential with RNAi technology is expected to revolutionize the modern agriculture for meeting the growing challenges is discussed.

  6. Transcriptome analysis and RNA interference of cockroach phototransduction indicate three opsins and suggest a major role for TRPL channels

    Directory of Open Access Journals (Sweden)

    Andrew S French

    2015-07-01

    Full Text Available Our current understanding of insect phototransduction is based on a small number of species, but insects occupy many different visual environments. We created the retinal transcriptome of a nocturnal insect, the cockroach, Periplaneta americana to identify proteins involved in the earliest stages of compound eye phototransduction, and test the hypothesis that different visual environments are reflected in different molecular contributions to function. We assembled five novel mRNAs: two green opsins, one UV opsin, and one each TRP and TRPL ion channel homologs. One green opsin mRNA (pGO1 was 100-1000 times more abundant than the other opsins (pGO2 and pUVO, while pTRPL mRNA was 10 times more abundant than pTRP, estimated by transcriptome analysis or quantitative PCR (qPCR. Electroretinograms were used to record photoreceptor responses. Gene-specific in vivo RNA interference (RNAi was achieved by injecting long (596-708 bp double-stranded RNA into head hemolymph, and verified by qPCR. RNAi of the most abundant green opsin reduced both green opsins by more than 97% without affecting UV opsin, and gave a maximal reduction of 75% in ERG amplitude seven days after injection that persisted for at least 19 days. RNAi of pTRP and pTRPL genes each specifically reduced the corresponding mRNA by 90%. Electroretinogram reduction by pTRPL RNAi was slower than for opsin, reaching 75% attenuation by 21 days, without recovery at 29 days. pTRP RNAi attenuated ERG much less; only 30% after 21 days. Combined pTRP plus pTRPL RNAi gave only weak evidence of any cooperative interactions. We conclude that silencing retinal genes by in vivo RNAi using long dsRNA is effective, that visible light transduction in Periplaneta is dominated by pGO1, and that pTRPL plays a major role in cockroach phototransduction.

  7. RNA interference of GADD153 protects photoreceptors from endoplasmic reticulum stress-mediated apoptosis after retinal detachment.

    Directory of Open Access Journals (Sweden)

    Hong Zhu

    Full Text Available BACKGROUND: Apoptosis of photoreceptors plays a critical role in the vision loss caused by retinal detachment (RD. Pharmacologic inhibition of photoreceptor cell death may prevent RD. This study investigated the role of GADD153 that participates in endoplasmic reticulum (ER stress-mediated apoptosis of photoreceptor cells after RD. METHODS: Retinal detachment was created in Wistar rats by subretinal injection of hyaluronic acid. The rats were then randomly divided into four groups: normal control group, RD group, GADD153 RNAi group and vehicle group. RNA interference of GADD153 was performed using short hairpin RNA (shRNA. Expressions of GADD153 mRNA and protein were examined by RT-PCR and Western blotting analysis, respectively. GADD153 protein distribution in the retinal cells was observed using immunofluorescence confocal laser scanning microscopy. Apoptosis of retinal cells was determined by TdT-mediated fluorescein-16-dUTP nick-end labeling (TUNEL assay. RESULTS: Lentivirus GADD153 shRNA with the most effective silencing effect was chosen for in vivo animal study and was successfully delivered into the retinal tissues. GADD153 mRNA and protein expressions in GADD153 RNAi group were significantly lower than those in the RD group. Silencing of GADD153 by RNAi protected photoreceptors from ER stress-induced apoptosis. CONCLUSION: ER stress-mediated pathway is involved in photoreceptor cell apoptosis after RD. GADD153 is a key regulatory molecule regulating ER-stress pathways and plays a crucial role in the apoptosis of photoreceptor cells after RD.

  8. The conserved SNARE SEC-22 localizes to late endosomes and negatively regulates RNA interference in Caenorhabditis elegans.

    Science.gov (United States)

    Zhao, Yani; Holmgren, Benjamin T; Hinas, Andrea

    2017-03-01

    Small RNA pathways, including RNA interference (RNAi), play crucial roles in regulation of gene expression. Initially considered to be cytoplasmic, these processes have later been demonstrated to associate with membranes. For example, maturation of late endosomes/multivesicular bodies (MVBs) is required for efficient RNAi, whereas fusion of MVBs to lysosomes appears to reduce silencing efficiency. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion and are thus at the core of membrane trafficking. In spite of this, no SNARE has previously been reported to affect RNAi. Here, we demonstrate that in Caenorhabditis elegans, loss of the conserved SNARE SEC-22 results in enhanced RNAi upon ingestion of double-stranded RNA. Furthermore, SEC-22 overexpression inhibits RNAi in wild-type animals. We find that overexpression of SEC-22 in the target tissue (body wall muscle) strongly suppresses the sec-22(-) enhanced RNAi phenotype, supporting a primary role for SEC-22 in import of RNAi silencing signals or cell autonomous RNAi. A functional mCherry::SEC-22 protein localizes primarily to late endosomes/MVBs and these compartments are enlarged in animals lacking sec-22 SEC-22 interacts with late endosome-associated RNA transport protein SID-5 in a yeast two-hybrid assay and functions in a sid-5-dependent manner. Taken together, our data indicate that SEC-22 reduces RNAi efficiency by affecting late endosome/MVB function, for example, by promoting fusion between late endosomes/MVBs and lysosomes. To our knowledge, this is the first report of a SNARE with a function in small RNA-mediated gene silencing. © 2017 Zhao et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  9. [Effect of RNA interference of CD59 gene on proliferation of non-small cell lung cancer cell line GLC-P in vitro].

    Science.gov (United States)

    Wu, Weidong; Liu, Dan; Hou, Wenjin; Yi, Yongsheng; Wang, Yuejun; Lin, Weijia

    2015-06-01

    To investigate the effect of CD59 gene inhibition mediated by RNA interference on the proliferation and apoptosis of non-small cell lung cancer (NSCLC) GLC-P cells in vitro. Recombinant plasmids for RNA interference of CD59 gene were constructed and transfected into GLC-P cells via lipofectamine 2000. The stably transfected cells were examined with real-time RT-PCR, MTT assay and enzyme-linked immunosorbent assay to investigate the changes in cell proliferation and apoptosis. Compared with the control cells, the cells transfected with CD59-siRNA showed significantly decreased expression levels of CD59 mRNA (PRNA interference-mediated CD59 silencing can strongly inhibit the proliferation and induce apoptosis in GLC-P cells, which shed light on a potentially new target for targeted gene therapy of NSCLC.

  10. Inhibition of influenza A virus matrix and nonstructural gene expression using RNA interference.

    Science.gov (United States)

    McMillen, Cynthia M; Beezhold, Donald H; Blachere, Francoise M; Othumpangat, Sreekumar; Kashon, Michael L; Noti, John D

    2016-10-01

    Influenza antiviral drugs that use protein inhibitors can lose their efficacy as resistant strains emerge. As an alternative strategy, we investigated the use of small interfering RNA molecules (siRNAs) by characterizing three siRNAs (M747, M776 and M832) targeting the influenza matrix 2 gene and three (NS570, NS595 and NS615) targeting the nonstructural protein 1 and 2 genes. We also re-examined two previously reported siRNAs, M331 and M950, which target the matrix 1 and 2 genes. Treatment with M331-, M776-, M832-, and M950-siRNAs attenuated influenza titer. M776-siRNA treated cells had 29.8% less infectious virus than cells treated with the previously characterized siRNA, M950. NS570-, NS595- and NS615-siRNAs reduced nonstructural protein 1 and 2 expression and enhanced type I interferon expression by 50%. Combination siRNA treatment attenuated 20.9% more infectious virus than single siRNA treatment. Our results suggest a potential use for these siRNAs as an effective anti-influenza virus therapy. Published by Elsevier Inc.

  11. VEGFR2 inhibition by RNA interference affects cell proliferation, migration, invasion, and response to radiation in Calu-1 cells.

    Science.gov (United States)

    Liu, Y; Qiao, Y; Hu, C; Liu, L; Zhou, L; Liu, B; Chen, H; Jiang, X

    2016-02-01

    To investigate the role of the vascular endothelial growth factor receptor 2 (VEGFR2) in the proliferation, migration, invasion, and radiation-induced apoptosis of the non-small cell lung cancer (NSCLC) cell line Calu-1. VEGFR2 gene was silenced by RNA interference in Calu-1 cells, and the expression of VEGFR2 was measured by qRT-PCR and Western blot analysis. The cells were divided into control, VEGF-treated, VEGFR2 knockdown, and VEGFR2 knockdown and VEGF-treated groups. A CCK8 assay and Transwell assay were performed to assess cell proliferation, migration, and invasion, respectively, after VEGFR2 knockdown. Western blot assays were used to detect signaling proteins downstream of VEGFR2. Cells in the groups listed above were also subjected to radiation treatment, followed by apoptosis analysis. (1) RNA interference of VEGFR2 in Calu-1 cells reduced VEGFR2 mRNA (P < 0.01) and protein levels (P < 0.01). (2) VEGFR2 knockdown inhibited proliferation (P < 0.05), migration (P < 0.05), and invasion (P < 0.05) in Calu-1 cells. (3) VEGFR2 knockdown blocked the phosphorylation of protein kinase B (Akt, also known as PKB), extracellular regulated kinase (ERK) 1/2, and p38 mitogen-activated protein kinase (p38 MAPK) to various extent (P < 0.05), but did not change their total protein expression. (4) Knockdown of VEGFR2 suppressed HIF-1α protein synthesis (P < 0.05), and exacerbated apoptosis induced by radiation (P < 0.05). VEGFR2 gene knockdown significantly suppressed a number of cellular activities in Calu-1 cells and increased radiation-induced cell death.

  12. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K, E-mail: Jamboor.vishwanatha@unthsc.edu [Department of Molecular Biology and Immunology and Institute for Cancer Research, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-11-04

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high ({approx}97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  13. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    Science.gov (United States)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.

    2011-11-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  14. A method for stable gene knock-down by RNA interference in larvae of the salmon louse (Lepeophtheirus salmonis).

    Science.gov (United States)

    Eichner, Christiane; Nilsen, Frank; Grotmol, Sindre; Dalvin, Sussie

    2014-05-01

    The salmon louse (Lepeophtheirus salmonis), an ectoparasitic copepod of salmonid fish, is a major threat to aquaculture in Norway, Ireland, Scotland and Canada. Due to rise in resistance against existing pesticides, development of novel drugs or vaccines is necessary. Posttranscriptional gene silencing by RNA interference (RNAi), when established in a high throughput system is a potential method for evaluation of molecular targets for new medical compounds or vaccine antigens. Successful use of RNAi has been reported in several stages of salmon lice. However, when we employed a previously described protocol for planktonic stages, no reproducible down-regulation of target genes was gained. In the present study, we describe a robust method for RNAi, where nauplius larvae are soaked in seawater added double stranded RNA (dsRNA). In order to test for when dsRNA may be introduced, and for the efficacy and duration of RNAi, we performed a series of experiments on accurately age determined larvae, ranging from the hatching egg to the copepodid with a salmon louse coatomer and a putative prostaglandin E synthase gene. Presumptive knock-down was monitored by real time PCR. Significant gene silencing was obtained only when nauplius I larvae were exposed to dsRNA during the period in which they molted to nauplius II. A knock down effect could be detected 2days after soaking, and it remained stable until the last measurement, on day 12. Soaking nauplius I larvae, knock-down was verified for six additional genes with a putative role in molting. For one chitinase, a loss-of-function phenotype with abnormal swimming was obtained. Hence, RNAi, induced in the nauplius, may facilitate studies of the molecular biology of the louse, such as the function of specific genes in developmental processes and physiology, host recognition, host-parasite interaction, and, in extension, the engineering of novel medicines. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. RNA interference suppressing PLCE1 gene expression decreases invasive power of human bladder cancer T24 cell line.

    Science.gov (United States)

    Ou, Liping; Guo, Yongcan; Luo, Chunli; Wu, Xiaohou; Zhao, Yi; Cai, Xiaozhong

    2010-07-15

    Mutational activation of the ras proto-oncogenes is frequently found in cancers. The phospholipase C epsilon gene (PLCE1) encodes a novel ras-related protein (R-Ras) effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion, because R-Ras is coprecipitated with the PLCE1 protein and can increase its activity. The nature of downstream signaling pathways from Ras involved in bladder cancer remains poorly understood. We aimed to construct a small hairpin RNA (shRNA) expression plasmid against the PLCE1 gene and to observe the inhibition of human bladder carcinoma cell T24 migration by RNA interference suppressing the expression of PLCE1. Two PLCE1 plasmids (P1 and P2) were constructed and inserted into T24 cells. Reverse transcriptase-polymerase chain reaction and Western blot analyses were performed to investigate inhibition of PLCE1 expression after plasmid transfection. Invasive power of the T24 cell line was measured before and after transfection by a membrane invasion culture system (transwell chamber), gelatin enzymography, and immunocytochemistry of cells. The RT-PCR analysis of BCL2 mRNA levels among different groups of T24 cell line indicated that expression of BCL2 mRNA was lower in the two positive plasmid-transfected cell groups than in the blank control or HK-A groups. Silencing of PLCE1 might downregulate the level of MMP and BCL2 gene expression, decreasing the invasive power of bladder cancer T24 cells and thus inhibiting tumor development. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  16. Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells.

    Directory of Open Access Journals (Sweden)

    Jaclyn C Scott

    2010-10-01

    Full Text Available The exogenous RNA interference (RNAi pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (siRNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2 cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

  17. Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells.

    Science.gov (United States)

    Scott, Jaclyn C; Brackney, Doug E; Campbell, Corey L; Bondu-Hawkins, Virginie; Hjelle, Brian; Ebel, Greg D; Olson, Ken E; Blair, Carol D

    2010-10-26

    The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

  18. A whole-genome RNA interference screen for human cell factors affecting myxoma virus replication.

    Science.gov (United States)

    Teferi, Wondimagegnehu M; Dodd, Kristopher; Maranchuk, Rob; Favis, Nicole; Evans, David H

    2013-04-01

    Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes ("hits") and nonsignificant genes ("nonhits") of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G(1), or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G(1)/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-D-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy.

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

    Science.gov (United States)

    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.

  20. Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

    Science.gov (United States)

    Hu, Zongli; Parekh, Urvi; Maruta, Natsumi; Trusov, Yuri; Botella, Jimmy

    2015-01-01

    Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Derived RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1 and OPR) in the hemi-biotrophic fungus Fusarium oxysporum f. sp. conglutinans. Expression of double stranded RNA molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75%, 83% and 72% reduction for FOW2, FRP1 and OPR respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and FOW2 between 45-70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

  1. Analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through a RNA interference screen.

    Directory of Open Access Journals (Sweden)

    Camille Stegen

    Full Text Available Viruses are strictly dependent on cells to propagate and many incorporate host proteins in their viral particles, but the significance of this incorporation is poorly understood. Recently, we performed the first comprehensive characterization of the mature herpes simplex virus type 1 (HSV-1 in which up to 49 distinct cellular proteins were identified by mass spectrometry. In the present study, we sought to identify if these cellular factors are relevant for the HSV-1 life cycle. To this end, we performed a small interfering RNA functional screen and found that 15 of these host proteins altered HSV-1 proliferation in cell culture, without any significant effect on cell viability. Moreover, the siRNA used had no negative consequences for Adenovirus type 5 propagation (with one exception indicating that the modulation was specific for HSV-1 and not merely due to unhealthy cells. The positive host proteins include several Rab GTPases and other intracellular transport components as well as proteins involved in signal transduction, gene regulation and immunity. Remarkably, in most cases when virions were depleted for one of the above proteins, they replicated more poorly in subsequent infections in wild type cells. This highlights for the first time that both the cellular and virion-associated pools of many of these proteins actively contribute to viral propagation. Altogether, these findings underscore the power and biological relevance of combining proteomics and RNA interference to identify novel host-pathogen interactions.

  2. Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

    Directory of Open Access Journals (Sweden)

    Zongli eHu

    2015-01-01

    Full Text Available Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Derived RNA interference (HD-RNAi technology to partially silence three different genes (FOW2, FRP1 and OPR in the hemi-biotrophic fungus Fusarium oxysporum f. sp. conglutinans. Expression of double stranded RNA molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75%, 83% and 72% reduction for FOW2, FRP1 and OPR respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and FOW2 between 45-70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

  3. [Expression of Jagged1 mRNA in human epithelial ovarian carcinoma tissues and effect of RNA interference of Jagged1 on growth of xenograft in nude mice].

    Science.gov (United States)

    Liu, G Y; Gao, Z H; Li, L; Song, T T; Sheng, X G

    2016-06-25

    To investigate the expression of Jagged1 in human epithelial ovarian carcinoma tissues and the effect of Jagged1 on growth of xenograft in nude mice. (1) Forty-eight cases of ovarian cancer and 30 cases of patients with benign epithelial ovarian tumor in the Henan Province Xinxiang Central Hospital during Feb. 2011 to Mar. 2014 were enrolled in this study. The mRNA expression of Jagged1, Notch1 and the downstream target genes Hes1, Hey1 were analyzed by using realtime PCR method. (2) The ovarian cancer xenograft models in nude mice were constructed by injecting SKOV3 cells in axillary subcutaneouswere. The nude mice were randomly divided into Jagged1 interference group, blank plasmid group and control group. Each group had 10 mice. They were transfected with pcDNA3.1(+)-siRNA-Jagged1, blank plasmid pDC3.1 and phosphate buffer, respectively. The tumor volumes and tumor masses were measured 14 days after transfection and the inhibition rate was calculated. The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues after transfection in each group was detected by using realtime PCR technique and the relative protein expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues was detected by utilizing western blot method. (1) The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in ovarian cancer tissues were higher than benign ovarian tumor tissues, the differences were statistically significant (Pinterference group, which were significantly lower than that in the blank plasmid group [(842±88) mm(3) and (4.4±0.8) g, respectively] and that in the control group [(851±90) mm(3) and (4.5±0.9) g, respectively; Pinterference group, which was significantly higher than that in the blank plasmid group and that in the control group (2.2% and 0, respectively), the differences were statistically significant (Pinterference group were lower than that in the other two groups, the differences were statistically significant (P0.05). Jagged1

  4. Targeting CCl4 -induced liver fibrosis by RNA interference-mediated inhibition of cyclin E1 in mice.

    Science.gov (United States)

    Bangen, Jörg-Martin; Hammerich, Linda; Sonntag, Roland; Baues, Maike; Haas, Ute; Lambertz, Daniela; Longerich, Thomas; Lammers, Twan; Tacke, Frank; Trautwein, Christian; Liedtke, Christian

    2017-10-01

    Initiation and progression of liver fibrosis requires proliferation and activation of resting hepatic stellate cells (HSCs). Cyclin E1 (CcnE1) is the regulatory subunit of the cyclin-dependent kinase 2 (Cdk2) and controls cell cycle re-entry. We have recently shown that genetic inactivation of CcnE1 prevents activation, proliferation, and survival of HSCs and protects from liver fibrogenesis. The aim of the present study was to translate these findings into preclinical applications using an RNA interference (RNAi)-based approach. CcnE1-siRNA (small interfering RNA) efficiently inhibited CcnE1 gene expression in murine and human HSC cell lines and in primary HSCs, resulting in diminished proliferation and increased cell death. In C57BL/6 wild-type (WT) mice, delivery of stabilized siRNA using a liposome-based carrier targeted approximately 95% of HSCs, 70% of hepatocytes, and 40% of CD45+ cells after single injection. Acute CCl4 -mediated liver injury in WT mice induced endogenous CcnE1 expression and proliferation of surviving hepatocytes and nonparenchymal cells, including CD45+ leukocytes. Pretreatment with CcnE1-siRNA reverted CcnE1 induction to baseline levels of healthy mice, which was associated with reduced liver injury, diminished proliferation of hepatocytes and leukocytes, and attenuated overall inflammatory response. For induction of liver fibrosis, WT mice were challenged with CCl4 for 4-6 weeks. Co-treatment with CcnE1-siRNA once a week was sufficient to continuously block CcnE1 expression and cell-cycle activity of hepatocytes and nonparenchymal cells, resulting in significantly ameliorated liver fibrosis and inflammation. Importantly, CcnE1-siRNA also prevented progression of liver fibrosis if applied after onset of chronic liver injury. Therapeutic targeting of CcnE1 in vivo using RNAi is feasible and has high antifibrotic activity. (Hepatology 2017;66:1242-1257). © 2017 by the American Association for the Study of Liver Diseases.

  5. Post-transcriptional inhibition of hepatitis C virus replication through small interference RNA

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-03-01

    Full Text Available Abstract Background Hepatitis C Virus (HCV infection is a major health problem throughout world that causes acute and chronic infection which resulted in liver fibrosis, hepatocellular carcinoma and death. The only therapy currently available for HCV infection is the combination of pegylated interferon alpha (PEG-IFN α and ribavirin. This therapy can effectively clear the virus infection in only 50% of infected individuals. Hence, there is a dire need to develop antiviral agents against HCV. Results This study was design to examine the ability of exogenous small interfering RNAs (siRNAs to block the replication of HCV in human liver cells. In the present study six 21-bp siRNAs were designed against different regions of HCV non-structural genes (NS2, NS3 serine protease/helicase, NS4Band NS5B RNA dependent RNA polymerase. siRNAs were labeled as NS2si241, NS3si-229, NS3si-858, NS4Bsi-166, NS5Bsi-241 and NS5Bsi-1064. We found that siRNAs against HCV NS2- NS5B efficiently inhibit HCV replication in Huh-7 cells. Our results demonstrated that siRNAs directed against HCV NS3 (NS3si-229 and NS3si-858 showed 58% and 88% reduction in viral titer respectively. Moreover, NS4Bsi-166 and NS5Bsi-1064 exhibited a dramatic reduction in HCV viral RNA and resulted in greater than 90% inhibition at a 20 μM concentration, while NS2si-241 showed 27% reduction in viral titer. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggest that siRNAs targeting against HCV non-structural genes (NS2-NS5B efficiently inhibit HCV replication and combination of these siRNAs of different targets and interferon will be better option to treat HCV infection throughout the world.

  6. Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug.

    Science.gov (United States)

    Bansal, Raman; Mittapelly, Priyanka; Chen, Yuting; Mamidala, Praveen; Zhao, Chaoyang; Michel, Andy

    2016-01-01

    The brown marmorated stink bug (Halyomorpha halys) has emerged as one of the most important invasive insect pests in the United States. Functional genomics in H. halys remains unexplored as molecular resources in this insect have recently been developed. To facilitate functional genomics research, we evaluated ten common insect housekeeping genes (RPS26, EF1A, FAU, UBE4A, ARL2, ARP8, GUS, TBP, TIF6 and RPL9) for their stability across various treatments in H. halys. Our treatments included two biotic factors (tissues and developmental stages) and two stress treatments (RNAi injection and starvation). Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). The qRT-PCR results indicated ARP8 and UBE4A exhibit the most stable expression across tissues and developmental stages, ARL2 and FAU for dsRNA treatment and TBP and UBE4A for starvation treatment. Following the dsRNA treatment, all genes except GUS showed relatively stable expression. To demonstrate the utility of validated reference genes in accurate gene expression analysis and to explore gene silencing in H. halys, we performed RNAi by administering dsRNA of target gene (catalase) through microinjection. A successful RNAi response with over 90% reduction in expression of target gene was observed.

  7. Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug.

    Directory of Open Access Journals (Sweden)

    Raman Bansal

    Full Text Available The brown marmorated stink bug (Halyomorpha halys has emerged as one of the most important invasive insect pests in the United States. Functional genomics in H. halys remains unexplored as molecular resources in this insect have recently been developed. To facilitate functional genomics research, we evaluated ten common insect housekeeping genes (RPS26, EF1A, FAU, UBE4A, ARL2, ARP8, GUS, TBP, TIF6 and RPL9 for their stability across various treatments in H. halys. Our treatments included two biotic factors (tissues and developmental stages and two stress treatments (RNAi injection and starvation. Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper and a web-based tool (RefFinder. The qRT-PCR results indicated ARP8 and UBE4A exhibit the most stable expression across tissues and developmental stages, ARL2 and FAU for dsRNA treatment and TBP and UBE4A for starvation treatment. Following the dsRNA treatment, all genes except GUS showed relatively stable expression. To demonstrate the utility of validated reference genes in accurate gene expression analysis and to explore gene silencing in H. halys, we performed RNAi by administering dsRNA of target gene (catalase through microinjection. A successful RNAi response with over 90% reduction in expression of target gene was observed.

  8. Global effects of the CSR-1 RNA interference pathway on transcriptional landscape

    Science.gov (United States)

    Cecere, Germano; Hoersch, Sebastian; O’Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

    2014-01-01

    Argonaute proteins and their small RNA co-factors short interfering RNAs (siRNAs) are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) antisense to germline transcripts and associates with chromatin in a siRNA-dependent manner. However, its role in gene expression regulation remains controversial. Here, we used a genome-wide profiling of nascent RNA transcripts to demonstrate that the CSR-1 RNAi pathway promotes sense-oriented Pol II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. Based on these findings, we propose that the CSR-1 pathway has a role in maintaining the directionality of active transcription thereby propagating the distinction between transcriptionally active and silent genomic regions. PMID:24681887

  9. Small regulatory RNAs of the RNA interference (RNAi) pathway as a prophylactic treatment against fish pathogenic viruses

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Hajiabadi, Seyed Amir Hossein Jalali; Kristensen, Lasse Bøgelund Juel

    2011-01-01

    Small RNAs acting in the recently discovered gene regulatory mechanism called RNA interference has a potential as diagnostic signatures of disease and immunological state and when produced synthetically as prophylactic treatment of such diseases. In the RNAi mechanism the cell produces different...... small RNAs which inhibit gene expression through more or less specific interaction with messenger RNAs resulting in repression of translation to protein. In this way cells can turn of genes of specific pathways thereby leading to altered physiological stages of tissues and possibly of whole organisms....... The mechanism can be programmed with several types of small double stranded RNAs - the type of which defines the destiny of the target. One such class of regulatory RNAs called microRNAs are upregulated due to various physiological responses of the cell and they suppress many genes simultaneously believed...

  10. Identification and RNA Interference of the Pheromone Biosynthesis Activating Neuropeptide (PBAN) in the Common Cutworm Moth Spodoptera litura (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Lu, Qin; Huang, Ling-Yan; Chen, Peng; Yu, Jin-Feng; Xu, Jin; Deng, Jian-Yu; Ye, Hui

    2015-06-01

    Spodoptera litura F. is one of the most destructive insect pests of many agricultural crops and notorious for developing insecticide resistance. Developing environmental friendly control methods such as novel pheromone and RNAi-related control strategies is imperative to control this pest. In the present study, the full-length cDNA encoding the diapause hormone and pheromone biosynthesis activating neuropeptide (DH-PBAN) was identified and characterized in S. litura. This 809-bp transcript contains a 573-nucleotide ORF encoding a 191-amino acid protein, from which five putative neuropeptides, including PBAN, DH, and α-, β-, and γ-subesophageal ganglion neuropeptides, were derived. Phylogenetic analysis showed that both the whole protein and each of the five neuropeptides have high similarities to those of DH-PBANs from other insect orders particularly Lepidoptera. Females treated with TKYFSPRLamide (the active core fragment of PBAN) produced significantly more four types of pheromone compounds (A; B; C; D) than controls. RNA interference by injection of PBAN dsRNA significantly reduced the relative expression levels of this gene in adult females (approximately reduced by 60%). As a consequence, females treated with PBAN dsRNA produced significantly less four types of pheromone compounds (A; B; C; D) than controls. These results suggest that PBAN function in activating sex pheromone biosynthesis and the RNAi of DH-PBAN gene can be induced by the injection of dsRNA into the body cavity in S. litura. This study suggests the possibility of novel pheromone-related pest control strategies based on RNAi techniques. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference.

    Directory of Open Access Journals (Sweden)

    Philippa M Beard

    Full Text Available Vaccinia virus (VACV is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.

  12. Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening

    Science.gov (United States)

    McDonough, Justin A.; Newton, Hayley J.; Klum, Scott; Swiss, Rachel; Agaisse, Hervé; Roy, Craig R.

    2013-01-01

    ABSTRACT Coxiella burnetii is an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used by C. burnetii to translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required for C. burnetii intracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication of C. burnetii was measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogated C. burnetii replication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles within C. burnetii-infected cells. Silencing of the STX17 gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containing C. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed for C. burnetii replication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate that C. burnetii vacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur during C. burnetii infection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication. PMID:23362322

  13. Targeting HMGB1 inhibits ovarian cancer growth and metastasis by lentivirus-mediated RNA interference.

    Science.gov (United States)

    Chen, Jie; Liu, Xiaoyan; Zhang, Jie; Zhao, Yueran

    2012-11-01

    High-mobility group box 1 (HMGB1), a nuclear and extracellular protein, is implicated in the development and progression of some types of cancers. However, no information is available to date regarding the function of HMGB1 in ovarian cancer. In this study, we performed cDNA microarray analysis and identified HMGB1 as a gene dramatically elevated in the highly invasive subclone S1 compared with the low invasive subclone S21 derived from the same cell line SKOV3. Then lentivirus vector with HMGB1 shRNA was constructed and infected the highly invasive cell line S1, A1, and HO8910PM. Real-time RT-PCR, Western blot, and IHC results confirmed the down-regulation of HMGB1 expression by its shRNA was about 80-90% at both the mRNA and protein levels. Knockdown of HMGB1 significantly suppressed ovarian cancer cell proliferation and induced cell cycle arrest at the G1/G0 phase, which was accompanied by decreased expressions of cyclin D1 and PCNA. Furthermore, knockdown of HMGB1 induced ovarian cancer cell apoptosis, which was mediated by increased expression of Bax and decreased expression of Bcl-2. Finally, knockdown of HMGB1 significantly inhibited ovarian cancer cell invasion and metastasis, which was regulated by decreased expressions of MMP2 and MMP9. Serum HMGB1 levels in patients with epithelial ovarian cancer were significantly higher than that in patients with benign ovarian tumor and healthy controls. These results indicate that HMGB1 is a newly identified gene associated with ovarian cancer growth and metastasis. HMGB1 may serve as a new therapeutic target for the treatment of ovarian cancer in the future. Copyright © 2012 Wiley Periodicals, Inc.

  14. Rational design of biosafe crop resistance to a range of nematodes using RNA interference.

    Science.gov (United States)

    Roderick, Hugh; Urwin, Peter E; Atkinson, Howard J

    2018-02-01

    Double-stranded RNA (dsRNA) molecules targeting two genes have been identified that suppress economically important parasitic nematode species of banana. Proteasomal alpha subunit 4 (pas-4) and Actin-4 (act-4) were identified from a survey of sequence databases and cloned sequences for genes conserved across four pests of banana, Radopholus similis, Pratylenchus coffeae, Meloidogyne incognita and Helicotylenchus multicinctus. These four species were targeted with dsRNAs containing exact 21 nucleotide matches to the conserved regions. Potential off-target effects were limited by comparison with Caenorhabditis, Drosophila, rat, rice and Arabidopsis genomes. In vitro act-4 dsRNA treatment of R. similis suppressed target gene expression by 2.3-fold, nematode locomotion by 66 ± 4% and nematode multiplication on carrot discs by 49 ± 5%. The best transgenic carrot hairy root lines expressing act-4 or pas-4 dsRNA reduced transcript message abundance of target genes in R. similis by 7.9-fold and fourfold and nematode multiplication by 94 ± 2% and 69 ± 3%, respectively. The same act-4 and pas-4 lines reduced P. coffeae target transcripts by 1.7- and twofold and multiplication by 50 ± 6% and 73 ± 8%. Multiplication of M. incognita on the pas-4 lines was reduced by 97 ± 1% and 99 ± 1% while target transcript abundance was suppressed 4.9- and 5.6-fold. There was no detectable RNAi effect on nontarget nematodes exposed to dsRNAs targeting parasitic nematodes. This work defines a framework for development of a range of nonprotein defences to provide broad resistance to pests and pathogens of crops. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  15. Regulation of gene expression in neuronal tissue by RNA interference and editing

    DEFF Research Database (Denmark)

    Venø, Morten Trillingsgaard

    in detailed data of both of these fine-tuning mechanisms in the embryonic development of the pig. Editing levels of transcripts examined are generally seen to increase through development, in agreement with editing of specific microRNA also examined in the Solexa sequencing study. Three studies examining......RNAs, causing these transgenic mice to be less prone to cocaine addictive behavior. Another study demonstrated that abolishing the expression of histone methylases, GLP and G9a, increases the expression level of a large number of miRNAs. A possible feed-back mechanism is suggested, since a subset of these mi...

  16. Effects and mechanism of downregulation of COX‑2 expression by RNA interference on proliferation and apoptosis of human breast cancer MCF‑7 cells.

    Science.gov (United States)

    Han, Hui; Yang, Sheng; Lin, Shun-Guo; Xu, Chun-Sen; Han, Zhong-Hua

    2014-12-01

    The aim of the present study was to investigate the effects of RNA interference with prostaglandin-endoperoxide synthase 2 (COX‑2) gene on the proliferation and apoptosis of breast cancer MCF‑7 cells, as well as the underlying mechanism. The present study constructed the eukaryotic expression vector of the targeted COX‑2 gene, transfected the MCF‑7 cells and screened the stably expressed clone. Changes in the COX‑2 gene expression in breast cancer MCF‑7 cells prior to and following transfection were examined; the proliferation and apoptosis of MCF‑7 cells were analyzed. Furthermore, changes in the protein levels of survivin, B-cell lymphoma 2 (Bcl‑2) and Bcl-2-associated X (Bax) genes were detected. RNA interference mediated by a lentiviral expression vector significantly decreased the protein expression levels of the COX‑2 gene, and therefore, the proliferation and growth of breast cancer MCF‑7 cells was significantly suppressed and the apoptotic rate increased. Of note, the mRNA and protein expression levels of survivin and Bcl‑2 decreased, while those of Bax increased following COX-2 silencing. RNA interference markedly deactivated the COX‑2 gene, suppressed the proliferation of breast cancer MCF‑7 cells, and, to a certain extent, enhanced the induced spontaneous apoptosis, which is regulated by the Bax gene. These results provided evidence for the potential applications of RNA interference of the targeted COX‑2 gene in gene therapy for the treatment of breast cancer.

  17. Evaluation of metaphylactic RNA interference to prevent equine herpesvirus type 1 infection in experimental herpesvirus myeloencephalopathy in horses.

    Science.gov (United States)

    Perkins, Gillian A; Van de Walle, Gerlinde R; Pusterla, Nicola; Erb, Hollis N; Osterrieder, Nikolaus

    2013-02-01

    To evaluate metaphylactic RNA interference to prevent equine herpesvirus type 1 (EHV-1) infection in experimental herpesvirus myeloencephalopathy in horses and to determine whether horses infected with a neuropathogenic strain of the virus that develop equine herpesvirus myeloencephalopathy (EHM) have differences in viremia. 13 seronegative horses. EHV-1 strain Ab4 was administered intranasally on day 0, and small interfering RNAs (siRNAs [EHV-1 specific siRNAs {n = 7} or an irrelevant siRNA {6}]) were administered intranasally 24 hours before and 12, 24, 36, and 48 hours after infection. Physical and neurologic examinations, nasal swab specimens, and blood samples were collected for virus isolation and quantitative PCR assay. Data from the study were combined with data from a previous study of 14 horses. No significant difference was detected in clinical variables, viremia, or detection of EHV-1 in nasal swab specimens of horses treated with the EHV-1 targeted siRNAs (sigB3-siOri2) versus controls. No significant differences in viremia were detected between horses that developed EHM and those that did not. Administration of siRNAs targeted against EHV-1 around the time of EHV-1 infection was not protective with this experimental design. Horses infected with the neuropathogenic EHV-1 strain Ab4 that developed EHM did not have a more pronounced viremia.

  18. RNA interference gene therapy in dominant retinitis pigmentosa and cone-rod dystrophy mouse models caused by GCAP1 mutations

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    Li eJiang

    2014-04-01

    Full Text Available RNA interference (RNAi knockdown is an efficacious therapeutic strategy for silencing genes causative for dominant retinal dystrophies. To test this, we used self-complementary (sc AAV2/8 vector to develop an RNAi-based therapy in two dominant retinal degeneration mouse models. The allele-specific model expresses transgenic bovine GCAP1(Y99C establishing a rapid RP-like phenotype, whereas the nonallele-specific model expresses mouse GCAP1(L151F producing a slowly progressing cone/rod dystrophy (CORD. The late onset GCAP1(L151F-CORD mimics the dystrophy observed in human GCAP1-CORD patients. Subretinal injection of scAAV2/8 carrying shRNA expression cassettes specific for bovine or mouse GCAP1 showed strong expression at one week post-injection. In both allele-specific (GCAP1(Y99C-RP and nonallele-specific (GCAP1(L151F-CORD models of dominant retinal dystrophy, RNAi-mediated gene silencing enhanced photoreceptor survival, delayed onset of degeneration and improved visual function. Such results provide a proof of concept toward effective RNAi-based gene therapy mediated by scAAV2/8 for dominant retinal disease based on GCAP1 mutation. Further, nonallele-specific RNAi knockdown of GCAP1 may prove generally applicable toward the rescue of any human GCAP1-based dominant cone-rod dystrophy.

  19. [Silenced NgR gene expression by RNA interference to promote rats facial nerve regeneration in vitro].

    Science.gov (United States)

    Shi, Yong; Zhou, Liang; Tian, Jie; Wang, Yang

    2014-05-01

    To suppress NgR gene expression in neural stem cells and observe differentiation of neural stem cells in vitro after interfered which provide nutritional support for the facial nerve repair in vivo. PCR amplification, restriction endonuclease digestion, T4DNA ligase connections were used to connected NgR with rector pGCsi, and constructed recombinant vector (NgR shRNA). Lipofectamine 2000 were used to transfect the NSC. The expression of NgR was examined by Western Blot. The proportion of neural stem cells transformed into neurons after transfection was tested by Immunocytochemistry. Neural stem cells were planted in PLGA tubes after transfected, and were scanned by electron microscopy. NgR shRNA plasmid was constructed and infected neural stem cells successfully. Western Blot showed that the expression of NgR decreased in neural stem cells after interference. Immunocytochemistry showed that the rate of the neural stem cells transformed into neurons after interfered was significantly higher (P facial nerve repair.

  20. Targeting SPARC by lentivirus-mediated RNA interference inhibits cervical cancer cell growth and metastasis

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    Chen Jie

    2012-10-01

    Full Text Available Abstract Background Secreted protein acidic and rich in cysteine (SPARC, a calcium-binding matricellular glycoprotein, is implicated in the progressions of some cancers. However, no information has been available to date regarding the function of SPARC in cervical cancer cell growth and metastasis. Methods In this study, we isolated and established high invasive subclones and low invasive subclones from human cervical cancer cell lines HeLa and SiHa by the limited dilution method. Real-time q-RT-PCR, Western Blot and ICC were performed to investigate SPARC mRNA and protein expressions in high invasive subclones and low invasive subclones. Then lentivirus vector with SPARC shRNA was constructed and infected the highly invasive subclones. Real-time q-RT-PCR, Western Blot and ICC were also performed to investigate the changes of SPARC expression after viral infection. In functional assays, effects of SPARC knockdown on the biological behaviors of cervical cancer cells were investigated. The mechanisms of SPARC in cervical cancer proliferation, apoptosis and invasion were also researched. Results SPARC was over-expressed in the highly invasive subclones compared with the low invasive subclones. Knockdown of SPARC significantly suppressed cervical cancer cell proliferation, and induced cell cycle arrest at the G1/G0 phase through the p53/p21 pathway, also caused cell apoptosis accompanied by the decreased ratio of Bcl-2/Bax, and inhibited cell invasion and metastasis accompanied by down-regulated MMP2 and MMP9 expressions and up-regulated E-cadherin expression. Conclusion SPARC is related to the invasive phenotype of cervical cancer cells. Knockdown of SPARC significantly suppresses cervical cancer cell proliferation, induces cell apoptosis and inhibits cell invasion and metastasis. SPARC as a promoter improves cervical cancer cell growth and metastasis.

  1. Large-scale RNA interference screening in mammalian cells identifies novel regulators of mutant huntingtin aggregation.

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    Tomoyuki Yamanaka

    Full Text Available In polyglutamine (polyQ diseases including Huntington's disease (HD, mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.

  2. RNA interference of chitin synthase genes inhibits chitin biosynthesis and affects larval performance in Leptinotarsa decemlineata (Say).

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    Shi, Ji-Feng; Mu, Li-Li; Chen, Xu; Guo, Wen-Chao; Li, Guo-Qing

    2016-01-01

    Dietary introduction of bacterially expressed double-stranded RNA (dsRNA) has great potential for management of Leptinotarsa decemlineata. Identification of the most attractive candidate genes for RNA interference (RNAi) is the first step. In the present paper, three complete chitin synthase cDNA sequences (LdChSAa, LdChSAb and LdChSB) were cloned. LdChSAa and LdChSAb, two splicing variants of LdChSA gene, were highly expressed in ectodermally-derived epidermal cells forming epidermis, trachea, foregut and hindgut, whereas LdChSB was mainly transcribed in midgut cells. Feeding bacterially expressed dsChSA (derived from a common fragment of LdChSAa and LdChSAb), dsChSAa, dsChSAb and dsChSB in the second- and fourth-instar larvae specifically knocked down their target mRNAs. RNAi of LdChSAa+LdChSAb and LdChSAa lowered chitin contents in whole body and integument samples, and thinned tracheal taenidia. The resulting larvae failed to ecdyse, pupate, or emerge as adults. Comparably, knockdown of LdChSAb mainly affected pupal-adult molting. The LdChSAb RNAi pupae did not completely shed the old larval exuviae, which caused failure of adult emergence. In contrast, silencing of LdChSB significantly reduced foliage consumption, decreased chitin content in midgut sample, damaged midgut peritrophic matrix, and retarded larval growth. As a result, the development of the LdChSB RNAi hypomorphs was arrested. Our data reveal that these LdChSs are among the effective candidate genes for an RNAi-based control strategy against L. decemlineata.

  3. Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system.

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    Ehlert, Erich M; Eggers, Ruben; Niclou, Simone P; Verhaagen, Joost

    2010-02-18

    After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2). We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents. RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.

  4. Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system

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    Verhaagen Joost

    2010-02-01

    Full Text Available Abstract Background After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs and several axon guidance molecules, including all members of the secreted (class 3 Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV mediated expression of short hairpin RNAs (shRNAs to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1 and Neuropilin 2 (Npn-2. Results We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents. Conclusions RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.

  5. Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica.

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    Gabriel Rinaldi

    2008-07-01

    Full Text Available The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite-host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC. We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP, and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth

  6. Expression and RNA Interference of Ribosomal Protein L5 Gene in Nilaparvata lugens (Hemiptera: Delphacidae).

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    Zhu, Jiajun; Hao, Peiying; Lu, Chaofeng; Ma, Yan; Feng, Yalin; Yu, Xiaoping

    2017-05-01

    The ribosomal proteins play important roles in the growth and development of organisms. This study aimed to explore the function of NlRPL5 (GenBank KX379234), a ribosomal protein L5 gene, in the brown planthopper Nilaparvata lugens. The open reading frame of NlRPL5 was cloned from N. lugens based on a previous transcriptome analysis. The results revealed that the open reading frame of NlRPL5 is of 900 bp, encoding 299 amino acid residues. The reverse transcription quantitative PCR results suggested that the expression of NlRPL5 gene was stronger in gravid females, but was relatively low in nymphs, males, and newly emerged females. The expression level of NlRPL5 in the ovary was about twofolds of that in the head, thorax, or fat body. RNAi of dsNlRPL5 resulted in a significant reduction of mRNA levels, ∼50% decrease in comparison with the dsGFP control at day 6. Treatment of dsNlRPL5 significantly restricted the ovarian development, and decreased the number of eggs laid on the rice (Oryza sativa) plants. This study provided a new clue for further study on the function and regulation mechanism of NlRPL5 in N. lugens. © The Author 2017. Published by Oxford University Press on behalf of the Entomological Society of America.

  7. Engineered disease resistance in cotton using RNA-interference to knock down cotton leaf curl kokhran virus-Burewala and cotton leaf curl Multan betasatellite

    Science.gov (United States)

    Cotton Leaf Curl virus Disease (CLCuD) has caused enormous losses in cotton (Gossypium hirsutum) production in Pakistan. RNA interference (RNAi) is an emerging technique that could knock out CLCuD by targeting different regions of the pathogen genome that are important for replication, transcription...

  8. RNA interference in the Asian Longhorned Beetle:Identification of Key RNAi Genes and Reference Genes for RT-qPCR

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    Asian longhorned beetle (ALB), Anoplophora glabripennis, is a serious invasive forest pest in several countries including the United States, Canada, and Europe. RNA interference (RNAi)technology is being developed as a novel method for pest management. Here, we identified the ALB core RNAi genes in...

  9. Analysis of small-sample clinical genomics studies using multi-parameter shrinkage: application to high-throughput RNA interference screening

    NARCIS (Netherlands)

    van de Wiel, M.; Menezes, R.; van Olst, E.; van Beusechem, V.W.

    2013-01-01

    High-throughput (HT) RNA interference (RNAi) screens are increasingly used for reverse genetics and drug discovery. These experiments are laborious and costly, hence sample sizes are often very small. Powerful statistical techniques to detect siRNAs that potentially enhance treatment are currently

  10. In vitro and in vivo inhibition of rabies virus replication by RNA interference

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    Ekaterina A. Durymanova Ono

    2013-09-01

    Full Text Available Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV (Mononegavirales: Rhabdoviridae: Lyssavirus. Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

  11. Identification of Drosophila mitotic genes by combining co-expression analysis and RNA interference.

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    Maria Patrizia Somma

    2008-07-01

    Full Text Available RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression-based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression-based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins.

  12. In vitro and in vivo inhibition of rabies virus replication by RNA interference.

    Science.gov (United States)

    Durymanova Ono, Ekaterina A; Iamamoto, Keila; Castilho, Juliana G; Carnieli, Pedro; de Novaes Oliveira, Rafael; Achkar, Samira M; Carrieri, Maria L; Kotait, Ivanete; Brandão, Paulo E

    2013-01-01

    Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

  13. Adeno-associated viruses serotype 2-mediated RNA interference efficiently inhibits rabies virus replication in vitro and in vivo.

    Science.gov (United States)

    Wu, Hong-Xia; Wang, Hua-Lei; Guo, Xiao-Feng; Yang, Yu-Jiao; Ma, Jin-Zhu; Wang, Tie-Cheng; Gao, Yu-Wei; Zhao, Yong-Kun; Yang, Song-Tao; Xia, Xian-Zhu

    2013-10-01

    To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RABV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RABV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RABV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication, inhibited RABV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies.

  14. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells

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    Liu X

    2016-04-01

    Full Text Available Xiaoxia Liu, Guiling Sun, Xiuju Sun Department of Nephrology, Affiliated Hospital of Weifang Medical University, Weifang, People’s Republic of China Abstract: This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP gene on renal cell cancer (RCC cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte–macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05. The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial

  15. An RNA interference screen uncovers a new molecule in stem cell self-renewal and long-term regeneration.

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    Chen, Ting; Heller, Evan; Beronja, Slobodan; Oshimori, Naoki; Stokes, Nicole; Fuchs, Elaine

    2012-04-04

    Adult stem cells sustain tissue maintenance and regeneration throughout the lifetime of an animal. These cells often reside in specific signalling niches that orchestrate the stem cell's balancing act between quiescence and cell-cycle re-entry based on the demand for tissue regeneration. How stem cells maintain their capacity to replenish themselves after tissue regeneration is poorly understood. Here we use RNA-interference-based loss-of-function screening as a powerful approach to uncover transcriptional regulators that govern the self-renewal capacity and regenerative potential of stem cells. Hair follicle stem cells provide an ideal model. These cells have been purified and characterized from their native niche in vivo and, in contrast to their rapidly dividing progeny, they can be maintained and passaged long-term in vitro. Focusing on the nuclear proteins and/or transcription factors that are enriched in stem cells compared with their progeny, we screened ∼2,000 short hairpin RNAs for their effect on long-term, but not short-term, stem cell self-renewal in vitro. To address the physiological relevance of our findings, we selected one candidate that was uncovered in the screen: TBX1. This transcription factor is expressed in many tissues but has not been studied in the context of stem cell biology. By conditionally ablating Tbx1 in vivo, we showed that during homeostasis, tissue regeneration occurs normally but is markedly delayed. We then devised an in vivo assay for stem cell replenishment and found that when challenged with repetitive rounds of regeneration, the Tbx1-deficient stem cell niche becomes progressively depleted. Addressing the mechanism of TBX1 action, we discovered that TBX1 acts as an intrinsic rheostat of BMP signalling: it is a gatekeeper that governs the transition between stem cell quiescence and proliferation in hair follicles. Our results validate the RNA interference screen and underscore its power in unearthing new molecules that

  16. [Silencing hypoxia inducible factor-2α gene by small interference RNA inhibits the growth of mammosphere cells in nude mice under hypoxic microenvironment].

    Science.gov (United States)

    Qu, Hong-bo; Fan, Yuan-ming; Han, Ming-li; Zeng, Ni; Zhu, Zhi-kun; Liu, Hong; Xie, Jia; Wu, Cheng-yi; Tang, Wei-xue

    2013-04-16

    To explore the effects of silencing hypoxia inducible factor-2α (HIF-2α) by small interference RNA on the growth of mammosphere cells in nude mice under hypoxic microenvironment. The empty and interference vectors were transfected into MCF-7 cell. Then G418 was added to screen the positive cells to obtain stable cell line. The empty and interference vectors were inoculated subcutaneously into left and right back near hind limb of nude mice. The volume and weight of tumors were calculated respectively. The expressions of HIF-2α, CD44, OCT-4 and KLF-4 protein in xenograft tumor tissues were detected by Western blot. The expression vector of HIF-2α-siRNA was successfully established. The formation of mammosphere was lowered by silencing HIF-2α gene expression. In contract to empty vector group cell, there were obvious decreases in the volumes and weights of tumors in interference group (P interference group (0.42 ± 0.01) was much lower than that of the empty vector group (0.89 ± 0.03, P interference group (0.21 ± 0.01) was much lower than the empty vector group (0.78 ± 0.03, P interference group (0.42 ± 0.01)was much lower than the empty vector group (0.68 ± 0.03, P interference group (0.34 ± 0.01) was much lower than the empty vector group (0.72 ± 0.03, P < 0.05). Silencing HIF-2α gene can effectively inhibit the growth of breast cancer stem cells in nude mice under hypoxic microenvironment. Its mechanism may be through inhibited capacity for self-renewal and proliferation of breast cancer stem cells in vivo through the down-regulated expressions of markers associated with stem cells. HIF-2α is expected to become a new target for gene therapy of breast cancer.

  17. An interference-free and label-free sandwich-type magnetic silicon microsphere -rGO-based probe for fluorescence detection of microRNA.

    Science.gov (United States)

    Li, Shiyu; He, Kui; Liao, Rong; Chen, Chunyan; Chen, Xiaoming; Cai, Changqun

    2017-11-01

    An interference-free and label-free sensing platform was developed for the highly sensitive detection of microRNA-21 (miRNA-21) in vitro by magnetic silicon microsphere (MNP)-reduced graphene oxide (rGO)-based sandwich probe. In this method, DNA capture probes (P1) were connected with MNPs at the 5' end and hybridized with completely complementary target miRNA. Subsequently, rGO was retained and induced the fluorescence quenching in the supernatant. Through the magnetic separation, the supernatant environment was simplified and the interference to analytical signal was eliminated. When DNA capture probe-modified magnetic silicon microspheres (MNP-P1) were adsorbed through rGO in the absence of a target and formed a sandwich structure, the formed nanostructure was easily removed from the solution by a magnetic field and the fluorescence intensity was maximally recovered. This proposed strategy, which both overcame the expensive and cumbersome fluorescent labeling, and eliminated interference to analytical signal for guaranteeing high signal-to-background ratio, exhibited high sensitivity with a detection limit as low as 0.098nM and special selectivity toward miRNA-21. The method was potentially applicable for not only detection of miRNA-21 but also various biomarker analyses just by changing capture probes. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. [Construction a lentiviral vector for RNA interference of glutathione peroxidase 2 gene and its effect on HepG2 cell apoptosis].

    Science.gov (United States)

    Cao, Jiangping; Tang, Liujun; Zhang, Jianhong; Zhan, Yiqun; Yang, Xiaoming; Ge, Changhui

    2015-06-01

    To construct a RNA interference lentiviral vector for human glutathione peroxidase 2 (GPX2) gene and observe the effect of GPX2 knockdown on cell apoptosis. The sequence of the small interfering RNA (siRNA) for GPX2 interference was inserted into the pSicoR vector. HepG2 cells were infected by the packaged si-GPX2 lentivirus and the expression of GPX2 in the infected cells was detected by both RT-PCR and Western blotting. Changes of cell apoptosis following the infection were analyzed by flow cytometry. The lentiviral particles pSicoR-GPX2 were successfully packaged. The expression of GPX2 in the infected cells was obviously down-regulated at both RNA and protein levels. GPX2 knockdown caused increased apoptosis rate, increased Bax expression and lowered Bcl-2 expression in HepG2 cells. We have successfully constructed the lentiviral vector for RNA interference of human GPX2 gene.

  19. Systematic Identification and Assessment of Therapeutic Targets for Breast Cancer Based on Genome-Wide RNA Interference Transcriptomes

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2017-02-01

    Full Text Available With accumulating public omics data, great efforts have been made to characterize the genetic heterogeneity of breast cancer. However, identifying novel targets and selecting the best from the sizeable lists of candidate targets is still a key challenge for targeted therapy, largely owing to the lack of economical, efficient and systematic discovery and assessment to prioritize potential therapeutic targets. Here, we describe an approach that combines the computational evaluation and objective, multifaceted assessment to systematically identify and prioritize targets for biological validation and therapeutic exploration. We first establish the reference gene expression profiles from breast cancer cell line MCF7 upon genome-wide RNA interference (RNAi of a total of 3689 genes, and the breast cancer query signatures using RNA-seq data generated from tissue samples of clinical breast cancer patients in the Cancer Genome Atlas (TCGA. Based on gene set enrichment analysis, we identified a set of 510 genes that when knocked down could significantly reverse the transcriptome of breast cancer state. We then perform multifaceted assessment to analyze the gene set to prioritize potential targets for gene therapy. We also propose drug repurposing opportunities and identify potentially druggable proteins that have been poorly explored with regard to the discovery of small-molecule modulators. Finally, we obtained a small list of candidate therapeutic targets for four major breast cancer subtypes, i.e., luminal A, luminal B, HER2+ and triple negative breast cancer. This RNAi transcriptome-based approach can be a helpful paradigm for relevant researches to identify and prioritize candidate targets for experimental validation.

  20. Targeting the pseudorabies virus DNA polymerase processivity factor UL42 by RNA interference efficiently inhibits viral replication.

    Science.gov (United States)

    Wang, Yi-Ping; Huang, Li-Ping; Du, Wen-Juan; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

    2016-08-01

    RNA interference (RNAi) is a conserved gene-silencing mechanism in which small interfering RNAs (siRNAs) induce the sequence-specific degradation of homologous RNAs. It has been shown to be a novel and effective antiviral therapy against a wide range of viruses. The pseudorabies virus (PRV) processivity factor UL42 can enhance the catalytic activity of the DNA polymerase and is essential for viral replication, thus it may represent a potential drug target of antiviral therapy against PRV infection. Here, we synthesized three siRNAs (siR-386, siR-517, and siR-849) directed against UL42 and determined their antiviral activities in cell culture. We first examined the kinetics of UL42 expression and found it was expressed with early kinetics during PRV replication. We verified that siR-386, siR-517, and siR-849 efficiently inhibited UL42 expression in an in vitro transfection system, thereby validating their inhibitory effects. Furthermore, we confirmed that these three siRNAs induced potent inhibitory effects on UL42 expression after PRV infection, comparable to the positive control siRNA, siR-1046, directed against the PRV DNA polymerase, the UL30 gene product, which is essential for virus replication. In addition, PRV replication was markedly reduced upon downregulation of UL42 expression. These results indicate that UL42-targeted RNAi efficiently inhibits target gene expression and impairs viral replication. This study provides a new clue for the design of an intervention strategy against herpesviruses by targeting their processivity factors. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Identification of a thioredoxin peroxidase gene involved in resistance to nucleopolyhedrovirus infection in Helicoverpa armigera with RNA interference.

    Science.gov (United States)

    Zhang, Songdou; Shen, Zhongjian; Li, Zhen; Wu, Fengming; Zhang, Boyu; Liu, Yanjun; Zhang, Qingwen; Liu, Xiaoxia

    2015-11-01

    Thioredoxin peroxidases (Tpxs) play a crucial role in protection against oxidative damage in several insect species. However, studies on the characteristics and functions of Tpxs in Helicoverpa armigera are lacking. In this study, a novel 2-Cys Tpx gene from H. armigera (HaTpx) was identified. Sequence analysis revealed that HaTpx is highly conserved and shares two catalysis regions (VCP) with other insect species. HaTpx mRNA was found to be expressed in an age-dependent manner and was ubiquitous in all tissues examined. Hormone treatment showed that the expression of HaTpx is clearly induced by 20-hydroxyecdysone but repressed by Juvenile hormone. Additionally, extreme temperature, ultraviolet light, mechanical injury, Escherichia coli, Metarhizium anisopliae, nucleopolyhedrovirus (NPV) infection, and H2O2 treatment markedly induced HaTpx gene expression. Reactive oxygen species (ROS) levels in hemocytes and MDA concentrations in the hemolymph after NPV infection were evaluated, and the results indicated that NPV infection causes excessive ROS generation. After knockdown of HaTpx by RNA interference, the expression of three antioxidant genes (Cu/ZnSOD, Trx, and TrxR) was increased, whereas two antioxidant genes (CAT and GPX) showed decreased expression. Moreover, the susceptibility of H. armigera to NPV infection increased after HaTpx knockdown. These results indicated that HaTpx contributes to the susceptibility of H. armigera to NPV, and the results also provide a theoretical basis for a novel strategy for developing new chemicals and microbial pesticides that target HaTpx gene for controlling H. armigera. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Review of the RNA Interference Pathway in Molluscs Including Some Possibilities for Use in Bivalves in Aquaculture

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    Leigh Owens

    2015-03-01

    Full Text Available Generalised reviews of RNA interference (RNAi in invertebrates, and for use in aquaculture, have taken for granted that RNAi pathways operate in molluscs, but inspection of such reviews show little specific evidence of such activity in molluscs. This review was to understand what specific research had been conducted on RNAi in molluscs, particularly with regard to aquaculture. There were questions of whether RNAi in molluscs functions similarly to the paradigm established for most eukaryotes or, alternatively, was it more similar to the ecdozoa and how RNAi may relate to disease control in aquaculture? RNAi in molluscs appears to have been only investigated in about 14 species, mostly as a gene silencing phenomenon. We can infer that microRNAs including let-7 are functional in molluscs. The genes/proteins involved in the actual RNAi pathways have only been rudimentarily investigated, so how homologous the genes and proteins are to other metazoa is unknown. Furthermore, how many different genes for each activity in the RNAi pathway are also unknown? The cephalopods have been greatly overlooked with only a single RNAi gene-silencing study found. The long dsRNA-linked interferon pathways seem to be present in molluscs, unlike some other invertebrates and could be used to reduce disease states in aquaculture. In particular, interferon regulatory factor genes have been found in molluscs of aquacultural importance such as Crassostrea, Mytilus, Pinctada and Haliotis. Two possible aquaculture scenarios are discussed, zoonotic norovirus and ostreid herpesvirus 1 to illustrate the possibilities. The entire field of RNAi in molluscs looks ripe for scientific exploitation and practical application.

  3. Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference.

    Science.gov (United States)

    Tants, Jan-Niklas; Fesser, Stephanie; Kern, Thomas; Stehle, Ralf; Geerlof, Arie; Wunderlich, Christoph; Juen, Michael; Hartlmüller, Christoph; Böttcher, Romy; Kunzelmann, Stefan; Lange, Oliver; Kreutz, Christoph; Förstemann, Klaus; Sattler, Michael

    2017-12-01

    RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing we demonstrate that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. RNA interference of Marlin-1/Jakmip1 results in abnormal morphogenesis and migration of cortical pyramidal neurons.

    Science.gov (United States)

    Vidal, René L; Fuentes, Patricio; Valenzuela, José Ignacio; Alvarado-Diaz, Carlos P; Ramírez, Omar A; Kukuljan, Manuel; Couve, Andrés

    2012-08-01

    The formation of the nervous systems requires processes that coordinate proliferation, differentiation and migration of neuronal cells, which extend axons, generate dendritic branching and establish synaptic connections during development. The structural organization and dynamic remodeling of the cytoskeleton and its association to the secretory pathway are critical determinants of cell morphogenesis and migration. Marlin-1 (Jakmip1) is a microtubule-associated protein predominantly expressed in neurons and lymphoid cells. Marlin-1 participates in polarized secretion in lymphocytes, but its functional association with the neuronal cytoskeleton and its contribution to brain development have not been explored. Combining in vitro and in vivo approaches we show that Marlin-1 contributes to the establishment of neuronal morphology. Marlin-1 associates to the cytoskeleton in neurites, is required for the maintenance of an intact Golgi apparatus and its depletion produces the down-regulation of kinesin-1, a plus-end directed molecular motor with a central function in morphogenesis and migration. RNA interference of Marlin-1 in vivo results in abnormal migration of newborn pyramidal neurons during the formation of the cortex. Our results support the involvement of Marlin-1 in the acquisition of the complex architecture and migration of pyramidal neurons, two fundamental processes for the laminar layering of the cortex. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Silencing of Entamoeba histolytica Glucosamine 6-Phosphate Isomerase by RNA Interference Inhibits the Formation of Cyst-Like Structures

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    Hugo Aguilar-Díaz

    2013-01-01

    Full Text Available Encystment is an essential process in the biological cycle of the human parasite Entamoeba histolytica. In the present study, we evaluated the participation of E. histolytica Gln6Pi in the formation of amoeba cyst-like structures by RNA interference assay. Amoeba trophozoites transfected with two Gln6Pi siRNAs reduced the expression of the enzyme in 85%, which was confirmed by western blot using an anti-Gln6Pi antibody. The E. histolytica Gln6Pi knockdown with the mix of both siRNAs resulted in the loss of its capacity to form cyst-like structures (CLSs and develop a chitin wall under hydrogen peroxide treatment, as evidenced by absence of both resistance to detergent treatment and calcofluor staining. Thus, only 5% of treated trophozoites were converted to CLS, from which only 15% were calcofluor stained. These results represent an advance in the understanding of chitin biosynthesis in E. histolytica and provide insight into the encystment process in this parasite, which could allow for the developing of new control strategies for this parasite.

  6. Normalization of Overexpressed α-Synuclein Causing Parkinson's Disease By a Moderate Gene Silencing With RNA Interference

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    Masaki Takahashi

    2015-01-01

    Full Text Available The α-synuclein (SNCA gene is a responsible gene for Parkinson's disease (PD; and not only nucleotide variations but also overexpression of SNCA appears to be involved in the pathogenesis of PD. A specific inhibition against mutant SNCA genes carrying nucleotide variations may be feasible by a specific silencing such as an allele-specific RNA interference (RNAi; however, there is no method for restoring the SNCA overexpression to a normal level. Here, we show that an atypical RNAi using small interfering RNAs (siRNAs that confer a moderate level of gene silencing is capable of controlling overexpressed SNCA genes to return to a normal level; named “expression-control RNAi” (ExCont-RNAi. ExCont-RNAi exhibited little or no significant off-target effects in its treated PD patient's fibroblasts that carry SNCA triplication. To further assess the therapeutic effect of ExCont-RNAi, PD-model flies that carried the human SNCA gene underwent an ExCont-RNAi treatment. The treated PD-flies demonstrated a significant improvement in their motor function. Our current findings suggested that ExCont-RNAi might be capable of becoming a novel therapeutic procedure for PD with the SNCA overexpression, and that siRNAs conferring a moderate level of gene silencing to target genes, which have been abandoned as useless siRNAs so far, might be available for controlling abnormally expressed disease-causing genes without producing adverse effects.

  7. Knockdown of USP39 by lentivirus-mediated RNA interference suppresses the growth of oral squamous cell carcinoma.

    Science.gov (United States)

    Li, Ke-Yi; Zhang, Jie; Jiang, Li-Cheng; Zhang, Bin; Xia, Chun-Peng; Xu, Kai; Chen, Hai-Ying; Yang, Qiao-Zhi; Liu, Shu-Wei; Zhu, Hong

    2016-01-01

    Oral squamous cell carcinoma (OSCC) is a frequently diagnosed life-threatening oral cancer worldwide and has become one of the leading causes of cancer-related mortality. However, the pathogenesis of this disease is very limited. In this study, we aimed to investigate the functional relationship between OSCC and a potential tumor related gene ubiquitin-specific proteases 39 (USP39). The lentivirus-based RNA interference was utilized to knock down USP39 expression in human OSCC CAL27 cells. The effect of USP39 on cell proliferation was detected by MTT and colony formation assays. The results uncovered that the proliferation rate was significantly decreased in specific USP39-targeting lentivirus infected cells compared to control lentivirus infected cells. The colony formation capacity was also attenuated in CAL27 cells after USP39 knockdown. Moreover, knockdown of USP39 arrested CAL27 cells in S and G1/M phases of the cell cycle. Furthermore, USP39 silencing induced apoptosis of CAL27 cells via activations of Caspase 3 and PARP. In conclusion, the inhibition of USP39 in CAL27 cells suppressed cell growth probably via induction cell cycle arrest and apoptosis. USP39 might act as an oncogenic factor in OSCC and could be a potential molecular target for OSCC gene therapy.

  8. Functional analysis of a chitinase gene during the larval-nymph transition in Panonychus citri by RNA interference.

    Science.gov (United States)

    Xia, Wen-Kai; Shen, Xiao-Min; Ding, Tian-Bo; Niu, Jin-Zhi; Zhong, Rui; Liao, Chong-Yu; Feng, Ying-Cai; Dou, Wei; Wang, Jin-Jun

    2016-09-01

    Chitinases are hydrolytic enzymes that are required for chitin degradation and reconstruction in arthropods. In this study, we report a cDNA sequence encoding a putative chitinase (PcCht1) from the citrus red mite, Panonychus citri. The PcCht1 (564 aa) possessed a signal peptide, a conserver domain, and a chitin-binding domain. Structural and phylogenetic analyses found that PcCht1 had high sequence similarity to chitinases in Tetranychus urticae. Real-time quantitative PCR analyses showed that the transcript levels of PcCht1 peaked periodically in larval and nymph stages. Moreover, significant increase of PcCht1 transcript level in the larvae was observed upon the exposure of diflubenzuron. In contrast, exposures of the larvae to diflubenzuron resulted in the decreased chitin content. Furthermore, through a feeding-based RNA interference approach, we were able to reduce the PcCht1 transcript level by 59.7 % in the larvae, and consequently the treated larvae showed a very low molting rate compared with the control. Our results expanded the understanding of the important role of PcCht1 in the growth and development of P. citri.

  9. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato.

    Science.gov (United States)

    Mamta; Reddy, K R K; Rajam, M V

    2016-02-01

    Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a devastating agricultural insect pest with broad spectrum of host range, causing million dollars crop loss annually. Limitations in the present conventional and transgenic approaches have made it crucial to develop sustainable and environmental friendly methods for crop improvement. In the present study, host-induced RNA interference (HI-RNAi) approach was used to develop H. armigera resistant tobacco and tomato plants. Chitinase (HaCHI) gene, critically required for insect molting and metamorphosis was selected as a potential target. Hair-pin RNAi construct was prepared from the conserved off-target free partial HaCHI gene sequence and was used to generate several HaCHI-RNAi tobacco and tomato plants. Northern hybridization confirmed the production of HaCHI gene-specific siRNAs in HaCHI-RNAi tobacco and tomato lines. Continuous feeding on leaves of RNAi lines drastically reduced the target gene transcripts and consequently, affected the overall growth and survival of H. armigera. Various developmental deformities were also manifested in H. armigera larvae after feeding on the leaves of RNAi lines. These results demonstrated the role of chitinase in insect development and potential of HI-RNAi for effective management of H. armigera.

  10. Effects of RNA interference combined with ultrasonic irradiation and SonoVue microbubbles on expression of STAT3 gene in keratinocytes of psoriatic lesions.

    Science.gov (United States)

    Ran, Li-Wei; Wang, Hao; Lan, Dong; Jia, Hong-Xia; Yu, Si-Si

    2017-04-01

    The most effective sequence of small interfering RNA (siRNA) silencing STAT3 of psoriatic keratinocytes (KCs) was screened out, and the effects of the most effective siRNA combined with ultrasonic irradiation and SonoVue microbubbles on the expression of STAT3 of KCs and the dose- and time-response were investigated. Three chemically-synthetic siRNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs, and the effects on STAT3 expression were detected, then the most effective siRNA was selected for the subsequent experiments. The negative controls of siRNA (siRNA-NC) labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles were transfected into KCs, then the optimal parameters of ultrasonic irradiation were determined. The most effective siRNA carried by Li-pofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and SonoVue microbubbles was transfected into KCs, and the dose- and time-response of RNA interference was determined. The effect of RNA interference by the most effective siRNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group) was compared with that only carried by Li-pofectamine 3000 (L group). The results showed that siRNA-3 achieved the highest silencing efficacy. 0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation. The siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression at mRNA and protein levels in dose- and time-dependent manners determined at 100 nmol/L with maximum downregulation on mRNA at 48 h, and on protein at 72 h after transfection. The LUS group achieved the highest silencing efficacy. It was concluded that siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3

  11. Silencing of RhoA and RhoC expression by RNA interference suppresses human colorectal carcinoma growth in vivo

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    Wang Haibo

    2010-09-01

    Full Text Available Abstract Background RhoA and RhoC have been proved to be over-expressed in many solid cancers, including colorectal cancer. The reduction of RhoA and RhoC expression by RNA interference (RNAi resulted growth inhibition of cancer cells. The present study was to evaluate the effect of silencing of RhoA and RhoC expression by RNAi on growth of human colorectal carcinoma (CRC in tumor-bearing nude mice in vivo. Methods To establish HCT116 cell transplantable model, the nude mice were subcutaneously inoculated with 1.0 × 107 HCT116 cells and kept growing till the tumor xenografts reached 5-7 mm in diameter. Then the mice were randomly assigned to three groups(seven mice in each group: (1 normal saline(NS group, (2replication-defective recombinant adenovirus carrying the negative control shRNA (Ad-HK group and (3replication-defective recombinant adenovirus carrying the 4-tandem linked RhoA and RhoC shRNAs (Ad-RhoA-RhoC group. Ad-HK (4 × 108 pfu, 30 ul/mouse, Ad-RhoA-RhoC (4 × 108 pfu, 30 ul/mouse or PBS (30 ul/mouse was injected intratumorally four times once every other day. The weight and volumes of tumor xenografts were recorded. The levels of RhoA and RhoC mRNA transcripts and proteins in tumor xenografts were detected by reverse quantitative transcription polymerase chain reaction (QRT-PCR and immunohistochemical staining respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay was used to detect the death of cells. Results The xenografts in mice could be seen at 5th day from the implantation of HCT116 cells and all had reached 5-7 mm in size at 9th day. After injection intratumorally, the growth speed of tumor xenografts in Ad-RhoA-RhoC group was significantly delayed compared with those in NS and Ad-HK group(P RhoA and RhoC reduced more in Ad-RhoA-RhoC group than those in NS and Ad-HK group. The relative RhoA and RhoC mRNA transcripts were decreased to 48% and 43% respectively (P RhoA and Rho

  12. Highly activated RNA silencing via strong induction of dicer by one virus can interfere with the replication of an unrelated virus

    Science.gov (United States)

    Chiba, Sotaro; Suzuki, Nobuhiro

    2015-01-01

    Viruses often coinfect single host organisms in nature. Depending on the combination of viruses in such coinfections, the interplay between them may be synergistic, apparently neutral with no effect on each other, or antagonistic. RNA silencing is responsible for many cases of interference or cross-protection between viruses, but such antagonistic interactions are usually restricted to closely related strains of the same viral species. In this study, we present an unprecedented example of RNA silencing-mediated one-way interference between unrelated viruses in a filamentous model fungus, Cryphonectria parasitica. The replication of Rosellinia necatrix victorivirus 1 (RnVV1; Totiviridae) was strongly impaired by coinfection with the prototypic member of the genus Mycoreovirus (MyRV1) or a mutant of the prototype hypovirus (Cryphonectria hypovirus 1, CHV1) lacking the RNA silencing suppressor (CHV1-Δp69). This interference was associated with marked transcriptional induction of key genes in antiviral RNA silencing, dicer-like 2 (dcl2) and argonaute-like 2 (agl2), following MyRV1 or CHV1-Δp69 infection. Interestingly, the inhibition of RnVV1 replication was reproduced when the levels of dcl2 and agl2 transcripts were elevated by transgenic expression of a hairpin construct of an endogenous C. parasitica gene. Disruption of dcl2 completely abolished the interference, whereas that of agl2 did not always lead to its abolishment, suggesting more crucial roles of dcl2 in antiviral defense. Taken altogether, these results demonstrated the susceptible nature of RnVV1 to the antiviral silencing in C. parasitica activated by distinct viruses or transgene-derived double-stranded RNAs and provide insight into the potential for broad-spectrum virus control mediated by RNA silencing. PMID:26283371

  13. Interferência por RNA: uma nova alternativa para terapia nas doenças reumáticas RNA interference: a new alternative for rheumatic diseases therapy

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    Natália Regine de França

    2010-12-01

    Full Text Available A interferência por RNA (RNAi é um mecanismo de silenciamento gênico pós-transcricional conservado durante a evolução. Esse mecanismo, recentemente descrito, é mediado por pequenos RNAs de fita dupla (dsRNAs capazes de reconhecer especificamente uma sequência de mRNA-alvo e mediar sua clivagem ou repressão traducional. O emprego da RNAi como uma ferramenta de terapia gênica tem sido muito estudado, especialmente em infecções virais, câncer, desordens genéticas herdadas, doenças cardiovasculares e mesmo em doenças reumáticas. Aliados aos dados do genoma humano, os conhecimentos do silenciamento gênico mediado por RNAi podem permitir a determinação funcional de praticamente qualquer gene expresso em uma célula e sua implicação para o funcionamento e homeostase celular. Vários estudos terapêuticos in vitro e in vivo em modelos de doenças autoimunes vêm sendo realizados com resultados encorajadores. As vias de quebra de tolerância e inflamação são alvos potenciais para terapia com RNAi em doenças inflamatórias e autoimunes. Nesta revisão vamos recordar os princípios básicos da RNAi e discutir os aspectos que levaram ao desenvolvimento de propostas terapêuticas baseadas em RNAi, começando pelos estudos in vitro de desenvolvimento de ferramentas e identificação de alvos, chegando até os estudos pré-clínicos de disponibilização da droga in vivo, e testes em células humanas e modelos animais de doenças autoimunes. Por fim, vamos revisar os últimos avanços da experiência clínica da terapia com RNAiRNA interference (RNAi is a post-transcriptional gene silencing mechanism preserved during evolution. This mechanism, recently described, is mediated by small double-stranded RNAs (dsRNAs that can specifically recognize a target mRNA sequence and mediate its cleavage or translational repression. The use of RNAi as a tool for gene therapy has been extensively studied, especially in viral infections, cancer

  14. Double-stranded RNA-mediated interference of dumpy genes in Bursaphelenchus xylophilus by feeding on filamentous fungal transformants.

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    Wang, Meng; Wang, Diandong; Zhang, Xi; Wang, Xu; Liu, Wencui; Hou, Xiaomeng; Huang, Xiaoyin; Xie, Bingyan; Cheng, Xinyue

    2016-05-01

    RNA interference (RNAi) is a valuable tool for studying gene function in vivo and provides a functional genomics platform in a wide variety of organisms. The pinewood nematode, Bursaphelenchus xylophilus, is a prominent invasive plant-parasitic nematode and has become a serious worldwide threat to forest ecosystems. Presently, the complete genome sequence of B. xylophilus has been published, and research involving genome-wide functional analyses is likely to increase. In this study, we describe the construction of an effective silencing vector, pDH-RH, which contains a transcriptional unit for a hairpin loop structure. Utilising this vector, double-stranded (ds)RNAs with sequences homologous to the target genes can be expressed in a transformed filamentous fungus via Agrobacterium tumefaciens-mediated transformation technology, and can subsequently induce the knockdown of target gene mRNA expression in B. xylophilus by allowing the nematode to feed on the fungal transformants. Four dumpy genes (Bx-dpy-2, 4, 10 and 11) were used as targets to detect RNAi efficiency. By allowing the nematode to feed on target gene-transformed Fusarium oxysporum strains, target transcripts were knocked down 34-87% compared with those feeding on the wild-type strain as determined by real-time quantitative PCR (RT-qPCR). Morphological RNAi phenotypes were observed, displaying obviously reduced body length; weak dumpy or small (short and thin) body size; or general abnormalities. Moreover, compensatory regulation and non-specific silencing of dpy genes were found in B. xylophilus. Our results indicate that RNAi delivery by feeding in B. xylophilus is a successful technique. This platform may provide a new opportunity for undertaking RNAi-based, genome-wide gene functional studies in vitro in B. xylophilus. Moreover, as B. xylophilus feeds on endophytic fungi when a host has died, RNAi feeding technology will offer the prospect for developing a novel control strategy for the nematode

  15. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.

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    Jie Chen

    Full Text Available BACKGROUND: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1 and membrane-bound (Tre-2 trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. PRINCIPAL FINDINGS: The membrane-bound trehalase of Spodoptera exigua (SeTre-2 was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1 and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. CONCLUSIONS: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2

  16. Role of Halloween genes in ecdysteroids biosynthesis of the swimming crab (Portunus trituberculatus): Implications from RNA interference and eyestalk ablation.

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    Xie, Xi; Liu, Zhiye; Liu, Mingxin; Tao, Tian; Shen, Xiquan; Zhu, Dongfa

    2016-09-01

    Molting, including metamorphosis molting in arthropods are controlled by the ecdysteroids that are synthesized and secreted by the crustacean Y-organ (YO) or the insect prothoracic gland (PG). The Halloween genes encoding the enzymes mainly involved in the biosynthesis of ecdysteroids are well studied in insects but not in crustaceans. Given the importance of Halloween genes in ecdysteroids biosynthesis, we have previously reported the cDNA cloning of disembodied (Dib) in P. trituberculatus. Here, cDNA sequences of another two Halloween genes, Spook (Spo) and Shadow (Sad), were further identified and characterized. The predicted amino acid sequences for these two Halloween genes of Portunus trituberculatus were compared to those of several other arthropods, and several typical domains of the cytochrome P450 mono-oxygenase (CYP) were identified. Similar to the tissue distribution of Dib, the Spo and Sad also showed high specificity to the YO. RNA interference (RNAi) of these 3 genes indicated they all play essential role in ecdysteroids biosynthesis. To investigate the relationships of the Halloween genes to the eyestalk neuropeptides such as molt-inhibiting hormone (MIH), effects of eyestalk ablation (ESA) on the expression of Dib, Spo and Sad were detected. Expression of Dib and Sad, but not Spo, was significantly induced by ESA. The result indicated that the inhibition of MIH in ecdysteroids biosynthesis may be partly through the transcriptional regulation of certain Halloween genes, such as Dib and Sad, while the Spo might not be the target for MIH signal. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. A single-cross, RNA interference-based genetic tool for examining the long-term maintenance of homeostatic plasticity

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    Douglas J. Brusich

    2015-03-01

    Full Text Available Homeostatic synaptic plasticity (HSP helps neurons and synapses maintain physiologically appropriate levels of output. The fruit fly Drosophila melanogaster larval neuromuscular junction (NMJ is a valuable model for studying HSP. Here we introduce a genetic tool that allows fruit fly researchers to examine the lifelong maintenance of HSP with a single cross. The tool is a fruit fly stock that combines the GAL4/UAS expression system with RNA interference (RNAi-based knock down of a glutamate receptor subunit gene. With this stock, we uncover important new information about the maintenance of HSP. We address an open question about the role that presynaptic CaV2-type Ca2+ channels play in NMJ homeostasis. Published experiments have demonstrated that hypomorphic missense mutations in the CaV2 α1a subunit gene cacophony (cac can impair homeostatic plasticity at the NMJ. Here we report that reducing cac expression levels by RNAi is not sufficient to impair homeostatic plasticity. The presence of wild-type channels appears to support HSP – even when total CaV2 function is severely reduced. We also conduct an RNAi- and electrophysiology-based screen to identify new factors required for sustained homeostatic signaling throughout development. We uncover novel roles in HSP for Drosophila homologs of Cysteine string protein (CSP and Phospholipase Cβ (Plc21C. We characterize those roles through follow-up genetic tests. We discuss how CSP, Plc21C, and associated factors could modulate presynaptic CaV2 function, presynaptic Ca2+ handling, or other signaling processes crucial for sustained homeostatic regulation of NMJ function throughout development. Our findings expand the scope of signaling pathways and processes that contribute to the durable strength of the NMJ.

  18. Inhibiting adenoid cystic carcinoma cells growth and metastasis by blocking the expression of ADAM 10 using RNA interference

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    Zhang Zhiyuan

    2010-12-01

    Full Text Available Abstract Background Adenoid cystic carcinoma is one of the most common types of salivary gland cancers. The poor long-term prognosis for patients with adenoid cystic carcinoma is mainly due to local recurrence and distant metastasis. Disintegrin and metalloprotease 10 (ADAM 10 is a transmembrane protein associated with metastasis in a number of diverse of cancers. The aim of this study was to analyze the relationship between ADAM 10 and the invasive and metastatic potentials as well as the proliferation capability of adenoid cystic carcinoma cells in vitro and in vivo. Methods Immunohistochemistry and Western blot analysis were applied to detect ADAM 10 expression levels in metastatic cancer tissues, corresponding primary adenoid cystic carcinoma tissues, adenoid cystic carcinoma cell lines with high metastatic potential, and adenoid cystic carcinoma cell lines with low metastatic potential. RNA interference was used to knockdown ADAM 10 expression in adenoid cystic carcinoma cell lines with high metastatic potential. Furthermore, the invasive and metastatic potentials as well as the proliferation capability of the treated cells were observed in vitro and in vivo. Results It was observed that ADAM 10 was expressed at a significantly higher level in metastatic cancer tissues and in adenoid cystic carcinoma cell lines with high metastatic potential than in corresponding primary adenoid cystic carcinomas and adenoid cystic carcinoma cell lines with low metastatic potential. Additionally, silencing of ADAM 10 resulted in inhibition of cell growth and invasion in vitro as well as inhibition of cancer metastasis in an experimental murine model of lung metastases in vivo. Conclusions These studies suggested that ADAM 10 plays an important role in regulating proliferation and metastasis of adenoid cystic carcinoma cells. ADAM 10 is potentially an important therapeutic target for the prevention of tumor metastases in adenoid cystic carcinoma.

  19. The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved Nucleotide Analog Interference Probing (trNAIP)

    Science.gov (United States)

    Soares, Emilie; Schwartz, Annie; Nollmann, Marcello; Margeat, Emmanuel; Boudvillain, Marc

    2014-01-01

    Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2′-hydroxyl group of the incoming 3′-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3′-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, ‘test to run’ iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems. PMID:25016524

  20. Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells

    Directory of Open Access Journals (Sweden)

    Canto Tomas

    2004-08-01

    Full Text Available Abstract Background RNA interference (RNAi in animals and post-transcriptional gene silencing (PTGS in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system. Results Here we demonstrate that both transient and constitutive expression of the Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein. Conclusion These results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi.

  1. Human health and ecological risk assessments for SmartStax PRO (MON 89034 x TC1507 x MON 87411 x DAS-59122-7), a plant-incorporated protectant intended to control corn rootworm through ribonucleic acid (RNA) interference

    Science.gov (United States)

    The use of RNA interference (RNAi) gene silencing technology, particularly RNAi for pesticidal purposes to control macroorganism pests, is a relatively recent innovation. Post-transcriptional silencing of gene function is a very rapid process where double-stranded RNA (dsRNA) dir...

  2. Functional peptide nanocarriers for delivery of novel anti-RelA RNA interference agents as a topical treatment of atopic dermatitis.

    Science.gov (United States)

    Kanazawa, Takanori; Hamasaki, Tomohiro; Endo, Takahiro; Tamano, Kuniko; Sogabe, Kana; Seta, Yasuo; Ohgi, Tadaaki; Okada, Hiroaki

    2015-07-15

    Small interfering RNAs (siRNAs) are a potential treatment of atopic dermatitis (AD) because they can specifically silence the gene expression of AD-related factors. However, siRNA alone cannot exert a sufficiently strong therapeutic effect due to low delivery efficiency to the target tissues and cells; simply increasing the amount used is not possible due to the possibility of off-target effects. We previously reported a novel class of therapeutic RNA interference (RNAi) agents called nkRNA(®) and PnkRNA(®), which have been shown to be effective in several disease models, have greater resistance to nuclease degradation than canonical siRNAs, and do not induce any immunotoxicity. In the present study, we describe a non-invasive and effective transdermal RNAi therapeutic system for atopic dermatitis that uses the functional cell-penetrating stearoyl-oligopeptide OK-102 as a cytoplasm-responsive nanocarrier for nkRNA(®) and PnkRNA(®). The two RNAi agents were targeted against RelA, a subclass of NF-κB (nuclear factor kappa B), and, as part of OK-102 complexes, they strongly silenced RelA mRNA in macrophage cells and demonstrated a significant therapeutic effect in a mouse model of AD. It was shown that OK-102-complexed RNAi agents were an efficient therapeutic system for AD and caused no adverse reactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. RNA interference-mediated targeting of DKK1 gene expression in Ishikawa endometrial carcinoma cells causes increased tumor cell invasion and migration.

    Science.gov (United States)

    Yi, Nuo; Liao, Qin-Ping; Li, Zhen-Hua; Xie, Bao-Jiang; Hu, Yu-Hong; Yi, Wei; Liu, Min

    2013-09-01

    The Wnt signaling pathway plays an essential role in tumor invasion and migration. DKK1 functions as an important inhibitor of the pathway and represents a promising target for cancer therapy. The aim of the present study was to determine the role of DKK1 in endometrial carcinoma (EC) cell invasion and migration using RNA interference (RNAi) technology. Ishikawa EC cells were transfected at high efficiency with specific DKK1 siRNA. RT-PCR and western blot analysis were used to determine the mRNA and protein levels of DKK1, β-catenin and metalloproteinase 14 (MMP14) in siRNA-treated and -untreated cells. In addition, the invasion and migration of the EC cells were detected by invasion and migration assays. Transient transfection of DKK1 siRNA significantly inhibited the mRNA and protein levels of DKK1. Markedly increased cell invasion and migration was observed following treatment with DKK1 siRNA when compared with the negative control siRNA-treated and siRNA-untreated cells. The knockdown of DKK1 also elevated the mRNA and protein levels of β-catenin and MMP14 involved in the Wnt signaling pathway, indicating that targeting this gene may promote intracellular Wnt signal transduction and thus, accelerate EC cell invasion and migration in vitro. The RNAi-mediated targeting of DKK1 gene expression in Ishikawa EC cells resulted in increased tumor cell invasion and migration. DKK1 was identified as an inhibitor of EC cell invasion and migration via its novel role in the Wnt signaling pathway. Targeting DKK1 may therefore represent an effective anti-invasion and -migration strategy for the treatment of EC.

  4. Transarterial embolization combined with RNA interference targeting hypoxia-inducible factor-1α for hepatocellular carcinoma: a preliminary study of rat model.

    Science.gov (United States)

    Ni, Jia-Yan; Xu, Lin-Feng; Wang, Wei-Dong; Huang, Qiao-Sheng; Sun, Hong-Liang; Chen, Yao-Ting

    2017-02-01

    To study whether transarterial embolization (TAE) with RNA interference (RNAi) targeting hypoxia-inducible factor-1α (HIF-1α) can improve efficacy of TAE in treating hepatocellular carcinoma (HCC). CBRH-7919 rat hepatoma cell line was used and HCC models of rats were constructed. The siRNA transfection compound was made by mixing specific siRNA and Lipofectamine 2000™. Delivery and transfection of siRNA were administered by injecting iodized oil emulsion (diluted lipiodol and siRNA) via hepatic artery. The expression levels of mRNA and protein were detected using the real-time reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and western blotting assays, respectively. In vitro experiment, the specific HIF-1α-siRNA was proved to inhibit expression levels of HIF-1α and vascular endothelial growth factor (VEGF) effectively. In animal study, real-time RT-PCR assay showed the average relative mRNA expressions of HIF-1α were 0.31 ± 0.01, 0.65 ± 0.03, 0.46 ± 0.005, and 1.00 ± 0.00 in TAE + siRNA, siRNA, TAE, and control groups, respectively. Western blotting assay showed the average relative protein expressions of HIF-1α were 0.13 ± 0.02, 0.87 ± 0.02, 0.39 ± 0.02, and 1.02 ± 0.01 in TAE + siRNA, siRNA, TAE, and control groups, respectively. Compared with control, TAE, and siRNA groups, TAE + siRNA can significantly inhibit protein expressions of HIF-1α and VEGF (P HIF-1α < 0.001; P VEGF < 0.001). Overall survival of rats underwent TAE + siRNA was significantly longer than that of rats treated with TAE monotherapy (P = 0.001). This animal study showed TAE combined with HIF-1α-RNAi could significantly improve efficacy of TAE in treating HCC by inhibiting expressions of HIF-1α and VEGF after TAE treatment.

  5. Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus

    Science.gov (United States)

    2010-01-01

    Background Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III) directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences. Results CRISPR spacer #1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer #1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae. Conclusions The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer #1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of a potential impact of

  6. Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus

    Directory of Open Access Journals (Sweden)

    Lovley Derek R

    2010-07-01

    Full Text Available Abstract Background Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences. Results CRISPR spacer #1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer #1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae. Conclusions The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer #1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of

  7. Noncoding subgenomic flavivirus RNA is processed by the mosquito RNA interference machinery and determines West Nile virus transmission by Culex pipiens mosquitoes

    NARCIS (Netherlands)

    Göertz, G.P.; Fros, J.J.; Miesen, P.; Vogels, C.B.F.; Bent, van der M.L.; Geertsema, C.; Koenraadt, C.J.M.; Rij, van R.P.; Oers, van M.M.; Pijlman, G.P.

    2016-01-01

    Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5=-3= exoribonuclease

  8. Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes

    NARCIS (Netherlands)

    Goertz, G.P.; Fros, J.J.; Miesen, P.; Vogels, C.B.F.; Bent, M.L. van der; Geertsema, C.; Koenraadt, C.J.M.; Rij, R.P. van; Oers, M.M. van; Pijlman, G.P.

    2016-01-01

    Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5'-3' exoribonuclease

  9. Expression profiling and cross-species RNA interference (RNAi of desiccation-induced transcripts in the anhydrobiotic nematode Aphelenchus avenae

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    Culleton Bridget A

    2010-01-01

    Full Text Available Abstract Background Some organisms can survive extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living mycophagous nematode Aphelenchus avenae can be induced to enter anhydrobiosis by pre-exposure to moderate reductions in relative humidity (RH prior to extreme desiccation. This preconditioning phase is thought to allow modification of the transcriptome by activation of genes required for desiccation tolerance. Results To identify such genes, a panel of expressed sequence tags (ESTs enriched for sequences upregulated in A. avenae during preconditioning was created. A subset of 30 genes with significant matches in databases, together with a number of apparently novel sequences, were chosen for further study. Several of the recognisable genes are associated with water stress, encoding, for example, two new hydrophilic proteins related to the late embryogenesis abundant (LEA protein family. Expression studies confirmed EST panel members to be upregulated by evaporative water loss, and the majority of genes was also induced by osmotic stress and cold, but rather fewer by heat. We attempted to use RNA interference (RNAi to demonstrate the importance of this gene set for anhydrobiosis, but found A. avenae to be recalcitrant with the techniques used. Instead, therefore, we developed a cross-species RNAi procedure using A. avenae sequences in another anhydrobiotic nematode, Panagrolaimus superbus, which is amenable to gene silencing. Of 20 A. avenae ESTs screened, a significant reduction in survival of desiccation in treated P. superbus populations was observed with two sequences, one of which was novel, while the other encoded a glutathione peroxidase. To confirm a role for glutathione peroxidases in anhydrobiosis, RNAi with cognate sequences from P. superbus was performed and was also shown to reduce desiccation tolerance in this species. Conclusions This study has identified and characterised the

  10. shRNA interference of NLRP3 inflammasome alleviate ischemia reperfusion-induced myocardial damage through autophagy activation.

    Science.gov (United States)

    Meng, Zhu; Song, Mei-Yan; Li, Chuan-Fang; Zhao, Jia-Qi

    2017-12-16

    remarkably suppressed by NLRP3 KO. Taken together, our study indicated that shRNA interference of NLRP3 inflammasome attenuated myocardial I/R injury via autophagy activation. These findings demonstrated that NLRP3 KO may a promising therapy in myocardial I/R injury. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Strand Analysis, a free online program for the computational identification of the best RNA interference (RNAi targets based on Gibbs free energy

    Directory of Open Access Journals (Sweden)

    Tiago Campos Pereira

    2007-01-01

    Full Text Available The RNA interference (RNAi technique is a recent technology that uses double-stranded RNA molecules to promote potent and specific gene silencing. The application of this technique to molecular biology has increased considerably, from gene function identification to disease treatment. However, not all small interfering RNAs (siRNAs are equally efficient, making target selection an essential procedure. Here we present Strand Analysis (SA, a free online software tool able to identify and classify the best RNAi targets based on Gibbs free energy (deltaG. Furthermore, particular features of the software, such as the free energy landscape and deltaG gradient, may be used to shed light on RNA-induced silencing complex (RISC activity and RNAi mechanisms, which makes the SA software a distinct and innovative tool.

  12. Replication competent HIV-1 viruses that express intragenomic microRNA reveal discrete RNA-interference mechanisms that affect viral replication

    OpenAIRE

    Klase Zachary; Houzet Laurent; Jeang Kuan-Teh

    2011-01-01

    Abstract Background It remains unclear whether retroviruses can encode and express an intragenomic microRNA (miRNA). Some have suggested that processing by the Drosha and Dicer enzymes might preclude the viability of a replicating retroviral RNA genome that contains a cis-embedded miRNA. To date, while many studies have shown that lentiviral vectors containing miRNAs can transduce mammalian cells and express the inserted miRNA efficiently, no study has examined the impact on the replication o...

  13. Replication competent HIV-1 viruses that express intragenomic microRNA reveal discrete RNA-interference mechanisms that affect viral replication.

    Science.gov (United States)

    Klase, Zachary; Houzet, Laurent; Jeang, Kuan-Teh

    2011-11-23

    It remains unclear whether retroviruses can encode and express an intragenomic microRNA (miRNA). Some have suggested that processing by the Drosha and Dicer enzymes might preclude the viability of a replicating retroviral RNA genome that contains a cis-embedded miRNA. To date, while many studies have shown that lentiviral vectors containing miRNAs can transduce mammalian cells and express the inserted miRNA efficiently, no study has examined the impact on the replication of a lentivirus such as HIV-1 after the deliberate intragenomic insertion of a bona fide miRNA. We have constructed several HIV-1 molecular clones, each containing a discrete cellular miRNA positioned in Nef. These retroviral genomes express the inserted miRNA and are generally replication competent in T-cells. The inserted intragenomic miRNA was observed to elicit two different consequences for HIV-1 replication. First, the expression of miRNAs with predicted target sequences in the HIV-1 genome was found to reduce viral replication. Second, in one case, where an inserted miRNA was unusually well-processed by Drosha, this processing event inhibited viral replication. This is the first study to examine in detail the replication competence of HIV-1 genomes that express cis-embedded miRNAs. The results indicate that a replication competent retroviral genome is not precluded from encoding and expressing a viral miRNA.

  14. RNA interference targeting hypoxia-inducible factor 1α via a novel multifunctional surfactant attenuates glioma growth in an intracranial mouse model.

    Science.gov (United States)

    Gillespie, David L; Aguirre, Maria T; Ravichandran, Sandhya; Leishman, Lisa L; Berrondo, Claudia; Gamboa, Joseph T; Wang, Libo; King, Rose; Wang, Xuli; Tan, Mingqian; Malamas, Anthony; Lu, Zheng-Rong; Jensen, Randy L

    2015-02-01

    High-grade gliomas are the most common form of adult brain cancer, and patients have a dismal survival rate despite aggressive therapeutic measures. Intratumoral hypoxia is thought to be a main contributor to tumorigenesis and angiogenesis of these tumors. Because hypoxia-inducible factor 1α (HIF-1α) is the major mediator of hypoxia-regulated cellular control, inhibition of this transcription factor may reduce glioblastoma growth. Using an orthotopic mouse model with U87-LucNeo cells, the authors used RNA interference to knock down HIF-1α in vivo. The small interfering RNA (siRNA) was packaged using a novel multifunctional surfactant, 1-(aminoethyl) iminobis[N-(oleicylcysteinylhistinyl-1-aminoethyl)propionamide] (EHCO), a nucleic acid carrier that facilitates cellular uptake and intracellular release of siRNA. Stereotactic injection was used to deliver siRNA locally through a guide-screw system, and delivery/uptake was verified by imaging of fluorescently labeled siRNA. Osmotic pumps were used for extended siRNA delivery to model a commonly used human intracranial drug-delivery technique, convection-enhanced delivery. Mice receiving daily siRNA injections targeting HIF-1α had a 79% lower tumor volume after 50 days of treatment than the controls. Levels of the HIF-1 transcriptional targets vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT-1), c-MET, and carbonic anhydrase-IX (CA-IX) and markers for cell growth (MIB-1 and microvascular density) were also significantly lower. Altering the carrier EHCO by adding polyethylene glycol significantly increased the efficacy of drug delivery and subsequent survival. Treating glioblastoma with siRNA targeting HIF-1α in vivo can significantly reduce tumor growth and increase survival in an intracranial mouse model, a finding that has direct clinical implications.

  15. RNA interference in the Asian Longhorned Beetle:Identification of Key RNAi Genes and Reference Genes for RT-qPCR.

    Science.gov (United States)

    Rodrigues, Thais B; Dhandapani, Ramesh Kumar; Duan, Jian J; Palli, Subba Reddy

    2017-08-21

    Asian Longhorned Beetle (ALB) Anoplophora glabripennis is a serious invasive forest pest in several countries including the United States, Canada, and Europe. RNA interference (RNAi) technology is being developed as a novel method for pest management. Here, we identified the ALB core RNAi genes including those coding for Dicer, Argonaute, and double-stranded RNA-binding proteins (dsRBP) as well as for proteins involved in dsRNA transport and the systemic RNAi. We also compared expression of six potential reference genes that could be used to normalize gene expression and selected gapdh and rpl32 as the most reliable genes among different tissues and stages of ALB. Injection of double-stranded RNA (dsRNA) targeting gene coding for inhibitor of apoptosis (IAP) into larvae and adults resulted in a significant knockdown of this gene and caused the death of 90% of the larvae and 100% of adults. No mortality of both larvae and adults injected with dsRNA targeting gene coding for green fluorescence protein (GFP, as a negative control) was observed. These data suggest that functional RNAi machinery exists in ALB and a potential RNAi-based method could be developed for controlling this insect.

  16. A Halloween gene shadow is a potential target for RNA-interference-based pest management in the small brown planthopper Laodelphax striatellus.

    Science.gov (United States)

    Wan, Pin-Jun; Jia, Shuang; Li, Na; Fan, Jin-Mei; Li, Guo-Qing

    2015-02-01

    Laodelphax striatellus is an economically important rice pest in China. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. The cytochrome P450 monooxygenase Shadow (Sad) plays a critical role in ecdysteroidogenesis. Here, tests were conducted to establish whether Lssad was a potential target gene for RNA-interference-based management of L. striatellus. Lssad was cloned and characterised. LsSad had Helix-C, Helix-I, Helix-K, PERF and haem-binding motifs. Lssad is expressed at a higher level in the thorax, where prothoracic glands are located, compared with the level in the head or abdomen. It showed two expression peaks in day 2 and day 4-5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Oral delivery of double-stranded RNA (dsRNA) of Lssad at the nymph stage successfully knocked down the expression of the target gene, reduced the expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development in a dose-dependent manner. Ingestion of 20-hydroxyecdysone in Lssad-dsRNA-exposed nymphs did not increase Lssad expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. The ecdysteroidogenic pathway is conserved in L. striatellus. Lssad can serve as a possible target for dsRNA-based pesticides for planthopper control. © 2014 Society of Chemical Industry.

  17. CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells.

    Science.gov (United States)

    Bellec, Jessica; Bacchetta, Marc; Losa, Davide; Anegon, Ignacio; Chanson, Marc; Nguyen, Tuan Huy

    2015-01-01

    Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation. Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells. shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion. We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

  18. One cell model establishment to inhibit CaMKIIγ mRNA expression in the dorsal root ganglion neuron by RNA interfere.

    Science.gov (United States)

    Wen, Xianjie; Li, Xiaohong; Liang, Hua; Yang, Chenxiang; Zhong, Jiying; Wang, Hanbing; Liu, Hongzhen

    2017-09-01

    CaMKIIγ in dorsal root ganglion neurons is closely related to the neuropathic pain, neuron injury induced by local anesthetics. To get great insight into the function of CaMKIIγ in dorsal root ganglion neurons, we need one cell model to specially inhibit the CaMKIIγ mRNA expression. The present study was aimed to establish one cell model to specially inhibit the CaMKIIγ mRNA expression. We designed the CaMKIIγ shRNA sequence and connected with pYr-1.1 plasmid. The ligation product of the CaMKIIγshRNA and pYr-1.1 plasmid was recombined with pAd/PL-DEST vector into pAD-CaMKIIγ-shRNA. adenovirus vector. pAD-CaMKIIγ-shRNA. adenovirus vector infected the dorsal root ganglion neuron to inhibit the CaMKIIγ mRNA expression in vitro. The pAD-CaMKIIγ-shRNA adenovirus vector was verified to be correct by the digestion, sequence. And pAD-CaMKIIγ-shRNA. adenovirus vector can infect the DRG cells to inhibit the CaMKIIγ mRNA or protein expression by the real-time polymerase chain reaction (PCR) or western blotting. Those results showed that we successfully constructed one adenovirus vector that can infect the dorsal root ganglion neuron to inhibit the CaMKIIγ mRNA and protein expression. That will supply with one cell model for the CaMKIIγ function study.

  19. Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

    Directory of Open Access Journals (Sweden)

    Anesti Anna-Maria

    2010-09-01

    Full Text Available Abstract Background Delivery of small interfering RNA (siRNA to tumours remains a major obstacle for the development of RNA interference (RNAi-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV OncoVEXGM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. Methods To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA or artificial microRNA (miRNA against the reporter genes green fluorescent protein (eGFP and β-galactosidase (lacZ. These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. Results Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. Conclusions This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen

  20. α-Fetoprotein promoter-driven Cre/LoxP-switched RNA interference for hepatocellular carcinoma tissue-specific target therapy.

    Directory of Open Access Journals (Sweden)

    Yuan-Fei Peng

    Full Text Available RNA interference (RNAi has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1 was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3 and non-HCC cell lines (L-02, Hela and SW1116 were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5 was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.The AFP-miRNA system could silence target gene (Beclin 1 but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1 in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA was successfully established. The system provides a usable tool for HCC-specific RNAi

  1. Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

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    Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong (Yale); (Cornell); (Tsinghua)

    2012-10-10

    Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.

  2. Inhibition of CD147 expression by RNA interference reduces proliferation, invasion and increases chemosensitivity in cancer stem cell-like HT-29 cells.

    Science.gov (United States)

    Chen, Jie; Pan, Yuqin; He, Bangshun; Ying, Houqun; Wang, Feng; Sun, Huiling; Deng, Qiwen; Liu, Xian; Lin, Kang; Peng, Hongxin; Cho, William C; Wang, Shukui

    2015-10-01

    The association between CD147 and cancer stem cells (CSCs) provides a new angle for cancer treatments. The aim of this study was to investigate the biological roles of CD147 in colorectal CSCs. The Oct4-green fluorescent protein (GFP) vector was used to isolate CSCs and pYr-mir30-shRNA was used to generate short hairpin RNA (shRNA) specifically for CD147. After RNA interference (RNAi), CD147 was evaluated by reverse transcription‑quantitative PCR and western blot analysis, and its biological functions were assessed by MTT and invasion assays. The results showed that the differentiation of isolated CSC-like HT-29 cells was blocked and these cells were highly positive for CD44 and CD147. RNAi-mediated CD147 silencing reduced the expression of CD147 at both mRNA and protein levels. Moreover, the activities of proliferation and invasion were decreased obviously in CSCs. Knockdown of CD147 increased the chemosensitivity of CSC-like cells to gemcitabine, cisplatin, docetaxel at 0.1, 1 and 10 µM respectively, however, there was no significant difference among the three groups to paclitaxel at 10 µM. In conclusion, these results suggest that CD147 plays an important role in colorectal CSCs and might be regarded as a novel CSC-specific targeted strategy against colorectal cancer.

  3. Design and validation of small interfering RNA on respiratory syncytial virus M2-2 gene: A potential approach in RNA interference on viral replication.

    Science.gov (United States)

    Chin, V K; Atika Aziz, Nur A; Hudu, Shuaibu A; Harmal, Nabil S; Syahrilnizam, A; Jalilian, Farid A; Zamberi, S

    2016-10-01

    Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and young children globally and is a significant pathogen of the elderly and immunocompromised. The M2-2 protein of respiratory syncytial virus (RSV) is particularly important in regulation of viral RNA transcription and replication that could be a potential anti-viral candidate against RSV infection. In this study, we designed and validated siRNAs that specifically target the RSV M2-2 gene. Four siRNAs targeting different regions of the M2-2 gene were designed using web tool. In-vitro evaluation of silencing effect was performed by using RSV infected Vero cell line. Viral M2-2 linked GFP recombinant plasmid was co-transfected with non-targeted siRNA, Pooled siRNA, siRNA 1, siRNA 2, siRNA 3 and siRNA 4 using synthetic cationic polymer. The silencing effect of M2-2 gene at the protein level was measured both qualitatively and quantitatively by using fluorescence microscopy and flow cytometry. Meanwhile, the silencing effect at the mRNA level was assessed by using RT-qPCR. This study showed that all four designed siRNAs can effectively and efficiently silence M2-2 gene. siRNA 2 showed the highest (98%) silencing effect on protein level and siRNA 4 with 83.1% at the mRNA level. The viral assay showed no significant cytopathic effects observed after 6days post-infection with siRNAs. In conclusion, this study showed the effectiveness of siRNA in silencing M2-2 gene both at the protein and mRNA level which could potentially be used as a novel therapeutic agent in the treatment of RSV infection. However, further study is warranted to investigate the silencing effect of M2-2 protein and inhibition of RSV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Interference of a speB 5' untranslated region partial deletion with mRNA degradation in Streptococcus pyogenes.

    Science.gov (United States)

    Chen, Z; Mashburn-Warren, L; Merritt, J; Federle, M J; Kreth, J

    2017-10-01

    The 5' untranslated region (5' UTR) of an mRNA molecule embeds important determinants that modify its stability and translation efficiency. In Streptococcus pyogenes, a strict human pathogen, a gene encoding a secreted protease (speB) has a large 5' UTR with unknown functions. Here we describe that a partial deletion of the speB 5' UTR caused a general accumulation of mRNA in the stationary phase, and that the mRNA accumulation was due to retarded mRNA degradation. The phenotype was observed in several M serotypes harboring the partial deletion of the speB 5' UTR. The phenotype was triggered by the production of the truncated speB 5' UTR, but not by the disruption of the intact speB 5' UTR. RNase Y, a major endoribonuclease, was previously shown to play a central role in bulk mRNA turnover in stationary phase. However, in contrast to our expectations, we observed a weaker interaction between the truncated speB 5' UTR and RNase Y compared with the wild-type, which suggests that other unidentified RNA degrading components are required for the pleiotropic effects observed from the speB UTR truncation. Our study demonstrates how S. pyogenes uses distinct mRNA degradation schemes in exponential and stationary growth phases. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Inhibitors of MyD88-dependent proinflammatory cytokine production identified utilizing a novel RNA interference screening approach.

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    John S Cho

    2009-09-01

    Full Text Available The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha. How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.We report here the development a completely random short-hairpin RNA (shRNA library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses.

  6. Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2-/- gammac-/- mice: functional inactivation of p53 in developing T cells

    NARCIS (Netherlands)

    Gimeno, Ramon; Weijer, Kees; Voordouw, Arie; Uittenbogaart, Christel H.; Legrand, Nicolas; Alves, Nuno L.; Wijnands, Erwin; Blom, Bianca; Spits, Hergen

    2004-01-01

    Tumor suppressor p53 plays an important role in regulating cell cycle progression and apoptosis. Here we applied RNA interference to study the role of p53 in human hematopoietic development in vivo. An siRNA construct specifically targeting the human tumor-suppressor gene p53 was introduced into

  7. Iron(II) supramolecular helicates interfere with the HIV-1 Tat-TAR RNA interaction critical for viral replication

    Science.gov (United States)

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-07-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat-TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

  8. Distal-less and dachshund pattern both plesiomorphic and apomorphic structures in chelicerates: RNA interference in the harvestman Phalangium opilio (Opiliones).

    Science.gov (United States)

    Sharma, Prashant P; Schwager, Evelyn E; Giribet, Gonzalo; Jockusch, Elizabeth L; Extavour, Cassandra G

    2013-01-01

    The discovery of genetic mechanisms that can transform a morphological structure from a plesiomorphic (=primitive) state to an apomorphic (=derived) one is a cardinal objective of evolutionary developmental biology. However, this objective is often impeded for many lineages of interest by limitations in taxonomic sampling, genomic resources, or functional genetic methods. In order to investigate the evolution of appendage morphology within Chelicerata, the putative sister group of the remaining arthropods, we developed an RNA interference (RNAi) protocol for the harvestman Phalangium opilio. We silenced the leg gap genes Distal-less (Dll) and dachshund (dac) in the harvestman via zygotic injections of double-stranded RNA (dsRNA), and used in situ hybridization to confirm RNAi efficacy. Consistent with the conserved roles of these genes in patterning the proximo-distal axis of arthropod appendages, we observed that embryos injected with Dll dsRNA lacked distal parts of appendages and appendage-like structures, such as the labrum, the chelicerae, the pedipalps, and the walking legs, whereas embryos injected with dac dsRNA lacked the medial podomeres femur and patella in the pedipalps and walking legs. In addition, we detected a role for these genes in patterning structures that do not occur in well-established chelicerate models (spiders and mites). Dll RNAi additionally results in loss of the preoral chamber, which is formed from pedipalpal and leg coxapophyses, and the ocularium, a dorsal outgrowth bearing the eyes. In one case, we observed that an embryo injected with dac dsRNA lacked the proximal segment of the chelicera, a plesiomorphic podomere that expresses dac in wild-type embryos. This may support the hypothesis that loss of the cheliceral dac domain underlies the transition to the two-segmented chelicera of derived arachnids. © 2013 Wiley Periodicals, Inc.

  9. RNA interference-mediated vascular endothelial growth factor-C reduction suppresses malignant progression and enhances mitomycin C sensitivity of bladder cancer T24 cells.

    Science.gov (United States)

    Zhang, Hui-hui; Qi, Fan; Shi, Ying-rui; Miao, Jian-guang; Zhou, Mi; He, Wei; Chen, Min-feng; Li, Yuan; Zu, Xiong-bing; Qi, Lin

    2012-06-01

    Vascular endothelial growth factor-C (VEGF-C) has been found to be significantly associated with lymphangiogenesis and regional lymph node metastasis in various human tumors. The present work was aimed to explore the role of VEGF-C in malignant progression of human bladder cancer T24 cell line. First, the expression of VEGF-C in T24 cells was detected by western blotting. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was employed to measure the cellular proliferation after treatment with various concentrations of recombinant human VEGF-C (rhVEGF-C). Then, lentivirus vector-based RNA interference (RNAi) was used to inhibit VEGF-C expression of T24 cells. The alterations of T24 cells regarding proliferation, invasiveness, and the apoptosis induced by mitomycin C (MMC) were evaluated. The results showed that the proliferation rate of T24 cells rose from 27.3% to 65.0%, with increasing rhVEGF-C concentration. T24 cells stably transfected with VEGF-C small interference RNA showed 85% reduction in VEGF-C mRNA expression (p T24 cells induced by MMC (p T24 cells, which is due to suppression of apoptosis and facilitation of migration, accompanied with upregulation of p38 MAPK and Akt phosphorylation. RNAi targeting VEGF-C could effectively suppress malignant progression and enhance chemosensitivity of T24 cells. Thus, inhibition of VEGF-C expression is a potential and promising therapeutic strategy for bladder cancer.

  10. The NS3 and NS4A genes as the targets of RNA interference inhibit replication of Japanese encephalitis virus in vitro and in vivo.

    Science.gov (United States)

    Yuan, Lei; Wu, Rui; Liu, Hanyang; Wen, Xintian; Huang, Xiaobo; Wen, Yiping; Ma, Xiaoping; Yan, Qigui; Huang, Yong; Zhao, Qin; Cao, Sanjie

    2016-12-15

    Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that can cause acute encephalitis with a high fatality rate. RNA interference (RNAi) is a powerful tool to silence gene expression and a potential therapy for virus infection. In this study, the antiviral ability of eight shRNA expression plasmids targeting different sites of the NS3 and NS4A genes of JEV was determined in BHK21 cells and mice. The pGP-NS3-3 and pGP-NS4A-4 suppressed 93.9% and 82.0% of JEV mRNA in cells, respectively. The virus titer in cells was reduced approximately 950-fold by pretreating with pGP-NS3-4, and 640-fold by pretreating with pGP-NS4A-4. The results of western blot and immunofluorescence analysis showed JEV E protein and viral load in cells were remarkably inhibited by shRNA expression plasmids. The viral load in brains of mice pretreated with pGP-NS3-4 or pGP-NS4A-4 were reduced approximately 2400-fold and 800-fold, respectively, and the survival rate of mice challenged with JEV were 70% and 50%, respectively. However, the antiviral ability of shRNA expression plasmids was decreased over time. This study indicates that RNAi targeting of the NS3 and NS4A genes of JEV can sufficiently inhibit the replication of JEV in vitro and in vivo, and NS3 and NS4A genes might be potential targets of molecular therapy for JEV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Knockdown of hMex-3A by small RNA interference suppresses cell proliferation and migration in human gastric cancer cells.

    Science.gov (United States)

    Jiang, Hong; Zhang, Xuemei; Luo, Jinhong; Dong, Chunyan; Xue, Junli; Wei, Wei; Chen, Jingde; Zhou, Jun; Gao, Yong; Yang, Changqing

    2012-09-01

    RNA-binding proteins (RBPs) play essential roles in RNA metabolism, regulating RNA splicing, transport, surveillance, decay and translation. The aberrant expression of RBPs leads to gene expression alteration and frequently causes various diseases, such as cancer. In this study, we are the first to provide evidence that hMex-3A, a RBP that belongs to the human Mex-3 family with two K-homology RNA-binding domains, is involved in the regulation of tumorigenesis. We show that the silencing of hMex-3A by small interference RNA effectively inhibits cell proliferation in SNU-16 and AGS gastric cancer cells. Flow cytometry analysis confirmed this effect on SNU-16 cell growth and indicated that hMex-3A may function in the G1/M phase. Notably, hMex-3A knockdown also reduced the colony formation ability of SNU-16 and AGS cells in soft agar, implying that hMex-3A is required for cell transformation. Furthermore, the hMex-3A knockdown markedly affected the migratory ability of BCG-823 cells by transwell chamber and wound healing assays. Clinical relevance analysis using 22 paired gastric cancer specimens by quantitative real-time PCR showed that hMex-3A was significantly upregulated (63.6%) in cancer tissues compared with matched adjacent non-cancerous tissues. Taken together, these results suggest that hMex-3A functions as an oncogene candidate in the development and metastasis of gastric cancer; thus it may serve as a potential target for the therapy of tumors.

  12. RNA Interference: A New Mechanism by Which FMRP Acts in the Normal Brain? What Can Drosophila Teach Us?

    Science.gov (United States)

    Siomi, Haruhiko; Ishizuka, Akira; Siomi, Mikiko C.

    2004-01-01

    Fragile X syndrome is the most common heritable form of mental retardation caused by loss-of-function mutations in the "FMR1" gene. The "FMR1" gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. Recent work in "Drosophila melanogaster" has shown that the fly homolog of…

  13. Targeting PPM1D by lentivirus-mediated RNA interference inhibits the tumorigenicity of bladder cancer cells

    Directory of Open Access Journals (Sweden)

    W. Wang

    2014-12-01

    Full Text Available Protein phosphatase magnesium/manganese-dependent 1D (PPM1D is a p53-induced phosphatase that functions as a negative regulator of stress response pathways and has oncogenic properties. However, the functional role of PPM1D in bladder cancer (BC remains largely unknown. In the present study, lentivirus vectors carrying small hairpin RNA (shRNA targeting PPM1D were used to explore the effects of PPM1D knockdown on BC cell proliferation and tumorigenesis. shRNA-mediated knockdown of PPM1D significantly inhibited cell growth and colony forming ability in the BC cell lines 5637 and T24. Flow cytometric analysis showed that PPM1D silencing increased the proportion of cells in the G0/G1 phase. Downregulation of PPM1D also inhibited 5637 cell tumorigenicity in nude mice. The results of the present study suggest that PPM1D plays a potentially important role in BC tumorigenicity, and lentivirus-mediated delivery of shRNA against PPM1D might be a promising therapeutic strategy for the treatment of BC.

  14. Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs.

    Science.gov (United States)

    Coffman, Stephanie R; Lu, Jinfeng; Guo, Xunyang; Zhong, Jing; Jiang, Hongshan; Broitman-Maduro, Gina; Li, Wan-Xiang; Lu, Rui; Maduro, Morris; Ding, Shou-Wei

    2017-03-21

    Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors.IMPORTANCE The helicase and C-terminal domains of mammalian RLRs sense intracellular viral RNAs to initiate the interferon-regulated innate immunity against RNA virus infection. Both of the domains from

  15. Lentivirus mediated RNA interference of EMMPRIN (CD147) gene inhibits the proliferation, matrigel invasion and tumor formation of breast cancer cells.

    Science.gov (United States)

    Yang, Jing; Wang, Rong; Li, Hongjiang; Lv, Qing; Meng, Wentong; Yang, Xiaoqin

    2016-07-08

    Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) or cluster of differentiation 147 (CD147), a glycoprotein enriched on the plasma membrane of tumor cells, promotes proliferation, invasion, metastasis, and survival of malignant tumor cells. In this study, we sought to examine the expression of EMMPRIN in breast tumors, and to identify the potential roles of EMMPRIN on breast cancer cells. EMMPRIN expression in breast cancer tissues was assessed by immunohistochemistry. We used a lentivirus vector-based RNA interference (RNAi) approach expressing short hairpin RNA (shRNA) to knockdown EMMPRIN gene in breast cancer cell lines MDA-MB-231 and MCF-7. In vitro, Cell proliferative, invasive potential were determined by Cell Counting Kit (CCK-8), cell cycle analysis and matrigel invasion assay, respectively. In vivo, tumorigenicity was monitored by inoculating tumor cells into breast fat pad of female nude mice. EMMPRIN was over-expressed in breast tumors and breast cancer cell lines. Down-regulation of EMMPRIN by lentivirus vector-based RNAi led to decreased cell proliferative, decreased matrigel invasion in vitro, and attenuated tumor formation in vivo. High expression of EMMPRIN plays a crucial role in breast cancer cell proliferation, matrigel invasion and tumor formation.

  16. Effective Silencing of Sry Gene with RNA Interference in Developing Mouse Embryos Resulted in Feminization of XY Gonad

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    Ning Wu

    2012-01-01

    Full Text Available Delivering siRNA or shRNA into the developing embryos is still a main challenge to use of RNAi in mammalian systems. Here we analyze several factors influencing RNAi-mediated silencing of Sry gene, which is a tightly controlled spatiotemporal expressed gene and only shortly expressed in developing mouse embryo gonad. A Sry gene-specific shRNAs expression vector (pSilencer4.1/Sry565 was constructed. The shRNA constructs were mixed with polyethylenimines (PEIs to form a complex and then injected into pregnant mice though tail vein. Our results showed that Sry gene was downregulated significantly in developing embryos. Further study revealed that knocking-down of Sry expression resulted in feminization of gonad development in mouse embryos and the expression level of Sox9 and Wt1 gene was also significantly changed by downregulation of Sry. The transfection efficiency is associated with the amount of plasmid DNA injection, injection time, injection speed, and volume. Our studies suggest that transplacental RNAi could be implemented by tail vein injection of plasmid vector into pregnant mice.

  17. Elongation Factor 1β' Gene from Spodoptera exigua: Characterization and Function Identification through RNA Interference

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    Li-Na Zhao

    2012-06-01

    Full Text Available Elongation factor (EF is a key regulation factor for translation in many organisms, including plants, bacteria, fungi, animals and insects. To investigate the nature and function of elongation factor 1β' from Spodoptera exigua (SeEF-1β', its cDNA was cloned. This contained an open reading frame of 672 nucleotides encoding a protein of 223 amino acids with a predicted molecular weight of 24.04 kDa and pI of 4.53. Northern blotting revealed that SeEF-1β' mRNA is expressed in brain, epidermis, fat body, midgut, Malpighian tubules, ovary and tracheae. RT-PCR revealed that SeEF-1β' mRNA is expressed at different levels in fat body and whole body during different developmental stages. In RNAi experiments, the survival rate of insects injected with SeEF-1β' dsRNA was 58.7% at 36 h after injection, which was significantly lower than three control groups. Other elongation factors and transcription factors were also influenced when EF-1β' was suppressed. The results demonstrate that SeEF-1β' is a key gene in transcription in S. exigua.

  18. Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing's sarcoma cells.

    Science.gov (United States)

    Sancéau, Josiane; Truchet, Sandrine; Bauvois, Brigitte

    2003-09-19

    Enhanced expression of (pro)matrix metalloproteinase-9 (MMP-9) is associated with human tumor invasion and/or metastasis. COH cells derived from a highly invasive and metastatic Ewing's sarcoma constitutively express proMMP-9. Transfection of a double stranded RNA that targets the MMP-9 mRNA into COH cells depleted the corresponding mRNA and protein as demonstrated by reverse transcriptase-PCR, enzyme-linked immunosorbent assay, and gelatin zymography. proMMP-9 extinction resulted in the following: (i) decreased spreading on extracellular matrix (fibronectin, laminin, collagen IV)-coated surfaces, (ii) inhibition of migration toward fibronectin, and (iii) induced aggregation, which was specifically disrupted by a function-blocking E-cadherin antibody. MMP-9 knockdown concomitantly resulted in increased levels of surface E-cadherin, redistribution at the plasma membrane of beta-catenin, and its physical association with E-cadherin. Moreover, induction of E-cadherin-mediated adhesion was associated with RhoA activation and changes in paxillin cytoskeleton. Finally, an inhibitor of gelatinolytic activity of pro-MMP9 did not reduce COH cell migration confirming that the enzymatic property of COH MMP-9 was not required for migration toward fibronectin. Overall, our observations define a novel critical role for proMMP-9 in providing a cellular switch between stationary and migratory cell phases.

  19. RNA interference depletion of the Halloween gene disembodied implies its potential application for management of planthopper Sogatella furcifera and Laodelphax striatellus.

    Science.gov (United States)

    Wan, Pin-Jun; Jia, Shuang; Li, Na; Fan, Jin-Mei; Li, Guo-Qing

    2014-01-01

    Sogatella furcifera and Laodelphax striatellus are economically important rice pests in China by acting as vectors of several rice viruses, sucking the phloem sap and blocking the phloem vessels. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. A cytochrome P450 monooxygenase CYP302A1 (22-hydroxylase), encoded by the Halloween gene disembodied (dib), plays a critical role in ecdysteroidogenesis. The objective of this study is to test whether dib genes are potential targets for RNA interference-based management of S. furcifera and L. striatellus. We cloned and characterized Sfdib and Lsdib. The open reading frame regions of dib genes were generated and used for designing and constructing dsRNA fragments. Experiments were conducted using oral delivery of dsdib to investigate the effectiveness of RNAi in S. furcifera and L. striatellus nymphs. Real-time quantitative reverse transcriptase-PCR analysis demonstrated that continuous ingestion of dsdib at the concentration of 0.01, 0.05 and 0.50 mg/ml diminished Sfdib expression levels by 35.9%, 45.1% and 66.2%, and ecdysone receptor (SfEcR) gene mRNA levels by 34.0%, 36.2% and 58.5% respectively in S. furcifera, and decreased Lsdib expression level by 18.8%, 35.8% and 56.7%, and LsEcR mRNA levels by 25.2%, 46.8% and 68.8% respectively in L. striatellus. The reduction in dib and EcR transcript abundance resulted in observable phenotypes. The development of nymphs was impaired and the survival was negatively affected. Our data will enable the development of new insect control strategies and functional analysis of vital genes in S. furcifera and L. striatellus nymphs.

  20. RNA interference depletion of the Halloween gene disembodied implies its potential application for management of planthopper Sogatella furcifera and Laodelphax striatellus.

    Directory of Open Access Journals (Sweden)

    Pin-Jun Wan

    Full Text Available Sogatella furcifera and Laodelphax striatellus are economically important rice pests in China by acting as vectors of several rice viruses, sucking the phloem sap and blocking the phloem vessels. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. A cytochrome P450 monooxygenase CYP302A1 (22-hydroxylase, encoded by the Halloween gene disembodied (dib, plays a critical role in ecdysteroidogenesis. The objective of this study is to test whether dib genes are potential targets for RNA interference-based management of S. furcifera and L. striatellus. We cloned and characterized Sfdib and Lsdib. The open reading frame regions of dib genes were generated and used for designing and constructing dsRNA fragments. Experiments were conducted using oral delivery of dsdib to investigate the effectiveness of RNAi in S. furcifera and L. striatellus nymphs. Real-time quantitative reverse transcriptase-PCR analysis demonstrated that continuous ingestion of dsdib at the concentration of 0.01, 0.05 and 0.50 mg/ml diminished Sfdib expression levels by 35.9%, 45.1% and 66.2%, and ecdysone receptor (SfEcR gene mRNA levels by 34.0%, 36.2% and 58.5% respectively in S. furcifera, and decreased Lsdib expression level by 18.8%, 35.8% and 56.7%, and LsEcR mRNA levels by 25.2%, 46.8% and 68.8% respectively in L. striatellus. The reduction in dib and EcR transcript abundance resulted in observable phenotypes. The development of nymphs was impaired and the survival was negatively affected. Our data will enable the development of new insect control strategies and functional analysis of vital genes in S. furcifera and L. striatellus nymphs.

  1. RNA interference-mediated silencing of a Halloween gene spookier affects nymph performance in the small brown planthopper Laodelphax striatellus.

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    Jia, Shuang; Wan, Pin-Jun; Zhou, Li-Tao; Mu, Li-Li; Li, Guo-Qing

    2015-04-01

    Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier (spok, cyp307a2) has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier (Lsspok, Lscyp307a2) in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth-instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth instars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  2. RNA Interference (RNAi) as a Potential Tool for Control of Mycotoxin Contamination in Crop Plants: Concepts and Considerations.

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    Majumdar, Rajtilak; Rajasekaran, Kanniah; Cary, Jeffrey W

    2017-01-01

    Mycotoxin contamination in food and feed crops is a major concern worldwide. Fungal pathogens of the genera Aspergillus. Fusarium , and Penicillium are a major threat to food and feed crops due to production of mycotoxins such as aflatoxins, 4-deoxynivalenol, patulin, and numerous other toxic secondary metabolites that substantially reduce the value of the crop. While host resistance genes are frequently used to introgress disease resistance into elite germplasm, either through traditional breeding or transgenic approaches, such resistance is often compromised by the evolving pathogen over time. RNAi-based host-induced gene silencing of key genes required by the pathogen for optimal growth, virulence and/or toxin production, can serve as an alternative, pre-harvest approach for disease control. RNAi represents a robust and efficient tool that can be used in a highly targeted, tissue specific manner to combat mycotoxigenic fungi infecting crop plants. Successful transgenic RNAi implementation depends on several factors including (1) designing vectors to produce double-stranded RNAs (dsRNAs) that will generate small interfering RNA (siRNA) species for optimal gene silencing and reduced potential for off-target effects; (2) availability of ample target siRNAs at the infection site; (3) efficient uptake of siRNAs by the fungus; (4) siRNA half-life and (5) amplification of the silencing effect. This review provides a critical and comprehensive evaluation of the published literature on the use of RNAi-based approaches to control mycotoxin contamination in crop plants. It also examines experimental strategies used to better understand the mode of action of RNAi with the aim of eliminating mycotoxin contamination, thereby improving food and feed safety.

  3. Lung-specific RNA interference of coupling factor 6, a novel peptide, attenuates pulmonary arterial hypertension in rats.

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    Yin, Jie; You, Shuling; Li, Nannan; Jiao, Shouhai; Hu, Hesheng; Xue, Mei; Wang, Ye; Cheng, Wenjuan; Liu, Ju; Xu, Min; Yan, Suhua; Li, Xiaolu

    2016-08-04

    Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease associated with high morbidity and mortality rates. However, the exact regulatory mechanism of PAH is unknown. Although coupling factor 6 (CF6) is known to function as a repressor, its role in PAH has not been explored. Here, we investigated the involvement of endogenous CF6 in the development of PAH. PAH was induced with monocrotaline (MCT), as demonstrated by significant increases in pulmonary artery pressure and vessel wall thickness. The adeno-associated virus (AAV) carrying CF6 short hairpin RNA (shRNA) or control vector (2×10(10) gp) was intratracheally transfected into the lungs of rats 2 weeks before or after MCT injection. A 2-6-fold increase in CF6 was observed in the lungs and circulation of the MCT-injected rats as confirmed by qRT-PCR and ELISA. Immunohistochemistry analysis revealed a small quantity of CF6 localized to endothelial cells (ECs) under physiological conditions spread to surrounding tissues in a paracrine manner in PAH lungs. Notably, CF6 shRNA effectively inhibited CF6 expression, abolished lung macrophage infiltration, reversed endothelial dysfunction and vascular remodeling, and ameliorated the severity of pulmonary hypertension and right ventricular dysfunction at 4 weeks both as a pretreatment and rescue intervention. In addition, the circulating and lung levels of 6-keto-PGF1a, a stable metabolite of prostacyclin, were reversed by CF6 inhibition, suggesting that the effect of CF6 inhibition may partly be mediated through prostacyclin. CF6 contributes to the pathogenesis of PAH, probably in association with downregulation of prostacyclin. The blockage of CF6 might be applied as a novel therapeutic approach for PAH and PA remodeling.

  4. Use of RNA Interference by In Utero Electroporation to Study Cortical Development: The Example of the Doublecortin Superfamily

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    Raanan Greenman

    2012-11-01

    Full Text Available The way we study cortical development has undergone a revolution in the last few years following the ability to use shRNA in the developing brain of the rodent embryo. The first gene to be knocked-down in the developing brain was doublecortin (Dcx. Here we will review knockdown experiments in the developing brain and compare them with knockout experiments, thus highlighting the advantages and disadvantages using the different systems. Our review will focus on experiments relating to the doublecortin superfamily of proteins.

  5. Downregulating galectin-3 inhibits proinflammatory cytokine production by human monocyte-derived dendritic cells via RNA interference.

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    Chen, Swey-Shen; Sun, Liang-Wu; Brickner, Howard; Sun, Pei-Qing

    2015-03-01

    Galectin-3 (Gal-3), a β-galactoside-binding lectin, serves as a pattern-recognition receptor (PRR) of dendritic cells (DCs) in regulating proinflammatory cytokine production. Galectin-3 (Gal-3) siRNA downregulates expression of IL-6, IL-1β and IL-23 p19, while upregulates IL-10 and IL-12 p35 in TLR/NLR stimulated human MoDCs. Furthermore, Gal-3 siRNA-treated MoDCs enhanced IFN-γ production in SEB-stimulated CD45RO CD4 T-cells, but attenuated IL-17A and IL-5 production by CD4 T-cells. Addition of neutralizing antibodies against Gal-3, or recombinant Gal-3 did not differentially modulate IL-23 p19 versus IL-12 p35. The data indicate that intracellular Gal-3 acts as cytokine hub of human DCs in responding to innate immunity signals. Gal-3 downregulation reprograms proinflammatory cytokine production by MoDCs that inhibit Th2/Th17 development. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. A RNA interference screen identifies the protein phosphatase 2A subunit PR55gamma as a stress-sensitive inhibitor of c-SRC.

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    Pieter J A Eichhorn

    2007-12-01

    Full Text Available Protein Phosphatase type 2A (PP2A represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55gamma and PR55delta as inhibitors of c-Jun NH(2-terminal kinase (JNK activation by UV irradiation. We show that PR55gamma binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55gamma and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55gamma.

  7. The lethal giant larvae Gene in Tribolium castaneum: Molecular Properties and Roles in Larval and Pupal Development as Revealed by RNA Interference

    Directory of Open Access Journals (Sweden)

    Da Xiao

    2014-04-01

    Full Text Available We identified and characterized the TcLgl gene putatively encoding lethal giant larvae (Lgl protein from the red flour beetle (Tribolium castaneum. Analyses of developmental stage and tissue-specific expression patterns revealed that TcLgl was constitutively expressed. To examine the role of TcLgl in insect development, RNA interference was performed in early (1-day larvae, late (20-day larvae, and early (1-day pupae. The early larvae injected with double-stranded RNA of TcLgl (dsTcLgl at 100, 200, and 400 ng/larva failed to pupate, and 100% mortality was achieved within 20 days after the injection or before the pupation. The late larvae injected with dsTcLgl at these doses reduced the pupation rates to only 50.3%, 36.0%, and 18.2%, respectively. The un-pupated larvae gradually died after one week, and visually unaffected pupae failed to emerge into adults and died during the pupal stage. Similarly, when early pupae were injected with dsTcLgl at these doses, the normal eclosion rates were reduced to only 22.5%, 18.0%, and 11.2%, respectively, on day 7 after the injection, and all the adults with abnormal eclosion died in two days after the eclosion. These results indicate that TcLgl plays an essential role in insect development, especially during their metamorphosis.

  8. RNA Interference of the Ecdysone Receptor Genes EcR and USP in Grain Aphid (Sitobion avenae F.) Affects Its Survival and Fecundity upon Feeding on Wheat Plants.

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    Yan, Ting; Chen, Hongmei; Sun, Yongwei; Yu, Xiudao; Xia, Lanqin

    2016-12-14

    RNA interference (RNAi) has been widely used in functional genomics of insects and received intensive attention in the development of RNAi-based plants for insect control. Ecdysone receptor (EcR) and ultraspiracle protein (USP) play important roles in molting, metamorphosis, and reproduction of insects. EcR and USP orthologs and their function in grain aphid (Sitobion avenae F.) have not been documented yet. Here, RT-PCR, qRT-PCR, dsRNA feeding assay and aphid bioassay were employed to isolate EcR and USP orthologs in grain aphid, investigate their expression patterns, and evaluate the effect of RNAi on aphid survival and fecundity, and its persistence. The results indicated that SaEcR and SaUSP exhibited similar expression profiles at different developmental stages. Oral administration of dsRNAs of SaEcR and dsSaUSP significantly decreased the survival of aphids due to the down-regulation of these two genes, respectively. The silencing effect was persistent and transgenerational, as demonstrated by the reduced survival and fecundity due to knock-down of SaEcR and SaUSP in both the surviving aphids and their offspring, even after switching to aphid-susceptible wheat plants. Taken together, our results demonstrate that SaEcR and SaUSP are essential genes in aphid growth and development, and could be used as RNAi targets for wheat aphid control.

  9. Mitochondrial uncoupling proteins regulate angiotensin‐converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies

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    Maubaret, Cecilia; Pedersen‐Bjergaard, Ulrik; Brull, David J.; Gohlke, Peter; Payne, John R.; World, Michael; Thorsteinsson, Birger; Humphries, Steve E.; Montgomery, Hugh E.

    2015-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin‐converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole‐body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3‐55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8‐fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. PMID:27347560

  10. Assessment of the evolutionary origin and possibility of CRISPR-Cas (CASS) mediated RNA interference pathway in Vibrio cholerae O395.

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    Chakraborty, Sajib; Waise, T M Zaved; Hassan, Faizule; Kabir, Yearul; Smith, Mark A; Arif, Mohammad

    2009-01-01

    Bacteria have developed several defense mechanisms against bacteriophages over evolutionary time, but the concept of prokaryotic RNA interference mediated defense mechanism against phages and other invading genetic elements has emerged only recently. Clustered regularly interspaced short palindromic repeats (CRISPR) together with closely associated genes (cas genes) constitute the CASS system that is believed to provide a RNAi-like defense mechanism against bacteriophages within the host bacterium. However, a CASS mediated RNAi-like pathway in enteric pathogens such as Vibrio cholerae O395 or Escherichia coli O157 have not been reported yet. This study specifically was designed to investigate the possibility and evolutionary origin of CASS mediated RNAi-like pathway in the genome of a set of enteric pathogens, especially V. cholerae. The results showed that V. cholerae O395 and also other related enteric pathogens have the essential CASS components (CRISPR and cas genes) to mediate a RNAi-like pathway. The functional domains of a V. cholerae Cas3 protein, which is believed to act as a prokaryotic Dicer, was revealed and compared with the domains of eukaryotic Dicer proteins. Extensive homology in several functional domains provides significant evidence that the Cas3 protein has the essential domains to play a vital role in RNAi like pathway in V. cholerae. The secondary structure of the pre-siRNA for V. cholerae O395 was determined and its thermodynamic stability also reinforced the previous findings and signifies the probability of a RNAi-like pathway in V. cholerae O395.

  11. The chitin biosynthesis pathway in Entamoeba and the role of glucosamine-6-P isomerase by RNA interference.

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    Samanta, Sintu Kumar; Ghosh, Sudip K

    2012-11-01

    Entamoeba histolytica, the causative agent of amoebiasis, infects through its cyst form. A thick chitin wall protects the cyst from the harsh environment outside of the body. It is known that chitin is synthesized only during encystation, but the chitin synthesis pathway (CSP) of Entamoeba is not well characterized. In this report, we have identified the genes involved in chitin biosynthesis from the Entamoeba genome database and verified their expression profile at the transcriptional level in encysting Entamoeba invadens. Semi-quantitative RT-PCR (sqRT-PCR) analysis showed that all the chitin pathway genes are entirely absent or transcribed at low levels in trophozoites. The mRNA expression of most of the CSP genes reached their maximum level between 9 and 12h after the in vitro initiation of encystation. Double-stranded RNA-mediated silencing of glucosamine-6-P isomerase (Gln6Pi) reduced chitin synthesis to 62-64%, which indicates that Gln6Pi might be a key enzyme for regulating chitin synthesis in Entamoeba. The study of different enzymes involved in glycogen metabolism revealed that stored glycogen is converted to glucose during encystation. It is clear from the sqRT-PCR analysis that the rate of glycolysis decreases as encystation proceeds. Encystation up-regulates the expression of glycogen phosphorylase, which is responsible for glycogen degradation. The significant decrease in chitin synthesis in encysting cells treated with a specific inhibitor of glycogen phosphorylase indicates that the glucose obtained from the degradation of stored glycogen in trophozoites might be one of the major sources of glucose for chitin synthesis. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Micro RNA-550a interferes with vitamin D metabolism in peripheral B cells of patients with diabetes.

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    He, Jinggui; Guo, Xiyun; Liu, Zhi-Qiang; Yang, Ping-Chang; Yang, Shaobo

    2016-12-01

    The pathogenesis of diabetes is to be further investigated. Vitamin D3 (VitD3) can improve diabetes. Micro RNAs (miR) are involved in regulating cell activities. This study tests a hypothesis that miR-550a interferes with the metabolism of VitD3 in peripheral B cells. In this study, blood samples were collected from patients with diabetes and healthy persons. The B cells were isolated from the blood samples to be treated with tumor necrosis factor (TNF)-α. The B cells were then collected and analyzed for the expression of miR-550a and cyp27b1. The results showed that B cells from healthy subjects were capable of converting VitD metabolite calcidiol to calcitriol, which was impaired in B cells collected from diabetic patients. The diabetic patients showed lower bone mineral density than that in healthy subject. The miR-550a was negatively correlated with bone mineral density and the Levels of cyp27b1 in peripheral B cells of patients with diabetes. In vitro study showed that TNF-α increased miR-550a expression and inhibited the expression of cyp27b1 in B cells. miR-550a mediated the effects of TNF-α on inducing chromatin remodeling at the cyp27b1 gene locus. In conclusion, miR-550a mediates the TNF-α-induced suppression of cyp27b1 expression in peripheral B cells of patients with diabetes, which can be blocked by inhibition of miR-550a. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity?

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    Clerzius Guerline

    2005-10-01

    Full Text Available Abstract Increasing evidence indicates that RNA interference (RNAi may be used to provide antiviral immunity in mammalian cells. Human micro (miRNAs can inhibit the replication of a primate virus, whereas a virally-encoded miRNA from HIV inhibits its own replication. Indirect proof comes from RNAi suppressors encoded by mammalian viruses. Influenza NS1 and Vaccinia E3L proteins can inhibit RNAi in plants, insects and worms. HIV-1 Tat protein and Adenovirus VA RNAs act as RNAi suppressors in mammalian cells. Surprisingly, many RNAi suppressors are also inhibitors of the interferon (IFN-induced protein kinase R (PKR but the potential overlap between the RNAi and the IFN pathways remains to be determined. The link between RNAi as an immune response and the IFN pathway may be formed by a cellular protein, TRBP, which has a dual role in HIV replication and RNAi. TRBP has been isolated as an HIV-1 TAR RNA binding protein that increases HIV expression and replication by inhibiting PKR and by increasing translation of structured RNAs. A recent report published in the Journal of Virology shows that the poor replication of HIV in astrocytes is mainly due to a heightened PKR response that can be overcome by supplying TRBP exogenously. In two recent papers published in Nature and EMBO Reports, TRBP is now shown to interact with Dicer and to be required for RNAi mediated by small interfering (si and micro (miRNAs. The apparent discrepancy between TRBP requirement in RNAi and in HIV replication opens the hypotheses that RNAi may be beneficial for HIV-1 replication or that HIV-1 may evade the RNAi restriction by diverting TRBP from Dicer and use it for its own benefit.

  14. Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression

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    Aftab Ahmad

    2017-09-01

    Full Text Available Cotton leaf curl virus disease (CLCuD is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu and the βC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB. The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vβ, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vβ transgene. Transgenic cotton plants and non-transgenic (susceptible test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vβ-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vβ-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.

  15. Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling

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    Craigon Marie

    2009-08-01

    Full Text Available Abstract Background Interferons (IFNs are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs. Here we have used targeted RNA interference to suppress the expression of a number of key genes associated with IFN signalling in murine macrophages prior to stimulation with interferon-gamma. Genome-wide changes in transcript abundance caused by siRNA activity were measured using exon-level microarrays in the presence or absence of IFNγ. Results Transfection of murine bone-marrow derived macrophages (BMDMs with a non-targeting (control siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000 prior to stimulation with IFNγ. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. Network-based analysis of these data revealed six siRNAs to cause a marked shift in the macrophage transcriptome in the presence or absence IFNγ. These six siRNAs targeted the Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2 transcripts. The perturbation of the transcriptome by the six siRNAs was highly similar in each case and affected the expression of over 600 downstream transcripts. Regulated transcripts were clustered based on co-expression into five major groups corresponding to transcriptional networks associated with the type I and II IFN response, cell cycle regulation, and NF-KB signalling. In addition we have observed a significant non-specific immune stimulation of cells transfected with siRNA using Lipofectamine2000, suggesting use of this reagent in BMDMs, even at low concentrations, is enough to induce a type I IFN response. Conclusion Our results provide evidence that the type I IFN response in murine BMDMs is dependent on Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2, and that siRNAs targeted to these genes results in perturbation of key transcriptional networks associated

  16. [Effects of silencing H-ras gene by RNA interference on cetuximab-sensitivity of cetuximab-resistant human nasopharyngeal carcinoma cells].

    Science.gov (United States)

    Zuo, Qiang; Luo, Rong-Cheng

    2011-08-01

    To explore the changes of the sensitivity of cetuximab-resistant human nasopharyngeal carcinoma (hNPC) cells 5-8F/Erbitux to cetuximab by silencing H-ras gene with RNA interference (RNAi). The 5-8F/Erbitux cells were induced by stepwise exposure to increasing doses of cetuximab. Western blot was conducted to detect the protein levels of H-ras and K-ras. Real-time PCR was employed to detect the expression of H-ras and K-ras. H-ras-shRNA plasmids (shRNA vector carrying the H-ras gene) were constructed and transferred into 5-8F/Erbitux cells. The gene and protein expression levels of H-ras and the changes of the sensitivity of 5-8F/Erbitux cells to cetuximab after transfection were measured, respectively. After treatment with cetuximab for 3 and 5 days, the resistance index (RI) of the 5-8F/Erbitux cells was 1.2 and 1.1, and the protein levels of H-ras and K-ras in 5-8F/Erbitux cells were 0.798 +/- 0.019 and 0.190 +/- 0.011, respectively, significantly higher than that in the 5-8F cells (Pgene expressions of H-ras and K-ras in 5-8F/Erbitux cells were 1.260 +/- 0.114 and 0.850 +/- 0.006, respectively. Compared with 5-8F cells, the former was higher (P = 0.016) and the latter was lower (P = 0.000). After transfection with H-ras-shRNA plasmid, the 5-8F/Erbitux cells showed reduced levels of H-ras gene and protein, and the cell apoptosis and inhibition rates increased significantly (Pras siRNA can reverse cetuximab-resistance of 5-8F/Erbitux cells through down-regulation of H-ras gene expression, indicating that the generation of cetuximab-resistance in 5-8F/Erbitux cells is associated with amplification and overexpression of the H-ras gene.

  17. RNA interference effectively degrades mRNA and inhibits protein expression of GBV-C E2 gene in Huh7 cells.

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    Cao, Ming-Mei; Li, Gang; Ren, Hao; Pan, Wei; Zhao, Ping; Qi, Zhong-Tian

    2009-12-01

    The GB virus C/hepatitis G virus (GBV-C/HGV) is a Flaviviridae member that despite its nonpathogenicity, has become of great interest given that it could inhibit the replication of the human immunodeficiency virus (HIV). Therefore, a better knowledge of the viral protein E2 has become our aim. In this study, a GBV-C model cell system (HuhEG) which expressing a fusion protein of the GBV-C E2 protein and enhanced green fluorescent protein (EGFP) stably was established. And the expression of these proteins was silenced effectively by the two E2 gene-specific siRNAs and an EGFP gene-specific siRNA. This inhibition is sequence-specific and extensive (90%). This HuhEG/specific siRNAs system can provide an approach for investigating the association between GBV-C E2 and HIV replication, which may be of potential value in the development of novel prophylactic or therapeutic agents for HIV infection.

  18. Stimulation of molt by RNA interference of the molt-inhibiting hormone in the crayfish Cherax quadricarinatus.

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    Pamuru, Ramachandra R; Rosen, Ohad; Manor, Rivka; Chung, J Sook; Zmora, Nilli; Glazer, Lilah; Aflalo, Eliahu D; Weil, Simy; Tamone, Sherry L; Sagi, Amir

    2012-09-01

    In crustaceans, molting is known to be under the control of neuropeptide hormones synthesized and secreted from the eyestalk ganglia. While the role of molt-inhibiting hormone (MIH) in regulating molting has been described in several species using classical methods, an in vivo specific MIH targeted manipulation has not been described yet. In the present study, an MIH cDNA was isolated and sequenced from the eyestalk ganglia of the Australian freshwater red claw crayfish Cherax quadricarinatus (Cq) by 5' and 3' RACE. We analyzed the putative Cq-MIH based on sequence homology, a three dimensional structure model and transcript's tissue specificity. We further examined the involvement of Cq-MIH in the control of molt in the crayfish through RNAi by in vivo injections of Cq-MIH double-stranded RNA, which resulted in, similarly to eyestalk ablation, acceleration of molt cycles. This acceleration was reflected by a significant reduction (up to 32%) in molt interval and an increased rate in molt mineralization index (MMI), which correlated with the induction of ecdysteroid hormones compared to control. Altogether, this study provides a proof of function for the involvement of the Cq-MIH gene in molt regulation in the crayfish. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Micro RNA-19a interferes with IL-10 expression in peripheral dendritic cells of patients with nasal polyposis.

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    Luo, Xiang-Qian; Shao, Jian-Bo; Xie, Rui-Di; Zeng, Lu; Li, Xiao-Xi; Qiu, Shu-Qi; Geng, Xiao-Rui; Yang, Li-Tao; Li, Lin-Jing; Liu, Da-Bo; Liu, Zhi-Gang; Yang, Ping-Chang

    2017-07-25

    The pathogenesis of nasal polyp is to be further investigated. Micro RNA (miR) plays a role in the development of allergic inflammation. Interleukin (IL)-10-producing dendritic cells (DC) have immune tolerogenic properties. This study test a hypothesis that miR-17-92 cluster is associated with suppressing IL-10 in peripheral DC. In this study, peripheral blood samples were obtained from 26 patients with nasal polyp. The CD11c DCs were isolated from the blood samples and analyzed for the expression of IL-10. We observed that, as compared with healthy subjects, the IL-10 expression in peripheral DC was significantly lower in polyp patients. The levels of miR-19a, but not the rest 5 members of the miR-17-92 cluster, were markedly higher in DCs in polyp group. Exposure to recombinant IL-4 suppressed the IL-10 expression in DCs, which was abolished by blocking histone deacetylase-11 or knocking down the miR-19a gene in DCs. We conclude that miR-19a plays a critical role in the suppression of IL-10 in peripheral DCs, which may be a target in the immune therapy for nasal polyp.

  20. Intrathecal injection of lentivirus-mediated glial cell line-derived neurotrophic factor RNA interference relieves bone cancer-induced pain in rats.

    Science.gov (United States)

    Meng, Fu-Fen; Xu, Yang; Dan, Qi-Qin; Wei, La; Deng, Ying-Jie; Liu, Jia; He, Mu; Liu, Wei; Xia, Qing-Jie; Zhou, Fiona H; Wang, Ting-Hua; Wang, Xi-Yan

    2015-04-01

    Bone cancer pain is a common symptom in cancer patients with bone metastases and the underlying mechanisms are largely unknown. The aim of this study is to explore the endogenous analgesic mechanisms to develop new therapeutic strategies for bone-cancer induced pain (BCIP) as a result of metastases. MRMT-1 tumor cells were injected into bilateral tibia of rats and X-rays showed that the area suffered from bone destruction, accompanied by an increase in osteoclast numbers. In addition, rats with bone cancer showed apparent mechanical and thermal hyperalgesia at day 28 after intratibial MRMT-1 inoculation. However, intrathecal injection of morphine or lentivirus-mediated glial cell line-derived neurotrophic factor RNAi (Lvs-siGDNF) significantly attenuated mechanical and thermal hyperalgesia, as shown by increases in paw withdrawal thresholds and tail-flick latencies, respectively. Furthermore, Lvs-siGDNF interference not only substantially downregulated GDNF protein levels, but also reduced substance P immunoreactivity and downregulated the ratio of pERK/ERK, where its activation is crucial for pain signaling, in the spinal dorsal horn of this model of bone-cancer induced pain. In this study, Lvs-siGDNF gene therapy appeared to be a beneficial method for the treatment of bone cancer pain. As the effect of Lvs-siGDNF to relieve pain was similar to morphine, but it is not a narcotic, the use of GDNF RNA interference may be considered as a new therapeutic strategy for the treatment of bone cancer pain in the future. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  1. Lentivirus-Mediated RNA Interference Targeting RhoA Slacks the Migration, Proliferation, and Myelin Formation of Schwann Cells.

    Science.gov (United States)

    Wen, Jinkun; Qian, Changhui; Pan, Mengjie; Wang, Xianghai; Li, Yuanyuan; Lu, Yanmeng; Zhou, Zhitao; Yan, Qing; Li, Lixia; Liu, Zhongying; Wu, Wutian; Guo, Jiasong

    2017-03-01

    RhoA, a member of Rho GTPases family, is known to play an important role in remodeling actin cytoskeleton. During the development of the peripheral nervous system (PNS), Schwann cells undergo proliferation, migration, and radial sorting and finally wrap the related axons compactly to form myelin sheath. All these processes involve actin cytoskeletal remodeling. However, the role of RhoA on Schwann cell during development is still unclear. To address this question, we first used a lentiviral vector-mediated short hairpin (sh) RNA targeting RhoA to knock down the expression of RhoA in the cultured Schwann cells in vitro. Effects of RhoA on Schwann cell proliferation and migration were examined by BrdU assay and transwell assay, respectively. Results of the present study indicated that downregulated RhoA expression in cultured Schwann cells significantly slacked the cells' capabilities of migration and proliferation. Then, we investigated the role of RhoA in the developing rat sciatic nerves. Immunohistology and Western blotting showed that RhoA was mainly expressed in Schwann cells in the sciatic nerves and was peaked at 2 weeks postnatal then kept in low level up to 8 weeks. In the subjected rats whose sciatic nerves were microinjected with lentiviral vectors at postnatal 3 days, we found that the lentiviruses mainly transfected Schwann cells, and the RhoA expression in the transfected Schwann cells was significantly knocked down. Four weeks after lentivirus microinjection, immunohistology and transmission electron microscopy illustrated that RhoA knockdown resulted in hypomyelination and significant decrease of the thickness of myelin in the transfected area. Overall data of current study suggested that RhoA plays a critical role in Schwann cell biology and is essential for myelination in developing peripheral nerve.

  2. [Effect of RNA Interference-silenced TAK1 on Kasumi-1 cell Proliferation Inhibition Induced by As2O3 and Its Mechanism].

    Science.gov (United States)

    Liu, Sha; Yuan, Fang-Fang; Mi, Rui-Hua; Wang, Xiao-Jiao; Fan, Rui-Hua; Wei, Xu-Dong

    2017-04-01

    To explore the effect of transforming growth factor-β activated kinase-1(TAK1) gene silenced by RNA interference on proliferation inhibition of Kasumi-1 cells induced by As2O3 and its mechanism. The experiments were divided into 4 groups, including control group(Kasumi-1 cells treated with non-specific siRNA), TAK1 specific siRNA treated group (Kasumi 1 treated with TAK specific siRNA), As2O3 treated group (Kasumi 1 cells treated with As2O3) and combined treated group (Kasumi 1 cells treated with TAK1 specific siRNA plus As2O3). The proliferation inhibition rate of Kasami 1 cells was detected by CCK-8 method, the apoptotic rate of cells was detected by flow eytometry, the expressions of TAK1, phosphorylated c-Jun N-terminal kinase(p-JNK) and apoptosis-related proteins were detected by Western blot. As2O3 could inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 20 µmol/L with IC50 of (3.79±0.36) µmol/L at 24 h, and also inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 10 µmol/L with IC50 of (2.38±0.17) µmol/L at 48 h, but then the inhibitory effect reached plateau. After treating Kasumi-1 cells with TAK1 siRNA and 3.5 µmol/L As2O3 for 24 h, the proliferation inhibition rate was (10.86±1.64)% and (49.80±2.19)%, meanwhile the apoptosis rate was (8.47±0.75)% and (24.78±2.14)%, all significantly higher than those in control group (P<0.05, P<0.01). The proliferation inhibition rate and apoptosis rate of the combined treated group were significantly higher than that in control and single treated groups (P<0.05, P<0.01), TAK1 silencing and 3.5 µmol/L of As2O3 could decrease the expression of TAK1, p-JNK, c-Fos, c-Jun and BCL-2 in different degrees, and increase the expression levels of BAX and the activated (cleaved) caspase-3, 9 with statistically significant differences as compared with control group (P< 0.05). When Kasumi-1 cells were treated with TAK1 specific siRNA plus As2O3 for 24 h, protein

  3. RNA interference against cancer/testis genes identifies dual specificity phosphatase 21 as a potential therapeutic target in human hepatocellular carcinoma.

    Science.gov (United States)

    Deng, Qing; Li, Kun-Yu; Chen, Hui; Dai, Ji-Hong; Zhai, Yang-Yang; Wang, Qun; Li, Niu; Wang, Yu-Ping; Han, Ze-Guang

    2014-02-01

    Cancer/testis (CT) antigens have been considered therapeutic targets for treating cancers. However, a central question is whether their expression contributes to tumorigenesis or if they are functionally irrelevant by-products derived from the process of cellular transformation. In any case, these CT antigens are essential for cancer cell survival and may serve as potential therapeutic targets. Recently, the cell-based RNA interference (RNAi) screen has proven to be a powerful approach for identifying potential therapeutic targets. In this study we sought to identify new CT antigens as potential therapeutic targets for human hepatocellular carcinoma (HCC), and 179 potential CT genes on the X chromosome were screened through a bioinformatics analysis of gene expression profiles. Then an RNAi screen against these potential CT genes identified nine that were required for sustaining the survival of Focus and PLC/PRF/5 cells. Among the nine genes, the physiologically testis-restricted dual specificity phosphatase 21 (DUSP21) encoding a dual specificity phosphatase was up-regulated in 39 (33%) of 118 human HCC specimens. Ectopic DUSP21 had no obvious impact on proliferation and colony formation in HCC cells. However, DUSP21 silencing significantly suppressed cell proliferation, colony formation, and in vivo tumorigenicity in HCC cells. The administration of adenovirus-mediated RNAi and an atelocollagen/siRNA mixture against endogenous DUSP21 significantly suppressed xenograft HCC tumors in mice. Further investigations showed that DUSP21 knockdown led to arrest of the cell cycle in G1 phase, cell senescence, and expression changes of some factors with functions in the cell cycle and/or senescence. Furthermore, the antiproliferative role of DUSP21 knockdown is through activation of p38 mitogen-activated protein kinase in HCC. DUSP21 plays an important role in sustaining HCC cell proliferation and may thus act as a potential therapeutic target in HCC treatment. © 2013 by

  4. Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study.

    Science.gov (United States)

    Geisbert, Thomas W; Lee, Amy C H; Robbins, Marjorie; Geisbert, Joan B; Honko, Anna N; Sood, Vandana; Johnson, Joshua C; de Jong, Susan; Tavakoli, Iran; Judge, Adam; Hensley, Lisa E; Maclachlan, Ian

    2010-05-29

    We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures, they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever. A combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP) 24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV. Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection. This complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might also be useful for treatment of other emerging viral infections. Defense Threat Reduction Agency. Copyright 2010 Elsevier Ltd. All rights reserved.

  5. RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman (TYLCV-OM) and its associated betasatellite.

    Science.gov (United States)

    Ammara, Um e; Mansoor, Shahid; Saeed, Muhammad; Amin, Imran; Briddon, Rob W; Al-Sadi, Abdullah Mohammed

    2015-03-04

    Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus (family Geminiviridae) is responsible for heavy yield losses for tomato production around the globe. In Oman at least five distinct begomoviruses cause disease in tomato, including TYLCV. Unusually, TYLCV infections in Oman are sometimes associated with a betasatellite (Tomato leaf curl betasatellite [ToLCB]; a symptom modulating satellite). RNA interference (RNAi) can be used to develop resistance against begomoviruses at either the transcriptional or post-transcriptional levels. A hairpin RNAi (hpRNAi) construct to express double-stranded RNA homologous to sequences of the intergenic region, coat protein gene, V2 gene and replication-associated gene of Tomato yellow leaf curl virus-Oman (TYLCV-OM) was produced. Initially, transient expression of the hpRNAi construct at the site of virus inoculation was shown to reduce the number of plants developing symptoms when inoculated with either TYLCV-OM or TYLCV-OM with ToLCB-OM to Nicotiana benthamiana or tomato. Solanum lycopersicum L. cv. Pusa Ruby was transformed with the hpRNAi construct and nine confirmed transgenic lines were obtained and challenged with TYLCV-OM and ToLCB-OM by Agrobacterium-mediated inoculation. For all but one line, for which all plants remained symptomless, inoculation with TYLCV-OM led to a proportion (≤25%) of tomato plants developing symptoms of infection. For inoculation with TYLCV-OM and ToLCB-OM all lines showed a proportion of plants (≤45%) symptomatic. However, for all infected transgenic plants the symptoms were milder and virus titre in plants was lower than in infected non-transgenic tomato plants. These results show that RNAi can be used to develop resistance against geminiviruses in tomato. The resistance in this case is not immunity but does reduce the severity of infections and virus titer. Also, the betasatellite may compromise resistance, increasing the proportion of plants which ultimately show symptoms.

  6. Molecular characterization and developmental expression of vitellogenin in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference and eyestalk ablation on ovarian maturation.

    Science.gov (United States)

    Bai, Hongkun; Qiao, Hui; Li, Fajun; Fu, Hongtuo; Sun, Shengming; Zhang, Wenyi; Jin, Shubo; Gong, Yongsheng; Jiang, Sufei; Xiong, Yiwei

    2015-05-10

    Vitellogenin (Vg) is the precursor of yolk protein, which functions as a nutritive resource that is important for embryonic growth and gonad development. In this study, the cDNA encoding the Vg gene from the oriental river prawn Macrobrachium nipponense was cloned using expressed sequence tag (EST) analysis and the rapid amplification of cDNA ends (RACE) approach. The transcript encoded 2536 amino acids with an estimated molecular mass of 286.810 kDa. Quantitative real-time PCR indicated high expression of Mn-Vg in the female ovary, hemocytes, and hepatopancreas. As ovaries developed, the expression level of Mn-Vg increased in both the hepatopancreas and ovary. In the hepatopancreas, the expression level rose more slowly at the early stage of vitellogenesis and reached the peak more rapidly compared to the expression pattern in ovary. The observed changes in Mn-Vg expression level at different development stages suggest the role of nutrient source in embryonic and larval development. Eyestalk ablation caused the Mn-Vg expression level to increase significantly compared to eyestalk-intact groups during the ovary development stages. Ablation accelerated ovary maturation by removing hormone inhibition of Mn-Vg in the hepatopancreas and ovary. In adult females, Mn-Vg dsRNA injection resulted in decreased expression of Mn-Vg in both the hepatopancreas and ovary, and two injection treatment dramatically delayed ovary maturation. Vg RNA interference down-regulated the vitellogenin receptor (VgR) expression level in the ovary, which illustrates the close relationship between Vg and VgR in the process of vitellogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. In silico cloning and annotation of genes involved in the digestion, detoxification and RNA interference mechanism in the midgut of Bactrocera dorsalis [Hendel (Diptera: Tephritidae)].

    Science.gov (United States)

    Shen, G-M; Dou, W; Huang, Y; Jiang, X-Z; Smagghe, G; Wang, J-J

    2013-08-01

    As the second largest organ in insects, the insect midgut is the major tissue involved in the digestion of food and detoxification of xenobiotics, such as insecticides, and the first barrier and target for oral RNA interference (RNAi). In this study, we performed a midgut-specific transcriptome analysis in the oriental fruit fly, Bactrocera dorsalis, an economically important worldwide pest, with many populations showing high levels of insecticide resistance. Using high-throughput sequencing, 52 838 060 short reads were generated and assembled to 25 236 unigenes with a mean length of 758 bp. Interestingly, 34 unique sequences encoding digestion enzymes were newly described and these included aminopeptidase and trypsin, genes associated with Bacillus thuringiensis resistance and fitness cost. Second, 41 transcripts were annotated to particular detoxification genes such as glutathione S-transferases, carboxylesterases and cytochrome P450s, and the subsequent phylogenetic analysis indicated homology with tissue-specific and insecticide resistance-related genes of Drosophila melanogaster. Third, we identified the genes involved in the mechanism of RNAi and the uptake of double-stranded RNA. The sequences encoding Dicer-2, R2D2, AGO2, and Eater were confirmed, but SID and SR-CI were absent in the midgut transcriptome. In conclusion, the results provide basic molecular information to better understand the mechanisms of food digestion, insecticide resistance and oral RNAi in this important pest insect in agriculture. Specific genes in these systems can be used in the future as potential targets for pest control, for instance, with RNAi technology. © 2013 Royal Entomological Society.

  8. RNA interference-mediated knockdown of the Halloween gene Spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus.

    Science.gov (United States)

    Van Ekert, E; Wang, M; Miao, Y-G; Brent, C S; Hull, J J

    2016-10-01

    Ecdysteroids play a critical role in coordinating insect growth, development and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNA interference (RNAi)-mediated knockdown of the CYP307B1 Halloween gene (Spookiest) in the western tarnished plant bug, Lygus hesperus. Transcripts for Ly. hesperus Spookiest (LhSpot) were amplified from all life stages and correlated well with timing of the pre-moult ecdysteroid pulse. In adults, LhSpot was amplified from heads of both genders as well as female reproductive tissues. Heterologous expression of a LhSpot fluorescent chimera in cultured insect cells co-localized with a fluorescent marker of the endoplasmic reticulum/secretory pathway. RNAi-mediated knockdown of LhSpot in fifth instars reduced expression of ecdysone-responsive genes E74 and E75, and prevented adult development. This developmental defect was rescued following application of exogenous 20-hydroxyecdysone but not exogenous 7-dehydrocholesterol. The unequivocal RNAi effects on Ly. hesperus development and the phenotypic rescue by 20-hydroxyecdysone are causal proof of the involvement of LhSpot in ecdysteroid biosynthesis and related developmental processes, and may provide an avenue for development of new control measures against Ly. hesperus. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  9. RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes expression prolongs the shelf life of Eksotika (Carica papaya L.) papaya fruit.

    Science.gov (United States)

    Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom; Yeong, Wee Chien; Pillai, Vilasini

    2014-06-19

    The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

  10. RNA Interference of 1-Aminocyclopropane-1-carboxylic Acid Oxidase (ACO1 and ACO2 Genes Expression Prolongs the Shelf Life of Eksotika (Carica papaya L. Papaya Fruit

    Directory of Open Access Journals (Sweden)

    Rogayah Sekeli

    2014-06-01

    Full Text Available The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6. Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

  11. Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins.

    Science.gov (United States)

    Shigemitsu, Takanari; Ozaki, Shinji; Saito, Yuhi; Kuroda, Masaharu; Morita, Shigeto; Satoh, Shigeru; Masumura, Takehiro

    2012-03-01

    Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.

  12. Novel genes participating in the formation of prismatic and nacreous layers in the pearl oyster as revealed by their tissue distribution and RNA interference knockdown.

    Directory of Open Access Journals (Sweden)

    Daisuke Funabara

    Full Text Available In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein, 000118, 000133 and 000411 (MSI60, which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers.

  13. Cyclooxygenase-2 Silencing for the Treatment of Colitis: A Combined In Vivo Strategy Based on RNA Interference and Engineered Escherichia Coli

    Science.gov (United States)

    Spisni, Enzo; Valerii, Maria C; De Fazio, Luigia; Cavazza, Elena; Borsetti, Francesca; Sgromo, Annamaria; Candela, Marco; Centanni, Manuela; Rizello, Fernando; Strillacci, Antonio

    2015-01-01

    Nonpathogenic-invasive Escherichia coli (InvColi) bacteria are suitable for genetic transfer into mammalian cells and may act as a vehicle for RNA Interference (RNAi) in vivo. Cyclooxygenase-2 (COX-2) is overexpressed in ulcerative colitis (UC) and Crohn's disease (CD), two inflammatory conditions of the colon and small intestine grouped as inflammatory bowel disease (IBD). We engineered InvColi strains for anti-COX-2 RNAi (InvColishCOX2), aiming to investigate the in vivo feasibility of a novel COX-2 silencing strategy in a murine model of colitis induced by dextran sulfate sodium (DSS). Enema administrations of InvColishCOX2 in DSS-treated mice led to COX-2 downregulation, colonic mucosa preservation, reduced colitis disease activity index (DAI) and increased mice survival. Moreover, DSS/InvColishCOX2-treated mice showed lower levels of circulating pro-inflammatory cytokines and a reduced colitis-associated shift of gut microbiota. Considering its effectiveness and safety, we propose our InvColishCOX2 strategy as a promising tool for molecular therapy in intestinal inflammatory diseases. PMID:25393372

  14. Human microRNA hsa-miR-125a-5p interferes with expression of hepatitis B virus surface antigen

    Science.gov (United States)

    Potenza, Nicoletta; Papa, Umberto; Mosca, Nicola; Zerbini, Francesca; Nobile, Valentina; Russo, Aniello

    2011-01-01

    MicroRNAs are small non-coding RNAs that modulate gene expression at post-transcriptional level, playing a crucial role in cell differentiation and development. Recently, some reports have shown that a limited number of mammalian microRNAs are also involved in anti-viral defense. In this study, the analysis of the hepatitis B virus (HBV) genome by the computer program MiRanda led to the identification of seven sites that are potential targets for human liver microRNAs. These sites were found to be clustered in a 995-bp segment within the viral polymerase ORF and the overlapping surface antigen ORF, and conserved among the most common HBV subtypes. The HBV genomic targets were then subjected to a validation test based on cultured hepatic cells (HepG2, HuH-7 and PLC/PRF/5) and luciferase reporter genes. In this test, one of the selected microRNAs, hsa-miR-125a-5p, was found to interact with the viral sequence and to suppress the reporter activity markedly. The microRNA was then shown to interfere with the viral translation, down-regulating the expression of the surface antigen. Overall, these results support the emerging concept that some mammalian microRNAs play a role in virus-host interaction. Furthermore, they provide the basis for the development of new strategies for anti-HBV intervention. PMID:21317190

  15. RNA interference in adult Ascaris suum – an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite

    Science.gov (United States)

    McCoy, Ciaran J.; Warnock, Neil D.; Atkinson, Louise E.; Atcheson, Erwan; Martin, Richard J.; Robertson, Alan P.; Maule, Aaron G.; Marks, Nikki J.; Mousley, Angela

    2015-01-01

    The sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance. PMID:26149642

  16. Lentivirus-mediated short hairpin RNA interference targeting TNF-alpha in macrophages inhibits particle-induced inflammation and osteolysis in vitro and in vivo.

    Science.gov (United States)

    Qin, Chu-Qiang; Huang, Dong-Sheng; Zhang, Chi; Song, Bin; Huang, Jian-Bin; Ding, Yue

    2016-10-18

    Aseptic loosening is a significant impediment to joint implant longevity. Prosthetic wear particles are postulated to play a central role in the onset and progression of periprosthetic osteolysis, leading to aseptic loosening of the prosthesis. We investigated the inhibitory effects of a lentivirus-mediated short hairpin RNA that targets the TNF-alpha gene on the particle-induced inflammatory and osteolytic changes via macrophages both in vitro and in vivo. An siRNA sequence targeting the mouse TNF-alpha gene from four candidates, transcribed in vitro, was screened and identified. A lentivirus vector expressing short hairpin RNA (shRNA) was then constructed in order to facilitate efficient expression of TNF-alpha-siRNA. Lentivirus-mediated shRNA was transduced into cells of the mouse macrophage line RAW 264.7. Ceramic and titanium particles were introduced 24 h after lentivirus transduction to stimulate cells. TNF-alpha expression, represented by both mRNA and protein levels, was quantified with real-time PCR and ELISA at all time intervals. Lentivirus-mediated shRNA suspension was locally administered into the murine calvarial model, followed by local injection of particles. A multi-slice spiral CT scan was used to evaluate the osteolysis of the calvaria by detecting the width of the cranial sutures. Macrophages developed pseudopods when co-cultured with particles. Lentivirus-mediated shRNA was shown to effectively inhibit the expression of TNF-alpha at both the mRNA and protein levels in RAW 264.7. The multi-slice spiral CT scan showed that the lentivirus-mediated shRNA significantly suppressed osteolysis of mouse calvaria. Our investigation highlighted the results that lentivirus-mediated shRNA targeting the TNF-alpha gene successfully inhibited particle-induced inflammatory and osteolytic changes both in vitro and in vivo. Therefore, lentivirus-mediated gene therapy may provide a novel therapeutic approach to aseptic joint loosening.

  17. Inhibition of NF-kB 1 (NF-kBp50 by RNA interference in chicken macrophage HD11 cell line challenged with Salmonella enteritidis

    Directory of Open Access Journals (Sweden)

    Hsin-I Chiang

    2009-01-01

    Full Text Available The NF-kB pathway plays an important role in regulating the immunity response in animals. In this study, small interfering RNAs (siRNA were used to specifically inhibit NF-kB 1 expression and to elucidate the role of NF-kB in the signal transduction pathway of the Salmonella challenge in the chicken HD11 cell line. The cells were transfected with either NF-kB 1 siRNA, glyceraldehyde 3-phosphate dehydrogenase siRNA (positive control or the negative control siRNA for 24 h, followed by Salmonella enteritidis (SE challenge or non-challenge for 1 h and 4 h. Eight candidate genes related to the signal pathway of SE challenge were selected to examine the effect of NF-kB 1 inhibition on their expressions by mRNA quantification. The results showed that, with a 36% inhibition of NF-kB 1 expression, gene expression of both Toll-like receptor (TLR 4 and interleukin (IL-6 was consistently and significantly increased at both 1 h and 4 h following SE challenge, whereas the gene expression of MyD88 and IL-1β was increased at 1 h and 4 h, respectively. These findings suggest a likely inhibitory regulation by NF-kB 1, and could lay the foundation for studying the gene network of the innate immune response of SE infection in chickens.

  18. The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

    Energy Technology Data Exchange (ETDEWEB)

    Doi, Nobutaka [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Ogawa, Ryohei, E-mail: ogawa@med.u-toyama.ac.jp [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); Cui, Zheng-Guo [Department of Public Health, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Morii, Akihiro; Watanabe, Akihiko [Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Kanayama, Shinji; Yoneda, Yuko [New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Kondo, Takashi [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan)

    2015-05-01

    The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl{sub 2} confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype.

  19. Study of RNA interference inhibiting rat ovarian androgen biosynthesis by depressing 17alpha-hydroxylase/17, 20-lyase activity in vivo

    Directory of Open Access Journals (Sweden)

    Yang Xing

    2009-07-01

    Full Text Available Abstract Background 17alpha-hydroxylase/17, 20-lyase encoded by CYP17 is the key enzyme in androgen biosynthesis pathway. Previous studies demonstrated the accentuation of the enzyme in patients with polycystic ovary syndrome (PCOS was the most important mechanism of androgen excess. We chose CYP17 as the therapeutic target, trying to suppress the activity of 17alpha-hydroxylase/17, 20-lyase and inhibit androgen biosynthesis by silencing the expression of CYP17 in the rat ovary. Methods Three CYP17-targeting and one negative control oligonucleotides were designed and used in the present study. The silence efficiency of lentivirus shRNA was assessed by qRT-PCR, Western blotting and hormone assay. After subcapsular injection of lentivirus shRNA in rat ovary, the delivery efficiency was evaluated by GFP fluorescence and qPCR. Total RNA was extracted from rat ovary for CYP17 mRNA determination and rat serum was collected for hormone measurement. Results In total, three CYP17-targeting lentivirus shRNAs were synthesized. The results showed that all of them had a silencing effect on CYP17 mRNA and protein. Moreover, androstenedione secreted by rat theca interstitial cells (TIC in the RNAi group declined significantly compared with that in the control group. Two weeks after rat ovarian subcapsular injection of chosen CYP17 shRNA, the GFP fluorescence of frozen ovarian sections could be seen clearly under fluorescence microscope. It also showed that the GFP DNA level increased significantly, and its relative expression level was 7.42 times higher than that in the control group. Simultaneously, shRNA treatment significantly decreased CYP17 mRNA and protein levels at 61% and 54%, respectively. Hormone assay showed that all the levels of androstenedione, 17-hydroxyprogesterone and testosterone declined to a certain degree, but progesterone levels declined significantly. Conclusion The present study proves for the first time that ovarian androgen

  20. Downregulation of CD147 expression by RNA interference inhibits HT29 cell proliferation, invasion and tumorigenicity in vitro and in vivo.

    Science.gov (United States)

    Li, Rui; Pan, Yuqin; He, Bangshun; Xu, Yeqiong; Gao, Tianyi; Song, Guoqi; Sun, Huiling; Deng, Qiwen; Wang, Shukui

    2013-12-01

    We investigated the effect of CD147 silencing on HT29 cell proliferation and invasion. We constructed a novel short hairpin RNA (shRNA) expression vector pYr-mir30-shRNA. The plasmid was transferred to HT29 cells. The expression of CD147, MCT1 (lactate transporters monocarboxylate transporter 1) and MCT4 (lactate transporters monocarboxylate transporter 4) were monitored by quantitative PCR and western blotting, respectively. The MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) activities were determined by gelatin zymography assay, while the intracellular lactate concentration was determined by the lactic acid assay kit. WST-8 assay was used to determine the HT29 cell proliferation and the chemosensitivity. Invasion assay was used to determine the invasion of HT29 cells. In addition, we established a colorectal cancer model, and detected CD147 expression in vivo. The results showed that the expression of CD147 and MCT1 was significantly reduced at both mRNA and protein levels, and also the activity of MMP-2 and MMP-9 was reduced. The proliferation and invasion were decreased, but chemosensitivity to cisplatin was increased. In vivo, the CD147 expression was also significantly decreased, and reduced the tumor growth after CD147 gene silencing. The results demonstrated that silencing of CD147 expression inhibited the proliferation and invasion, suggesting CD147 silencing might be an adjuvant gene therapy strategy to chemotherapy.

  1. Therapeutic effect of Tat-LK15-mediated the interference of siRNA with nNOS expression in the spinal dorsal horn on neuropathic pain in rats

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    Yun RAO

    2017-10-01

    Full Text Available Objective To investigate the therapeutic effect of cell penetrating peptide Tat-LK15 mediating small interfering RNA (siRNA interference with the expression of neuronal nitric oxide synthase (nNOS in rat spinal dorsal horn on neuropathic pain. Methods The transfection reagent, Tat-LK15, was used to mediate the transfection of rat spinal dorsal horn (SDH neuronal cells with carboxyfluorescein (FAM, and then the transfection effect was observed under inverted fluorescence microscope. Fifty healthy male SD rats were randomly divided into 5 groups (n=10: control group, sham operation group (sham group, neuropathic pain group (SNL group, Tat-LK15-nNOS siRNA group (TS group and Tat-LK15-NC siRNA group (TN group. Neuropathic pain was induced by spinal nerve ligation (SNL, rats in control group did not receive operation and only the spinal nerve was exposed in sham group. Groups SNL, TS and TN were made into the models by SNL and implanted intrathecal catheter, intrathecal administration was performed from the 7th day after model establishment, and 10μl normal saline, 10μl TS complex (including 5μg siRNA and 10μl TN (including 5μg siRNA were injected intrathecally each day for 7 days. Paw withdrawal mechanical threshold (PWMT and paw withdrawal thermal latency (PWTL were measured at 1 day before (baseline and 3, 7, 10 and 14 days after model establishment. Then animals were sacrificed on the 14th day after the operation and the lumbar segment (L4-6 of the spinal cord was removed to detect the expressions of nNOS mRNA and protein using q-PCR and Western blotting analysis. Results Tat-LK15 effectively mediated FAM-siRNA into SDH neuronal cells. Compared with sham group, SNL significantly decreased PWMT and PWTL and increased expressions of nNOS mRNA and protein from the 3rd day (P<0.01, but there was no significant difference between the sham and control group. Tat-LK15-nNOS siRNA complex significantly increased PWMT and PWTL and down-regulated n

  2. New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Ruiz, S.; Schyth, Brian Dall; Encinas, P.

    2009-01-01

    Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect...... cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene...... of the viral hemorrhagic septicemia virus (VHSV), a rhabdovirus affecting fish. Eight siRNA sequences were first designed, synthesized and screened for inhibition of in vitro VHSV infectivity. Small hairpin (sh) DNAs corresponding to three selected siRNAs were then cloned into pRNA-CMV3.1/puro plasmids...

  3. Ran Involved in the Development and Reproduction Is a Potential Target for RNA-Interference-Based Pest Management in Nilaparvata lugens.

    Directory of Open Access Journals (Sweden)

    Kai-Long Li

    Full Text Available Ran (RanGTPase in insects participates in the 20-hydroxyecdysone signal transduction pathway in which downstream genes, FTZ-F1, Krüppel-homolog 1 (Kr-h1 and vitellogenin, are involved. A putative Ran gene (NlRan was cloned from Nilaparvata lugens, a destructive phloem-feeding pest of rice. NlRan has the typical Ran primary structure features that are conserved in insects. NlRan showed higher mRNA abundance immediately after molting and peaked in newly emerged female adults. Among the examined tissues ovary had the highest transcript level, followed by fat body, midgut and integument, and legs. Three days after dsNlRan injection the NlRan mRNA abundance in the third-, fourth-, and fifth-instar nymphs was decreased by 94.3%, 98.4% and 97.0%, respectively. NlFTZ-F1 expression levels in treated third- and fourth-instar nymphs were reduced by 89.3% and 23.8%, respectively. In contrast, NlKr-h1 mRNA levels were up-regulated by 67.5 and 1.5 folds, respectively. NlRan knockdown significantly decreased the body weights, delayed development, and killed >85% of the nymphs at day seven. Two apparent phenotypic defects were observed: (1 Extended body form, and failed to molt; (2 The cuticle at the notum was split open but cannot completely shed off. The newly emerged female adults from dsNlRan injected fifth-instar nymphs showed lower levels of NlRan and vitellogenin, lower weight gain and honeydew excretion comparing with the blank control, and no offspring. Those results suggest that NlRan encodes a functional protein that was involved in development and reproduction. The study established proof of concept that NlRan could serve as a target for dsRNA-based pesticides for N. lugens control.

  4. Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation reveals a close correlation between mRNA translation initiation and antigen presentation.

    Science.gov (United States)

    Apcher, Sebastien; Daskalogianni, Chrysoula; Manoury, Benedicte; Fåhraeus, Robin

    2010-10-14

    Viruses are known to employ different strategies to manipulate the major histocompatibility (MHC) class I antigen presentation pathway to avoid recognition of the infected host cell by the immune system. However, viral control of antigen presentation via the processes that supply and select antigenic peptide precursors is yet relatively unknown. The Epstein-Barr virus (EBV)-encoded EBNA1 is expressed in all EBV-infected cells, but the immune system fails to detect and destroy EBV-carrying host cells. This immune evasion has been attributed to the capacity of a Gly-Ala repeat (GAr) within EBNA1 to inhibit MHC class I restricted antigen presentation. Here we demonstrate that suppression of mRNA translation initiation by the GAr in cis is sufficient and necessary to prevent presentation of antigenic peptides from mRNAs to which it is fused. Furthermore, we demonstrate a direct correlation between the rate of translation initiation and MHC class I antigen presentation from a certain mRNA. These results support the idea that mRNAs, and not the encoded full length proteins, are used for MHC class I restricted immune surveillance. This offers an additional view on the role of virus-mediated control of mRNA translation initiation and of the mechanisms that control MHC class I restricted antigen presentation in general.

  5. RNA interference in adult Ascaris suum--an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite.

    Science.gov (United States)

    McCoy, Ciaran J; Warnock, Neil D; Atkinson, Louise E; Atcheson, Erwan; Martin, Richard J; Robertson, Alan P; Maule, Aaron G; Marks, Nikki J; Mousley, Angela

    2015-09-01

    The sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Influence of RNA interference on the mitochondrial subcellular localization of alpha-synuclein and on the formation of Lewy body-like inclusions in the cytoplasm of human embryonic kidney 293 cells induced by the overexpression of alpha-synuclein☆

    Science.gov (United States)

    Chen, Tao; Liao, Xiaoping; Wen, Guoqiang; Deng, Yidong; Guo, Min; Long, Zhigang; Ouyang, Feng

    2012-01-01

    The specific and effective α-synuclein RNA interference (RNAi) plasmids, and the α-synuclein-pEGFP recombinant plasmids were co-transfected into human embryonic kidney 293 (HEK293) cells using the lipofectamine method. Using an inverted fluorescence microscope, α-synuclein proteins were observed to aggregate in the cytoplasm and nucleus. Wild-type α-synuclein proteins co-localized with mitochondria. Hematoxylin-eosin staining revealed round eosinophilic bodies (Lewy body-like inclusions) in the cytoplasm of some cells transfected with α-synuclein-pEGFP plasmid. However, the formation of Lewy body-like inclusions was not observed following transfection with the RNAi pSYN-1 plasmid. RNAi blocked Lewy body-like inclusions in the cytoplasm of HEK293 cells induced by wild-type α-synuclein overexpression, but RNAi did not affect the subcellular localization of wild-type α-synuclein in mitochondria. PMID:25767480

  7. RNA interference knockdown of aminopeptidase N genes decrease the susceptibility of Chilo suppressalis larvae to Cry1Ab/Cry1Ac and Cry1Ca-expressing transgenic rice.

    Science.gov (United States)

    Qiu, Lin; Fan, Jinxing; Zhang, Boyao; Liu, Lang; Wang, Xiaoping; Lei, Chaoliang; Lin, Yongjun; Ma, Weihua

    2017-05-01

    Transgenic rice expressing Bacillus thuringiensis (Bt) Cry toxins are resistant to lepidopteran pests, such as Chilo suppressalis, a major insect pest of rice in Asia. Understanding how these toxins interact with their hosts is crucial to understanding their insecticidal action. In this study, knockdown of two aminopeptidase N genes (APN1 and APN2) by RNA interference resulted in decreased susceptibility of C. suppressalis larvae to the Bt rice varieties TT51 (Cry1Ab and Cry1Ac fusion genes) and T1C-19 (Cry1Ca), but not T2A-1 (Cry2Aa). This suggests that APN1 and APN2 are receptors for Cry1A and Cry1C toxins in C. suppressalis. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Identification of Restriction Factors by Human Genome-Wide RNA Interference Screening of Viral Host Range Mutants Exemplified by Discovery of SAMD9 and WDR6 as Inhibitors of the Vaccinia Virus K1L−C7L− Mutant

    Science.gov (United States)

    Sivan, Gilad; Ormanoglu, Pinar; Buehler, Eugen C.; Martin, Scott E.

    2015-01-01

    ABSTRACT RNA interference (RNAi) screens intended to identify host factors that restrict virus replication may fail if the virus already counteracts host defense mechanisms. To overcome this limitation, we are investigating the use of viral host range mutants that exhibit impaired replication in nonpermissive cells. A vaccinia virus (VACV) mutant with a deletion of both the C7L and K1L genes, K1L−C7L−, which abrogates replication in human cells at a step prior to late gene expression, was chosen for this strategy. We carried out a human genome-wide small interfering RNA (siRNA) screen in HeLa cells infected with a VACV K1L−C7L− mutant that expresses the green fluorescent protein regulated by a late promoter. This positive-selection screen had remarkably low background levels and resulted in the identification of a few cellular genes, notably SAMD9 and WDR6, from approximately 20,000 tested that dramatically enhanced green fluorescent protein expression. Replication of the mutant virus was enabled by multiple siRNAs to SAMD9 or WDR6. Moreover, SAMD9 and WDR6 clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout HeLa cell lines were permissive for replication of the K1L−C7L− mutant, in agreement with the siRNA data. Expression of exogenous SAMD9 or interferon regulatory factor 1 restricted replication of the K1L−C7L− mutant in the SAMD9−/− cells. Independent interactions of SAMD9 with the K1 and C7 proteins were suggested by immunoprecipitation. Knockout of WDR6 did not reduce the levels of SAMD9 and interactions of WDR6 with SAMD9, C7, and K1 proteins were not detected, suggesting that these restriction factors act independently but possibly in the same innate defense pathway. PMID:26242627

  9. Molecular cloning and RNA interference-mediated functional characterization of a Halloween gene spook in the white-backed planthopper Sogatella furcifera.

    Science.gov (United States)

    Jia, Shuang; Wan, Pin-Jun; Zhou, Li-Tao; Mu, Li-Li; Li, Guo-Qing

    2013-09-04

    Ecdysteroid hormones ecdysone and 20-hydroxyecdysone play fundamental roles in insect postembryonic development and reproduction. Five cytochrome P450 monooxygenases (CYPs), encoded by Halloween genes, have been documented to be involved in the ecdysteroidogenesis in insect species of diverse orders such as Diptera, Lepidoptera and Orthoptera. Up to now, however, the involvement of the Halloween genes in ecdysteroid synthesis has not been confirmed in hemipteran insect species. In the present paper, a Halloween gene spook (Sfspo, Sfcyp307a1) was cloned in the hemipteran Sogatella furcifera. SfSPO has three insect conserved P450 motifs, i.e., Helix-K, PERF and heme-binding motifs. Temporal and spatial expression patterns of Sfspo were evaluated by qPCR. Sfspo showed three expression peaks in late second-, third- and fourth-instar stages. In contrast, the expression levels were lower and formed three troughs in the newly-molted second-, third- and fourth-instar nymphs. On day 3 of the fourth-instar nymphs, Sfspo clearly had a high transcript level in the thorax where PGs were located. Dietary introduction of double-stranded RNA (dsRNA) of Sfspo into the second instars successfully knocked down the target gene, and greatly reduced expression level of ecdysone receptor (EcR) gene. Moreover, knockdown of Sfspo caused lethality and delayed development during nymphal stages. Furthermore, application of 20-hydroxyecdysone on Sfspo-dsRNA-exposed nymphs did not increase Sfspo expression, but could almost completely rescue SfEcR expression, and relieved the negative effects on nymphal survival and development. In S. furcifera, Sfspo was cloned and the conservation of SfSPO is valid. Thus, SfSPO is probably also involved in ecdysteroidogenesis for hemiptera.

  10. Systems biology approach to transplant tolerance: proof of concept experiments using RNA interference (RNAi) to knock down hub genes in Jurkat and HeLa cells in vitro.

    Science.gov (United States)

    Lwin, Wint Wah; Park, Ken; Wauson, Matthew; Gao, Qin; Finn, Patricia W; Perkins, David; Khanna, Ajai

    2012-07-01

    Systems biology is gaining importance in studying complex systems such as the functional interconnections of human genes [1]. To investigate the molecular interactions involved in T cell immune responses, we used databases of physical gene-gene interactions to constructed molecular interaction networks (interconnections) with R language algorithms. This helped to identify highly interconnected "hub" genes AT(1)P5C1, IL6ST, PRKCZ, MYC, FOS, JUN, and MAPK1. We hypothesized that suppression of these hub genes in the gene network would result in significant phenotypic effects on T cells and examined this in vitro. The molecular interaction networks were then analyzed and visualized with Cytoscape. Jurkat and HeLa cells were transfected with siRNA for the selected hub genes. Cell proliferation was measured using ATP luminescence and BrdU labeling, which were measured 36, 72, and 96 h after activation. Following T cell stimulation, we found a significant decrease in ATP production (P cells. However, HeLa cells showed a significant (P cell proliferation when the genes MAPK1, IL6ST, ATP5C1, JUN, and FOS were knocked down. In both Jurkat and HeLa cells, targeted gene knockdown using siRNA showed decreased cell proliferation and ATP production in both Jurkat and HeLa cells. However, Jurkat T cells and HELA cells use different hub genes to regulate activation responses. This experiment provides proof of principle of applying siRNA knockdown of T cell hub genes to evaluate their proliferative capacity and ATP production. This novel concept outlines a systems biology approach to identify hub genes for targeted therapeutics. Published by Elsevier Inc.

  11. Germline Defects Caused by Smed-boule RNA-Interference Reveal That Egg Capsule Deposition Occurs Independently of Fertilization, Ovulation, Mating, or the Presence of Gametes in Planarian Flatworms.

    Directory of Open Access Journals (Sweden)

    Jessica Kathryne Steiner

    2016-05-01

    Full Text Available Few animals are known to lay eggs in the absence of ovulation or copulation, as it is presumably energetically wasteful and subjected to negative selection. Characterization of Smed-boule, a member of the DAZ family of germline RNA-binding proteins, revealed that egg capsule (or capsule production and deposition occurs independently of the presence of gametes in the planarian flatworm Schmidtea mediterranea. Reduction of Smed-boule expression by RNA-interference (RNAi causes ablation of spermatogonial stem cells and the inability of ovarian germline stem cells to undergo oogenesis. Although animals subjected to Smed-boule RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin Smed-boule(RNAi and control planarians maintained in complete isolation, demonstrating that egg production in S. mediterranea occurs independently of ovulation, fertilization, or mating. Evidence suggests that this is a conserved feature amongst Platyhelminthes, and therefore relevant to the pathology and dissemination of parasitic flatworms. These findings demonstrate that Smed-boule functions at different stages during male and female germline stem cell development, and also demonstrate that egg capsule production by planarian flatworms occurs independently of signals produced by mating or ova.

  12. Germline Defects Caused by Smed-boule RNA-Interference Reveal That Egg Capsule Deposition Occurs Independently of Fertilization, Ovulation, Mating, or the Presence of Gametes in Planarian Flatworms.

    Science.gov (United States)

    Steiner, Jessica Kathryne; Tasaki, Junichi; Rouhana, Labib

    2016-05-01

    Few animals are known to lay eggs in the absence of ovulation or copulation, as it is presumably energetically wasteful and subjected to negative selection. Characterization of Smed-boule, a member of the DAZ family of germline RNA-binding proteins, revealed that egg capsule (or capsule) production and deposition occurs independently of the presence of gametes in the planarian flatworm Schmidtea mediterranea. Reduction of Smed-boule expression by RNA-interference (RNAi) causes ablation of spermatogonial stem cells and the inability of ovarian germline stem cells to undergo oogenesis. Although animals subjected to Smed-boule RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin Smed-boule(RNAi) and control planarians maintained in complete isolation, demonstrating that egg production in S. mediterranea occurs independently of ovulation, fertilization, or mating. Evidence suggests that this is a conserved feature amongst Platyhelminthes, and therefore relevant to the pathology and dissemination of parasitic flatworms. These findings demonstrate that Smed-boule functions at different stages during male and female germline stem cell development, and also demonstrate that egg capsule production by planarian flatworms occurs independently of signals produced by mating or ova.

  13. Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus

    Directory of Open Access Journals (Sweden)

    Chu Justin

    2010-02-01

    Full Text Available Abstract Background Dengue virus (DENV is the causative agent of Dengue fever and the life-threatening Dengue Haemorrhagic fever or Dengue shock syndrome. In the absence of anti-viral agents or vaccine, there is an urgent need to develop an effective anti-viral strategy against this medically important viral pathogen. The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites. Currently the involvements of human membrane trafficking host genes or factors that mediate the infectious cellular entry of dengue virus are not well defined. Results In this study, we have used a targeted small interfering RNA (siRNA library to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics and endosome trafficking that are important and essential for DENV infection. The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. Furthermore, DENV infection was shown to be sensitive to the disruption of human genes in regulating the early to late endosomal trafficking as well as the endosomal acidic pH. The importance and involvement of both actin and microtubule dynamics in mediating the infectious entry of DENV was also revealed in this study. Conclusions Together, the findings from this study have provided a detail profiling of the human membrane trafficking cellular genes and the mechanistic insight into the interplay of these host genes with DENV to initiate an infection, hence broadening our understanding on the entry pathway of this medically important viral pathogen. These data may also provide a new potential avenue for development of anti

  14. RNA interference targeting CD147 inhibits the proliferation, invasiveness, and metastatic activity of thyroid carcinoma cells by down-regulating glycolysis.

    Science.gov (United States)

    Huang, Peng; Chang, Shi; Jiang, Xiaolin; Su, Juan; Dong, Chao; Liu, Xu; Yuan, Zhengtai; Zhang, Zhipeng; Liao, Huijun

    2015-01-01

    A high rate of glycolytic flux, even in the presence of oxygen, is a key metabolic hallmark of cancer cells. Lactate, the end product of glycolysis, decreases the extracellular pH and contributes to the proliferation, invasiveness and metastasis of tumor cells. CD147 play a crucial role in tumorigenicity, invasion and metastasis; and CD147 also interacts strongly and specifically with monocarboxylate transporter1 (MCT1) that mediates the transport of lactate. The objective of this study was to determine whether CD147 is involved, via its association with MCT1 to transport lactate, in glycolysis, contributing to the progression of thyroid carcinoma. The expression levels of CD147 in surgical specimens of normal thyroid, nodular goiter (NG), well-differentiated thyroid carcinoma (WDTC), and undifferentiated thyroid carcinoma (UDTC) were determined using immunohistochemical techniques. The effects of CD147 silencing on cell proliferation, invasiveness, metastasis, co-localization with MCT1, glycolysis rate and extracellular pH of thyroid cancer cells (WRO and FRO cell lines) were measured after CD147 was knocked-down using siRNA targeting CD147. Immunohistochemical analysis of thyroid carcinoma (TC) tissues revealed significant increases in signal for CD147 compared with normal tissue or NG, while UDTC expressed remarkably higher levels of CD147 compared with WDTC. Furthermore, silencing of CD147 in TC cells clearly abrogated the expression of MCT1 and its co-localization with CD147 and dramatically decreased both the glycolysis rate and extracellular pH. Thus, cell proliferation, invasiveness, and metastasis were all significantly decreased by siRNA. These results demonstrate in vitro that the expression of CD147 correlates with the degree of dedifferentiation of thyroid cancer, and show that CD147 interacts with MCT1 to regulate tumor cell glycolysis, resulting in the progression of thyroid carcinoma.

  15. Identification of Restriction Factors by Human Genome-Wide RNA Interference Screening of Viral Host Range Mutants Exemplified by Discovery of SAMD9 and WDR6 as Inhibitors of the Vaccinia Virus K1L-C7L- Mutant.

    Science.gov (United States)

    Sivan, Gilad; Ormanoglu, Pinar; Buehler, Eugen C; Martin, Scott E; Moss, Bernard

    2015-08-04

    RNA interference (RNAi) screens intended to identify host factors that restrict virus replication may fail if the virus already counteracts host defense mechanisms. To overcome this limitation, we are investigating the use of viral host range mutants that exhibit impaired replication in nonpermissive cells. A vaccinia virus (VACV) mutant with a deletion of both the C7L and K1L genes, K1L(-)C7L(-), which abrogates replication in human cells at a step prior to late gene expression, was chosen for this strategy. We carried out a human genome-wide small interfering RNA (siRNA) screen in HeLa cells infected with a VACV K1L(-)C7L(-) mutant that expresses the green fluorescent protein regulated by a late promoter. This positive-selection screen had remarkably low background levels and resulted in the identification of a few cellular genes, notably SAMD9 and WDR6, from approximately 20,000 tested that dramatically enhanced green fluorescent protein expression. Replication of the mutant virus was enabled by multiple siRNAs to SAMD9 or WDR6. Moreover, SAMD9 and WDR6 clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout HeLa cell lines were permissive for replication of the K1L(-)C7L(-) mutant, in agreement with the siRNA data. Expression of exogenous SAMD9 or interferon regulatory factor 1 restricted replication of the K1L(-)C7L(-) mutant in the SAMD9(-/-) cells. Independent interactions of SAMD9 with the K1 and C7 proteins were suggested by immunoprecipitation. Knockout of WDR6 did not reduce the levels of SAMD9 and interactions of WDR6 with SAMD9, C7, and K1 proteins were not detected, suggesting that these restriction factors act independently but possibly in the same innate defense pathway. The coevolution of microbial pathogens with cells has led to an arms race in which the invader and host continuously struggle to gain the advantage. For this reason, traditional siRNA screens may fail to uncover important immune mechanisms if the pathogens

  16. Scavenger Receptor Class B, Type I, a CD36 Related Protein in Macrobrachium nipponense: Characterization, RNA Interference, and Expression Analysis with Different Dietary Lipid Sources

    Directory of Open Access Journals (Sweden)

    Zhili Ding

    2016-01-01

    Full Text Available The scavenger receptor class B, type I (SR-BI, is a member of the CD36 superfamily comprising transmembrane proteins involved in mammalian and fish lipid homeostasis regulation. We hypothesize that this receptor plays an important role in Macrobrachium nipponense lipid metabolism. However, little attention has been paid to SR-BI in commercial crustaceans. In the present study, we report a cDNA encoding M. nipponense scavenger receptor class B, type I (designated as MnSR-BI, obtained from a hepatopancreas cDNA library. The complete MnSR-BI coding sequence was 1545 bp, encoding 514 amino acid peptides. The MnSR-BI primary structure consisted of a CD36 domain that contained two transmembrane regions at the N- and C-terminals of the protein. SR-BI mRNA expression was specifically detected in muscle, gill, ovum, intestine, hepatopancreas, stomach, and ovary tissues. Furthermore, its expression in the hepatopancreas was regulated by dietary lipid sources, with prawns fed soybean and linseed oils exhibiting higher expression levels. RNAi-based SR-BI silencing resulted in the suppression of its expression in the hepatopancreas and variation in the expression of lipid metabolism-related genes. This is the first report of SR-BI in freshwater prawns and provides the basis for further studies on SR-BI in crustaceans.

  17. RNA Interference of Endochitinases in the Sugarcane Endophyte Trichoderma virens 223 Reduces Its Fitness as a Biocontrol Agent of Pineapple Disease

    Science.gov (United States)

    Romão-Dumaresq, Aline S.; de Araújo, Welington Luiz; Talbot, Nicholas J.; Thornton, Christopher R.

    2012-01-01

    The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-ß-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-ß-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-ß-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte. PMID:23110120

  18. RNA interference of caveolin-1 via lentiviral vector inhibits growth of hypopharyngeal squamous cell carcinoma FaDu cells In Vitro and In Vivo.

    Science.gov (United States)

    Zhao, Xuening; Ma, Chao; Cai, Xiaolan; Lei, Dapeng; Liu, Dayu; Xu, Fenglei; Jin, Tong; Liu, Jun; Pan, Xinliang

    2011-01-01

    To investigate the effects of caveolin-1 (CAV1) on the growth of hypopharyngeal squamous cell carcinoma (HSCC) FaDu cells in vitro and in vivo. A CAV1-RNAi-lentivirus construct was transfected into FaDu cells and expression of caveolin-1 was tested by RT-PCR and western blotting analysis. Cell apoptosis was analyzed by transferase-medisated dUTP nick-end labeling (TUNEL) assay. Tumor inhibition effects were investigated by injecting rCAV1-RNAi-lentivirus construct into tumors created with FaDu cells in the HSCC mouse model, with the empty-vector lentivirus as a control. CAV1 expression in xenografts was tested by RT-PCR and immunohistochemistry. RT-PCR and western blot analysis demonstrated successful construction of the CAV1-RNAi-lentivirus construct producing small hairpin RNA. The average weights and volumes of tumor in mice treated with CAV1-RNAi-lentivirus were lower than in mice with control treatment (P < 0.05). RT-PCR revealed weak positive expression of CAV1 in CAV1-construct-treated xenografts and immunohistochemistry confirmed lower CAV1 expression than in controls.(P < 0.05). In addition, downregulation of CAV1 increased cell apoptosis in vitro. The growth of HSCCs could be inhibited by recombinant CAV1-RNAi-lentivirus in vitro and in vivo.

  19. RNA interference of endochitinases in the sugarcane endophyte Trichoderma virens 223 reduces its fitness as a biocontrol agent of pineapple disease.

    Directory of Open Access Journals (Sweden)

    Aline S Romão-Dumaresq

    Full Text Available The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-ß-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-ß-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-ß-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte.

  20. Messenger RNA transcripts

    Science.gov (United States)

    Dan Cullen

    2004-01-01

    In contrast to DNA, messenger RNA (mRNA) in complex substrata is rarely analyzed, in large part because labile RNA molecules are difficult to purify. Nucleic acid extractions from fungi that colonize soil are particularly difficult and plagued by humic substances that interfere with Taq polymerase (Tebbe and Vahjen 1993 and references therein). Magnetic capture...

  1. Kinome-Wide RNA Interference Screen Reveals a Role for PDK1 in Acquired Resistance to CDK4/6 Inhibition in ER-Positive Breast Cancer.

    Science.gov (United States)

    Jansen, Valerie M; Bhola, Neil E; Bauer, Joshua A; Formisano, Luigi; Lee, Kyung-Min; Hutchinson, Katherine E; Witkiewicz, Agnieszka K; Moore, Preston D; Estrada, Mónica Valéria; Sánchez, Violeta; Ericsson, Paula G; Sanders, Melinda E; Pohlmann, Paula R; Pishvaian, Michael J; Riddle, David A; Dugger, Teresa C; Wei, Wenyi; Knudsen, Erik S; Arteaga, Carlos L

    2017-05-01

    Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) small-molecule inhibitors in breast cancer arises through mechanisms that are yet uncharacterized. In this study, we used a kinome-wide siRNA screen to identify kinases that, when downregulated, yield sensitivity to the CDK4/6 inhibitor ribociclib. In this manner, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a key modifier of ribociclib sensitivity in estrogen receptor-positive MCF-7 breast cancer cells. Pharmacologic inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, another CDK4/6 inhibitor, synergistically inhibited proliferation and increased apoptosis in a panel of ER-positive breast cancer cell lines. Ribociclib-resistant breast cancer cells selected by chronic drug exposure displayed a relative increase in the levels of PDK1 and activation of the AKT pathway. Analysis of these cells revealed that CDK4/6 inhibition failed to induce cell-cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E, and D1, activated phospho-CDK2, and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and to restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib, in combination with GSK2334470 or the PI3Kα inhibitor alpelisib, decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K-PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors. Cancer Res; 77(9); 2488-99. ©2017 AACR. ©2017 American Association for Cancer Research.

  2. RNA interference of a trehalose-6-phosphate synthase gene reveals its roles during larval-pupal metamorphosis in Bactrocera minax (Diptera: Tephritidae).

    Science.gov (United States)

    Xiong, Ke-Cai; Wang, Jia; Li, Jia-Hao; Deng, Yu-Qing; Pu, Po; Fan, Huan; Liu, Ying-Hong

    2016-01-01

    Trehalose is the major blood sugar in insects, which plays a crucial role as an instant source of energy and the starting substrate for chitin biosynthesis. In insects, trehalose is synthesized by catalysis of an important enzyme, trehalose-6-phosphate synthase (TPS). In the present study, a trehalose-6-phosphate synthase gene from Bactrocera minax (BmTPS) was cloned and characterized. BmTPS contained an open reading frame of 2445 nucleotides encoding a protein of 814 amino acids with a predicted molecular weight of 92.05kDa. BmTPS was detectable in all developmental stages of Bactrocera minax and expressed higher in the final- (third-) instar larvae. Tissue-specific expression patterns of BmTPS showed that it was mainly expressed in the fat body. The 20-hydroxyecdysone (20E) induced the expression of BmTPS and three genes in the chitin biosynthesis pathway. Moreover, injection of double-stranded RNA into third-instar larvae successfully silenced the transcription of BmTPS in B. minax, and thereby decreased the activity of TPS and trehalose content. Additionally, silencing of BmTPS inhibited the expression of three key genes in the chitin biosynthesis pathway and exhibited 52% death and abnormal phenotypes. The findings demonstrate that BmTPS is indispensable for larval-pupal metamorphosis. Besides, the establishment of RNAi experimental system in B. minax would lay a solid foundation for further investigation of molecular biology and physiology of this pest. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Cloning, expression analysis, and RNA interference study of a HORMA domain containing autophagy-related gene 13 (ATG13) from the coleopteran beetle, Tenebrio molitor

    Science.gov (United States)

    Lee, Jung Hee; Jo, Yong Hun; Patnaik, Bharat Bhusan; Park, Ki Beom; Tindwa, Hamisi; Seo, Gi Won; Chandrasekar, Raman; Lee, Yong Seok; Han, Yeon Soo

    2015-01-01

    Autophagy is a process that is necessary during starvation, as it replenishes metabolic precursors by eliminating damaged organelles. Autophagy is mediated by more than 35 autophagy-related (Atg) proteins that participate in the nucleation, elongation, and curving of the autophagosome membrane. In a pursuit to address the role of autophagy during development and immune resistance of the mealworm beetle, Tenebrio molitor, we screened ATG gene sequences from the whole-larva transcriptome database. We identified a homolog of ATG13 gene in T. molitor (designated as TmATG13) that comprises a cDNA of 1176 bp open reading frame (ORF) encoding a protein of 391 amino acids. Analyses of the structure-specific features of TmAtg13 showed an intrinsically disordered middle and C-terminal region that was rich in regulatory phosphorylation sites. The N-terminal Atg13 domain had a HORMA (Hop1, Rev7, and Mad2) fold containing amino acid residues conserved across the Atg13 insect orthologs. A quantitative reverse-transcription-polymerase chain reaction analysis revealed that TmATG13 was expressed ubiquitously during all developmental stages of the insect. TmATG13 mRNA expression was high in the fat body and gut of the larval and adult stages of the insect. The TmATG13 transcripts were expressed at a high level until 6 days of ovarian development, followed by a significant decline. Silencing of ATG13 transcripts in T. molitor larvae showed a reduced survivability of 39 and 38% in response to Escherichia coli and Staphylococcus aureus infection. Furthermore, the role of TmAtg13 in initiating autophagy as a part of the host cell autophagic complex of the host cells against the intracellular pathogen Listeria monocytogenes is currently under study and will be critical to unfold the structure-function relationships. PMID:26136688

  4. Genomes of parasitic nematodes (Meloidogyne hapla, Meloidogyne incognita, Ascaris suum and Brugia malayi) have a reduced complement of small RNA interference pathway genes: knockdown can reduce host infectivity of M. incognita.

    Science.gov (United States)

    Iqbal, Sadia; Fosu-Nyarko, John; Jones, Michael G K

    2016-07-01

    The discovery of RNA interference (RNAi) as an endogenous mechanism of gene regulation in a range of eukaryotes has resulted in its extensive use as a tool for functional genomic studies. It is important to study the mechanisms which underlie this phenomenon in different organisms, and in particular to understand details of the effectors that modulate its effectiveness. The aim of this study was to identify and compare genomic sequences encoding genes involved in the RNAi pathway of four parasitic nematodes: the plant parasites Meloidogyne hapla and Meloidogyne incognita and the animal parasites Ascaris suum and Brugia malayi because full genomic sequences were available-in relation to those of the model nematode Caenorhabditis elegans. The data generated was then used to identify some potential targets for control of the root knot nematode, M. incognita. Of the 84 RNAi pathway genes of C. elegans used as model in this study, there was a 42-53 % reduction in the number of effectors in the parasitic nematodes indicating substantial differences in the pathway between species. A gene each from six functional groups of the RNAi pathway of M. incognita was downregulated using in vitro RNAi, and depending on the gene (drh-3, tsn-1, rrf-1, xrn-2, mut-2 and alg-1), subsequent plant infection was reduced by up to 44 % and knockdown of some genes (i.e. drh-3, mut-2) also resulted in abnormal nematode development. The information generated here will contribute to defining targets for more robust nematode control using the RNAi technology.

  5. IETS and quantum interference

    DEFF Research Database (Denmark)

    Jørgensen, Jacob Lykkebo; Gagliardi, Alessio; Pecchia, Alessandro

    2014-01-01

    Destructive quantum interference in single molecule electronics is an intriguing phenomenon; however, distinguishing quantum interference effects from generically low transmission is not trivial. In this paper, we discuss how quantum interference effects in the transmission lead to either low...... suppressed when quantum interference effects dominate. That is, we expand the understanding of propensity rules in inelastic electron tunneling spectroscopy to molecules with destructive quantum interference....

  6. Deletion of fucose residues in plant N-glycans by repression of the GDP-mannose 4,6-dehydratase gene using virus-induced gene silencing and RNA interference.

    Science.gov (United States)

    Matsuo, Kouki; Matsumura, Takeshi

    2011-02-01

    Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant-produced glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a) ) epitope, i.e., Galβ(1-3)[Fucα(1-4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant-specific core α-1,3-fucose and α-1,4-fucose residues in the Le(a) epitopes by repressing the Guanosine 5'-diphosphate (GDP)-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus-induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose-free N-glycans found in total soluble protein from GMD gene-repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild-type plants. A small amount of putative galactose substitution in N-glycans from the NbGMD gene-repressed plants was observed, similar to what has been previously reported GMD-knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) with fucose-deleted N-glycans was successfully produced in NbGMD-RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  7. Viral counterdefense on RNA silencing : analysis of RNA silencing suppressors from arthropod-borne negative strand RNA plant viruses

    NARCIS (Netherlands)

    Schnettler, E.

    2010-01-01

    This thesis describes that RNA silencing suppressor (RSS) proteins encoded by negative-stranded RNA plant viruses are able to interfere with different RNA silencing pathways in a variety of organisms by interacting with double stranded (ds)RNA molecules. These RSS proteins are able to counteract the

  8. Conducted interference, challenges and interference cases

    NARCIS (Netherlands)

    Leferink, Frank Bernardus Johannes

    2015-01-01

    Conducted interference has become increasingly problematic in the past few years, especially within the 2-150 kHz band. The high penetration of non-linear loads, combined with distributed generation, will influence the voltage profile, i.e. the power quality. New technologies will introduce new

  9. Dark Matter Interference

    DEFF Research Database (Denmark)

    Del Nobile, Eugenio; Kouvaris, Christoforos; Sannino, Francesco

    2012-01-01

    We study different patterns of interference in WIMP-nuclei elastic scattering that can accommodate the DAMA and CoGeNT experiments via an isospin violating ratio $f_n/f_p=-0.71$. We study interference between the following pairs of mediators: Z and Z', Z' and Higgs, and two Higgs fields. We show ...

  10. Quantum Interference of Molecules

    Indian Academy of Sciences (India)

    IAS Admin

    GENERAL │ ARTICLE. Quantum Interference of Molecules. Probing the Wave Nature of Matter. Anu Venugopalan. Keywords. Matter waves, wave-particle du- ality, electron interference, decoherence. Anu Venugopalan is on the faculty of the School of. Basic and Applied. Sciences, GGS. Indraprastha University,. Delhi.

  11. Interference of kallikrein 1b26 (klk1b26 translation by microRNA specifically expressed in female mouse submandibular glands: an additional mechanism for sexual dimorphism of klk1b26 protein in the glands

    Directory of Open Access Journals (Sweden)

    Kurihara Kinji

    2011-11-01

    Full Text Available Abstract Background Mouse kallikrein 1b26 (klk1b26 protein is more abundant in male submandibular glands (SMGs than in female ones. This sexual dimorphism has been thought to be due to increased mRNA synthesis stimulated by androgen. However, the klk1b26 protein level in female SMG is far less than that expected from the mRNA level, suggesting an additional mechanism for down-regulation of klk1b26 expression in female SMGs. Methods We examined the effects of small non-coding RNAs in mouse SMGs on in vitro translation of klk1b26 using a reticulocyte lysate system and reverse transcription (RT-PCR for klk1b26 mRNA. Statistical analyses were performed with a computer package (Microsoft Excel. Results The microRNA (miRNA preparation from female SMGs, but not male SMGs, interfered with the in vitro translation of the klk1b26 protein and inhibited the RT-PCR for klk1b26 mRNA with forward primers targeting its 5'-terminal region (between the 15th and 40th nucleotide from the 5'-terminal. The miRNA preparation from castrated mouse SMGs showed the inhibitory effect on the klk1b26 translation, but that from a 5α-dihydrotestosterone-treated female mouse SMGs did not. Synthetic miRNAs (miR-325 and miR-1497a, which have partial complementarity with klk1b26 mRNA at its 5'-terminal region (15th to 40th nucleotide position from the 5'-terminal, also interfered with the in vitro klk1b26 translation. When the female miRNA preparation was incubated with a 30-nucleotide-long single-strand oligoDNA (named [15th-44th]ssDNA, whose sequence corresponded to the 15th to 44th position from the 5'-terminal of klk1b26 mRNA prior to the addition into the in vitro translation system, the inhibitory effect of the miRNA preparation on klk1b26 translation disappeared, while [15th-44th]ssDNA itself had no effect on the translation. Preincubation of the miRNA preparation with another single-strand DNA ([169th-198th]ssDNA, whose sequence corresponded with 169th to 198th position

  12. Reinitiated viral RNA-dependent RNA polymerase resumes replication at a reduced rate

    NARCIS (Netherlands)

    Vilfan, I.D.; Candelli, A.; Hage, S.; Aalto, A.P.; Poranen, M.M.; Bamford, D.H.; Dekker, N.H.

    2008-01-01

    RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important

  13. Collectivity from interference

    Science.gov (United States)

    Blok, Boris; Jäkel, Christian D.; Strikman, Mark; Wiedemann, Urs Achim

    2017-12-01

    In hadronic collisions, interference between different production channels affects momentum distributions of multi-particle final states. As this QCD interference does not depend on the strong coupling constant α s , it is part of the no-interaction baseline that needs to be controlled prior to searching for other manifestations of collective dynamics, e.g., in the analysis of azimuthal anisostropy coefficients v n at the LHC. Here, we introduce a model that is based on the QCD theory of multi-parton interactions and that allows one to study interference effects in the production of m particles in hadronic collisions with N parton-parton interactions ("sources"). In an expansion in powers of 1/( N c 2 - 1) and to leading order in the number of sources N , we calculate interference effects in the m-particle spectra and we determine from them the second and fourth order cumulant momentum anisotropies v n {2} and v n {4}. Without invoking any azimuthal asymmetry and any density dependent non-linear dynamics in the incoming state, and without invoking any interaction in the final state, we find that QCD interference alone can give rise to values for v n {2} and v n {4}, n even, that persist unattenuated for increasing number of sources, that may increase with increasing multiplicity and that agree with measurements in proton-proton (pp) collisions in terms of the order of magnitude of the signal and the approximate shape of the transverse momentum dependence. We further find that the non-abelian features of QCD interference can give rise to odd harmonic anisotropies. These findings indicate that the no-interaction baseline including QCD interference effects can make a sizeable if not dominant contribution to the measured v n coefficients in pp collisions. Prospects for analyzing QCD interference contributions further and their possible relevance for proton-nucleus and nucleus-nucleus collisions are discussed shortly.

  14. Plasmonic optical interference.

    Science.gov (United States)

    Choi, Dukhyun; Shin, Chang Kyun; Yoon, Daesung; Chung, Deuk Seok; Jin, Yong Wan; Lee, Luke P

    2014-06-11

    Understanding optical interference is of great importance in fundamental and analytical optical design for next-generation personal, industrial, and military applications. So far, various researches have been performed for optical interference phenomena, but there have been no reports on plasmonic optical interference. Here, we report that optical interference could be effectively coupled with surface plasmons, resulting in enhanced optical absorption. We prepared a three-dimensional (3D) plasmonic nanostructure that consists of a plasmonic layer at the top, a nanoporous dielectric layer at the center, and a mirror layer at the bottom. The plasmonic layer mediates strong plasmonic absorption when the constructive interference pattern is matched with the plasmonic component. By tailoring the thickness of the dielectric layer, the strong plasmonic absorption can facilely be controlled and covers the full visible range. The plasmonic interference in the 3D nanostructure thus creates brilliant structural colors. We develop a design equation to determine the thickness of the dielectric layer in a 3D plasmonic nanostructure that could create the maximum absorption at a given wavelength. It is further demonstrated that the 3D plasmonic nanostructure can be realized on a flexible substrate. Our 3D plasmonic nanostructures will have a huge impact on the fields of optoelectronic systems, biochemical optical sensors, and spectral imaging.

  15. RNA Interference of Interferon Regulatory Factor-1 Gene Expression in THP-1 Cell Line Leads to Toll-Like Receptor-4 Overexpression/Activation As Well As Up-modulation of Annexin-II

    Directory of Open Access Journals (Sweden)

    Christos I. Maratheftis

    2007-12-01

    Full Text Available Interferon regulatory factor-1 (IRF-1 is a candidate transcription factor for the regulation of the Toll-like receptor-4 (TLR-4 gene. Using a small interfering RNAbased (siRNA process to silence IRF-1 gene expression in the leukemic monocytic cell line THP-1, we investigated whether such a modulation would alter TLR-4 expression and activation status in these cells. The siIRF-1 cells expressed elevated levels of TLR-4 mRNA and protein compared to controls by 90% and 77%, respectively. ICAM.1 protein expression and apoptosis levels were increased by 8.35- and 4.25-fold, respectively. The siIRF-1 cells overexpressed Bax mRNA compared to controls. Proteomic analysis revealed upmodulation of the Annexin-II protein in siIRF-1 THP-1 cells. Myelodysplastic syndrome (MDS patients with an absence of full-length IRF-1 mRNA also overexpressed Annexin-II. It is plausible that this overexpression may lead to the activation of TLR-4 contributing to the increased apoptosis characterizing MDS.

  16. Binaural Interference: Quo Vadis?

    Science.gov (United States)

    Jerger, James; Silman, Shlomo; Silverman, Carol; Emmer, Michele

    2017-04-01

    The reality of the phenomenon of binaural interference with speech recognition has been debated for two decades. Research has taken one of two avenues; group studies or case reports. In group studies, a sample of the elderly population is tested on speech recognition under three conditions; binaural, monaural right and monaural left. The aim is to determine the percent of the sample in which the expected outcome (binaural score-better-than-either-monaural score) is reversed (i.e., one of the monaural scores is better than the binaural score). This outcome has been commonly used to define binaural interference. The object of group studies is to answer the "how many" question, what is the prevalence of binaural interference in the sample. In case reports the binaural interference conclusion suggested by the speech recognition tests is not accepted until it has been corroborated by other independent diagnostic audiological measures. The aim is to attempt to determine the basis for the findings, to answer the "why" question. This article is at once tutorial, editorial and a case report. We argue that it is time to accept the reality of the phenomenon of binaural interference, to eschew group statistical approaches in search of an answer to the "how many" question, and to focus on individual case reports in search of an answer to the "why" question. American Academy of Audiology.

  17. Deep Sequencing of HIV-1 RNA and DNA in Newly Diagnosed Patients with Baseline Drug Resistance Showed No Indications for Hidden Resistance and Is Biased by Strong Interference of Hypermutation.

    Science.gov (United States)

    Dauwe, Kenny; Staelens, Delfien; Vancoillie, Leen; Mortier, Virginie; Verhofstede, Chris

    2016-06-01

    Deep sequencing of plasma RNA or proviral DNA may be an interesting alternative to population sequencing for the detection of baseline transmitted HIV-1 drug resistance. Using a Roche 454 GS Junior HIV-1 prototype kit, we performed deep sequencing of the HIV-1 protease and reverse transcriptase genes on paired plasma and buffy coat samples from newly diagnosed HIV-1-positive individuals. Selection was based on the outcome of population sequencing and included 12 patients with either a revertant amino acid at codon 215 of the reverse transcriptase or a singleton resistance mutation, 4 patients with multiple resistance mutations, and 4 patients with wild-type virus. Deep sequencing of RNA and DNA detected 6 and 43 mutations, respectively, that were not identified by population sequencing. A subsequently performed hypermutation analysis, however, revealed hypermutation in 61.19% of 3,188 DNA reads with a resistance mutation. The removal of hypermutated reads dropped the number of additional mutations in DNA from 43 to 17. No hypermutation evidence was found in the RNA reads. Five of the 6 additional RNA mutations and all additional DNA mutations, after full exclusion of hypermutation bias, were observed in the 3 individuals with multiple resistance mutations detected by population sequencing. Despite focused selection of patients with T215 revertants or singleton mutations, deep sequencing failed to identify the resistant T215Y/F or M184V or any other resistance mutation, indicating that in most of these cases there is no hidden resistance and that the virus detected at diagnosis by population sequencing is the original infecting variant. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Retroactive Interference and Forgetting

    Directory of Open Access Journals (Sweden)

    Vinishaa Ankala

    2011-01-01

    Full Text Available Retroactive interference is the amount of information that can be forgotten by a person over time due to newly learned material. In this paper we establish a relationship between the amount of information forgotten by college students while they read and watch television and the time taken to forget it. We equate these numerical equations to solve for the unknown constants. By doing so, we can find the exact equation and also the amount of forgetting information due to retroactive interference.

  19. Cellular delivery of siRNA mediated by fusion-active virosomes

    NARCIS (Netherlands)

    Huckriede, Anke; De Jonge, Jorgen; Holtrop, Marijke; Wilschut, Jan

    2007-01-01

    RNA interference is expected to have considerable potential for the development of novel specific therapeutic strategies. However, successful application of RNA interference in vivo will depend on the availability of efficient delivery systems for the introduction of small-interfering RNA (siRNA)

  20. Quantum interference in polyenes.

    Science.gov (United States)

    Tsuji, Yuta; Hoffmann, Roald; Movassagh, Ramis; Datta, Supriyo

    2014-12-14

    The explicit form of the zeroth Green's function in the Hückel model, approximated by the negative of the inverse of the Hückel matrix, has direct quantum interference consequences for molecular conductance. We derive a set of rules for transmission between two electrodes attached to a polyene, when the molecule is extended by an even number of carbons at either end (transmission unchanged) or by an odd number of carbons at both ends (transmission turned on or annihilated). These prescriptions for the occurrence of quantum interference lead to an unexpected consequence for switches which realize such extension through electrocyclic reactions: for some specific attachment modes the chemically closed ring will be the ON position of the switch. Normally the signs of the entries of the Green's function matrix are assumed to have no physical significance; however, we show that the signs may have observable consequences. In particular, in the case of multiple probe attachments - if coherence in probe connections can be arranged - in some cases new destructive interference results, while in others one may have constructive interference. One such case may already exist in the literature.

  1. Localization of Interference Fringes.

    Science.gov (United States)

    Simon, J. M.; Comastri, Silvia A.

    1980-01-01

    Discusses a proof for determining the localized fringes position arrived at when one considers the interference of two extended sources when one is able to observe fringes only at certain points in space. Shows how the localized fringes may be found in a device used to observe Newton's rings. (Author/CS)

  2. Laser Interference Lithography

    NARCIS (Netherlands)

    van Wolferen, Hendricus A.G.M.; Abelmann, Leon; Hennessy, Theodore C.

    In this chapter we explain how submicron gratings can be prepared by Laser Interference Lithography (LIL). In this maskless lithography technique, the standing wave pattern that exists at the intersection of two coherent laser beams is used to expose a photosensitive layer. We show how to build the

  3. RNA topology

    OpenAIRE

    Frank-Kamenetskii, Maxim D.

    2013-01-01

    A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases.

  4. Binaural interference: effects of temporal interferer fringe and interstimulus interval.

    Science.gov (United States)

    Camalier, Corrie R; Grantham, D Wesley; Bernstein, Leslie R

    2014-02-01

    Binaural interference refers to the phenomenon in which the potency of binaural cues conveyed by a "target" stimulus occupying one spectral region is degraded by the presence of an "interferer" stimulus occupying a spectral region remote from the target. It is typified by conditions in which thresholds for detection of interaural temporal difference conveyed by a high-frequency target are elevated when the target is accompanied by a spectrally remote low-frequency interferer. This study explored effects of temporal relations between targets and interferers on binaural interference. In the first experiment, duration by which the interferer preceded and/or trailed the target (onset and offset "fringes") was varied. Results indicated binaural interference decreased with total duration of the temporal fringe, but did not depend on whether that duration was composed of onset, offset, or onset + offset fringes. In the second experiment, binaural interference was measured as a function of the interstimulus interval (ISI) between the two presentations of the target. Results indicated that shorter ISIs increased thresholds in both the interferer and no-interferer conditions, but did not affect binaural interference. These results suggest that the mechanisms underlying the effects of manipulations of the interferer temporal fringe and manipulation of the ISI are essentially independent.

  5. Interference Rejection and Management

    Science.gov (United States)

    2009-07-01

    there has been some work relating to NBI suppression in coded OFDM systems [12, 45, 57]. In this work, the prediction-error filter ( PEF ) [30, 60] is...error filter ( PEF ) is considered for this system as a means for removing the interference in the time domain, thereby avoiding the spectral leakage that...occurs after demodulation (see block diagram given in Fig. 9.6). The PEF is a well-studied structure that uses the correlation between past samples to

  6. Diboson interference resurrection

    Science.gov (United States)

    Panico, Giuliano; Riva, Francesco; Wulzer, Andrea

    2018-01-01

    High-energy diboson processes at the LHC are potentially powerful indirect probes of heavy new physics, whose effects can be encapsulated in higher-dimensional operators or in modified Standard Model couplings. An obstruction however comes from the fact that leading new physics effects often emerge in diboson helicity amplitudes that are anomalously small in the Standard Model. As such, the formally leading Standard Model/New Physics interference contribution cancels in inclusive measurements. This paper describes a solution to this problem.

  7. Totally Asynchronous Interference Channels

    CERN Document Server

    Moshksar, Kamyar

    2010-01-01

    This paper addresses an interference channel consisting of $\\mathbf{n}$ active users sharing $u$ frequency sub-bands. Users are asynchronous meaning there exists a mutual delay between their transmitted codes. A stationary model for interference is considered by assuming the starting point of an interferer's data is uniformly distributed along the codeword of any user. The spectrum is divided to private and common bands each containing $v_{\\mathrm{p}}$ and $v_{\\mathrm{c}}$ frequency sub-bands respectively. We consider a scenario where all transmitters are unaware of the number of active users and the channel gains. The optimum $v_{\\mathrm{p}}$ and $v_{\\mathrm{c}}$ are obtained such that the so-called outage capacity per user is maximized. If $\\Pr\\{\\mathbf{n}\\leq 2\\}=1$, upper and lower bounds on the mutual information between the input and output of the channel for each user are derived using a genie-aided technique. The proposed bounds meet each other as the code length grows to infinity yielding a closed ex...

  8. Diboson interference resurrection

    Directory of Open Access Journals (Sweden)

    Giuliano Panico

    2018-01-01

    Full Text Available High-energy diboson processes at the LHC are potentially powerful indirect probes of heavy new physics, whose effects can be encapsulated in higher-dimensional operators or in modified Standard Model couplings. An obstruction however comes from the fact that leading new physics effects often emerge in diboson helicity amplitudes that are anomalously small in the Standard Model. As such, the formally leading Standard Model/New Physics interference contribution cancels in inclusive measurements. This paper describes a solution to this problem.

  9. The intention interference effect.

    Science.gov (United States)

    Cohen, Anna-Lisa; Kantner, Justin; Dixon, Roger A; Lindsay, D Stephen

    2011-01-01

    Intentions have been shown to be more accessible (e.g., more quickly and accurately recalled) compared to other sorts of to-be-remembered information; a result termed an intention superiority effect (Goschke & Kuhl, 1993). In the current study, we demonstrate an intention interference effect (IIE) in which color-naming performance in a Stroop task was slower for words belonging to an intention that participants had to remember to carry out (Do-the-Task condition) versus an intention that did not have to be executed (Ignore-the-Task condition). In previous work (e.g., Cohen et al., 2005), having a prospective intention in mind was confounded with carrying a memory load. In Experiment 1, we added a digit-retention task to control for effects of cognitive load. In Experiment 2, we eliminated the memory confound in a new way, by comparing intention-related and control words within each trial. Results from both Experiments 1 and 2 revealed an IIE suggesting that interference is very specific to the intention, not just to a memory load.

  10. Graphene quantum interference photodetector

    Directory of Open Access Journals (Sweden)

    Mahbub Alam

    2015-03-01

    Full Text Available In this work, a graphene quantum interference (QI photodetector was simulated in two regimes of operation. The structure consists of a graphene nanoribbon, Mach–Zehnder interferometer (MZI, which exhibits a strongly resonant transmission of electrons of specific energies. In the first regime of operation (that of a linear photodetector, low intensity light couples two resonant energy levels, resulting in scattering and differential transmission of current with an external quantum efficiency of up to 5.2%. In the second regime of operation, full current switching is caused by the phase decoherence of the current due to a strong photon flux in one or both of the interferometer arms in the same MZI structure. Graphene QI photodetectors have several distinct advantages: they are of very small size, they do not require p- and n-doped regions, and they exhibit a high external quantum efficiency.

  11. Graphene quantum interference photodetector.

    Science.gov (United States)

    Alam, Mahbub; Voss, Paul L

    2015-01-01

    In this work, a graphene quantum interference (QI) photodetector was simulated in two regimes of operation. The structure consists of a graphene nanoribbon, Mach-Zehnder interferometer (MZI), which exhibits a strongly resonant transmission of electrons of specific energies. In the first regime of operation (that of a linear photodetector), low intensity light couples two resonant energy levels, resulting in scattering and differential transmission of current with an external quantum efficiency of up to 5.2%. In the second regime of operation, full current switching is caused by the phase decoherence of the current due to a strong photon flux in one or both of the interferometer arms in the same MZI structure. Graphene QI photodetectors have several distinct advantages: they are of very small size, they do not require p- and n-doped regions, and they exhibit a high external quantum efficiency.

  12. How the RNA isolation method can affect microRNA microarray results

    DEFF Research Database (Denmark)

    Podolska, Agnieszka; Kaczkowski, Bogumil; Litman, Thomas

    2011-01-01

    microarray analysis on porcine brain tissue. One method is a phenol-guanidine isothiocyanate-based procedure that permits isolation of total RNA. The second method, miRVana™ microRNA isolation, is column based and recovers the small RNA fraction alone. We found that microarray analyses give different results......The quality of RNA is crucial in gene expression experiments. RNA degradation interferes in the measurement of gene expression, and in this context, microRNA quantification can lead to an incorrect estimation. In the present study, two different RNA isolation methods were used to perform microRNA...... that depend on the RNA fraction used, in particular because some microRNAs appear very sensitive to the RNA isolation method. We conclude that precautions need to be taken when comparing microarray studies based on RNA isolated with different methods....

  13. Quantum Interference in Graphene Nanoconstrictions.

    Science.gov (United States)

    Gehring, Pascal; Sadeghi, Hatef; Sangtarash, Sara; Lau, Chit Siong; Liu, Junjie; Ardavan, Arzhang; Warner, Jamie H; Lambert, Colin J; Briggs, G Andrew D; Mol, Jan A

    2016-07-13

    We report quantum interference effects in the electrical conductance of chemical vapor deposited graphene nanoconstrictions fabricated using feedback controlled electroburning. The observed multimode Fabry-Pérot interferences can be attributed to reflections at potential steps inside the channel. Sharp antiresonance features with a Fano line shape are observed. Theoretical modeling reveals that these Fano resonances are due to localized states inside the constriction, which couple to the delocalized states that also give rise to the Fabry-Pérot interference patterns. This study provides new insight into the interplay between two fundamental forms of quantum interference in graphene nanoconstrictions.

  14. Use of small RNA as antiaging cosmeceuticals.

    Science.gov (United States)

    Zhang, Pingjing; Chen, Jianxin; Li, Tiejun; Zhu, York Yuanyuan

    2013-01-01

    Over the past two decades, RNA interference (RNAi) has achieved great improvements in medicine, which has benefited the development of innovative cosmeceutical products, particular, to antiaging cosmeceuticals. A variety of ongoing research has tried to employ small RNAs-small interference RNA and microRNA as new cosmeceutical ingredients. Furthermore, several skin care companies have released new small RNA products in cosmetic market. In this review, we will describe the latest and most advanced approaches and strategies of using small RNA as antiaging cosmetics, including investigations on aging-related genes that small RNA target, method of delivering them, and challenges in the development of RNAi-based therapeutics for skin care cosmeceuticals. It is certain that advancement in this direction will evolve a new landscape for innovative antiaging cosmeceuticals.

  15. Industrial interference and radio astronomy

    Science.gov (United States)

    Jessner, A.

    2013-07-01

    The interferer - victim scenario is described for the case of industrial interference affecting radio astronomical observatories. The sensitivity of radio astronomical receivers and their interference limits are outlined. EMC above 30 MHz is a serious problem for Radio Astronomy. Interferer (CISPR) and victim (ITU-R RA 769) standards are not harmonised. The emissions from the interferer and their spectral characteristics are not defined sufficiently well by CISPR standards. The required minimum coupling losses (MCL) between an industrial device and radio astronomical antenna depends on device properties but is shown to exceed 140 dB in most cases. Spatial separation of a few km is insufficient on its own, the terrain must shield > 30-40 dB, additional mitigations such as extra shielding or suppression of high frequency emissions may be necessary. A case by case compatibility analysis and tailored EMC measures are required for individual installations. Aggregation of many weak rfi emitters can become serious problem. If deployment densities are high enough, the emission constraints can even exceed those for a single interferer at a short distance from the radio observatory. Compatibility studies must account not only for the single interferer but also for many widely distributed interference sources.

  16. Output Interference in Recognition Memory

    Science.gov (United States)

    Criss, Amy H.; Malmberg, Kenneth J.; Shiffrin, Richard M.

    2011-01-01

    Dennis and Humphreys (2001) proposed that interference in recognition memory arises solely from the prior contexts of the test word: Interference does not arise from memory traces of other words (from events prior to the study list or on the study list, and regardless of similarity to the test item). We evaluate this model using output…

  17. Characterizing Destructive Quantum Interference in Electron Transport

    OpenAIRE

    Sam-ang, Panu; Reuter, Matthew G.

    2017-01-01

    Destructive quantum interference in electron transport through molecules provides an unconventional route for suppressing electric current. In this work we introduce "interference vectors" for each interference and use them to characterize the interference. An interference vector may be an orbital of the bare molecule, in which case the interference is very sensitive to perturbation. In contrast, an interference vector may be a combination of multiple molecular orbitals, leading to more robus...

  18. A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Garrett, Roger Antony; Shah, Shiraz Ali

    2013-01-01

    Recent studies on CRISPR-based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon Sulfolobus islandicus REY15A encodes three interference modules, one of type IA and two of type IIIB...... targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S.¿islandicus REY15A which cooperate functionally by sharing a single Cas6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers....

  19. Communications in interference limited networks

    CERN Document Server

    2016-01-01

    This book offers means to handle interference as a central problem of operating wireless networks. It investigates centralized and decentralized methods to avoid and handle interference as well as approaches that resolve interference constructively. The latter type of approach tries to solve the joint detection and estimation problem of several data streams that share a common medium. In fact, an exciting insight into the operation of networks is that it may be beneficial, in terms of an overall throughput, to actively create and manage interference. Thus, when handled properly, "mixing" of data in networks becomes a useful tool of operation rather than the nuisance as which it has been treated traditionally. With the development of mobile, robust, ubiquitous, reliable and instantaneous communication being a driving and enabling factor of an information centric economy, the understanding, mitigation and exploitation of interference in networks must be seen as a centrally important task.

  20. Application of RNA interference methodology to investigate and ...

    Indian Academy of Sciences (India)

    Anhui Province Key Laboratory of Crop Biology, School of Life Science, Anhui Agricultural University, Hefei 230036, People's Republic of China; School of Horticulture, Anhui Agricultural University, Hefei 230036, People's Republic of China; School of Plant Protection, Anhui Agricultural University, Hefei 230036, People's ...

  1. RNA interference in plant parasitic nematodes | Karakas | African ...

    African Journals Online (AJOL)

    African Journal of Biotechnology. Journal Home · ABOUT THIS JOURNAL · Advanced Search · Current Issue · Archives · Journal Home > Vol 7, No 15 (2008) >. Log in or Register to get access to full text downloads. Username, Password, Remember me, or Register · Download this PDF file. The PDF file you selected should ...

  2. expression by RNA interference suppresses human lung cancer cell ...

    African Journals Online (AJOL)

    DR TONUKARI NYEROVWO

    2012-02-16

    Feb 16, 2012 ... genes can function in forming tetramers in the cell membrane to facilitate the ... respiratory tract. Lung carcinomas, especially adeno- carcinomas, can produce AQP3, possibly in connection with their functional and/or biological nature, although, the detailed ..... Progress on the structure and function of ...

  3. Application of RNA interference in treating human diseases

    Indian Academy of Sciences (India)

    cancer-associated genomic regions or fragile sites (Calin et al. 2004). The genes encoding mir-15 and mir-16 are ..... Also, DiGeorge syndrome is a rare congen- ital disease whose symptoms vary but include .... Cai X., Hagedorn C. H. and Cullen B. R. 2004 Human microRNAs are processed from capped, polyadenylated ...

  4. RNA interference-mediated control of Aspergillus flavus in maize

    Science.gov (United States)

    Introduction: Aflatoxigenic Aspergillus flavus is a frequent contaminant of agricultural commodities such as corn, peanut, tree nuts and cottonseed. Ingestion of foods, especially corn, contaminated with aflatoxins has been implicated in acute toxicoses while chronic, low-level exposure can lead to...

  5. Inducing RNA interference in the arbovirus vector, Culicoides sonorensis

    Science.gov (United States)

    Biting midges in the genus Culicoides are important vectors of arboviral diseases, including Epizootic Hemorrhagic Disease, Bluetongue, and likely Schmallenberg, which cause significant economic burden worldwide. Research on these vectors has been hindered by the lack of a sequenced genome, the diff...

  6. Intervention of radiation‐induced skin fibrosis by RNA interference

    DEFF Research Database (Denmark)

    Nawroth, Isabel

    Radiation therapy is a cornerstone of cancer treatment for many types of cancer, but is often limited by normal tissue toxicity. One of the most common and dose‐limiting long‐term adverse effects is radiation‐induced fibrosis (RIF), which is characterized by restricted tissue flexibility, reduced...

  7. Application of RNA interference in treating human diseases

    Indian Academy of Sciences (India)

    Interestingly, mechanisms for gene silencing also protect the genome from transposons and viruses. In this paper, we first review RNAi mechanism and then focus on some of its applications in biomedical research such as treatment for HIV, viral hepatitis, cardiovascular and cerebrovascular diseases, metabolic disease, ...

  8. Biochemical and Structural Studies of RNA Modification and Repair

    Science.gov (United States)

    Chan, Chio Mui

    2009-01-01

    RNA modification, RNA interference, and RNA repair are important events in the cell. This thesis presents three projects related to these three fields. By using both biochemical and structural methods, we characterized enzymatic activities of pseudouridine synthase TruD, solved the structure of "A. aeolicus" GidA, and reconstituted a novel…

  9. Preparation and isolation of siRNA-loaded extracellular vesicles

    NARCIS (Netherlands)

    Vader, Pieter; Mäger, Imre; Lee, Yi; Nordin, Joel Z.; Andaloussi, Samir E L; Wood, Matthew J A

    2017-01-01

    RNA interference (RNAi) has tremendous potential for specific silencing of disease-causing genes. Its clinical usage however critically depends on the development of carrier systems that can transport the RNAimediating small interfering RNA (siRNA) molecules to the cytosol of target cells. Recent

  10. Construction of lentiviral shRNA expression vector targeting ...

    African Journals Online (AJOL)

    The optimal interfering target was then selected, while the titer of lentiviral packing PLD2-shRNA was 3.47 × 104 TU/ml and the virus was successfully packaged. PCR and sequencing analyses revealed that lentiviral shRNA vectors of three targeting PLD2 gene were successfully constructed. Key words: RNA interference ...

  11. Interference in motor learning - is motor interference sensory?

    DEFF Research Database (Denmark)

    Jensen, Jesper Lundbye; Petersen, Tue Hvass; Rothwell, John C

    Skill gained after a short period of practice in one motor task can be abolished if a second task is learned shortly afterwards, but not all motor activities cause interference. After all it is not necessary to remain completely still after practicing a task for learning to occur. Here we ask which...... mechanisms determine whether or not interference occurs. We hypothesised that interference requires the same neural circuits to be engaged in the two tasks and provoke competing processes of synaptic plasticity. To test this, subjects learned a ballistic ankle plantarflexion task. Early motor memory...... learning of the primary task, no interference was observed. Previous studies have suggested that primary motor cortex (M1) may be involved in early motor memory consolidation. 1Hz Repetitive Transcranial Magnetic Stimulation (rTMS) of corticospinal motor output at intensities below ankle movement threshold...

  12. The quantum interference effect transistor.

    Science.gov (United States)

    Stafford, Charles A; Cardamone, David M; Mazumdar, Sumit

    2007-10-24

    We give a detailed discussion of the quantum interference effect transistor (QuIET), a proposed device which exploits the interference between electron paths through aromatic molecules to modulate the current flow. In the off state, perfect destructive interference stemming from the molecular symmetry blocks the current, while in the on state, the current is allowed to flow by locally introducing either decoherence or elastic scattering. Details of a model calculation demonstrating the efficacy of the QuIET are presented, and various fabrication scenarios are proposed, including the possibility of using conducting polymers to connect the QuIET with multiple leads.

  13. RNA genetics

    Energy Technology Data Exchange (ETDEWEB)

    Domingo, E. (Instituto de Biologia Molecular, Facultad de Ciencias, Universidad Autonoma de Madrid, Canto Blanco, Madrid (ES)); Holland, J.J. (California Univ., San Diego, La Jolla, CA (USA). Dept. of Biology); Ahlquist, P. (Wisconsin Univ., Madison, WI (USA). Dept. of Plant Pathology)

    1988-01-01

    This book contains the proceedings on RNA gentics: Variability of RNA genomes, Volume III. Topics covered include: High error rate, population equilibrium, and evolution of RNA replication systems; Influenza viruses; High rate of nutation and evolution; and Sequence space and quasi species distribution.

  14. Multipolar interference effects in nanophotonics

    CERN Document Server

    Liu, Wei

    2016-01-01

    Scattering of electromagnetic waves by an arbitrary nanoscale object can be characterized by a multipole decomposition of the electromagnetic field that allows to describe the scattering intensity and radiation pattern through interferences of dominating excited multipole modes. In modern nanophotonics, both generation and interference of multipole modes start to play an indispensable role, and they enable nanoscale manipulation of light with many related applications. Here we review the multipolar interference effects in metallic, metal-dielectric, and dielectric nanostructures, and suggest a comprehensive view on many phenomena involving the interferences of electric, magnetic and toroidal multipoles, which drive a number of recently discussed effects in nanophotonics such as unidirectional scattering, effective optical antiferromagnetism, generalized Kerker scattering with controlled angular patterns, generalized Brewster angle, and nonradiating optical anapoles. We further discuss other types of possible ...

  15. Quantum Erasure: Quantum Interference Revisited

    OpenAIRE

    Walborn, Stephen P.; Cunha, Marcelo O. Terra; Pádua, Sebastião; Monken, Carlos H.

    2005-01-01

    Recent experiments in quantum optics have shed light on the foundations of quantum physics. Quantum erasers - modified quantum interference experiments - show that quantum entanglement is responsible for the complementarity principle.

  16. Liposomal siRNA for ovarian cancer.

    Science.gov (United States)

    Mangala, Lingegowda S; Han, Hee Dong; Lopez-Berestein, Gabriel; Sood, Anil K

    2009-01-01

    Discovery of RNA interference (RNAi) has been one of the most important findings in the last ten years. In recent years, small interfering RNA (siRNA)-mediated gene silencing is beginning to show substantial promise as a new treatment modality in preclinical studies because of its robust gene selective silencing. However, until recently, delivery of siRNA in vivo was a major impediment to its use as a therapeutic modality. We have used a neutral liposome, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), for highly efficient in vivo siRNA delivery. Using siRNA tagged with Alexa-555, incorporated in DOPC liposomes, we have demonstrated efficient intra-tumoral delivery following either intraperitoneal or intravenous injection. Furthermore, EphA2-targeted siRNA in DOPC liposomes showed significant target modulation and anti-tumor efficacy.

  17. Secondary siRNAs result from unprimed RNA synthesis and form a distinct class.

    NARCIS (Netherlands)

    Sijen, L.M.T.; Steiner, F.A.; Thijssen, K.L.; Plasterk, R.H.A.

    2007-01-01

    In Caenorhabditis elegans, an effective RNA interference (RNAi) response requires the production of secondary short interfering RNAs (siRNAs) by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22-nucleotide primary siRNA. Several

  18. Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

    Science.gov (United States)

    Lakatos, Lóránt; Csorba, Tibor; Pantaleo, Vitantonio; Chapman, Elisabeth J; Carrington, James C; Liu, Yu-Ping; Dolja, Valerian V; Calvino, Lourdes Fernández; López-Moya, Juan José; Burgyán, József

    2006-01-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. PMID:16724105

  19. Characterizing destructive quantum interference in electron transport

    Science.gov (United States)

    Sam-ang, Panu; Reuter, Matthew G.

    2017-05-01

    Destructive quantum interference in electron transport through molecules provides an unconventional route for suppressing electric current. In this work we introduce ‘interference vectors’ for each interference and use them to characterize the interference. An interference vector may be a combination of multiple molecular orbitals (MOs), leading to more robust interference that is likelier to be experimentally observable. In contrast, an interference vector may itself be a MO, in which case the interference is not robust and will be harder to detect. Our characterization scheme quantifies these two possibilities through the degree of rotation and also assigns an order to each interference that describes the shape of the Landauer-Büttiker transmission function around the interference. Several examples are then presented, showcasing the generality of our theory and characterization scheme, which is not limited to specific classes of molecules or particular molecule-electrode coupling patterns.

  20. Interference in ballistic motor learning - is motor interference really sensory?

    DEFF Research Database (Denmark)

    Lundbye-Jensen, Jesper; Petersen, Tue Hvass; Rothwell, John C

    Skill gained after a short period of practice in one motor task can be abolished if a second task is learned shortly afterwards. We hypothesised that interference requires the same circuits to be engaged in the two tasks and provoke competing processes of synaptic plasticity. To test this, subjects...

  1. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.

    2015-01-01

    RNA modification has attracted increasing interest as it is realized that epitranscriptomics is important in disease development. In type 2 diabetes we have suggested that high urinary excretion of 8-oxo-2'-Guanosine (8oxoGuo), as a measure of global RNA oxidation, is associated with poor survival.......9 significant hazard ratio for death compared with the quartile with the lowest 8oxoGuo excretion when adjusted for age, sex, BMI, smoker status, s-HbA1c, urine protein excretion and s-cholesterol. We conclude that it is now established that RNA oxidation is an independent risk factor for death in type 2...... diabetes. In agreement with our previous finding, DNA oxidation did not show any prognostic value. RNA oxidation represents oxidative stress intracellularly, presumably predominantly in the cytosol. The mechanism of RNA oxidation is not clear, but hypothesized to result from mitochondrial dysfunction...

  2. A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Garrett, Roger Antony; Shah, Shiraz Ali

    2013-01-01

    targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S.¿islandicus REY15A which cooperate functionally by sharing a single Cas6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers.......Recent studies on CRISPR-based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon Sulfolobus islandicus REY15A encodes three interference modules, one of type IA and two of type IIIB....... Earlier we showed that type IA activity eliminated plasmid vectors carrying matching protospacers with specific CCN PAM sequences. Here we demonstrate that interference-mediated by one type IIIB module Cmr-a, and a Csx1 protein, efficiently eliminated plasmid vectors carrying matching protospacers...