Control of thermoacoustic instability with a drum-like silencer

NASA Astrophysics Data System (ADS)

Zhang, Guangyu; Wang, Xiaoyu; Li, Lei; Jing, Xiaodong; Sun, Xiaofeng

2017-10-01

Theoretical investigation is carried out by a novel method of controlling thermoacoustic instability with a drum-like silencer. It is shown that by decreasing the frequency of thermoacoustic system, the instability can be suppressed with the help of drum-like silencer. The purely reactive silencer, which is composed of a flexible membrane and a backing cavity, is usually known as a noise control device that works effectively in low frequency bandwidth without any aerodynamic loss. In present research, the silencer is exploited in a Rijke tube, as a means of decreasing the natural frequency of the system, and consequently changing the resonance period of the system. The "transfer element method" (TEM) is used to consider the interactions between the acoustic waves and the flexible membranes of the silencer. The effects of all possible properties of the silencer on the growth rate and resonance frequency of the thermoacoustic system are explored. According to the calculation results, it is found that for some properties of the silencer, the resonance frequencies are greatly decreased and then the phase difference between the unsteady heat release and the pressure fluctuation is increased. Consequently, the instability is suppressed with some dissipation that can not be able to control its onset in the original system. Therefore, when the damping is low, but not zero, it is effective to control thermoacoustic instability with this technique.

Virus-induced gene silencing (VIGS) in barley seedling leaves

USDA-ARS?s Scientific Manuscript database

Virus-induced gene silencing (VIGS) is one of the most potent reverse genetics technologies for gene functional characterization. This method exploits a dsRNA-mediated antiviral defense mechanism in plants. Using this method allows researchers to generate rapid phenotypic data in a relatively rapid ...

A mode matching method for modeling dissipative silencers lined with poroelastic materials and containing mean flow.

PubMed

Nennig, Benoit; Perrey-Debain, Emmanuel; Ben Tahar, Mabrouk

2010-12-01

A mode matching method for predicting the transmission loss of a cylindrical shaped dissipative silencer partially filled with a poroelastic foam is developed. The model takes into account the solid phase elasticity of the sound-absorbing material, the mounting conditions of the foam, and the presence of a uniform mean flow in the central airway. The novelty of the proposed approach lies in the fact that guided modes of the silencer have a composite nature containing both compressional and shear waves as opposed to classical mode matching methods in which only acoustic pressure waves are present. Results presented demonstrate good agreement with finite element calculations provided a sufficient number of modes are retained. In practice, it is found that the time for computing the transmission loss over a large frequency range takes a few minutes on a personal computer. This makes the present method a reliable tool for tackling dissipative silencers lined with poroelastic materials.

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

PubMed

Lim, Hyoun-Sub; Vaira, Anna Maria; Domier, Leslie L; Lee, Sung Chul; Kim, Hong Gi; Hammond, John

2010-06-20

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS. Published by Elsevier Inc.

Gene silencing pathway RNA-dependent RNA polymerase of Neurospora crassa: yeast expression and crystallization of selenomethionated QDE-1 protein.

PubMed

Laurila, Minni R L; Salgado, Paula S; Makeyev, Eugene V; Nettelship, Joanne; Stuart, David I; Grimes, Jonathan M; Bamford, Dennis H

2005-01-01

The RNA-dependent RNA polymerase, QDE-1, is a component of the RNA silencing pathway in Neurospora crassa. The enzymatically active carboxy-terminal fragment QDE-1 DeltaN has been expressed in Saccharomyces cerevisiae in the presence and absence of selenomethionine (SeMet). The level of SeMet incorporation was estimated by mass spectrometry to be approximately 98%. Both native and SeMet proteins were crystallized in space group P2(1) with unit cell parameters a=101.2, b=122.5, c=114.4A, beta=108.9 degrees , and 2 molecules per asymmetric unit. The native and SeMet crystals diffract to 2.3 and 3.2A, respectively, the latter are suitable for MAD structure determination.

Personalized gene silencing therapeutics for Huntington disease.

PubMed

Kay, C; Skotte, N H; Southwell, A L; Hayden, M R

2014-07-01

Gene silencing offers a novel therapeutic strategy for dominant genetic disorders. In specific diseases, selective silencing of only one copy of a gene may be advantageous over non-selective silencing of both copies. Huntington disease (HD) is an autosomal dominant disorder caused by an expanded CAG trinucleotide repeat in the Huntingtin gene (HTT). Silencing both expanded and normal copies of HTT may be therapeutically beneficial, but preservation of normal HTT expression is preferred. Allele-specific methods can selectively silence the mutant HTT transcript by targeting either the expanded CAG repeat or single nucleotide polymorphisms (SNPs) in linkage disequilibrium with the expansion. Both approaches require personalized treatment strategies based on patient genotypes. We compare the prospect of safe treatment of HD by CAG- and SNP-specific silencing approaches and review HD population genetics used to guide target identification in the patient population. Clinical implementation of allele-specific HTT silencing faces challenges common to personalized genetic medicine, requiring novel solutions from clinical scientists and regulatory authorities. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Functional Genomic Analysis of Cotton Genes with Agrobacterium-Mediated Virus-Induced Gene Silencing

PubMed Central

Gao, Xiquan; Shan, Libo

2015-01-01

Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses. PMID:23386302

Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.

PubMed

Gao, Xiquan; Shan, Libo

2013-01-01

Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses.

Low Immunogenic Endothelial Cells Maintain Morphological and Functional Properties Required for Vascular Tissue Engineering.

PubMed

Lau, Skadi; Eicke, Dorothee; Carvalho Oliveira, Marco; Wiegmann, Bettina; Schrimpf, Claudia; Haverich, Axel; Blasczyk, Rainer; Wilhelmi, Mathias; Figueiredo, Constança; Böer, Ulrike

2018-03-01

The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue-engineered vascular grafts containing autologous cells are considered ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC, but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leukocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood and umbilical cord blood and vein. EC from all three sources were transduced by 93.1% ± 4.8% and effectively, HLA I-silenced by up to 67% compared to nontransduced (NT) cells or transduced with a nonspecific short hairpin RNA, respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier, as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low-density lipoprotein, and to form capillary-like tube structures in three-dimensional fibrin gels similar to NT cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC toward long and widely branched vascular networks necessary for prevascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological and functional properties required for vascular tissue engineering. This extends the spectrum of available cell sources from autologous to allogeneic sources, thereby accelerating the generation of tissue-engineered vascular grafts in acute clinical cases.

The Relationship between Organizational Climate and the Organizational Silence of Administrative Staff in Education Department

ERIC Educational Resources Information Center

Pozveh, Asghar Zamani; Karimi, Fariba

2016-01-01

The aim of the present study was to determine the relationship between organizational climate and the organizational silence of administrative staff in Education Department in Isfahan. The research method was descriptive and correlational-type method. The study population was administrative staff of Education Department in Isfahan during the…

Hearing Silence: Toward a Mixed-Method Approach for Studying Genres' Exclusionary Potential

ERIC Educational Resources Information Center

Randazzo, Chalice

2015-01-01

Traditional Rhetorical Genre Study (RGS) methods are not well adapted to study exclusion because excluded information and people are typically absent from the genre, and some excluded information is simply unrelated to the genre because of genre conventions or social context. Within genre-based silences, how can scholars differentiate between an…

Agrobacterium-mediated virus-induced gene silencing assay in cotton.

PubMed

Gao, Xiquan; Britt, Robert C; Shan, Libo; He, Ping

2011-08-20

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation(1). To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions. Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation(2,3). Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies(3,4). As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation. In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development(6), and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves(7), providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

PubMed Central

Gao, Xiquan; Britt Jr., Robert C.; Shan, Libo; He, Ping

2011-01-01

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions. Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation. In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton. PMID:21876527

RNA therapeutics targeting osteoclast-mediated excessive bone resorption

PubMed Central

Wang, Yuwei; Grainger, David W

2011-01-01

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing technique developed with dramatically increasing utility for both scientific and therapeutic purposes. Short interfering RNA (siRNA) is currently exploited to regulate protein expression relevant to many therapeutic applications, and commonly used as a tool for elucidating disease-associated genes. Osteoporosis and their associated osteoporotic fragility fractures in both men and women are rapidly becoming a global healthcare crisis as average life expectancy increases worldwide. New therapeutics are needed for this increasing patient population. This review describes the diversity of molecular targets suitable for RNAi-based gene knock-down in osteoclasts to control osteoclast-mediated excessive bone resorption. We identify strategies for developing targeted siRNA delivery and efficient gene silencing, and describe opportunities and challenges of introducing siRNA as a therapeutic approach to hard and connective tissue disorders. PMID:21945356

Development of Agrobacterium-Mediated Virus-Induced Gene Silencing and Performance Evaluation of Four Marker Genes in Gossypium barbadense

PubMed Central

Pang, Jinhuan; Zhu, Yue; Li, Qing; Liu, Jinzhi; Tian, Yingchuan; Liu, Yule; Wu, Jiahe

2013-01-01

Gossypium barbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species). These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G . barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G . barbadense . In this study, we had successfully introduced a virus-induced gene silencing (VIGS) system into three cultivars of G . barbadense by inserting marker genes into the tobacco rattle virus (TRV) vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G . barbadense . The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G . barbadense , and help to contribute desirable traits for breeding of G . barbadense and G. hirsutum. PMID:24023833

HMGA1 silencing reduces stemness and temozolomide resistance in glioblastoma stem cells.

PubMed

Colamaio, Marianna; Tosti, Nadia; Puca, Francesca; Mari, Alessia; Gattordo, Rosaria; Kuzay, Yalçın; Federico, Antonella; Pepe, Anna; Sarnataro, Daniela; Ragozzino, Elvira; Raia, Maddalena; Hirata, Hidenari; Gemei, Marica; Mimori, Koshi; Del Vecchio, Luigi; Battista, Sabrina; Fusco, Alfredo

2016-10-01

Glioblastoma multiforme (GBM) develops from a small subpopulation of stem-like cells, which are endowed with the ability to self-renew, proliferate and give rise to progeny of multiple neuroepithelial lineages. These cells are resistant to conventional chemo- and radiotherapy and are hence also responsible for tumor recurrence. HMGA1 overexpression has been shown to correlate with proliferation, invasion, and angiogenesis of GBMs and to affect self-renewal of cancer stem cells from colon cancer. The role of HMGA1 in GBM tumor stem cells is not completely understood. We have investigated the role of HMGA1 in brain tumor stem cell (BTSC) self-renewal, stemness and resistance to temozolomide by shRNA- mediated HMGA1 silencing. We first report that HMGA1 is overexpressed in a subset of BTSC lines from human GBMs. Then, we show that HMGA1 knockdown reduces self-renewal, sphere forming efficiency and stemness, and sensitizes BTSCs to temozolomide. Interestingly, HMGA1 silencing also leads to reduced tumor initiation ability in vivo. These results demonstrate a pivotal role of HMGA1 in cancer stem cell gliomagenesis and endorse HMGA1 as a suitable target for CSC-specific GBM therapy.

Reconstruction of gene regulatory modules from RNA silencing of IFN-α modulators: experimental set-up and inference method.

PubMed

Grassi, Angela; Di Camillo, Barbara; Ciccarese, Francesco; Agnusdei, Valentina; Zanovello, Paola; Amadori, Alberto; Finesso, Lorenzo; Indraccolo, Stefano; Toffolo, Gianna Maria

2016-03-12

Inference of gene regulation from expression data may help to unravel regulatory mechanisms involved in complex diseases or in the action of specific drugs. A challenging task for many researchers working in the field of systems biology is to build up an experiment with a limited budget and produce a dataset suitable to reconstruct putative regulatory modules worth of biological validation. Here, we focus on small-scale gene expression screens and we introduce a novel experimental set-up and a customized method of analysis to make inference on regulatory modules starting from genetic perturbation data, e.g. knockdown and overexpression data. To illustrate the utility of our strategy, it was applied to produce and analyze a dataset of quantitative real-time RT-PCR data, in which interferon-α (IFN-α) transcriptional response in endothelial cells is investigated by RNA silencing of two candidate IFN-α modulators, STAT1 and IFIH1. A putative regulatory module was reconstructed by our method, revealing an intriguing feed-forward loop, in which STAT1 regulates IFIH1 and they both negatively regulate IFNAR1. STAT1 regulation on IFNAR1 was object of experimental validation at the protein level. Detailed description of the experimental set-up and of the analysis procedure is reported, with the intent to be of inspiration for other scientists who want to realize similar experiments to reconstruct gene regulatory modules starting from perturbations of possible regulators. Application of our approach to the study of IFN-α transcriptional response modulators in endothelial cells has led to many interesting novel findings and new biological hypotheses worth of validation.

A novel VIGS method by agroinoculation of cotton seeds and application for elucidating functions of GhBI-1 in salt-stress response.

PubMed

Zhang, Jingxia; Wang, Furong; Zhang, Chuanyun; Zhang, Junhao; Chen, Yu; Liu, Guodong; Zhao, Yanxiu; Hao, Fushun; Zhang, Jun

2018-06-04

A VIGS method by agroinoculation of cotton seeds was developed for gene silencing in young seedlings and roots, and applied in functional analysis of GhBI-1 in response to salt stress. Virus-induced gene silencing (VIGS) has been widely used to investigate the functions of genes expressed in mature leaves, but not yet in young seedlings or roots of cotton (Gossypium hirsutum L.). Here, we developed a simple and effective VIGS method for silencing genes in young cotton seedlings and roots by soaking naked seeds in Agrobacterium cultures carrying tobacco rattle virus (TRV)-VIGS vectors. When the naked seeds were soaked in Agrobacterium cultures with an OD600 of 1.5 for 90 min, it was optimal for silencing genes effectively in young seedlings as clear photo-bleaching phenotype in the newly emerging leaves of pTRV:GhCLA1 seedlings were observed at 12-14 days post inoculation. Silencing of GhPGF (cotton pigment gland formation) by this method resulted in a 90% decrease in transcript abundances of the gene in roots at the early development stage. We further used the tool to investigate function of GhBI-1 (cotton Bax inhibitor-1) gene in response to salt stress and demonstrated that GhBI-1 might play a protective role under salt stress by suppressing stress-induced cell death in cotton. Our results showed that the newly established VIGS method is a powerful tool for elucidating functions of genes in cotton, especially the genes expressed in young seedlings and roots.

Characterizing virus-induced gene silencing at the cellular level with in situ multimodal imaging

DOE PAGES

Burkhow, Sadie J.; Stephens, Nicole M.; Mei, Yu; ...

2018-05-25

Reverse genetic strategies, such as virus-induced gene silencing, are powerful techniques to study gene function. Currently, there are few tools to study the spatial dependence of the consequences of gene silencing at the cellular level. Here, we report the use of multimodal Raman and mass spectrometry imaging to study the cellular-level biochemical changes that occur from silencing the phytoene desaturase ( pds) gene using a Foxtail mosaic virus (FoMV) vector in maize leaves. The multimodal imaging method allows the localized carotenoid distribution to be measured and reveals differences lost in the spatial average when analyzing a carotenoid extraction of themore » whole leaf. The nature of the Raman and mass spectrometry signals are complementary: silencing pds reduces the downstream carotenoid Raman signal and increases the phytoene mass spectrometry signal.« less

Characterizing virus-induced gene silencing at the cellular level with in situ multimodal imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burkhow, Sadie J.; Stephens, Nicole M.; Mei, Yu

Reverse genetic strategies, such as virus-induced gene silencing, are powerful techniques to study gene function. Currently, there are few tools to study the spatial dependence of the consequences of gene silencing at the cellular level. Here, we report the use of multimodal Raman and mass spectrometry imaging to study the cellular-level biochemical changes that occur from silencing the phytoene desaturase ( pds) gene using a Foxtail mosaic virus (FoMV) vector in maize leaves. The multimodal imaging method allows the localized carotenoid distribution to be measured and reveals differences lost in the spatial average when analyzing a carotenoid extraction of themore » whole leaf. The nature of the Raman and mass spectrometry signals are complementary: silencing pds reduces the downstream carotenoid Raman signal and increases the phytoene mass spectrometry signal.« less

An efficient method for gene silencing in human primary plasmacytoid dendritic cells: silencing of the TLR7/IRF-7 pathway as a proof of concept

PubMed Central

Smith, Nikaïa; Vidalain, Pierre-Olivier; Nisole, Sébastien; Herbeuval, Jean-Philippe

2016-01-01

Plasmacytoid dendritic cells (pDC) are specialized immune cells that produce massive levels of type I interferon in response to pathogens. Unfortunately, pDC are fragile and extremely rare, rendering their functional study a tough challenge. However, because of their central role in numerous pathologies, there is a considerable need for an efficient and reproducible protocol for gene silencing in these cells. In this report, we tested six different methods for siRNA delivery into primary human pDC including viral-based, lipid-based, electroporation, and poly-ethylenimine (PEI) technologies. We show that lipid-based reagent DOTAP was extremely efficient for siRNA delivery into pDC, and did not induce cell death or pDC activation. We successfully silenced Toll-Like Receptor 7 (TLR7), CXCR4 and IFN regulatory factor 7 (IRF-7) gene expression in pDC as assessed by RT-qPCR or cytometry. Finally, we showed that TLR7 or IRF-7 silencing in pDC specifically suppressed IFN-α production upon stimulation, providing a functional validation of our transfection protocol. PMID:27412723

Preparation of siRNA encapsulated nanoliposomes suitable for siRNA delivery by simply discontinuous mixing.

PubMed

Mokhtarieh, Amir Abbas; Lee, Jieun; Kim, Semi; Lee, Myung Kyu

2018-06-01

Previously a scalable and extrusion-free method has been developed for efficient liposomal encapsulation of DNA by twice stepwise mixing of lipids in ethanol and DNA solution using T-shape mixing chamber. In this study, we prepared nanoliposomes encapsulating siRNA by simply discontinuous mixing of lipids in ethanol/ether/water mixture and acidic siRNA solution without use of special equipment. The simple mixing siRNA/liposomal particles (siRNA/SMLs) prepared using ethanol/ether/water (3:1:1) mixture showed 120.4 ± 20.2 nm particle size, 0.174 ± 0.033 polydispersity and 86.5 ± 2.76% siRNA encapsulation rate. In addition, the SMLs almost completely protected the encapsulated siRNA from RNase A digestion. Coupling of anti-human epidermal growth factor receptor (EGFR) Fab' to siRNA/SMLs enhanced EGFR-specific cell penetration of SMLs and induced siRNA dependent gene silencing. Unexpectedly, the Cy5.5-labeled Fab' showed almost no in vivo targeting to the xenografted A549 tumors in SCID-NOD mice. However, multiple injection of the unmodified siRNA/SMLs accumulated in the tumors and induced siRNA-dependent in vivo gene silencing. These results demonstrate that the siRNA/SMLs can be used as a siRNA delivery tool for gene therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery

PubMed Central

Hammond, Thomas M.; Xiao, Hua; Boone, Erin C.; Perdue, Tony D.; Pukkila, Patricia J.; Shiu, Patrick K. T.

2011-01-01

In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species. PMID:22384347

Biotechnological application of functional genomics towards plant-parasitic nematode control.

PubMed

Li, Jiarui; Todd, Timothy C; Lee, Junghoon; Trick, Harold N

2011-12-01

Plant-parasitic nematodes are primary biotic factors limiting the crop production. Current nematode control strategies include nematicides, crop rotation and resistant cultivars, but each has serious limitations. RNA interference (RNAi) represents a major breakthrough in the application of functional genomics for plant-parasitic nematode control. RNAi-induced suppression of numerous genes essential for nematode development, reproduction or parasitism has been demonstrated, highlighting the considerable potential for using this strategy to control damaging pest populations. In an effort to find more suitable and effective gene targets for silencing, researchers are employing functional genomics methodologies, including genome sequencing and transcriptome profiling. Microarrays have been used for studying the interactions between nematodes and plant roots and to measure both plants and nematodes transcripts. Furthermore, laser capture microdissection has been applied for the precise dissection of nematode feeding sites (syncytia) to allow the study of gene expression specifically in syncytia. In the near future, small RNA sequencing techniques will provide more direct information for elucidating small RNA regulatory mechanisms in plants and specific gene silencing using artificial microRNAs should further improve the potential of targeted gene silencing as a strategy for nematode management. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Biominetic High Density Lipoproteins for the Delivery of Therapeutic Oligonucleotides

NASA Astrophysics Data System (ADS)

Tripathy, Sushant

Advances in nanotechnology have brought about novel inorganic and hybrid nanoparticles with unique physico-chemical properties that make them suitable for a broad range of applications---from nano-circuitry to drug delivery. A significant part of those advancements have led to ground-breaking discoveries that have changed the approaches to formulation of therapeutics against diseases, such as cancer. Now-a-days the focus does not lie solely on finding a candidate small-molecule therapeutic with minimal adverse effects, but researchers are looking up to nanoparticles to improve biodistribution and biocompatibility profile of clinically proven therapeutics. The plethora of conjugation chemistries offered by currently extant inorganic nanoparticles have, in recent years, led to great leaps in the field of biomimicry---a modality that promises high biocompatibility. Further, in the pursuit of highly specific therapeutic molecules, researchers have turned to silencing oligonucleotides and some have already brought together the strengths of nanoparticles and silencing oligonucleotides in search of an efficacious therapy for cancer with minimal adverse effects. This dissertation work focuses on such a biomimetic platform---a gold nanoparticle based high density lipoprotein biomimetic (HDL NP), for the delivery of therapeutic oligonucleotides. The first chapter of this body of work introduces the molecular target of the silencing oligonucleotides---VEGFR2, and its role in the progression of solid tumor cancers. The background information also covers important aspects of natural high density lipoproteins (HDL), especially their innate capacity to bind and deliver exogenous and endogenous silencing oligonucleotides to tissues that express their high affinity receptor SRB1. We subsequently describe the synthesis of the biomimetic HDL NP and its oligonucleotide conjugates, and establish their biocompatibility. Further on, experimental data demonstrate the efficacy of silencing oligonucleotides conjugated HDL NPs in regulating the expression and function of VEGFR2 in cultured endothelial cells. Finally, the efficacy of the conjugates in two animal models of angiogenesis is presented.

Phenotyping of VIGS-mediated gene silencing in rice using a vector derived from a DNA virus.

PubMed

Kant, Ravi; Dasgupta, Indranil

2017-07-01

Target genes in rice can be optimally silenced if inserted in antisense or hairpin orientation in the RTBV-derived VIGS vector and plants grown at 28 °C and 80% humidity after inoculation. Virus induced gene silencing (VIGS) is a method used to transiently silence genes in dicot as well as monocot plants. For the important monocot species rice, the Rice tungro bacilliform virus (RTBV)-derived VIGS system (RTBV-VIGS), which uses agroinoculation to initiate silencing, has not been standardized for optimal use. Here, using RTBV-VIGS, three sets of conditions were tested to achieve optimal silencing of the rice marker gene phytoene desaturase (pds). The effect of orientation of the insert in the RTBV-VIGS plasmid (sense, antisense and hairpin) on the silencing of the target gene was then evaluated using rice magnesium chelatase subunit H (chlH). Finally, the rice Xa21 gene, conferring resistance against bacterial leaf blight disease (BLB) was silenced using RTBV-VIGS system. In each case, real-time PCR-based assessment indicated approximately 40-80% fall in the accumulation levels of the transcripts of pds, chlH and Xa21. In the case of pds, the appearance of white streaks in the emerging leaves, and for chlH, chlorophyll levels and F v /F m ratio were assessed as phenotypes for silencing. For Xa21, the resistance levels to BLB were assessed by measuring the lesion length and the percent diseased areas of leaves, following challenge inoculation with Xanthomonas oryzae. In each case, the RTBV-MVIGS system gave rise to a discernible phenotype indicating the silencing of the respective target gene using condition III (temperature 28 °C, humidity 80% and 1 mM MES and 20 µM acetosyringone in secondary agrobacterium culture), which revealed the robustness of this gene silencing system for rice.

An Improved Ivermectin-activated Chloride Channel Receptor for Inhibiting Electrical Activity in Defined Neuronal Populations*

PubMed Central

Lynagh, Timothy; Lynch, Joseph W.

2010-01-01

The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity. Several neuronal silencing methods have been developed, with the bacterial light-activated halorhodopsin and the invertebrate allatostatin-activated G protein-coupled receptor proving the most successful to date. However, both techniques may be difficult to implement clinically due to their requirement for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches the brain following systemic administration. However, the limitations of this method include poor functional expression, possibly due to the requirement to coexpress two different subunits in individual neurons, and the nonhuman origin of GluClR. Here, we describe the development of a modified human α1 glycine receptor as an improved ivermectin-gated silencing receptor. The crucial development was the identification of a mutation, A288G, which increased ivermectin sensitivity almost 100-fold, rendering it similar to that of GluClR. Glycine sensitivity was eliminated via the F207A mutation. Its large unitary conductance, homomeric expression, and human origin may render the F207A/A288G α1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue appears essential for exquisite ivermectin sensitivity. PMID:20308070

Prediction of the diffuse-field transmission loss of interior natural-ventilation openings and silencers.

PubMed

Bibby, Chris; Hodgson, Murray

2017-01-01

The work reported here, part of a study on the performance and optimal design of interior natural-ventilation openings and silencers ("ventilators"), discusses the prediction of the acoustical performance of such ventilators, and the factors that affect it. A wave-based numerical approach-the finite-element method (FEM)-is applied. The development of a FEM technique for the prediction of ventilator diffuse-field transmission loss is presented. Model convergence is studied with respect to mesh, frequency-sampling and diffuse-field convergence. The modeling technique is validated by way of predictions and the comparison of them to analytical and experimental results. The transmission-loss performance of crosstalk silencers of four shapes, and the factors that affect it, are predicted and discussed. Performance increases with flow-path length for all silencer types. Adding elbows significantly increases high-frequency transmission loss, but does not increase overall silencer performance which is controlled by low-to-mid-frequency transmission loss.

In Vitro Formation of Plant RNA-Induced Silencing Complexes Using an Extract of Evacuolated Tobacco Protoplasts.

PubMed

Iki, Taichiro; Ishikawa, Masayuki; Yoshikawa, Manabu

2017-01-01

Small RNA-mediated gene silencing is involved in a variety of biological processes among many eukaryotic organisms. The silencing effector, generally referred to as RNA-induced silencing complex (RISC), comprises an ARGONAUTE (AGO) protein and a small single-stranded guide RNA in its core. RISCs recognize target genes containing sequences complementary to the guide RNA and repress their expression transcriptionally or posttranscriptionally. In vitro systems that recapitulate RISC assembly are useful not only to decipher the molecular mechanisms underlying the assembly process itself but also to dissect the downstream silencing pathways mediated by RISCs. Here, we describe a method for in vitro plant RISC assembly, which relies on an extract of evacuolated protoplasts derived from Nicotiana tabacum BY-2 suspension-cultured cells. In this extract, synthetic duplexes of small RNAs are incorporated into AGO proteins that are synthesized by in vitro translation, and then duplex unwinding and selective strand elimination result in formation of mature RISCs.

Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli

PubMed Central

Nakashima, Nobutaka; Tamura, Tomohiro; Good, Liam

2006-01-01

Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control. PMID:17062631

Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli.

PubMed

Nakashima, Nobutaka; Tamura, Tomohiro; Good, Liam

2006-01-01

Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control.

Gene Silencing in Crustaceans: From Basic Research to Biotechnologies

PubMed Central

Sagi, Amir; Manor, Rivka; Ventura, Tomer

2013-01-01

Gene silencing through RNA interference (RNAi) is gaining momentum for crustaceans, both in basic research and for commercial development. RNAi has proven instrumental in a growing number of crustacean species, revealing the functionality of novel crustacean genes essential among others to development, growth, metabolism and reproduction. Extensive studies have also been done on silencing of viral transcripts in crustaceans, contributing to the understanding of the defense mechanisms of crustaceans and strategies employed by viruses to overcome these. The first practical use of gene silencing in aquaculture industry has been recently achieved, through manipulation of a crustacean insulin-like androgenic gland hormone. This review summarizes the advancements in the use of RNAi in crustaceans, and assesses the advantages of this method, as well as the current hurdles that hinder its large-scale practice. PMID:24705266

Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7

PubMed Central

Curtis, Helen J.; Wood, Matthew J.A.

2017-01-01

Abstract We evaluate a knockdown-replacement strategy mediated by mirtrons as an alternative to allele-specific silencing using spinocerebellar ataxia 7 (SCA7) as a model. Mirtrons are introns that form pre-microRNA hairpins after splicing, producing RNAi effectors not processed by Drosha. Mirtron mimics may therefore avoid saturation of the canonical processing pathway. This method combines gene silencing mediated by an artificial mirtron with delivery of a functional copy of the gene such that both elements of the therapy are always expressed concurrently, minimizing the potential for undesirable effects and preserving wild-type function. This mutation- and single nucleotide polymorphism-independent method could be crucial in dominant diseases that feature both gain- and loss-of-function pathologies or have a heterogeneous genetic background. Here we develop mirtrons against ataxin 7 with silencing efficacy comparable to shRNAs, and introduce silent mutations into an ataxin 7 transgene such that it is resistant to their effect. We successfully express the transgene and one mirtron together from a single construct. Hence, we show that this method can be used to silence the endogenous allele of ataxin 7 and replace it with an exogenous copy of the gene, highlighting the efficacy and transferability across patient genotypes of this approach. PMID:28575281

Sina and Sinb genes in triticale do not determine grain hardness contrary to their orthologs Pina and Pinb in wheat

PubMed Central

2013-01-01

Background Secaloindoline a (Sina) and secaloindoline b (Sinb) genes of hexaploid triticale (x Triticosecale Wittmack) are orthologs of puroindoline a (Pina) and puroindoline b (Pinb) in hexaploid wheat (Triticum aestivum L.). It has already been proven that RNA interference (RNAi)-based silencing of Pina and Pinb genes significantly decreased the puroindoline a and puroindoline b proteins in wheat and essentially increased grain hardness (J Exp Bot 62:4025-4036, 2011). The function of Sina and Sinb in triticale was tested by means of RNAi silencing and compared to wheat. Results Novel Sina and Sinb alleles in wild-type plants of cv. Wanad were identified and their expression profiles characterized. Alignment with wheat Pina-D1a and Pinb-D1a alleles showed 95% and 93.3% homology with Sina and Sinb coding sequences. Twenty transgenic lines transformed with two hpRNA silencing cassettes directed to silence Sina or Sinb were obtained by the Agrobacterium-mediated method. A significant decrease of expression of both Sin genes in segregating progeny of tested T1 lines was observed independent of the silencing cassette used. The silencing was transmitted to the T4 kernel generation. The relative transcript level was reduced by up to 99% in T3 progeny with the mean for the sublines being around 90%. Silencing of the Sin genes resulted in a substantial decrease of secaloindoline a and secaloindoline b content. The identity of SIN peptides was confirmed by mass spectrometry. The hardness index, measured by the SKCS (Single Kernel Characterization System) method, ranged from 22 to 56 in silent lines and from 37 to 49 in the control, and the mean values were insignificantly lower in the silent ones, proving increased softness. Additionally, the mean total seed protein content of silenced lines was about 6% lower compared with control lines. Correlation coefficients between hardness and transcript level were weakly positive. Conclusions We documented that RNAi-based silencing of Sin genes resulted in significant decrease of their transcripts and the level of both secaloindoline proteins, however did not affect grain hardness. The unexpected, functional differences of Sin genes from triticale compared with their orthologs, Pin of wheat, are discussed. PMID:24279512

Sina and Sinb genes in triticale do not determine grain hardness contrary to their orthologs Pina and Pinb in wheat.

PubMed

Gasparis, Sebastian; Orczyk, Waclaw; Nadolska-Orczyk, Anna

2013-11-26

Secaloindoline a (Sina) and secaloindoline b (Sinb) genes of hexaploid triticale (x Triticosecale Wittmack) are orthologs of puroindoline a (Pina) and puroindoline b (Pinb) in hexaploid wheat (Triticum aestivum L.). It has already been proven that RNA interference (RNAi)-based silencing of Pina and Pinb genes significantly decreased the puroindoline a and puroindoline b proteins in wheat and essentially increased grain hardness (J Exp Bot 62:4025-4036, 2011). The function of Sina and Sinb in triticale was tested by means of RNAi silencing and compared to wheat. Novel Sina and Sinb alleles in wild-type plants of cv. Wanad were identified and their expression profiles characterized. Alignment with wheat Pina-D1a and Pinb-D1a alleles showed 95% and 93.3% homology with Sina and Sinb coding sequences. Twenty transgenic lines transformed with two hpRNA silencing cassettes directed to silence Sina or Sinb were obtained by the Agrobacterium-mediated method. A significant decrease of expression of both Sin genes in segregating progeny of tested T1 lines was observed independent of the silencing cassette used. The silencing was transmitted to the T4 kernel generation. The relative transcript level was reduced by up to 99% in T3 progeny with the mean for the sublines being around 90%. Silencing of the Sin genes resulted in a substantial decrease of secaloindoline a and secaloindoline b content. The identity of SIN peptides was confirmed by mass spectrometry. The hardness index, measured by the SKCS (Single Kernel Characterization System) method, ranged from 22 to 56 in silent lines and from 37 to 49 in the control, and the mean values were insignificantly lower in the silent ones, proving increased softness. Additionally, the mean total seed protein content of silenced lines was about 6% lower compared with control lines. Correlation coefficients between hardness and transcript level were weakly positive. We documented that RNAi-based silencing of Sin genes resulted in significant decrease of their transcripts and the level of both secaloindoline proteins, however did not affect grain hardness. The unexpected, functional differences of Sin genes from triticale compared with their orthologs, Pin of wheat, are discussed.

RNA interference-based functional knockdown of the voltage-gated potassium channel Kv7.2 in dorsal root ganglion neurons after in vitro and in vivo gene transfer by adeno-associated virus vectors.

PubMed

Valdor, Markus; Wagner, Anke; Röhrs, Viola; Berg, Johanna; Fechner, Henry; Schröder, Wolfgang; Tzschentke, Thomas M; Bahrenberg, Gregor; Christoph, Thomas; Kurreck, Jens

2018-01-01

Activation of the neuronal potassium channel Kv7.2 encoded by the KCNQ2 gene has recently been shown to be an attractive mechanism to inhibit nociceptive transmission. However, potent, selective, and clinically proven activators of Kv7.2/Kv7.3 currents with analgesic properties are still lacking. An important prerequisite for the development of new drugs is a model to test the selectivity of novel agonists by abrogating Kv7.2/Kv7.3 function. Since constitutive knockout mice are not viable, we developed a model based on RNA interference-mediated silencing of KCNQ2. By delivery of a KCNQ2-specific short hairpin RNA with adeno-associated virus vectors, we completely abolished the activity of the specific Kv7.2/Kv7.3-opener ICA-27243 in rat sensory neurons. Results obtained in the silencing experiments were consistent between freshly prepared and cryopreserved dorsal root ganglion neurons, as well as in dorsal root ganglion neurons dissociated and cultured after in vivo administration of the silencing vector by intrathecal injections into rats. Interestingly, the tested associated virus serotypes substantially differed with respect to their transduction capability in cultured neuronal cell lines and primary dorsal root ganglion neurons and the in vivo transfer of transgenes by intrathecal injection of associated virus vectors. However, our study provides the proof-of-concept that RNA interference-mediated silencing of KCNQ2 is a suitable approach to create an ex vivo model for testing the specificity of novel Kv7.2/Kv7.3 agonists.

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS1[OPEN

PubMed Central

2018-01-01

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 (HSP70-1) inserts in Nicotiana benthamiana and maize (Zea mays). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70, silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors. PMID:29127260

An Eastern Learning Paradox: Paradoxes in Two Korean Mathematics Teachers' Pedagogy of Silence in the Classroom

ERIC Educational Resources Information Center

Lee, Kyeonghwa; Sriraman, Bharath

2013-01-01

Eastern philosophies of education such as Confucianism and Taosim advocate the use of silence in the teacher-pupil tradition of pedagogy. We investigate contemporary classrooms in Korea, and study whether teachers in Korea today incorporate this method implicitly or explicitly in their classrooms. Empirical data in the form of video-taped…

The Relationship between Organizational Trust and Organizational Silence with Job Satisfaction and Organizational Commitment of the Employees of University

ERIC Educational Resources Information Center

Fard, Parastoo Gashtasebi; Karimi, Fariba

2015-01-01

This study aimed to investigate the structural model between organizational trust and organizational silence with job satisfaction and organizational commitment of the employees of Islamic Azad University of Isfahan, (Khorasgan) branch. The study method is descriptive-correlation. The study population is the employees of Islamic Azad University of…

RNA interference as a method for target-site screening in the Western Corn Rootworm, Diabrotica virgifera virgifera

USDA-ARS?s Scientific Manuscript database

RNA interference (RNAi) is one of the most powerful and extraordinarily-specific means by which to silence genes. The ability of RNAi to silence genes makes it possible to ascertain function from genomic data, thereby making it an excellent choice for target-site screening. To test the efficacy of...

Breaking the Silence: A Phenomenological Exploration of Secondary Traumatic Stress in U.S. College Student Affairs Professionals

ERIC Educational Resources Information Center

Lynch, Robert Jason

2017-01-01

Breaking the Silence: A Phenomenological Exploration of Secondary Traumatic Stress in U.S. College Student Affairs Professionals is a qualitative-intensive mixed methods study using phenomenology and art-based research techniques to uncover the essence of secondary traumatic stress in U.S. college student affairs professionals. Researchers in the…

Effect of Hyp delivery system on PKCα activity: What will happen after pkcα gene silencing and Hyp photo-activation?

NASA Astrophysics Data System (ADS)

Misuth, Matus; Joniova, Jaroslava; Ferencakova, Michaela; Miskovsky, Pavol; Nadova, Zuzana

2015-08-01

Low density lipoproteins (LDL) are considered as suitable natural in vivo delivery system for hydrophobic photosensitizers (pts) such as hypericin (Hyp) and it was shown that over expression of LDL-receptors in tumor cells can be used for specific targeting. Activation of pts by irradiation results in a formation of reactive oxygen species (ROS) at the place of light application and starts destructive mechanism. PKCα plays a key role in the cell survival and its overexpression was observed in glioma cell lines. In the present study we aim to present the effectivity of the pts delivery in the glioma cells and consequences of silencing pkcα gene on cell death/survival after Hyp photo-activation. Pts can be delivered through two pathways: endocytosis - when cells are incubated with LDL/Hyp complex and Hyp transport through cellular membrane without any carrier. Preliminary results show that incubation of cells with or without LDL leads to PKCα activation. Photo-activated Hyp seems to be more effective in terms of apoptosis induction when compared to photo-activated LDL/Hyp complex. We have evaluated the influence of photo-activated Hyp on cell death in non-transfected and transfected (PKCα-) human glioma cells (U87-MG). Level of ROS production and type of cell death was notably affected by silencing pkca gene resulting in significant increase of necrosis after Hyp photo-activation.

Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea

PubMed Central

Ding, Zhong-Tao; Zhang, Zhi; Luo, Di; Zhou, Jin-Yan; Zhong, Juan; Yang, Jie; Xiao, Liang; Shu, Dan; Tan, Hong

2015-01-01

The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain. PMID:25955649

Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA.

PubMed

Kocan, Katherine M; Manzano-Roman, Raúl; de la Fuente, José

2007-05-01

RNA interference (RNAi) has become the most powerful experimental tool for the study of gene function in ticks. Subolesin, initially called 4D8, was found to be protective against tick infestations when used as a vaccine and was shown to be highly conserved among ixodid tick species at the nucleotide and protein levels. RNAi caused systemic silencing of subolesin and demonstrated that this protein is involved in regulation of tick feeding, reproduction, and development. Recently, these results were extended to the one-host tick Rhipicephalus (Boophilus) microplus in which injection of dsRNA into replete females resulted in transovarial silencing of subolesin expression in eggs and larvae. Herein, we report transovarial silencing of subolesin by RNAi in the three-host ticks, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis. Silencing of subolesin expression by RNAi in these tick species also affected subolesin expression in eggs and larvae. Transovarial RNAi appears to be a common mechanism in ixodid ticks and provides a simple method for the rapid characterization of tick genes involved in oviposition, embryogenesis, and larval development.

Investigating plasmodesmata genetics with virus-induced gene silencing and an agrobacterium-mediated GFP movement assay.

PubMed

Brunkard, Jacob O; Burch-Smith, Tessa M; Runkel, Anne M; Zambryski, Patricia

2015-01-01

Plasmodesmata (PD) are channels that connect the cytoplasm of adjacent plant cells, permitting intercellular transport and communication. PD function and formation are essential to plant growth and development, but we still know very little about the genetic pathways regulating PD transport. Here, we present a method for assaying changes in the rate of PD transport following genetic manipulation. Gene expression in leaves is modified by virus-induced gene silencing. Seven to ten days after infection with Tobacco rattle virus carrying a silencing trigger, the gene(s) of interest is silenced in newly arising leaves. In these new leaves, individual cells are then transformed with Agrobacterium to express GFP, and the rate of GFP diffusion via PD is measured. By measuring GFP diffusion both within the epidermis and between the epidermis and mesophyll, the assay can be used to study the effects of silencing a gene(s) on PD transport in general, or transport through secondary PD specifically. Plant biologists working in several fields will find this assay useful, since PD transport impacts plant physiology, development, and defense.

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS.

PubMed

Ding, Xin Shun; Mannas, Stephen W; Bishop, Bethany A; Rao, Xiaolan; Lecoultre, Mitchell; Kwon, Soonil; Nelson, Richard S

2018-01-01

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 ( HSP70-1 ) inserts in Nicotiana benthamiana and maize ( Zea mays ). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70 , silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors. © 2018 American Society of Plant Biologists. All Rights Reserved.

Virus-induced gene silencing in Rauwolfia species.

PubMed

Corbin, Cyrielle; Lafontaine, Florent; Sepúlveda, Liuda Johana; Carqueijeiro, Ines; Courtois, Martine; Lanoue, Arnaud; Dugé de Bernonville, Thomas; Besseau, Sébastien; Glévarec, Gaëlle; Papon, Nicolas; Atehortúa, Lucia; Giglioli-Guivarc'h, Nathalie; Clastre, Marc; St-Pierre, Benoit; Oudin, Audrey; Courdavault, Vincent

2017-07-01

Elucidation of the monoterpene indole alkaloid biosynthesis has recently progressed in Apocynaceae through the concomitant development of transcriptomic analyses and reverse genetic approaches performed by virus-induced gene silencing (VIGS). While most of these tools have been primarily adapted for the Madagascar periwinkle (Catharanthus roseus), the VIGS procedure has scarcely been used on other Apocynaceae species. For instance, Rauwolfia sp. constitutes a unique source of specific and valuable monoterpene indole alkaloids such as the hypertensive reserpine but are also well recognized models for studying alkaloid metabolism, and as such would benefit from an efficient VIGS procedure. By taking advantage of a recent modification in the inoculation method of the Tobacco rattle virus vectors via particle bombardment, we demonstrated that the biolistic-mediated VIGS approach can be readily used to silence genes in both Rauwolfia tetraphylla and Rauwolfia serpentina. After establishing the bombardment conditions minimizing injuries to the transformed plantlets, gene downregulation efficiency was evaluated at approximately a 70% expression decrease in both species by silencing the phytoene desaturase encoding gene. Such a gene silencing approach will thus constitute a critical tool to identify and characterize genes involved in alkaloid biosynthesis in both of these prominent Rauwolfia species.

Immunity-Associated Programmed Cell Death as a Tool for the Identification of Genes Essential for Plant Innate Immunity.

PubMed

Zhou, Bangjun; Zeng, Lirong

2018-01-01

Plants have evolved a sophisticated innate immune system to contend with potential infection by various pathogens. Understanding and manipulation of key molecular mechanisms that plants use to defend against various pathogens are critical for developing novel strategies in plant disease control. In plants, resistance to attempted pathogen infection is often associated with hypersensitive response (HR), a form of rapid programmed cell death (PCD) at the site of attempted pathogen invasion. In this chapter, we describe a method for rapid identification of genes that are essential for plant innate immunity. It combines virus-induced gene silencing (VIGS), a tool that is suitable for studying gene function in high-throughput, with the utilization of immunity-associated PCD, particularly HR-linked PCD as the readout of changes in plant innate immunity. The chapter covers from the design of gene fragment for VIGS, the agroinfiltration of the Nicotiana benthamian plants, to the use of immunity-associated PCD induced by twelve elicitors as the indicator of activation of plant immunity.

Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells

PubMed Central

Novobrantseva, Tatiana I; Borodovsky, Anna; Wong, Jamie; Klebanov, Boris; Zafari, Mohammad; Yucius, Kristina; Querbes, William; Ge, Pei; Ruda, Vera M; Milstein, Stuart; Speciner, Lauren; Duncan, Rick; Barros, Scott; Basha, Genc; Cullis, Pieter; Akinc, Akin; Donahoe, Jessica S; Narayanannair Jayaprakash, K; Jayaraman, Muthusamy; Bogorad, Roman L; Love, Kevin; Whitehead, Katie; Levins, Chris; Manoharan, Muthiah; Swirski, Filip K; Weissleder, Ralph; Langer, Robert; Anderson, Daniel G; de Fougerolles, Antonin; Nahrendorf, Matthias; Koteliansky, Victor

2012-01-01

Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells. PMID:23344621

RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells

PubMed Central

Maxwell, Michele M.; Pasinelli, Piera; Kazantsev, Aleksey G.; Brown, Robert H.

2004-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder resulting from selective death of motor neurons in the brain and spinal cord. In ≈25% of familial ALS cases, the disease is caused by dominantly acting point mutations in the gene encoding cytosolic Cu,Zn superoxide dismutase (SOD1). In cell culture and in rodent models of ALS, mutant SOD1 proteins exhibit dose-dependent toxicity; thus, agents that reduce mutant protein expression would be powerful therapeutic tools. A wealth of recent evidence has demonstrated that the mechanism of RNA-mediated interference (RNAi) can be exploited to achieve potent and specific gene silencing in vitro and in vivo. We have evaluated the utility of RNAi for selective silencing of mutant SOD1 expression in cultured cells and have identified small interfering RNAs capable of specifically inhibiting expression of ALS-linked mutant, but not wild-type, SOD1. We have investigated the functional effects of RNAi-mediated silencing of mutant SOD1 in cultured murine neuroblastoma cells. In this model, stable expression of mutant, but not wild-type, human SOD1 sensitizes cells to cytotoxic stimuli. We find that silencing of mutant SOD1 protects these cells against cyclosporin A-induced cell death. These results demonstrate a positive physiological effect caused by RNAi-mediated silencing of a dominant disease allele. The present study further supports the therapeutic potential of RNAi-based methods for the treatment of inherited human diseases, including ALS. PMID:14981234

Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger Nucleases1[W][OA

PubMed Central

Curtin, Shaun J.; Zhang, Feng; Sander, Jeffry D.; Haun, William J.; Starker, Colby; Baltes, Nicholas J.; Reyon, Deepak; Dahlborg, Elizabeth J.; Goodwin, Mathew J.; Coffman, Andrew P.; Dobbs, Drena; Joung, J. Keith; Voytas, Daniel F.; Stupar, Robert M.

2011-01-01

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform—a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting DICER-LIKE (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome. PMID:21464476

Eloquent silences: A musical and lexical analysis of conversation between oncologists and their patients.

PubMed

Bartels, Josef; Rodenbach, Rachel; Ciesinski, Katherine; Gramling, Robert; Fiscella, Kevin; Epstein, Ronald

2016-10-01

Silences in doctor-patient communication can be "connectional" and communicative, in contrast to silences that indicate awkwardness or distraction. Musical and lexical analyses can identify and characterize connectional silences in consultations between oncologists and patients. Two medical students and a professor of voice screened all 1211 silences over 2s in length from 124 oncology office visits. We developed a "strength of connection" taxonomy and examined ten connectional silences for lexical and musical features including pitch, volume, and speaker turn-taking rhythm. We identified connectional silences with good reliability. Typical dialog rhythms surrounding connectional silences are characterized by relatively equal turn lengths and frequent short vocalizations. We found no pattern of volume and pitch variability around these silences. Connectional silences occurred in a wide variety of lexical contexts. Particular patterns of dialog rhythm mark connectional silences. Exploring structures of connectional silence extends our understanding of the audio-linguistic conditions that mark patient-clinician connection. Communicating with an awareness of pitch, rhythm, and silence - in addition to lexical content - can facilitate shared understanding and emotional connection. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cell-autonomous-like silencing of GFP-partitioned transgenic Nicotiana benthamiana.

PubMed

Sohn, Seong-Han; Frost, Jennifer; Kim, Yoon-Hee; Choi, Seung-Kook; Lee, Yi; Seo, Mi-Suk; Lim, Sun-Hyung; Choi, Yeonhee; Kim, Kook-Hyung; Lomonossoff, George

2014-08-01

We previously reported the novel partitioning of regional GFP-silencing on leaves of 35S-GFP transgenic plants, coining the term "partitioned silencing". We set out to delineate the mechanism of partitioned silencing. Here, we report that the partitioned plants were hemizygous for the transgene, possessing two direct-repeat copies of 35S-GFP. The detection of both siRNA expression (21 and 24 nt) and DNA methylation enrichment specifically at silenced regions indicated that both post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) were involved in the silencing mechanism. Using in vivo agroinfiltration of 35S-GFP/GUS and inoculation of TMV-GFP RNA, we demonstrate that PTGS, not TGS, plays a dominant role in the partitioned silencing, concluding that the underlying mechanism of partitioned silencing is analogous to RNA-directed DNA methylation (RdDM). The initial pattern of partitioned silencing was tightly maintained in a cell-autonomous manner, although partitioned-silenced regions possess a potential for systemic spread. Surprisingly, transcriptome profiling through next-generation sequencing demonstrated that expression levels of most genes involved in the silencing pathway were similar in both GFP-expressing and silenced regions although a diverse set of region-specific transcripts were detected.This suggests that partitioned silencing can be triggered and regulated by genes other than the genes involved in the silencing pathway. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Systematic evaluation of markers used for the identification of human induced pluripotent stem cells

PubMed Central

Bharathan, Sumitha Prameela; Manian, Kannan Vrindavan; Aalam, Syed Mohammed Musheer; Palani, Dhavapriya; Deshpande, Prashant Ajit; Pratheesh, Mankuzhy Damodaran; Srivastava, Alok

2017-01-01

ABSTRACT Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4+ and TRA-1-60+ cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs). PMID:28089995

Double-stranded RNA delivery through soaking mediates silencing of the muscle protein 20 and increases mortality to the Asian citrus psyllid, Diaphorina citri.

PubMed

Yu, Xiudao; Gowda, Siddarame; Killiny, Nabil

2017-09-01

Asian citrus psyllid, Diaphorina citri Kuwayama, is the most important economic pest of citrus because it transmits Candidatus Liberibacter asiaticus (CLas), the causal agent of huanglongbing (HLB). Silencing genes by RNA interference (RNAi) is a promising approach for controlling D. citri. RNAi-based insect management strategies depend on the selection of suitable target genes. The muscle protein 20 gene DcMP20 was characterized from D. citri in an effort to impair proper muscle development through RNAi. Phylogenetic analysis showed that DcMP20 was more closely related to MP20 from Drosophila compared with its counterpart from other insect species. Developmental expression analysis revealed that transcription of DcMP20 was development dependent and reached a maximum level in the last instar (fourth-fifth) of the nymphal stage. The extent of RNAi in D. citri was dose dependent, with dsRNA-DcMP20 at 75 ng µL -1 being sufficient to knock down endogenous DcMP20 expression, which resulted in significant mortality and reduced body weight that positively correlated with the silencing of DcMP20. No effect was found when dsRNA-GFP or water was used, indicating the specific effect of dsRNA-DcMP20. Our results suggest that dsRNA can be delivered to D. citri through soaking, and DcMP20 is an effective RNAi target to be used in the management of D. citri. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

"Listening Silence" and Its Discursive Effects

ERIC Educational Resources Information Center

Applebaum, Barbara

2016-01-01

While researchers have studied how white silence protects white innocence and white ignorance, in this essay Barbara Applebaum explores a form of white silence that she refers to as "listening silence" in which silence protects white innocence but does not necessarily promote resistance to learning. White listening silence can appear to…

Identification of the TaBTF3 gene in wheat (Triticum aestivum L.) and the effect of its silencing on wheat chloroplast, mitochondria and mesophyll cell development.

PubMed

Ma, Hong-Zhen; Liu, Guo-Qin; Li, Cheng-Wei; Kang, Guo-Zhang; Guo, Tian-Cai

2012-10-05

The full-length cDNA (882bp) and DNA (1742bp) sequences encoding a basic transcription factor 3, designated as TaBTF3, were first isolated from common wheat (Triticum aestivum L.). Subcellular localization studies revealed that the TaBTF3 protein was mainly located in the cytoplasm and nucleus. In TaBTF3-silenced transgenic wheat seedlings obtained using the Virus-induced gene silencing (VIGS) method, the chlorophyll pigment content was markedly reduced. However, the malonaldehyde (MDA) and H(2)O(2) contents were enhanced, and the structure of the wheat mesophyll cell was seriously damaged. Furthermore, transcripts of the chloroplast- and mitochondrial-encoded genes were significantly reduced in TaBTF3-silenced transgenic wheat plants. These results suggest that the TaBTF3 gene might function in the development of the wheat chloroplast, mitochondria and mesophyll cell. This paper is the first report to describe the involvement of TaBTF3 in maintaining the normal plant mesophyll cell structure. Copyright © 2012 Elsevier Inc. All rights reserved.

Silence or the Sound of Limpid Water: Disability, Power, and the Educationalisation of Silence

ERIC Educational Resources Information Center

Verstraete, Pieter

2017-01-01

In this article the history of silence is looked at from an educational perspective. By closely examining the way three nineteenth-century authors--who all based their educational theories on concrete experiences with persons with disabilities--have related themselves to silence, it will be argued that silence has been educationalised. Silence has…

Metabolic Engineering of Wheat Provitamin A by Simultaneously Overexpressing CrtB and Silencing Carotenoid Hydroxylase (TaHYD).

PubMed

Zeng, Jian; Wang, Xiatian; Miao, Yingjie; Wang, Cheng; Zang, Mingli; Chen, Xi; Li, Miao; Li, Xiaoyan; Wang, Qiong; Li, Kexiu; Chang, Junli; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

2015-10-21

Increasing the provitamin A content in staple crops via carotenoid metabolic engineering is one way to address vitamin A deficiency. In this work a combination of methods was applied to specifically increase β-carotene content in wheat by metabolic engineering. Endosperm-specific silencing of the carotenoid hydroxylase gene (TaHYD) increased β-carotene content 10.5-fold to 1.76 μg g(-1) in wheat endosperm. Overexpression of CrtB introduced an additional flux into wheat, accompanied by a β-carotene increase of 14.6-fold to 2.45 μg g(-1). When the "push strategy" (overexpressing CrtB) and "block strategy" (silencing TaHYD) were combined in wheat metabolic engineering, significant levels of β-carotene accumulation were obtained, corresponding to an increase of up to 31-fold to 5.06 μg g(-1). This is the first example of successful metabolic engineering to specifically improve β-carotene content in wheat endosperm through a combination of methods and demonstrates the potential of genetic engineering for specific nutritional enhancement of wheat.

Functional analysis of a weak viral RNA silencing suppressor using two GFP variants as silencing inducers.

PubMed

Mann, Krin S; Dietzgen, Ralf G

2017-01-01

RNA silencing in plants can be triggered by the introduction of an exogenous gene. Green fluorescent protein (GFP) has been widely used as a visual reporter to study RNA silencing and viral-mediated suppression of RNA silencing in the model plant Nicotiana benthamiana. In transgenic N. benthamiana plants expressing an endoplasmic reticulum targeted GFP variant (16c) known as mGFP5, RNA silencing can be induced by ectopic over-expression of mGFP5. However, other GFP variants can also be used to induce GFP silencing in these plants. We compared the efficiency to induce local and systemic silencing of two commonly used GFP variants: enhanced GFP (eGFP) and mGFP5. Using lettuce necrotic yellows virus (LNYV) P protein to suppress GFP silencing, we demonstrate that eGFP gene, which is 76% identical at the nucleotide level to the endogenously expressed mGFP5 in 16c plants, triggers silencing more slowly and concurrently prolongs detectable silencing suppressor activity of the weak LNYV P suppressor, compared to the homologous mGFP5 gene. The use of eGFP as RNA silencing inducer in wild type or 16c plants appears to be a useful tool in identifying and analysing weak viral RNA silencing suppressor proteins whose activity might otherwise have been masked when challenged by a stronger RNA silencing response. We also show that reducing the dosage of strong dsRNA silencing inducers in conjunction with their homologous GFP targets facilitates the discovery and analysis of "weaker" RNA silencing suppressor activities. Copyright © 2016 Elsevier B.V. All rights reserved.

Spontaneous Behavior in Noise and Silence: A Possible New Measure to Assess Tinnitus in Guinea Pigs

PubMed Central

Heeringa, Amarins N.; Agterberg, Martijn J. H.; van Dijk, Pim

2014-01-01

This study describes two experiments that were conducted in search for a behavioral paradigm to test for tinnitus in guinea pigs. Conditioning paradigms are available to determine the presence of tinnitus in animals and are based on the assumption that tinnitus impairs their ability to detect silent intervals in continuous noise. Guinea pigs have not been subjected to these paradigms yet; therefore, we investigated whether guinea pigs could be conditioned in the two-way shuttle-box paradigm to respond to silent intervals in noise. Even though guinea pigs could be trained relatively easy to respond to the presence of a noise interval, training guinea pigs to silent intervals in noise was unsuccessful. Instead, it appeared that they became immobile when the continuous stimulus was suddenly stopped. This was confirmed by the next experiment, in which we subjected guinea pigs to alternating intervals of noise and silence with a random duration between 30 and 120 s. Indeed, guinea pigs were significantly longer immobile during silence compared to during noise. By interpreting immobility as a signature of perceiving silence, we hypothesized that the presence of tinnitus would reduce immobility in silence. Therefore, we unilaterally exposed one group of guinea pigs to an 11-kHz tone of 124 dB sound pressure level for 1 h. A subset of the exposed animals was significantly more active in silence, but also more active in noise, as compared to the control group. The increased mobility during silent intervals might represent tinnitus. However, the increased mobility in noise of this group implies that the observed behavior could have derived from, e.g., an overall increase in activity. Therefore, conducting validation experiments is very important before implementing this method as a new screening tool for tinnitus. Follow-up experiments are discussed to further elucidate the origin of the increased mobility in both silence and noise. PMID:25360130

Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

PubMed Central

2010-01-01

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 in human clinical trials. PMID:20836854

RNA Interference: Biology, Mechanism, and Applications

PubMed Central

Agrawal, Neema; Dasaradhi, P. V. N.; Mohmmed, Asif; Malhotra, Pawan; Bhatnagar, Raj K.; Mukherjee, Sunil K.

2003-01-01

Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes. PMID:14665679

An RNAi in silico approach to find an optimal shRNA cocktail against HIV-1

PubMed Central

2010-01-01

Background HIV-1 can be inhibited by RNA interference in vitro through the expression of short hairpin RNAs (shRNAs) that target conserved genome sequences. In silico shRNA design for HIV has lacked a detailed study of virus variability constituting a possible breaking point in a clinical setting. We designed shRNAs against HIV-1 considering the variability observed in naïve and drug-resistant isolates available at public databases. Methods A Bioperl-based algorithm was developed to automatically scan multiple sequence alignments of HIV, while evaluating the possibility of identifying dominant and subdominant viral variants that could be used as efficient silencing molecules. Student t-test and Bonferroni Dunn correction test were used to assess statistical significance of our findings. Results Our in silico approach identified the most common viral variants within highly conserved genome regions, with a calculated free energy of ≥ -6.6 kcal/mol. This is crucial for strand loading to RISC complex and for a predicted silencing efficiency score, which could be used in combination for achieving over 90% silencing. Resistant and naïve isolate variability revealed that the most frequent shRNA per region targets a maximum of 85% of viral sequences. Adding more divergent sequences maintained this percentage. Specific sequence features that have been found to be related with higher silencing efficiency were hardly accomplished in conserved regions, even when lower entropy values correlated with better scores. We identified a conserved region among most HIV-1 genomes, which meets as many sequence features for efficient silencing. Conclusions HIV-1 variability is an obstacle to achieving absolute silencing using shRNAs designed against a consensus sequence, mainly because there are many functional viral variants. Our shRNA cocktail could be truly effective at silencing dominant and subdominant naïve viral variants. Additionally, resistant isolates might be targeted under specific antiretroviral selective pressure, but in both cases these should be tested exhaustively prior to clinical use. PMID:21172023

Elucidating the role of highly homologous Nicotiana benthamiana ubiquitin E2 gene family members in plant immunity through an improved virus-induced gene silencing approach.

PubMed

Zhou, Bangjun; Zeng, Lirong

2017-01-01

Virus-induced gene silencing (VIGS) has been used in many plant species as an attractive post transcriptional gene silencing (PTGS) method for studying gene function either individually or at large-scale in a high-throughput manner. However, the specificity and efficiency for knocking down members of a highly homologous gene family have remained to date a significant challenge in VIGS due to silencing of off-targets. Here we present an improved method for the selection and evaluation of gene fragments used for VIGS to specifically and efficiently knock down members of a highly homologous gene family. Using this method, we knocked down twelve and four members, respectively of group III of the gene family encoding ubiquitin-conjugating enzymes (E2) in Nicotiana benthamiana . Assays using these VIGS-treated plants revealed that the group III E2s are essential for plant development, plant immunity-associated reactive oxygen species (ROS) production, expression of the gene NbRbohB that is required for ROS production, and suppression of immunity-associated programmed cell death (PCD) by AvrPtoB, an effector protein of the bacterial pathogen Pseudomons syringae . Moreover, functional redundancy for plant development and ROS production was found to exist among members of group III E2s. We have found that employment of a gene fragment as short as approximately 70 base pairs (bp) that contains at least three mismatched nucleotides to other genes within any 21-bp sequences prevents silencing of off-target(s) in VIGS. This improved approach in the selection and evaluation of gene fragments allows for specific and efficient knocking down of highly homologous members of a gene family. Using this approach, we implicated N. benthamiana group III E2s in plant development, immunity-associated ROS production, and suppression of multiple immunity-associated PCD by AvrPtoB. We also unraveled functional redundancy among group III members in their requirement for plant development and plant immunity-associated ROS production.

Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome

NASA Astrophysics Data System (ADS)

Lu, Rui; Folimonov, Alexey; Shintaku, Michael; Li, Wan-Xiang; Falk, Bryce W.; Dawson, William O.; Ding, Shou-Wei

2004-11-01

Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the 20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP- or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host. RNA interference | citrus tristeza virus | virus synergy | antiviral immunity

Distribution of a limited Sir2 protein pool regulates the strength of yeast rDNA silencing and is modulated by Sir4p.

PubMed Central

Smith, J S; Brachmann, C B; Pillus, L; Boeke, J D

1998-01-01

Transcriptional silencing in Saccharomyces cerevisiae occurs at the silent mating-type loci HML and HMR, at telomeres, and at the ribosomal DNA (rDNA) locus RDN1. Silencing in the rDNA occurs by a novel mechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2. SIR4, essential for other silenced loci, paradoxically inhibits rDNA silencing. In this study, we elucidate a regulatory mechanism for rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2 protein levels. The endogenous level of Sir2p was shown to be limiting for rDNA silencing. Furthermore, small changes in Sir2p levels altered rDNA silencing strength. In rDNA silencing phenotypes, sir2 mutations were shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediated through SIR2. Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reduced by overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p. This negative effect of SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibits the rDNA silencing function of SIR2. Finally, genetic manipulations of SIR4 previously shown to promote extended life span also resulted in enhanced rDNA silencing. We propose a simple model in which telomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein. PMID:9649515

Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

PubMed Central

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

Dorsal and ventral hippocampal adult-born neurons contribute to context fear memory.

PubMed

Huckleberry, Kylie A; Shue, Francis; Copeland, Taylor; Chitwood, Raymond A; Yin, Weiling; Drew, Michael R

2018-06-02

The hippocampus contains one of the few neurogenic niches within the adult brain-the subgranular zone of the dentate gyrus. The functional significance of adult-born neurons in this region has been characterized using context fear conditioning, a Pavlovian paradigm in which animals learn to associate a location with danger. Ablation or silencing of adult-born neurons impairs both acquisition and recall of contextual fear conditioning, suggesting that these neurons contribute importantly to hippocampal memory. Lesion studies indicate that CFC depends on neural activity in both the dorsal and ventral hippocampus, subregions with unique extrahippocampal connectivity and behavioral functions. Because most studies of adult neurogenesis have relied on methods that permanently ablate neurogenesis throughout the entire hippocampus, little is known about how the function of adult-born neurons varies along the dorsal-ventral axis. Using a Nestin-CreER T2 mouse line to target the optogenetic silencer Archaerhodopsin to adult-born neurons, we compared the contribution of dorsal and ventral adult-born neurons to acquisition, recall, and generalization of CFC. Acquisition of CFC was impaired when either dorsal or ventral adult-born neurons were silenced during training. Silencing dorsal or ventral adult-born neurons during test sessions decreased context-evoked freezing but did not impair freezing in a hippocampus-independent tone-shock freezing paradigm. Silencing adult-born neurons modestly reduced generalization of fear. Our data indicate that adult-born neurons in the dorsal and ventral hippocampus contribute to both memory acquisition and recall. The comparatively large behavioral effects of silencing a small number of adult-born neurons suggest that these neurons make a unique and powerful contribution to hippocampal function.

A Functional Element Necessary for Fetal Hemoglobin Silencing

PubMed Central

Sankaran, Vijay G.; Xu, Jian; Byron, Rachel; Greisman, Harvey A.; Fisher, Chris; Weatherall, David J.; Sabath, Daniel E.; Groudine, Mark; Orkin, Stuart H.; Premawardhena, Anuja; Bender, M.A.

2011-01-01

BACKGROUND An improved understanding of the regulation of the fetal hemoglobin genes holds promise for the development of targeted therapeutic approaches for fetal hemoglobin induction in the β-hemoglobinopathies. Although recent studies have uncovered trans-acting factors necessary for this regulation, limited insight has been gained into the cis-regulatory elements involved. METHODS We identified three families with unusual patterns of hemoglobin expression, suggestive of deletions in the locus of the β-globin gene (β-globin locus). We performed array comparative genomic hybridization to map these deletions and confirmed breakpoints by means of polymerase-chain-reaction assays and DNA sequencing. We compared these deletions, along with previously mapped deletions, and studied the trans-acting factors binding to these sites in the β-globin locus by using chromatin immunoprecipitation. RESULTS We found a new (δβ)0-thalassemia deletion and a rare hereditary persistence of fetal hemoglobin deletion with identical downstream breakpoints. Comparison of the two deletions resulted in the identification of a small intergenic region required for γ-globin (fetal hemoglobin) gene silencing. We mapped a Kurdish β0-thalassemia deletion, which retains the required intergenic region, deletes other surrounding sequences, and maintains fetal hemoglobin silencing. By comparing these deletions and other previously mapped deletions, we elucidated a 3.5-kb intergenic region near the 5′ end of the δ-globin gene that is necessary for γ-globin silencing. We found that a critical fetal hemoglobin silencing factor, BCL11A, and its partners bind within this region in the chromatin of adult erythroid cells. CONCLUSIONS By studying three families with unusual deletions in the β-globin locus, we identified an intergenic region near the δ-globin gene that is necessary for fetal hemoglobin silencing. (Funded by the National Institutes of Health and others.) PMID:21879898

Silence and voicing accumulations in Italian primary school teachers with and without voice disorders

PubMed Central

Bottalico, Pasquale; Graetzer, Simone; Astolfi, Arianna; Hunter, Eric J.

2016-01-01

Objectives The relationship between the silence and voicing accumulations of primary school teachers and the teachers’ clinical status was examined. The goal was to determine whether more voicing accumulations and fewer silence accumulations were measured for the vocally unhealthy subjects than for the healthy subjects, which would imply more vocal loading and fewer short-term recovery moments. Methods 26 Italian primary school teachers were allocated by clinicians to three groups: (1) with organic voice disorders, (2) with subjectively mild organic alteration and/or functional voice symptoms, and (3) normal voice quality and physiology. Continuous silence and voicing periods were measured with the APM3200 during the teachers’ 4-hour workdays. The accumulations were grouped into 7 time intervals, ranging from 0.03–0.9 s to 3.16–10 s, according to Italian prosody. The effects of group on silence and voicing accumulations were evaluated. Results Regarding silence accumulations, Group 1 accumulated higher values in intervals between 0.1 and 3.15 s than other groups, while Groups 2 and 3 did not differ from each other. Voicing accumulations between 0.17 and 3.15 s were higher for subjects with a structural disorder. A higher time dose was accumulated by these subjects (40.6%) than other subjects (Group 2, 31.9%; Group 3, 32.3%). Conclusions While previous research has suggested that a rest period of a few seconds may produce some vocal fatigue recovery, these results indicate that periods shorter than 3.16 s may not have an observable effect on recovery. The results provide insight into how vocal fatigue and vocal recovery may relate to voice disorders in occupational voice users. PMID:27316793

RNA silencing in the life cycle of soybean: multiple restriction systems and spatiotemporal variation associated with plant architecture.

PubMed

Mori, Ayumi; Sato, Hiroshi; Kasai, Megumi; Yamada, Tetsuya; Kanazawa, Akira

2017-06-01

The expression of transgenes introduced into a plant genome is sometimes suppressed by RNA silencing. Although local and systemic spread of RNA silencing have been studied, little is known about the mechanisms underlying spatial and temporal variation in transgene silencing between individual plants or between plants of different generations, which occurs seemingly stochastically. Here, we analyzed the occurrence, spread, and transmission of RNA silencing of the green fluorescent protein (GFP) gene over multiple generations of the progeny of a single soybean transformant. Observation of GFP fluorescence in entire plants of the T 3 -T 5 generations indicated that the initiation and subsequent spread of GFP silencing varied between individuals, although this GFP silencing most frequently began in the primary leaves. In addition, GFP silencing could spread into the outer layer of seed coat tissues but was hardly detectable in the embryos. These results are consistent with the notion that transgene silencing involves its reset during reproductive phase, initiation after germination, and systemic spread in each generation. GFP silencing was absent in the pulvinus, suggesting that its cortical cells inhibit cell-to-cell spread or induction of RNA silencing. The extent of GFP silencing could differ between the stem and a petiole or between petiolules, which have limited vascular bundles connecting them and thus deter long-distant movement of silencing. Taken together, these observations indicate that the initiation and/or spread of RNA silencing depend on specific features of the architecture of the plant in addition to the mechanisms that can be conserved in higher plants.

Silence and the Notion of the Commons.

ERIC Educational Resources Information Center

Franklin, Ursula

1994-01-01

Stresses the value of silence, the right to have silence, and how technology has manipulated the sound environment and therefore taken silence out of common availability. Discusses noise pollution and the manipulative use of sound for private gain. Suggests taking action to restore the right to silence. (LP)

The Effects of Vocational High School Teachers' Perceived Trust on Organizational Silence

ERIC Educational Resources Information Center

Saglam, Aycan Çiçek

2016-01-01

The objective of this research is to reveal the effects of vocational school teachers' perceived organizational trust on organizational silence. For this purpose, at first teachers' perception on sub-dimensions of organizational silence and organizational trust, which are respectively "acquiescent silence," "defensive silence,"…

Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses

PubMed Central

Lakatos, Lóránt; Szittya, György; Silhavy, Dániel; Burgyán, József

2004-01-01

RNA silencing is an evolutionarily conserved surveillance system that occurs in a broad range of eukaryotic organisms. In plants, RNA silencing acts as an antiviral system; thus, successful virus infection requires suppression of gene silencing. A number of viral suppressors have been identified so far; however, the molecular bases of silencing suppression are still poorly understood. Here we show that p19 of Cymbidium ringspot virus (CymRSV) inhibits RNA silencing via its small RNA-binding activity in vivo. Small RNAs bound by p19 in planta are bona fide double-stranded siRNAs and they are silencing competent in the in vitro RNA-silencing system. p19 also suppresses RNA silencing in the heterologous Drosophila in vitro system by preventing siRNA incorporation into RISC. During CymRSV infection, p19 markedly diminishes the amount of free siRNA in cells by forming p19–siRNA complexes, thus making siRNAs inaccessible for effector complexes of RNA-silencing machinery. Furthermore, the obtained results also suggest that the p19-mediated sequestration of siRNAs in virus-infected cells blocks the spread of the mobile, systemic signal of RNA silencing. PMID:14976549

Between the Unbearable Weight and Lightness of the Past. Banal Silence in Spain's Post-Dictatorship Memory Politics.

PubMed

Brescó de Luna, Ignacio

2018-05-04

This paper explores the role of silence in Spain's post-dictatorship memory politics. More specifically, the paper examines the forgotten Spanish colonial past in North Africa vis-à-vis the so-called pact of silence that accompanied Spain's transition to democracy after the Franco dictatorship. Drawing on various theoretical approaches in relation to collective memory, traditionally assumed associations between silence and forgetting are questioned. As is argued, silence may nurture and preserve memory just as it may also feed into forgetting. In the former case, silence typically enshrouds a living memory of a past that still weighs on the present, as the pact of silence in Spain clearly illustrates. In the latter case, silence signals a past perceived as already left behind and alien to society's current problems, as is the case of the Spanish colonial past in North Africa. In order to further explore the latter case, the notion of banal silence is introduced. Such notion points to cases where silence over certain contentious historical issues goes unnoticed by society, thus becoming naturalized. The paper concludes with some final reflexions on memory, banal silence and political change.

The system with zwitterionic lactose-based surfactant for complexation and delivery of small interfering ribonucleic acid—A structural and spectroscopic study

NASA Astrophysics Data System (ADS)

Skupin, Michalina; Sobczak, Krzysztof; Zieliński, Ryszard; Kozak, Maciej

2016-05-01

Systems suitable for the effective preparation of complexes with siRNA (small interfering RNA) are at the center of interest in the area of research work on the delivery of the RNA-based drugs (RNA-therapeutics). This article presents results of a study on the structural effects associated with siRNA complexation by a surfactant comprising a lactose group (N-(3-propanesulfone)-N-dodecyl-amino-beta-D-lactose hydrochloride, LA12). The double stranded siRNA oligomer (21 base pairs) used in this study is responsible for silencing a gene that can be important in the therapy of myotonic dystrophy type 1. The obtained siRNA/LA12 lipoplexes were studied using the methods of small angle scattering of synchrotron radiation, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, and electrophoretic mobility tests. Lipoplexes form in solution stable lamellar or cubic phases. The surfactant selected for the study shows much lower cytotoxicity and good complexation abilities of siRNA than dicationic or polycationic surfactants.

The system with zwitterionic lactose-based surfactant for complexation and delivery of small interfering ribonucleic acid—A structural and spectroscopic study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Skupin, Michalina; Sobczak, Krzysztof; Zieliński, Ryszard

Systems suitable for the effective preparation of complexes with siRNA (small interfering RNA) are at the center of interest in the area of research work on the delivery of the RNA-based drugs (RNA-therapeutics). This article presents results of a study on the structural effects associated with siRNA complexation by a surfactant comprising a lactose group (N-(3-propanesulfone)-N-dodecyl-amino-beta-D-lactose hydrochloride, LA12). The double stranded siRNA oligomer (21 base pairs) used in this study is responsible for silencing a gene that can be important in the therapy of myotonic dystrophy type 1. The obtained siRNA/LA12 lipoplexes were studied using the methods of small anglemore » scattering of synchrotron radiation, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, and electrophoretic mobility tests. Lipoplexes form in solution stable lamellar or cubic phases. The surfactant selected for the study shows much lower cytotoxicity and good complexation abilities of siRNA than dicationic or polycationic surfactants.« less

Ex vivo evaluation of antifibrotic compounds in skin scarring: EGCG and silencing of PAI-1 independently inhibit growth and induce keloid shrinkage.

PubMed

Syed, Farhatullah; Bagabir, Rania A; Paus, Ralf; Bayat, Ardeshir

2013-08-01

Keloid disease (KD) is a common fibroproliferative disorder of unknown etiopathogenesis. Its unique occurrence in human skin and lack of animal models pose challenges for KD research. The lack of a suitable model in KD and over-reliance on cell culture has hampered the progress in developing new treatments. Therefore, we evaluated the effect of two promising candidate antifibrotic therapies: (-)-epigallocatechin-3-gallate (EGCG) and plasminogen activator inhibitor-1 (PAI-1) silencing in a long-term human keloid organ culture (OC). Four millimeters of air-liquid interface (ALI) keloid explants on collagen gel matrix in serum-free medium (n=8 cases) were treated with different modalities (EGCG treatment; PAI-1 knockdown by short interfering RNA (siRNA) and application of dexamethasone (DEX) as control). Normal skin (n=6) was used as control (only for D0 keloid-untreated comparison). Besides routine histology and quantitative (immuno-) histomorphometry, the key phenotypic and growth parameters of KD were assessed. Results demonstrated that EGCG reduced keloid volume significantly (40% by week 4), increased apoptosis (≥40% from weeks 1 to 4), and decreased proliferation (≤17% in week 2). EGCG induced epidermal shrinkage, reduced collagen-I and -III at mRNA and protein levels, depleted 98% of keloid-associated mast cells, and reduced the percentage of both cellularity and blood vessel count by week 4. Knockdown of PAI-1 significantly reduced keloid volume by 28% in week 4, respectively, and reduced collagen-I and -III at both mRNA and protein levels. As expected, DEX increased keloid apoptosis, decreased keloid proliferation, and collagen synthesis, but induced connective tissue growth factor overexpression. In conclusion, using keloid OC model, we provide the first functional evidence for testing candidate antifibrotic compounds in KD. We show that EGCG and PAI-1 silencing effectively inhibits growth and induces shrinkage of human keloid tissue in situ. Therefore, the application of EGCG, PAI-1 silencing, and other emerging compounds tested using this model may provide effective treatment and potentially aid in the prevention of recurrence of KD following surgery.

RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles

NASA Astrophysics Data System (ADS)

Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Swee Chuan, Tjin; Yong, Ken-Tye

2015-09-01

RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications.

Poly(amidoamine) Dendrimer Nanocarriers and Their Aerosol Formulations for siRNA Delivery to the Lung Epithelium

PubMed Central

2015-01-01

Small interfering RNA (siRNA)-based therapies have great promise in the treatment of a number of prevalent pulmonary disorders including lung cancer, asthma and cystic fibrosis. However, progress in this area has been hindered due to the lack of carriers that can efficiently deliver siRNA to lung epithelial cells, and also due to challenges in developing oral inhalation (OI) formulations for the regional administration of siRNA and their carriers to the lungs. In this work we report the ability of generation four, amine-terminated poly(amidoamine) (PAMAM) dendrimer (G4NH2)–siRNA complexes (dendriplexes) to silence the enhanced green fluorescent protein (eGFP) gene on A549 lung alveolar epithelial cells stably expressing eGFP. We also report the formulation of the dendriplexes and their aerosol characteristics in propellant-based portable OI devices. The size and gene silencing ability of the dendriplexes was seen not to be a strong function of the N/P ratio. Silencing efficiencies of up to 40% are reported. Stable dispersions of the dendriplexes encapsulated in mannitol and also in a biodegradable and water-soluble co-oligomer were prepared in hydrofluoroalkane (HFA)-based pressurized metered-dose inhalers (pMDIs). Their aerosol characteristics were very favorable, and conducive to deep lung deposition, with respirable fractions of up to 77%. Importantly, siRNA formulated as dendriplexes in pMDIs was shown to keep its integrity after the particle preparation processes, and also after long-term exposures to HFA. The relevance of this study stems from the fact that this is the first work to report the formulation of inhalable siRNA with aerosol properties suitable to deep lung deposition using pMDIs devices that are the least expensive and most widely used portable inhalers. This study is relevant because, also for the first time, it shows that siRNA–G4NH2 dendriplexes can efficiently target lung alveolar epithelial A549 cells and silence genes even after siRNA has been exposed to the propellant environment. PMID:24811243

Changing Knowledge and Attitudes with a Middle School Mental Health Education Curriculum

PubMed Central

Wahl, Otto F.; Susin, Janet; Kaplan, Lorraine; Lax, Amy; Zatina, Dayna

2011-01-01

Purpose This research tested the effectiveness of a widely used mental health education curriculum in changing knowledge and attitudes about mental illness. Method Middle school students from four schools were provided the Breaking the Silence: Teaching the Next Generation About Mental Illness mental health instruction while students from other classes at the same schools received usual class instruction. Students completed questionnaires assessing knowledge, attitudes, and social distance preferences before, immediately after, and six weeks after the instruction was given. Results Students given the Breaking the Silence instruction showed improvements in knowledge about mental illness, attitudes toward mental illness, and willingness to interact with people with mental illnesses. Students in the comparison classes showed no changes. Conclusions Breaking the Silence was an effective means of improving the knowledge and attitudes of middle school students about mental illness. Implications An easy-to-administer and effective curriculum, Breaking the Silence is available to teachers and schools to help improve understanding and acceptance of people with mental illness. Such a curriculum, introduced during childhood and adolescence, may help to prevent the negative attitudes and misunderstanding that characterize adult perceptions of mental illness. PMID:21731851

MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

PubMed Central

Zhong, Bushuai; Zhang, Yanli; Yan, Yibo; Wang, Ziyu; Ying, Shijia; Huang, Mingrui; Wang, Feng

2014-01-01

Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats. PMID:25244645

Transcriptional silencing of a transgene by RNAi in the soma of C. elegans.

PubMed

Grishok, Alla; Sinskey, Jina L; Sharp, Phillip A

2005-03-15

The silencing of transgene expression at the level of transcription in the soma of Caenorhabditis elegans through an RNAi-dependent pathway has not been previously characterized. Most gene silencing due to RNAi in C. elegans occurs at the post-transcriptional level. We observed transcriptional silencing when worms containing the elt-2::gfp/LacZ transgene were fed RNA produced from the commonly used L4440 vector. The transgene and the vector share plasmid backbone sequences. This transgene silencing depends on multiple RNAi pathway genes, including dcr-1, rde-1, rde-4, and rrf-1. Unlike post-transcriptional gene silencing in worms, elt-2::gfp/LacZ silencing is dependent on the PAZ-PIWI protein Alg-1 and on the HP1 homolog Hpl-2. The latter is a chromatin silencing factor, and expression of the transgene is inhibited at the level of intron-containing precursor mRNA. This inhibition is accompanied by a decrease in the acetylation of histones associated with the transgene. This transcriptional silencing in the soma can be distinguished from transgene silencing in the germline by its inability to be transmitted across generations and its dependence on the rde-1 gene. We therefore define this type of silencing as RNAi-induced Transcriptional Gene Silencing (RNAi-TGS). Additional chromatin-modifying components affecting RNAi-TGS were identified in a candidate RNAi screen.

Practising Silence in Teaching

ERIC Educational Resources Information Center

Forrest, Michelle

2013-01-01

The concept "silence" has diametrically opposed meanings; it connotes peace and contemplation as well as death and oblivion. Silence can also be considered a practice. There is keeping the rule of silence to still the mind and find inner truth, as well as forcibly silencing in the sense of subjugating another to one's own purposes.…

Silencing Genes in the Heart.

PubMed

Fechner, Henry; Vetter, Roland; Kurreck, Jens; Poller, Wolfgang

2017-01-01

Silencing of cardiac genes by RNA interference (RNAi) has developed into a powerful new method to treat cardiac diseases. Small interfering (si)RNAs are the inducers of RNAi, but cultured primary cardiomyocytes and heart are highly resistant to siRNA transfection. This can be overcome by delivery of small hairpin (sh)RNAs or artificial microRNA (amiRNAs) by cardiotropic adeno-associated virus (AAV) vectors. Here we describe as example of the silencing of a cardiac gene, the generation and cloning of shRNA, and amiRNAs directed against the cardiac protein phospholamban. We further describe the generation of AAV shuttle plasmids with self complementary vector genomes, the production of AAV vectors in roller bottles, and their purification via iodixanol gradient centrifugation and concentration with filter systems. Finally we describe the preparation of primary neonatal rat cardiomyocytes (PNRC), the transduction of PNRC with AAV vectors, and the maintenance of the transduced cell culture.

Self-silencing and age as risk factors for sexually acquired HIV in midlife and older women.

PubMed

Jacobs, Robin J; Thomlison, Barbara

2009-02-01

Objectives. This study explores the contribution of psychosocial factors on sex behaviors of midlife and older women. Methods. A community-based sample of ethnically diverse women (N = 572) between the ages of 50 and 93 completed standardized measures of self-silencing, self-esteem, sensation seeking behavior, HIV-related stigma behavior, sexual assertiveness, and safer sex behaviors. Results. Results from the regression analysis indicated the model significantly predicted safer sex behaviors (p < .001), with self silencing(â = -.115, p < .05) and age (â = .173, p < .001) as significant predictors.Bivariate correlation analysis indicated an inverse correlation between HIV stigma (p < .05) and safer sex behaviors. Discussion. Implications for further study and practice are discussed to include considerations for development of age- and gender-appropriate prevention interventions assisting women with interpersonal processes combined with skills for active involvement in addressing high-risk sex behaviors.

Efficient transformation and artificial miRNA gene silencing in Lemna minor

PubMed Central

Cantó-Pastor, Alex; Mollá-Morales, Almudena; Ernst, Evan; Dahl, William; Zhai, Jixian; Yan, Yiheng; Meyers, Blake; Shanklin, John; Martienssen, Robert

2015-01-01

Lack of genetic tools in the Lemnaceae (duckweed) has impeded full implementation of this organism as model for biological research, despite its rapid doubling time, simple architecture and unusual metabolic characteristics. Here we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a Magnesium Chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic Lemna minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae. PMID:24989135

The RNA-induced silencing complex: a versatile gene-silencing machine.

PubMed

Pratt, Ashley J; MacRae, Ian J

2009-07-03

RNA interference is a powerful mechanism of gene silencing that underlies many aspects of eukaryotic biology. On the molecular level, RNA interference is mediated by a family of ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs), which can be programmed to target virtually any nucleic acid sequence for silencing. The ability of RISC to locate target RNAs has been co-opted by evolution many times to generate a broad spectrum of gene-silencing pathways. Here, we review the fundamental biochemical and biophysical properties of RISC that facilitate gene targeting and describe the various mechanisms of gene silencing known to exploit RISC activity.

Nine Instructional Exercises to Teach Silence.

ERIC Educational Resources Information Center

Crocker, Jim

1980-01-01

Outlines some recent theoretical discussions of silence as communication. Describes nine exercises the speech communication instructor can use to teach silence and shows which specific function of silence each exercise teaches. (JMF)

Another Piece of the "Silence in PBL" Puzzle: Students' Explanations of Dominance and Quietness as Complementary Group Roles

ERIC Educational Resources Information Center

Skinner, Vicki J.; Braunack-Mayer, Annette; Winning, Tracey A.

2016-01-01

A problem-based learning (PBL) assumption is that silence is incompatible with collaborative learning. Although sociocultural studies have reinterpreted silence as collaborative, we must understand how silence occurs in PBL groups. This essay presents students' explanations of dominance, leadership, and silence as PBL group roles. An ethnographic…

Development of transgenic Brassica juncea lines for reduced seed sinapine content by perturbing phenylpropanoid pathway genes

PubMed Central

Kajla, Sachin; Mukhopadhyay, Arundhati

2017-01-01

Sinapine is a major anti-nutritive compound that accumulates in the seeds of Brassica species. When ingested, sinapine imparts gritty flavuor in meat and milk of animals and fishy odor to eggs of brown egg layers, thereby compromising the potential use of the valuable protein rich seed meal. Sinapine content in Brassica juncea germplasm ranges from 6.7 to 15.1 mg/g of dry seed weight (DSW) which is significantly higher than the prescribed permissible level of 3.0 mg/g of DSW. Due to limited natural genetic variability, conventional plant breeding approach for reducing the sinapine content has largely been unsuccessful. Hence, transgenic approach for gene silencing was adopted by targeting two genes—SGT and SCT, encoding enzymes UDP- glucose: sinapate glucosyltransferase and sinapoylglucose: choline sinapoyltransferase, respectively, involved in the final two steps of sinapine biosynthetic pathway. These two genes were isolated from B. juncea and eight silencing constructs were developed using three different RNA silencing approaches viz. antisense RNA, RNAi and artificial microRNA. Transgenics in B. juncea were developed following Agrobacterium-mediated transformation. From a total of 1232 independent T0 transgenic events obtained using eight silencing constructs, 25 homozygous lines showing single gene inheritance were identified in the T2 generation. Reduction of seed sinapine content in these lines ranged from 15.8% to 67.2%; the line with maximum reduction had sinapine content of 3.79 mg/g of DSW. The study also revealed that RNAi method was more efficient than the other two methods used in this study. PMID:28787461

A role for the replication proteins PCNA, RF-C, polymerase epsilon and Cdc45 in transcriptional silencing in Saccharomyces cerevisiae.

PubMed Central

Ehrenhofer-Murray, A E; Kamakaka, R T; Rine, J

1999-01-01

Transcriptional silencing in the budding yeast Saccharomyces cerevisiae may be linked to DNA replication and cell cycle progression. In this study, we have surveyed the effect of 41 mutations in genes with a role in replication, the cell cycle, and DNA repair on silencing at HMR. Mutations in PCNA (POL30), RF-C (CDC44), polymerase epsilon (POL2, DPB2, DPB11), and CDC45 were found to restore silencing at a mutant HMR silencer allele that was still a chromosomal origin of replication. Replication timing experiments indicated that the mutant HMR locus was replicated late in S-phase, at the same time as wild-type HMR. Restoration of silencing by PCNA and CDC45 mutations required the origin recognition complex binding site of the HMR-E silencer. Several models for the precise role of these replication proteins in silencing are discussed. PMID:10545450

Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

PubMed

Han, Yao; Zhang, Bin; Qin, Xiaoting; Li, Mingyang; Guo, Yulong

2015-01-01

MIGS (miRNA-induced gene silencing) is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs). MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase) and PDS (phytoene desaturase) gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5'- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant functions.

Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes

PubMed Central

Han, Yao; Zhang, Bin; Qin, Xiaoting; Li, Mingyang; Guo, Yulong

2015-01-01

MIGS (miRNA-induced gene silencing) is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs). MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase) and PDS (phytoene desaturase) gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5′- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant functions. PMID:26658695

"The Silence Itself Is Enough of a Statement": The Day of Silence and LGBTQ Awareness Raising

ERIC Educational Resources Information Center

Woolley, Susan W.

2012-01-01

This ethnographic study of a high school gay-straight alliance club examines unintended consequences of silence during the Day of Silence, a day of action aimed at addressing anti-LGBTQ bias in schools. While this strategy calls for students to engage in intentional silences to raise awareness of anti-LGBTQ bias, it does not necessarily lead…

Ex vitro composite plants: an inexpensive, rapid method for root biology.

PubMed

Collier, Ray; Fuchs, Beth; Walter, Nathalie; Kevin Lutke, William; Taylor, Christopher G

2005-08-01

Plant transformation technology is frequently the rate-limiting step in gene function analysis in non-model plants. An important tool for root biologists is the Agrobacterium rhizogenes-derived composite plant, which has made possible genetic analyses in a wide variety of transformation recalcitrant dicotyledonous plants. The novel, rapid and inexpensive ex vitro method for producing composite plants described in this report represents a significant advance over existing composite plant induction protocols, which rely on expensive and time-consuming in vitro conditions. The utility of the new system is validated by expression and RNAi silencing of GFP in transgenic roots of composite plants, and is bolstered further by experimental disruption, via RNAi silencing, of endogenous plant resistance to the plant parasitic nematode Meloidogyne incognita in transgenic roots of Lycopersicon esculentum cv. Motelle composite plants. Critical parameters of the method are described and discussed herein.

A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation.

PubMed

Itaya, Asuka; Zhong, Xuehua; Bundschuh, Ralf; Qi, Yijun; Wang, Ying; Takeda, Ryuta; Harris, Ann R; Molina, Carlos; Nelson, Richard S; Ding, Biao

2007-03-01

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

Tobacco mosaic virus Movement Protein Enhances the Spread of RNA Silencing

PubMed Central

Vogler, Hannes; Kwon, Myoung-Ok; Dang, Vy; Sambade, Adrian; Fasler, Monika; Ashby, Jamie; Heinlein, Manfred

2008-01-01

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA. PMID:18389061

Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

NASA Astrophysics Data System (ADS)

Travella, Silvia; Keller, Beat

Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

Molecular tools for carotenogenesis analysis in the zygomycete Mucor circinelloides.

PubMed

Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M; Garre, Victoriano; López-García, Sergio; Navarro, Eusebio; Vila, Ana

2012-01-01

The carotene producer fungus Mucor circinelloides is the zygomycete more amenable to genetic manipulations by using molecular tools. Since the initial development of an effective procedure of genetic transformation, more than two decades ago, the availability of new molecular approaches such as gene replacement techniques and gene expression inactivation by RNA silencing, in addition to the sequencing of its genome, has made Mucor a valuable organism for the study of a number of processes. Here we describe in detail the main techniques and methods currently used to manipulate M. circinelloides, including transformation, gene replacement, gene silencing, RNAi, and immunoprecipitation.

Symbolic Action in India: Gandhi's Nonverbal Persuasion

ERIC Educational Resources Information Center

Merriam, Allen H.

1975-01-01

Examines symbolic action as a method of exerting public influence nonverbally through nonviolent behavior. Discusses Gandhi's persuasive tactics including fasting, propaganda tours, silence, clothing and adoption of symbols. (MH)

The Role of Small RNA-Based Epigenetic Silencing for Purifying Selection on Transposable Elements in Capsella grandiflora

PubMed Central

Horvath, Robert

2017-01-01

Abstract To avoid negative effects of transposable element (TE) proliferation, plants epigenetically silence TEs using a number of mechanisms, including RNA-directed DNA methylation. These epigenetic modifications can extend outside the boundaries of TE insertions and lead to silencing of nearby genes, resulting in a trade-off between TE silencing and interference with nearby gene regulation. Therefore, purifying selection is expected to remove silenced TE insertions near genes more efficiently and prevent their accumulation within a population. To explore how effects of TE silencing on gene regulation shapes purifying selection on TEs, we analyzed whole genome sequencing data from 166 individuals of a large population of the outcrossing species Capsella grandiflora. We found that most TEs are rare, and in chromosome arms, silenced TEs are exposed to stronger purifying selection than those that are not silenced by 24-nucleotide small RNAs, especially with increasing proximity to genes. An age-of-allele test of neutrality on a subset of TEs supports our inference of purifying selection on silenced TEs, suggesting that our results are robust to varying transposition rates. Our results provide new insights into the processes affecting the accumulation of TEs in an outcrossing species and support the view that epigenetic silencing of TEs results in a trade-off between preventing TE proliferation and interference with nearby gene regulation. We also suggest that in the centromeric and pericentromeric regions, the negative aspects of epigenetic TE silencing are missing. PMID:29036316

Segmental Versican Expression in the Trabecular Meshwork and Involvement in Outflow Facility

PubMed Central

Keller, Kate E.; Bradley, John M.; Vranka, Janice A.

2011-01-01

Purpose. Versican is a large proteoglycan with numerous chondroitin sulfate (CS) glycosaminoglycan (GAG) side chains attached. To assess versican's potential contributions to aqueous humor outflow resistance, its segmental distribution in the trabecular meshwork (TM) and the effect on outflow facility of silencing the versican gene were evaluated. Methods. Fluorescent quantum dots (Qdots) were perfused to label outflow pathways of anterior segments. Immunofluorescence with confocal microscopy and quantitative RT-PCR were used to determine versican protein and mRNA distribution relative to Qdot-labeled regions. Lentiviral delivery of shRNA-silencing cassettes to TM cells in perfused anterior segment cultures was used to evaluate the involvement of versican and CS GAG chains in outflow facility. Results. Qdot uptake by TM cells showed considerable segmental variability in both human and porcine outflow pathways. Regional levels of Qdot labeling were inversely related to versican protein and mRNA levels; versican levels were relatively high in sparsely Qdot-labeled regions and low in densely labeled regions. Versican silencing decreased outflow facility in human and increased facility in porcine anterior segments. However, RNAi silencing of ChGn, an enzyme unique to CS GAG biosynthesis, increased outflow facility in both species. The fibrillar pattern of versican immunostaining in the TM juxtacanalicular region was disrupted after versican silencing in perfusion culture. Conclusions. Versican appears to be a central component of the outflow resistance, where it may organize GAGs and other ECM components to facilitate and control open flow channels in the TM. However, the exact molecular organization of this resistance appears to differ between human and porcine eyes. PMID:21596823

Effect of Sirtuin-1 on Synaptic Plasticity in Nucleus Accumbens in a Rat Model of Heroin Addiction.

PubMed

Xia, Baijuan; Li, Yixin; Li, Rongrong; Yin, Dan; Chen, Xingqiang; Li, Jie; Liang, Wenmei

2018-06-05

BACKGROUND Synaptic plasticity plays an important role in the process of addiction. This study investigated the relationship between synaptic plasticity and changes in addictive behavior and examined the expression of synaptic plasticity-associated proteins and genes in the nucleus accumbens (NAc) region in different rat models. MATERIAL AND METHODS Heroin addiction, SIRT1-overexpression, and SIRT1-silenced rat models were established. Polymerase chain reaction gene chip technology, immunohistochemistry, Western blotting, and transmission electron microscopy were used to detect changes in synaptic plasticity-related gene and protein expression, and changes in the ultrastructure of synapses, in the NAc. RESULTS Naloxone withdrawal symptoms appeared in the SIRT1-overexpression group. In the SIRT1-silenced group the symptoms were reduced. Immunohistochemistry and Western blotting results showed that FOXO1 expression decreased in the heroin addiction (HA) group but increased in the SIRT1-silenced group (p<0.05). The expression of Cdk5, Nf-κB, PSD95, and Syn was enhanced in the HA group (p<0.05) and further increased in the SIRT1-overexpression group but were reduced in the SIRT1-silenced group (p<0.05). The number of synapses increased in the HA group (p<0.05) along with mitochondrial swelling in the presynaptic membrane and obscuring of the synaptic cleft. CONCLUSIONS SIRT1 and other synaptic plasticity-related genes in NAc are involved in the regulation of heroin addiction. SIRT1 overexpression can increase behavioral sensitization in the NAc of rats, and SIRT1 silencing might ease withdrawal symptoms and reduce conditioned place preferences.

Therapeutic silence of pleiotrophin by targeted delivery of siRNA and its effect on the inhibition of tumor growth and metastasis.

PubMed

Zha, Lisha; He, Lichun; Xie, Weidong; Cheng, Jin; Li, Tong; Mohsen, Mona O; Lei, Fan; Storni, Federico; Bachmann, Martin; Chen, Hongquan; Zhang, Yaou

2017-01-01

Pleiotrophin (PTN) is a secreted cytokine that is expressed in various cancer cell lines and human tumor such as colon cancer, lung cancer, gastric cancer and melanoma. It plays significant roles in angiogenesis, metastasis, differentiation and cell growth. The expression of PTN in the adult is limited to the hippocampus in an activity-dependent manner, making it a very attractive target for cancer therapy. RNA interference (RNAi) offers great potential as a new powerful therapeutic strategy based on its highly specific and efficient silencing of a target gene. However, efficient delivery of small interfering RNA (siRNA) in vivo remains a significant hurdle for its successful therapeutic application. In this study, we first identified, on a cell-based experiment, applying a 1:1 mixture of two PTN specific siRNA engenders a higher silencing efficiency on both mRNA and protein level than using any of them discretely at the same dose. As a consequence, slower melanoma cells growth was also observed for using two specific siRNA combinatorially. To establish a robust way for siRNA delivery in vivo and further investigate how silence of PTN affects tumor growth, we tested three different methods to deliver siRNA in vivo: first non-targeted in-vivo delivery of siRNA via jetPEI; second lung targeted delivery of siRNA via microbubble coated jetPEI; third tumor cell targeted delivery of siRNA via transferrin-polyethylenimine (Tf-PEI). As a result, we found that all three in-vivo siRNAs delivery methods led to an evident inhibition of melanoma growth in non-immune deficiency C57BL/6 mice without a measureable change of ALT and AST activities. Both targeted delivery methods showed more significant curative effect than jetPEI. The lung targeted delivery by microbubble coated jetPEI revealed a comparable therapeutic effect with Tf-PEI, indicating its potential application for target delivery of siRNA in vivo.

Therapeutic silence of pleiotrophin by targeted delivery of siRNA and its effect on the inhibition of tumor growth and metastasis

PubMed Central

Xie, Weidong; Cheng, Jin; Li, Tong; Mohsen, Mona O.; Lei, Fan; Storni, Federico; Bachmann, Martin; Chen, Hongquan; Zhang, Yaou

2017-01-01

Pleiotrophin (PTN) is a secreted cytokine that is expressed in various cancer cell lines and human tumor such as colon cancer, lung cancer, gastric cancer and melanoma. It plays significant roles in angiogenesis, metastasis, differentiation and cell growth. The expression of PTN in the adult is limited to the hippocampus in an activity-dependent manner, making it a very attractive target for cancer therapy. RNA interference (RNAi) offers great potential as a new powerful therapeutic strategy based on its highly specific and efficient silencing of a target gene. However, efficient delivery of small interfering RNA (siRNA) in vivo remains a significant hurdle for its successful therapeutic application. In this study, we first identified, on a cell-based experiment, applying a 1:1 mixture of two PTN specific siRNA engenders a higher silencing efficiency on both mRNA and protein level than using any of them discretely at the same dose. As a consequence, slower melanoma cells growth was also observed for using two specific siRNA combinatorially. To establish a robust way for siRNA delivery in vivo and further investigate how silence of PTN affects tumor growth, we tested three different methods to deliver siRNA in vivo: first non-targeted in-vivo delivery of siRNA via jetPEI; second lung targeted delivery of siRNA via microbubble coated jetPEI; third tumor cell targeted delivery of siRNA via transferrin-polyethylenimine (Tf-PEI). As a result, we found that all three in-vivo siRNAs delivery methods led to an evident inhibition of melanoma growth in non-immune deficiency C57BL/6 mice without a measureable change of ALT and AST activities. Both targeted delivery methods showed more significant curative effect than jetPEI. The lung targeted delivery by microbubble coated jetPEI revealed a comparable therapeutic effect with Tf-PEI, indicating its potential application for target delivery of siRNA in vivo. PMID:28562667

Motor responses and weight gaining in neonates through use of two methods of earmuff and receiving silence in NICU.

PubMed

Abdeyazdan, Z; Ghasemi, S; Marofi, M; Berjis, N

2014-01-01

With technological advances in NICUs the survival rate of preterm infants has been increased. Because NICU environment is a potent source of stress for infants, its modification is an essential measure to decrease infants' morbidity. The purposes of this study were to compare the effects of wearing earmuff and provision silence for infants on their motor responses and gaining weight. In a randomized clinical trial 96 preterm infants were enrolled. Their motor responses were evaluated for two consecutive days in the morning and afternoon shifts, in the groups of earmuff and silence, and at similar time points in the control group. Also their weight was measured at days 1 and 10. In the two intervention groups, means of motor responses in infants were significantly less than in the control group, and weight gain of infants was more than the control group. However weight gain was more pronounced in the earmuff group. Both interventions led to decreasing number of motor responses and improvement of weight gain pattern, but these effects were more pronounced in earmuff group; thus because implementation of silence in NICUs has many barriers, it is suggested to use earmuff for preterm infants in these units. This trial obtained IRCT registration number IRCT2012092010812N2.

A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation▿

PubMed Central

Itaya, Asuka; Zhong, Xuehua; Bundschuh, Ralf; Qi, Yijun; Wang, Ying; Takeda, Ryuta; Harris, Ann R.; Molina, Carlos; Nelson, Richard S.; Ding, Biao

2007-01-01

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures. PMID:17202210

An investigation of noise produced by unsteady gas flow through silencer elements

NASA Astrophysics Data System (ADS)

Mawhinney, Graeme Hugh

This thesis presents an investigation of the noise produced by unsteady gas flow through silencer elements. The central aim of the research project was to produce a tool for assistance in the design of the exhaust systems of diesel powered electrical generator sets, with the modelling techniques developed having a much wider application in reciprocating internal combustion engine exhaust systems. An automotive cylinder head was incorporated in a purpose built test rig to supply exhaust pulses, typical of those found in the exhaust system of four stroke diesel engines, to various experimental exhaust systems. Exhaust silencer elements evaluated included expansion, re- entrant, concentric tube resonator and absorptive elements. Measurements taken on the test rig included, unsteady superposition pressure in the exhaust ducting, cyclically averaged mass flow rate through the system and exhaust noise levels radiated into a semi-anechoic measurement chamber. The entire test rig was modelled using the 1D finite volume method developed previously developed at Queen's University Belfast. Various boundary conditions, developed over the years, were used to model the various silencer elements being evaluated. The 1D gas dynamic simulation thus estimated the mass flux history at the open end of the exhaust system. The mass flux history was then broken into its harmonic components and an acoustic radiation model was developed to model the sound pressure level produced by an acoustic monopole over a reflecting plane. The accuracy of the simulation technique was evaluated by correlation of measured and simulated superposition pressure and noise data. In general correlation of superposition pressure was excellent for all of the silencer elements tested. Predicted sound pressure level radiated from the open end of the exhaust tailpipe was seen to be accurate in the 100 Hz to 1 kHz frequency range for all of the silencer elements tested.

STAT3 Knockdown Reduces Pancreatic Cancer Cell Invasiveness and Matrix Metalloproteinase-7 Expression in Nude Mice

PubMed Central

Huang, Ke jian; Wu, Wei dong; Jiang, Tao; Cao, Jun; Feng, Zhen zhong; Qiu, Zheng jun

2011-01-01

Aims Transducer and activator of transcription-3 (STAT3) plays an important role in tumor cell invasion and metastasis. The aim of the present study was to investigate the effects of STAT3 knockdown in nude mouse xenografts of pancreatic cancer cells and underlying gene expression. Methods A STAT3 shRNA lentiviral vector was constructed and infected into SW1990 cells. qRT-PCR and western immunoblot were performed to detect gene expression. Nude mouse xenograft assays were used to assess changes in phenotypes of these stable cells in vivo. HE staining was utilized to evaluate tumor cell invasion and immunohistochemistry was performed to analyze gene expression. Results STAT3 shRNA successfully silenced expression of STAT3 mRNA and protein in SW1990 cells compared to control cells. Growth rate of the STAT3-silenced tumor cells in nude mice was significantly reduced compared to in the control vector tumors and parental cells-generated tumors. Tumor invasion into the vessel and muscle were also suppressed in the STAT3-silenced tumors compared to controls. Collagen IV expression was complete and continuous surrounding the tumors of STAT3-silenced SW1990 cells, whereas collagen IV expression was incomplete and discontinuous surrounding the control tumors. Moreover, microvessel density was significantly lower in STAT3-silenced tumors than parental or control tumors of SW1990 cells. In addition, MMP-7 expression was reduced in STAT3-silenced tumors compared to parental SW1990 xenografts and controls. In contrast, expression of IL-1β and IgT7α was not altered. Conclusion These data clearly demonstrate that STAT3 plays an important role in regulation of tumor growth, invasion, and angiogenesis, which could be act by reducing MMP-7 expression in pancreatic cancer cells. PMID:21991388

The Arabidopsis acetylated histone-binding protein BRAT1 forms a complex with BRP1 and prevents transcriptional silencing

PubMed Central

Zhang, Cui-Jun; Hou, Xiao-Mei; Tan, Lian-Mei; Shao, Chang-Rong; Huang, Huan-Wei; Li, Yong-Qiang; Li, Lin; Cai, Tao; Chen, She; He, Xin-Jian

2016-01-01

Transposable elements and other repetitive DNA sequences are usually subject to DNA methylation and transcriptional silencing. However, anti-silencing mechanisms that promote transcription in these regions are not well understood. Here, we describe an anti-silencing factor, Bromodomain and ATPase domain-containing protein 1 (BRAT1), which we identified by a genetic screen in Arabidopsis thaliana. BRAT1 interacts with an ATPase domain-containing protein, BRP1 (BRAT1 Partner 1), and both prevent transcriptional silencing at methylated genomic regions. Although BRAT1 mediates DNA demethylation at a small set of loci targeted by the 5-methylcytosine DNA glycosylase ROS1, the involvement of BRAT1 in anti-silencing is largely independent of DNA demethylation. We also demonstrate that the bromodomain of BRAT1 binds to acetylated histone, which may facilitate the prevention of transcriptional silencing. Thus, BRAT1 represents a potential link between histone acetylation and transcriptional anti-silencing at methylated genomic regions, which may be conserved in eukaryotes. PMID:27273316

The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1

PubMed Central

Osada, Shigehiro; Sutton, Ann; Muster, Nemone; Brown, Christine E.; Yates, John R.; Sternglanz, Rolf; Workman, Jerry L.

2001-01-01

It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ∼450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing. PMID:11731479

Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum

PubMed Central

2013-01-01

Background Hairpin RNA (hpRNA) transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms. However, in fungi, expression of hairpin RNA transcripts can induce post-transcriptional gene silencing, but in some species can also lead to transcriptional gene silencing, suggesting a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a powerful technique to understand gene function, particularly when gene knockouts are difficult to obtain. We investigated whether the plant pathogenic fungus Fusarium oxysporum possesses a functional gene silencing machinery and whether hairpin RNA transcripts can be employed to effectively induce gene silencing. Results Here we show that, in the phytopathogenic fungus F. oxysporum, hpRNA transgenes targeting either a β-glucuronidase (Gus) reporter transgene (hpGus) or the endogenous gene Frp1 (hpFrp) did not induce significant silencing of the target genes. Expression analysis suggested that the hpRNA transgenes are prone to transcriptional inactivation, resulting in low levels of hpRNA and siRNA production. However, the hpGus RNA can be efficiently transcribed by promoters acquired either by recombination with a pre-existing, actively transcribed Gus transgene or by fortuitous integration near an endogenous gene promoter allowing siRNA production. These siRNAs effectively induced silencing of a target Gus transgene, which in turn appeared to also induce secondary siRNA production. Furthermore, our results suggested that hpRNA transcripts without poly(A) tails are efficiently processed into siRNAs to induce gene silencing. A convergent promoter transgene, designed to express poly(A)-minus sense and antisense Gus RNAs, without an inverted-repeat DNA structure, induced consistent Gus silencing in F. oxysporum. Conclusions These results indicate that F. oxysporum possesses functional RNA silencing machineries for siRNA production and target mRNA cleavage, but hpRNA transgenes may induce transcriptional self-silencing due to its inverted-repeat structure. Our results suggest that F. oxysporum possesses a similar gene silencing pathway to other fungi like fission yeast, and indicate a need for developing more effective RNA silencing technology for gene function studies in this fungal pathogen. PMID:23819794

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli.

PubMed

Bernheim, Aude G; Libis, Vincent K; Lindner, Ariel B; Wintermute, Edwin H

2016-03-20

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Non-structural protein 1 from avian influenza virus H9N2 is an efficient RNA silencing suppressor with characteristics that differ from those of Tomato bushy stunt virus p19.

PubMed

Yu, Ru; Jing, Xiuli; Li, Wenjing; Xu, Jie; Xu, Yang; Geng, Liwei; Zhu, Changxiang; Liu, Hongmei

2018-06-01

Non-structural protein 1 (NS1) of influenza A virus is a multifunctional dimeric protein that contains a conserved N-terminal RNA binding domain. Studies have shown that NS1 suppresses RNA silencing and the NS1 proteins encoded by different influenza A virus strains exhibit differential RNA silencing suppression activities. In this study, we showed that the NS1 protein from avian influenza virus (AIV) H9N2 suppressed systemic RNA silencing induced by sense RNA or dsRNA. It resulted in more severe Potato virus X symptom, but could not reverse established systemic green fluorescent protein silencing in Nicotiana benthamiana. In addition, its systemic silencing suppression activity was much weaker than that of p19. The local silencing suppression activity of AIV H9N2 NS1 was most powerful at 7 dpi and was even stronger than that of p19. And the inhibition ability to RNA silencing of NS1 is stronger than that of p19 in human cells. Collectively, these results indicate that AIV H9N2 NS1 is an effective RNA silencing suppressor that likely targets downstream step(s) of dsRNA formation at an early stage in RNA silencing. Although NS1 and p19 both bind siRNA, their suppression mechanisms seem to differ because of differences in their suppression activities at various times post-infiltration and because p19 can reverse established systemic RNA silencing, but NS1 cannot.

Mutuality, Self-Silencing, and Disordered Eating in College Women

ERIC Educational Resources Information Center

Wechsler, Lisa S.; Riggs, Shelley A.; Stabb, Sally D.; Marshall, David M.

2006-01-01

The current study examined patterns of association among mutuality, self-silencing, and disordered eating in an ethnically diverse sample of college women (N = 149). Partner mutuality and overall self-silencing were negatively correlated and together were associated with six disordered eating indices. All four self-silencing subscales were…

After the Blackbird Whistles: Listening to Silence in Classrooms

ERIC Educational Resources Information Center

Schultz, Katherine

2010-01-01

Background/Context: Students spend a large part of their time in schools in silence. However, teachers tend to spend most of their time attending to student talk. Anthropological and linguistic research has contributed to an understanding of silence in particular communities, offering explanations for students' silence in school. This research…

47 CFR 80.304 - Watch requirement during silence periods.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 5 2010-10-01 2010-10-01 false Watch requirement during silence periods. 80... Safety Watches § 80.304 Watch requirement during silence periods. Each ship station operating on... safety messages, ship stations must not transmit during the silence periods on 2182 kHz. [69 FR 64673...

47 CFR 80.304 - Watch requirement during silence periods.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 5 2011-10-01 2011-10-01 false Watch requirement during silence periods. 80... Safety Watches § 80.304 Watch requirement during silence periods. Each ship station operating on... safety messages, ship stations must not transmit during the silence periods on 2182 kHz. [69 FR 64673...

Efficient transformation and artificial miRNA gene silencing in Lemna minor.

PubMed

Cantó-Pastor, A; Mollá-Morales, A; Ernst, E; Dahl, W; Zhai, J; Yan, Y; Meyers, B C; Shanklin, J; Martienssen, R

2015-01-01

Despite rapid doubling time, simple architecture and ease of metabolic labelling, a lack of genetic tools in the Lemnaceae (duckweed) has impeded the full implementation of this organism as a model for biological research. Here, we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via Agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a magnesium chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic L. minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Antiviral RNA silencing suppression activity of Tomato spotted wilt virus NSs protein.

PubMed

Ocampo Ocampo, T; Gabriel Peralta, S M; Bacheller, N; Uiterwaal, S; Knapp, A; Hennen, A; Ochoa-Martinez, D L; Garcia-Ruiz, H

2016-06-17

In addition to regulating gene expression, RNA silencing is an essential antiviral defense system in plants. Triggered by double-stranded RNA, silencing results in degradation or translational repression of target transcripts. Viruses are inducers and targets of RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressors that interfere with this process, such as the Tomato spotted wilt virus (TSWV) NSs protein. The mechanism by which NSs suppresses RNA silencing and its role in viral infection and movement remain to be determined. We cloned NSs from the Hawaii isolate of TSWV and using two independent assays show for the first time that this protein restored pathogenicity and supported the formation of local infection foci by suppressor-deficient Turnip mosaic virus and Turnip crinkle virus. Demonstrating the suppression of RNA silencing directed against heterologous viruses establishes the foundation to determine the means used by NSs to block this antiviral process.

PABP is not essential for microRNA-mediated translational repression and deadenylation in vitro

PubMed Central

Fukaya, Takashi; Tomari, Yukihide

2011-01-01

MicroRNAs silence their complementary target genes via formation of the RNA-induced silencing complex (RISC) that contains an Argonaute (Ago) protein at its core. It was previously proposed that GW182, an Ago-associating protein, directly binds to poly(A)-binding protein (PABP) and interferes with its function, leading to silencing of the target mRNAs. Here we show that Drosophila Ago1-RISC induces silencing via two independent pathways: shortening of the poly(A) tail and pure repression of translation. Our data suggest that although PABP generally modulates poly(A) length and translation efficiency, neither PABP function nor GW182–PABP interaction is a prerequisite for these two silencing pathways. Instead, we propose that each of the multiple functional domains within GW182 has a potential for silencing, and yet they need to act together in the context of full-length GW182 to exert maximal silencing. PMID:22117217

A two-step process for epigenetic inheritance in Arabidopsis

PubMed Central

Blevins, Todd; Pontvianne, Frédéric; Cocklin, Ross; Podicheti, Ram; Chandrasekhara, Chinmayi; Yerneni, Satwica; Braun, Chris; Lee, Brandon; Rusch, Doug; Mockaitis, Keithanne; Tang, Haixu; Pikaard, Craig S.

2014-01-01

Summary In Arabidopsis, multisubunit RNA polymerases IV and V orchestrate RNA-directed DNA methylation (RdDM) and transcriptional silencing, but what identifies the loci to be silenced is unclear. We show that heritable silent locus identity at a specific subset of RdDM targets requires HISTONE DEACETYLASE 6 (HDA6) acting upstream of Pol IV recruitment and siRNA biogenesis. At these loci, epigenetic memory conferring silent locus identity is erased in hda6 mutants such that restoration of HDA6 activity cannot restore siRNA biogenesis or silencing. Silent locus identity is similarly lost in mutants for the cytosine maintenance methyltransferase, MET1. By contrast, pol IV or pol V mutants disrupt silencing without erasing silent locus identity, allowing restoration of Pol IV or Pol V function to restore silencing. Collectively, these observations indicate that silent locus specification and silencing are separable steps that together account for epigenetic inheritance of the silenced state. PMID:24657166

Searching the Silent Smiles of Women Superintendents: Did You Say Something?

ERIC Educational Resources Information Center

Brunner, C. Cryss

While numerous feminist scholars have written about "silence," few have focused on the silence of women in powerful masculinized positions such as the superintendency. The purpose of the paper is to expose the silencing, the disallowing of voice in actual practice, or the "unnatural silence" (Olsen 1978) of women superintendents through a…

Choosing Silence for Equality in and through Schooling

ERIC Educational Resources Information Center

Lees, Helen E.

2016-01-01

This article considers silences and equality as combined from a theoretical perspective. Equality in and through chosen, deliberate and regular silence experience is seen as an equaliser: if no one is speaking no one can dominate. The article uses a bifurcated concept of silence: weak, negative forms and strong, positive forms. Only the strong…

A petunia ethylene-responsive element binding factor, PhERF2, plays an important role in antiviral RNA silencing

USDA-ARS?s Scientific Manuscript database

Virus-induced gene silencing (VIGS) is a useful technique for functional characterization of plant genes. However, the silencing efficiency of the VIGS system is variable largely depending on compatibility between the host and the virus. Antiviral RNA silencing is involved in plant antiviral defense...

Silence in Teaching and Learning: Perspectives of a Nepalese Graduate Student

ERIC Educational Resources Information Center

Bista, Krishna

2012-01-01

The nature of silence is complex in any classroom with international or domestic students. Instructors sometimes fail to recognize that the classroom silence of foreign students is unlike their native counterparts. With an insider perspective, this article explores the concept of silence among international students by examining the existing body…

"I Don't Want to Hear That!": Legitimating Whiteness through Silence in Schools

ERIC Educational Resources Information Center

Castagno, Angelina E.

2008-01-01

In this article, I examine the ways in which silences around race contribute to the maintenance and legitimation of Whiteness. Drawing on ethnographic data from two demographically different schools, I highlight patterns of racially coded language, teacher silence, silencing students' race talk, and the conflating of culture with race, equality…

The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fusaro, Adriana F.; CSIRO Plant Industry, Canberra, P.O. Box 1600, ACT 2601; Correa, Regis L.

The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0{sup PE}, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0{sup PE} has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemicmore » silencing. Like the P0s from poleroviruses, P0{sup PE} destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.« less

A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

PubMed Central

Ohno, Satoshi; Yoshikawa, Katsunori; Shimizu, Hiroshi; Tamura, Tomohiro

2014-01-01

We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination. PMID:24212579

Language matters: towards an understanding of silence and humour in medical education.

PubMed

Lingard, Lorelei

2013-01-01

This paper considers the state of the science regarding language matters in medical education, with particular attention to two informal language practices: silence and humour. Silence and humour pervade clinical training settings, although we rarely attend explicitly to them. This paper considers the treatment of these topics in our field to date and introduces a selection of the scholarship on silence and humour from other fields, including philosophy, sociology, anthropology, linguistics and rhetoric. Particular attention is paid to distilling the theoretical and methodological possibilities for an elaborated research agenda around silence and humour in medical education. These two language practices assume a variety of forms and serve a range of social functions. Episodes of silence and humour are intimately tied to their relational and institutional contexts. Power often figures centrally, although not predictably. A rich theoretical and methodological basis exists on which to elaborate a research agenda around silence and humour in medical education. Such research promises to reveal more fully the contributions of silence and humour to socialisation in clinical training settings. © Blackwell Publishing Ltd 2013.

Silencing of transposable elements may not be a major driver of regulatory evolution in primate iPSCs

PubMed Central

Zhao, Siming; Luo, Kaixuan; Pavlovic, Bryan J; Karimi, Mohammad M; Stephens, Matthew

2018-01-01

Transposable elements (TEs) comprise almost half of primate genomes and their aberrant regulation can result in deleterious effects. In pluripotent stem cells, rapidly evolving KRAB-ZNF genes target TEs for silencing by H3K9me3. To investigate the evolution of TE silencing, we performed H3K9me3 ChIP-seq experiments in induced pluripotent stem cells from 10 human and 7 chimpanzee individuals. We identified four million orthologous TEs and found the SVA and ERV families to be marked most frequently by H3K9me3. We found little evidence of inter-species differences in TE silencing, with as many as 82% of putatively silenced TEs marked at similar levels in humans and chimpanzees. TEs that are preferentially silenced in one species are a similar age to those silenced in both species and are not more likely to be associated with expression divergence of nearby orthologous genes. Our data suggest limited species-specificity of TE silencing across 6 million years of primate evolution. PMID:29648536

The physics of chromatin silencing: Bi-stability and front propagation

NASA Astrophysics Data System (ADS)

Sedighi, Mohammad

A mean-field dynamical model of chromatin silencing in budding yeast is provided and the conditions giving rise to two states: one silenced and another un-silenced, is studied. Based on these conditions, the space of control parameters is divided into two distinct regions of mono-stable and bi-stable solutions (the bifurcation diagram). Then, considering both the discrete and continuous versions of the model, the formation of a stable boundary between the silenced and un-silenced areas on DNA is investigated. As a result, a richer phase diagram is provided. The dynamics of the boundary is also studied under different conditions. Consequently, assuming negative feedback due to possible depletion of silencing proteins, the model explains a paradoxical epigenetic behavior of yeast that happens under some mutation. A stochastic treatment of the model is also considered to verify the results of the mean-field approximation and also to understand the role of intrinsic noise at single cell level. This model could be used as a general guide to discuss chromatin silencing in many organisms.

RNAi for functional genomics in plants.

PubMed

McGinnis, Karen M

2010-03-01

RNAi refers to several different types of gene silencing mediated by small, dsRNA molecules. Over the course of 20 years, the scientific understanding of RNAi has developed from the initial observation of unexpected expression patterns to a sophisticated understanding of a multi-faceted, evolutionarily conserved network of mechanisms that regulate gene expression in many organisms. It has also been developed as a genetic tool that can be exploited in a wide range of species. Because transgene-induced RNAi has been effective at silencing one or more genes in a wide range of plants, this technology also bears potential as a powerful functional genomics tool across the plant kingdom. Transgene-induced RNAi has indeed been shown to be an effective mechanism for silencing many genes in many organisms, but the results from multiple projects which attempted to exploit RNAi on a genome-wide scale suggest that there is a great deal of variation in the silencing efficacy between transgenic events, silencing targets and silencing-induced phenotype. The results from these projects indicate several important variables that should be considered in experimental design prior to the initiation of functional genomics efforts based on RNAi silencing. In recent years, alternative strategies have been developed for targeted gene silencing, and a combination of approaches may also enhance the use of targeted gene silencing for functional genomics.

RNA-mediated gene silencing signals are not graft transmissible from the rootstock to the scion in greenhouse-grown apple plants Malus sp.

PubMed

Flachowsky, Henryk; Tränkner, Conny; Szankowski, Iris; Waidmann, Sascha; Hanke, Magda-Viola; Treutter, Dieter; Fischer, Thilo C

2012-01-01

RNA silencing describes the sequence specific degradation of RNA targets. Silencing is a non-cell autonomous event that is graft transmissible in different plant species. The present study is the first report on systemic acquired dsRNA-mediated gene silencing of transgenic and endogenous gene sequences in a woody plant like apple. Transgenic apple plants overexpressing a hairpin gene construct of the gusA reporter gene were produced. These plants were used as rootstocks and grafted with scions of the gusA overexpressing transgenic apple clone T355. After grafting, we observed a reduction of the gusA gene expression in T355 scions in vitro, but not in T355 scions grown in the greenhouse. Similar results were obtained after silencing of the endogenous Mdans gene in apple that is responsible for anthocyanin biosynthesis. Subsequently, we performed grafting experiments with Mdans silenced rootstocks and red leaf scions of TNR31-35 in order to evaluate graft transmitted silencing of the endogenous Mdans. The results obtained suggested a graft transmission of silencing signals in in vitro shoots. In contrast, no graft transmission of dsRNA-mediated gene silencing signals was detectable in greenhouse-grown plants and in plants grown in an insect protection tent.

RNA-Mediated Gene Silencing Signals Are Not Graft Transmissible from the Rootstock to the Scion in Greenhouse-Grown Apple Plants Malus sp

PubMed Central

Flachowsky, Henryk; Tränkner, Conny; Szankowski, Iris; Waidmann, Sascha; Hanke, Magda-Viola; Treutter, Dieter; Fischer, Thilo C.

2012-01-01

RNA silencing describes the sequence specific degradation of RNA targets. Silencing is a non-cell autonomous event that is graft transmissible in different plant species. The present study is the first report on systemic acquired dsRNA-mediated gene silencing of transgenic and endogenous gene sequences in a woody plant like apple. Transgenic apple plants overexpressing a hairpin gene construct of the gusA reporter gene were produced. These plants were used as rootstocks and grafted with scions of the gusA overexpressing transgenic apple clone T355. After grafting, we observed a reduction of the gusA gene expression in T355 scions in vitro, but not in T355 scions grown in the greenhouse. Similar results were obtained after silencing of the endogenous Mdans gene in apple that is responsible for anthocyanin biosynthesis. Subsequently, we performed grafting experiments with Mdans silenced rootstocks and red leaf scions of TNR31-35 in order to evaluate graft transmitted silencing of the endogenous Mdans. The results obtained suggested a graft transmission of silencing signals in in vitro shoots. In contrast, no graft transmission of dsRNA-mediated gene silencing signals was detectable in greenhouse-grown plants and in plants grown in an insect protection tent. PMID:22949844

The molecular topography of silenced chromatin in Saccharomyces cerevisiae

PubMed Central

Thurtle, Deborah M.; Rine, Jasper

2014-01-01

Heterochromatin imparts regional, promoter-independent repression of genes and is epigenetically heritable. Understanding how silencing achieves this regional repression is a fundamental problem in genetics and development. Current models of yeast silencing posit that Sir proteins, recruited by transcription factors bound to the silencers, spread throughout the silenced region. To test this model directly at high resolution, we probed the silenced chromatin architecture by chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) of Sir proteins, histones, and a key histone modification, H4K16-acetyl. These analyses revealed that Sir proteins are strikingly concentrated at and immediately adjacent to the silencers, with lower levels of enrichment over the promoters at HML and HMR, the critical targets for transcriptional repression. The telomeres also showed discrete peaks of Sir enrichment yet a continuous domain of hypoacetylated histone H4K16. Surprisingly, ChIP-seq of cross-linked chromatin revealed a distribution of nucleosomes at silenced loci that was similar to Sir proteins, whereas native nucleosome maps showed a regular distribution throughout silenced loci, indicating that cross-linking captured a specialized chromatin organization imposed by Sir proteins. This specialized chromatin architecture observed in yeast informs the importance of a steric contribution to regional repression in other organisms. PMID:24493645

Identification of Strawberry vein banding virus encoded P6 as an RNA silencing suppressor.

PubMed

Feng, Mingfeng; Zuo, Dengpan; Jiang, Xizi; Li, Shuai; Chen, Jing; Jiang, Lei; Zhou, Xueping; Jiang, Tong

2018-07-01

RNA silencing is a common mechanism that plays a key role in antiviral defense. To overcome host defense responses, plant viruses encode silencing-suppressor proteins to target one or several key steps in the silencing machinery. Here, we report that the P6 protein encoded by Strawberry vein banding virus (SVBV) is an RNA silencing suppressor through Agrobacterium-mediated co-infiltration assays. SVBV P6 protein can suppress green fluorescent protein (GFP) gene silencing induced by single-stranded RNA but not by double-stranded RNA. The P6 protein can also inhibit systemic silencing of GFP through interfering the systemic spread of GFP silencing signal. Subcellular localization study indicated that P6 protein formed irregular bodies and distributed in both cytoplasm and nucleus of Nicotiana benthamiana cells. Furthermore, deletion analysis indicated that a nuclear localization signal (NLS, aa 402-426) in the P6 protein is responsible for the silencing suppression efficiency. In addition, expression of the P6 protein via a Potato virus X (PVX)-based vectors induced more severe mosaic symptoms in N. benthamiana leaves, and transgenic N. benthamiana plants expressing P6 showed obvious vein yellowing as well as severe mosaic symptoms in leaves. Taken together, our results demonstrates that SVBV P6 is a suppressor of RNA silencing, possibly acting at a upstream step for dsRNA generation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

A petunia ethylene-responsive element binding factor, PhERF2, plays an important role in antiviral RNA silencing

PubMed Central

Sun, Daoyang; Nandety, Raja Sekhar; Zhang, Yanlong; Reid, Michael S.; Niu, Lixin; Jiang, Cai-Zhong

2016-01-01

Virus-induced RNA silencing is involved in plant antiviral defense and requires key enzyme components, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonaute proteins (AGOs). However, the transcriptional regulation of these critical components is largely unknown. In petunia (Petunia hybrida), an ethylene-responsive element binding factor, PhERF2, is induced by Tobacco rattle virus (TRV) infection. Inclusion of a PhERF2 fragment in a TRV silencing construct containing reporter fragments of phytoene desaturase (PDS) or chalcone synthase (CHS) substantially impaired silencing efficiency of both the PDS and CHS reporters. Silencing was also impaired in PhERF2- RNAi lines, where TRV-PhPDS infection did not show the expected silencing phenotype (photobleaching). In contrast, photobleaching in response to infiltration with the TRV-PhPDS construct was enhanced in plants overexpressing PhERF2. Transcript abundance of the RNA silencing-related genes RDR2, RDR6, DCL2, and AGO2 was lower in PhERF2-silenced plants but higher in PhERF2-overexpressing plants. Moreover, PhERF2-silenced lines showed higher susceptibility to Cucumber mosaic virus (CMV) than wild-type (WT) plants, while plants overexpressing PhERF2 exhibited increased resistance. Interestingly, growth and development of PhERF2-RNAi lines were substantially slower, whereas the overexpressing lines were more vigorous than the controls. Taken together, our results indicate that PhERF2 functions as a positive regulator in antiviral RNA silencing. PMID:27099376

Silencing of the PiAvr3a effector-encoding gene from Phytophthora infestans by transcriptional fusion to a short interspersed element.

PubMed

Vetukuri, Ramesh R; Tian, Zhendong; Avrova, Anna O; Savenkov, Eugene I; Dixelius, Christina; Whisson, Stephen C

2011-12-01

Phytophthora infestans is the notorious oomycete causing late blight of potato and tomato. A large proportion of the P. infestans genome is composed of transposable elements, the activity of which may be controlled by RNA silencing. Accumulation of small RNAs is one of the hallmarks of RNA silencing. Here we demonstrate the presence of small RNAs corresponding to the sequence of a short interspersed retrotransposable element (SINE) suggesting that small RNAs might be involved in silencing of SINEs in P. infestans. This notion was exploited to develop novel tools for gene silencing in P. infestans by engineering transcriptional fusions of the PiAvr3a gene, encoding an RXLR avirulence effector, to the infSINEm retroelement. Transgenic P. infestans lines expressing either 5'-infSINEm::PiAvr3a-3' or 5'-PiAvr3a::SINEm-3' chimeric transcripts initially exhibited partial silencing of PiAvr3a. Over time, PiAvr3a either recovered wild type transcript levels in some lines, or became fully silenced in others. Introduction of an inverted repeat construct was also successful in yielding P. infestans transgenic lines silenced for PiAvr3a. In contrast, constructs expressing antisense or aberrant RNA transcripts failed to initiate silencing of PiAvr3a. Lines exhibiting the most effective silencing of PiAvr3a were either weakly or non-pathogenic on susceptible potato cv. Bintje. This study expands the repertoire of reverse genetics tools available for P. infestans research, and provides insights into a possible mode of variation in effector expression through spread of silencing from adjacent retroelements. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Identification and characterization of a silencer regulatory element in the 3'-flanking region of the murine CD46 gene.

PubMed Central

Nomura, M; Tsujimura, A; Begum, N A; Matsumoto, M; Wabiko, H; Toyoshima, K; Seya, T

2000-01-01

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3'-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8 kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein-silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46. PMID:11023821

Microarray Analysis of LTR Retrotransposon Silencing Identifies Hdac1 as a Regulator of Retrotransposon Expression in Mouse Embryonic Stem Cells

PubMed Central

Madej, Monika J.; Taggart, Mary; Gautier, Philippe; Garcia-Perez, Jose Luis; Meehan, Richard R.; Adams, Ian R.

2012-01-01

Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells. PMID:22570599

Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong Ruyi; Wu Jianxiang; Zhou Yijun

2009-04-25

Rice stripe virus (RSV) is a single-stranded (ss) RNA virus belonging to the genus Tenuivirus. RSV is present in many East Asian countries and causes severe diseases in rice fields, especially in China. In this study, we analyzed six proteins encoded by the virus for their abilities to suppress RNA silencing in plant using a green fluorescent protein (GFP)-based transient expression assay. Our results indicate that NS3 encoded by RSV RNA3, but not other five RSV encoded proteins, can strongly suppress local GFP silencing in agroinfiltrated Nicotiana benthamiana leaves. NS3 can reverse the GFP silencing, it can also prevent longmore » distance spread of silencing signals which have been reported to be necessary for inducing systemic silencing in host plants. The NS3 protein can significantly reduce the levels of small interfering RNAs (siRNAs) in silencing cells, and was found to bind 21-nucleotide ss-siRNA, siRNA duplex and long ssRNA but not long double-stranded (ds)-RNA. Both N and C terminal of the NS3 protein are critical for silencing suppression, and mutation of the putative nuclear localization signal decreases its local silencing suppression efficiency and blocks its systemic silencing suppression. The NS3-GFP fusion protein and NS3 were shown to accumulate predominantly in nuclei of onion, tobacco and rice cells through transient expression assay or immunocytochemistry and electron microscopy. In addition, transgenic rice and tobacco plants expressing the NS3 did not show any apparent alteration in plant growth and morphology, although NS3 was proven to be a pathogenicity determinant in the PVX heterogenous system. Taken together, our results demonstrate that RSV NS3 is a suppressor of RNA silencing in planta, possibly through sequestering siRNA molecules generated in cells that are undergoing gene silencing.« less

Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing.

PubMed

Hedil, Marcio; Sterken, Mark G; de Ronde, Dryas; Lohuis, Dick; Kormelink, Richard

2015-01-01

RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Using the system established, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSsTSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.

Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

PubMed Central

Hedil, Marcio; Sterken, Mark G.; de Ronde, Dryas; Lohuis, Dick; Kormelink, Richard

2015-01-01

RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Using the system established, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSsTSWV to map domains required for RSS activity and triggering of Tsw-governed resistance. PMID:26275304

Development of marker-free transgenic Jatropha curcas producing curcin-deficient seeds through endosperm-specific RNAi-mediated gene silencing.

PubMed

Gu, Keyu; Tian, Dongsheng; Mao, Huizhu; Wu, Lifang; Yin, Zhongchao

2015-10-08

Jatropha curcas L. is a potential biofuel plant and its seed oil is suitable for biodiesel production. Despite this promising application, jatropha seeds contain two major toxic components, namely phorbol esters and curcins. These compounds would reduce commercial value of seed cake and raise safety and environment concerns on jatropha plantation and processing. Curcins are Type I ribosome inactivating proteins. Several curcin genes have been identified in the jatropha genome. Among which, the Curcin 1 (C1) gene is identified to be specifically expressed in endosperm, whereas the Curcin 2A (C2A) is mainly expressed in young leaves. A marker-free RNAi construct carrying a β-estradiol-regulated Cre/loxP system and a C1 promoter-driven RNAi cassette for C1 gene was made and used to generate marker-free transgenic RNAi plants to specifically silence the C1 gene in the endosperm of J. curcas. Plants of transgenic line L1, derived from T0-1, carry two copies of marker-free RNAi cassette, whereas plants of L35, derived from T0-35, harbored one copy of marker-free RNAi cassette and three copies of closely linked and yet truncated Hpt genes. The C1 protein content in endosperm of L1 and L35 seeds was greatly reduced or undetectable, while the C2A proteins in young leaves of T0-1 and T0-35 plants were unaffected. In addition, the C1 mRNA transcripts were undetectable in the endosperm of T3 seeds of L1 and L35. The results demonstrated that the expression of the C1 gene was specifically down-regulated or silenced by the double-stranded RNA-mediated RNA interference generated from the RNAi cassette. The C1 promoter-driven RNAi cassette for the C1 gene in transgenic plants was functional and heritable. Both C1 transcripts and C1 proteins were greatly down-regulated or silenced in the endosperm of transgenic J. curcas. The marker-free transgenic plants and curcin-deficient seeds developed in this study provided a solution for the toxicity of curcins in jatropha seeds and addressed the safety concerns of the marker genes in transgenic plants on the environments.

Strategies for Improving siRNA-Induced Gene Silencing Efficiency

PubMed Central

Safari, Fatemeh; Rahmani Barouji, Solmaz; Tamaddon, Ali Mohammad

2017-01-01

Purpose: Human telomerase reverse transcriptase (hTERT) plays a crucial role in tumorigenesis and progression of cancers. Gene silencing of hTERT by short interfering RNA (siRNA) is considered as a promising strategy for cancer gene therapy. Various algorithms have been devised for designing a high efficient siRNA which is a significant issue in the clinical usage. Thereby, in the present study, the relation of siRNA designing criteria and the gene silencing efficiency was evaluated. Methods: The siRNA sequences were designed and characterized by using on line soft wares. Cationic co-polymer (polyethylene glycol-g-polyethylene imine (PEG-g-PEI)) was used for the construction of polyelectrolyte complexes (PECs) containing siRNAs. The cellular uptake of the PECs was evaluated. The gene silencing efficiency of different siRNA sequences was investigated and the effect of observing the rational designing on the functionality of siRNAs was assessed. Results: The size of PEG-g-PEI siRNA with N/P (Nitrogen/Phosphate) ratio of 2.5 was 114 ± 0.645 nm. The transfection efficiency of PECs was desirable (95.5% ± 2.4%.). The results of Real-Time PCR showed that main sequence (MS) reduced the hTERT expression up to 90% and control positive sequence (CPS) up to 63%. These findings demonstrated that the accessibility to the target site has priority than the other criteria such as sequence preferences and thermodynamic features. Conclusion: siRNA opens a hopeful window in cancer therapy which provides a convenient and tolerable therapeutic approach. Thereby, using the set of criteria and rational algorithms in the designing of siRNA remarkably affect the gene silencing efficiency. PMID:29399550

Strategies for Improving siRNA-Induced Gene Silencing Efficiency.

PubMed

Safari, Fatemeh; Rahmani Barouji, Solmaz; Tamaddon, Ali Mohammad

2017-12-01

Purpose: Human telomerase reverse transcriptase (hTERT) plays a crucial role in tumorigenesis and progression of cancers. Gene silencing of hTERT by short interfering RNA (siRNA) is considered as a promising strategy for cancer gene therapy. Various algorithms have been devised for designing a high efficient siRNA which is a significant issue in the clinical usage. Thereby, in the present study, the relation of siRNA designing criteria and the gene silencing efficiency was evaluated. Methods: The siRNA sequences were designed and characterized by using on line soft wares. Cationic co-polymer (polyethylene glycol-g-polyethylene imine (PEG-g-PEI)) was used for the construction of polyelectrolyte complexes (PECs) containing siRNAs. The cellular uptake of the PECs was evaluated. The gene silencing efficiency of different siRNA sequences was investigated and the effect of observing the rational designing on the functionality of siRNAs was assessed. Results: The size of PEG-g-PEI siRNA with N/P (Nitrogen/Phosphate) ratio of 2.5 was 114 ± 0.645 nm. The transfection efficiency of PECs was desirable (95.5% ± 2.4%.). The results of Real-Time PCR showed that main sequence (MS) reduced the hTERT expression up to 90% and control positive sequence (CPS) up to 63%. These findings demonstrated that the accessibility to the target site has priority than the other criteria such as sequence preferences and thermodynamic features. Conclusion: siRNA opens a hopeful window in cancer therapy which provides a convenient and tolerable therapeutic approach. Thereby, using the set of criteria and rational algorithms in the designing of siRNA remarkably affect the gene silencing efficiency.

Virus-induced gene silencing of the two squalene synthase isoforms of apple tree (Malus × domestica L.) negatively impacts phytosterol biosynthesis, plastid pigmentation and leaf growth.

PubMed

Navarro Gallón, Sandra M; Elejalde-Palmett, Carolina; Daudu, Dimitri; Liesecke, Franziska; Jullien, Frédéric; Papon, Nicolas; Dugé de Bernonville, Thomas; Courdavault, Vincent; Lanoue, Arnaud; Oudin, Audrey; Glévarec, Gaëlle; Pichon, Olivier; Clastre, Marc; St-Pierre, Benoit; Atehortùa, Lucia; Yoshikawa, Nobuyuki; Giglioli-Guivarc'h, Nathalie; Besseau, Sébastien

2017-07-01

The use of a VIGS approach to silence the newly characterized apple tree SQS isoforms points out the biological function of phytosterols in plastid pigmentation and leaf development. Triterpenoids are beneficial health compounds highly accumulated in apple; however, their metabolic regulation is poorly understood. Squalene synthase (SQS) is a key branch point enzyme involved in both phytosterol and triterpene biosynthesis. In this study, two SQS isoforms were identified in apple tree genome. Both isoforms are located at the endoplasmic reticulum surface and were demonstrated to be functional SQS enzymes using an in vitro activity assay. MdSQS1 and MdSQS2 display specificities in their expression profiles with respect to plant organs and environmental constraints. This indicates a possible preferential involvement of each isoform in phytosterol and/or triterpene metabolic pathways as further argued using RNAseq meta-transcriptomic analyses. Finally, a virus-induced gene silencing (VIGS) approach was used to silence MdSQS1 and MdSQS2. The concomitant down-regulation of both MdSQS isoforms strongly affected phytosterol synthesis without alteration in triterpene accumulation, since triterpene-specific oxidosqualene synthases were found to be up-regulated to compensate metabolic flux reduction. Phytosterol deficiencies in silenced plants clearly disturbed chloroplast pigmentation and led to abnormal development impacting leaf division rather than elongation or differentiation. In conclusion, beyond the characterization of two SQS isoforms in apple tree, this work brings clues for a specific involvement of each isoform in phytosterol and triterpene pathways and emphasizes the biological function of phytosterols in development and chloroplast integrity. Our report also opens the door to metabolism studies in Malus domestica using the apple latent spherical virus-based VIGS method.

Cancelation and its simulation using Matlab according to active noise control case study of automotive noise silencer

NASA Astrophysics Data System (ADS)

Alfisyahrin; Isranuri, I.

2018-02-01

Active Noise Control is a technique to overcome noisy with noise or sound countered with sound in scientific terminology i.e signal countered with signals. This technique can be used to dampen relevant noise in accordance with the wishes of the engineering task and reducing automotive muffler noise to a minimum. Objective of this study is to develop a Active Noise Control which should cancel the noise of automotive Exhaust (Silencer) through Signal Processing Simulation methods. Noise generator of Active Noise Control is to make the opponent signal amplitude and frequency of the automotive noise. The steps are: Firstly, the noise of automotive silencer was measured to characterize the automotive noise that its amplitude and frequency which intended to be expressed. The opposed sound which having similar character with the signal source should be generated by signal function. A comparison between the data which has been completed with simulation calculations Fourier transform field data is data that has been captured on the muffler (noise silencer) Toyota Kijang Capsule assembly 2009. MATLAB is used to simulate how the signal processing noise generated by exhaust (silencer) using FFT. This opponent is inverted phase signal from the signal source 180° conducted by Instruments of Signal Noise Generators. The process of noise cancelation examined through simulation using computer software simulation. The result is obtained that attenuation of sound (noise cancellation) has a difference of 33.7%. This value is obtained from the comparison of the value of the signal source and the signal value of the opponent. So it can be concluded that the noisy signal can be attenuated by 33.7%.

Unpacking the Unspoken: Silence in Collective Memory and Forgetting

ERIC Educational Resources Information Center

Vinitzky-Seroussi, Vered; Teeger, Chana

2010-01-01

Collective memory quite naturally brings to mind notions of mnemonic speech and representation. In this article, however, we propose that collective silences be thought of as a rich and promising arena through which to understand how groups deal with their collective pasts. In so doing, we explore two types of silence: overt silence and covert…

Teaching Basic Algebra Courses at the College Level

ERIC Educational Resources Information Center

Mallenby, Michel L.; Mallenby, Douglas W.

2004-01-01

Three dysfunctional behaviors of basic algebra students are described: Silence as Camouflage, Wing and a Prayer, and Ignorance is OK. These behavior patterns are explained, and beneficial teaching methods that address the weaknesses are presented.

Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group.

PubMed

Petrova, Natalya S; Chernikov, Ivan V; Meschaninova, Mariya I; Dovydenko, Iiya S; Venyaminova, Aliya G; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

2012-03-01

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5'-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

RNAi as an emerging approach to control Fusarium head blight disease and mycotoxin contamination in cereals

PubMed Central

Machado, Ana Karla; Brown, Neil A; Urban, Martin; Kanyuka, Kostya

2017-01-01

Abstract Fusarium graminearum is a major fungal pathogen of cereals worldwide, causing seedling, stem base and floral diseases, including Fusarium head blight (FHB). In addition to yield and quality losses, FHB contaminates cereal grain with mycotoxins, including deoxynivalenol, which are harmful to human, animal and ecosystem health. Currently, FHB control is only partially effective due to several intractable problems. RNA interference (RNAi) is a natural mechanism that regulates gene expression. RNAi has been exploited in the development of new genomic tools that allow the targeted silencing of genes of interest in many eukaryotes. Host‐induced gene silencing (HIGS) is a transgenic technology used to silence fungal genes in planta during attempted infection and thereby reduces disease levels. HIGS relies on the host plant's ability to produce mobile small interfering RNA molecules, generated from long double‐stranded RNA, which are complementary to targeted fungal genes. These molecules are transferred from the plant to invading fungi via an uncharacterised mechanism, to cause gene silencing. Here, we describe recent advances in RNAi‐mediated control of plant pathogenic fungi, highlighting the key advantages and disadvantages. We then discuss the developments and implications of combining HIGS with other methods of disease control. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:28967180

Angiogenic effects of apigenin on endothelial cells after hypoxia-reoxygenation via the caveolin-1 pathway

PubMed Central

Tu, Fengxia; Pang, Qiongyi; Chen, Xiang; Huang, Tingting; Liu, Meixia; Zhai, Qiongxiang

2017-01-01

In the present study, we aimed to elucidate whether apigenin contributes to the induction of angiogenesis and the related mechanisms in cell hypoxia-reoxygenation injury. The role of apigenin was examined in human umbilical vein endothelial cell (HUVEC) viability, migration and tube formation in vitro. To investigate the related mechanisms, we used caveolin-1 short interfering RNA. The viability of HUVECs was measured using Cell Counting Kit-8 assays, HUVEC migration was analyzed by crystal violet staining, and a tube formation assay was performed using the branch point method. Expression of caveolin-1, vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS) in HUVECs was examined by polymerase chain reaction and western blotting. Our data revealed that apigenin induced angiogenesis in vitro by increasing the tube formation ability of HUVECs, which was counteracted by caveolin-1 silencing. Compared to the NC group, Caveolin-1 and eNOS expression was upregulated by apigenin, whereas compared to the NC group, eNOS expression was increased upon caveolin-1 silencing. The expression of VEGF was increased by treatment with apigenin; however, compared to the NC group, caveolin-1 silencing did not affect VEGF expression, and apigenin did not increase VEGF expression in HUVECs after caveolin-1 silencing. These data suggest that apigenin may be a candidate therapeutic target for stroke recovery by promoting angiogenesis via the caveolin-1 signaling pathway. PMID:29039442

An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

PubMed

Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

2016-01-01

Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.

Host-Induced Silencing of Pathogenicity Genes Enhances Resistance to Fusarium oxysporum Wilt in Tomato.

PubMed

Bharti, Poonam; Jyoti, Poonam; Kapoor, Priya; Sharma, Vandana; Shanmugam, V; Yadav, Sudesh Kumar

2017-08-01

This study presents a novel approach of controlling vascular wilt in tomato by RNAi expression directed to pathogenicity genes of Fusarium oxysporum f. sp. lycopersici. Vascular wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici leads to qualitative and quantitative loss of the crop. Limitation in the existing control measures necessitates the development of alternative strategies to increase resistance in the plants against pathogens. Recent findings paved way to RNAi, as a promising method for silencing of pathogenicity genes in fungus and provided effective resistance against fungal pathogens. Here, two important pathogenicity genes FOW2, a Zn(II)2Cys6 family putative transcription regulator, and chsV, a putative myosin motor and a chitin synthase domain, were used for host-induced gene silencing through hairpinRNA cassettes of these genes against Fusarium oxysporum f. sp. lycopersici. HairpinRNAs were assembled in appropriate binary vectors and transformed into tomato plant targeting FOW2 and chsV genes, for two highly pathogenic strains of Fusarium oxysporum viz. TOFOL-IHBT and TOFOL-IVRI. Transgenic tomatoes were analyzed for possible attainment of resistance in transgenic lines against fungal infection. Eight transgenic lines expressing hairpinRNA cassettes showed trivial disease symptoms after 6-8 weeks of infection. Hence, the host-induced posttranscriptional gene silencing of pathogenicity genes in transgenic tomato plants has enhanced their resistance to vascular wilt disease caused by Fusarium oxysporum.

A petunia ethylene-responsive element binding factor, PhERF2, plays an important role in antiviral RNA silencing.

PubMed

Sun, Daoyang; Nandety, Raja Sekhar; Zhang, Yanlong; Reid, Michael S; Niu, Lixin; Jiang, Cai-Zhong

2016-05-01

Virus-induced RNA silencing is involved in plant antiviral defense and requires key enzyme components, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonaute proteins (AGOs). However, the transcriptional regulation of these critical components is largely unknown. In petunia (Petunia hybrida), an ethylene-responsive element binding factor, PhERF2, is induced by Tobacco rattle virus (TRV) infection. Inclusion of a PhERF2 fragment in a TRV silencing construct containing reporter fragments of phytoene desaturase (PDS) or chalcone synthase (CHS) substantially impaired silencing efficiency of both the PDS and CHS reporters. Silencing was also impaired in PhERF2- RNAi lines, where TRV-PhPDS infection did not show the expected silencing phenotype (photobleaching). In contrast, photobleaching in response to infiltration with the TRV-PhPDS construct was enhanced in plants overexpressing PhERF2 Transcript abundance of the RNA silencing-related genes RDR2, RDR6, DCL2, and AGO2 was lower in PhERF2-silenced plants but higher in PhERF2-overexpressing plants. Moreover, PhERF2-silenced lines showed higher susceptibility to Cucumber mosaic virus (CMV) than wild-type (WT) plants, while plants overexpressing PhERF2 exhibited increased resistance. Interestingly, growth and development of PhERF2-RNAi lines were substantially slower, whereas the overexpressing lines were more vigorous than the controls. Taken together, our results indicate that PhERF2 functions as a positive regulator in antiviral RNA silencing. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The double-stranded RNA binding protein RDE-4 can act cell autonomously during feeding RNAi in C. elegans

PubMed Central

Raman, Pravrutha; Zaghab, Soriayah M.; Traver, Edward C.

2017-01-01

Abstract Long double-stranded RNA (dsRNA) can silence genes of matching sequence upon ingestion in many invertebrates and is therefore being developed as a pesticide. Such feeding RNA interference (RNAi) is best understood in the worm Caenorhabditis elegans, where the dsRNA-binding protein RDE-4 initiates silencing by recruiting an endonuclease to process long dsRNA into short dsRNA. These short dsRNAs are thought to move between cells because muscle-specific rescue of rde-4 using repetitive transgenes enables silencing in other tissues. Here, we extend this observation using additional promoters, report an inhibitory effect of repetitive transgenes, and discover conditions for cell-autonomous silencing in animals with tissue-specific rescue of rde-4. While expression of rde-4(+) in intestine, hypodermis, or neurons using a repetitive transgene can enable silencing also in unrescued tissues, silencing can be inhibited wihin tissues that express a repetitive transgene. Single-copy transgenes that express rde-4(+) in body-wall muscles or hypodermis, however, enable silencing selectively in the rescued tissue but not in other tissues. These results suggest that silencing by the movement of short dsRNA between cells is not an obligatory feature of feeding RNAi in C. elegans. We speculate that similar control of dsRNA movement could modulate tissue-specific silencing by feeding RNAi in other invertebrates. PMID:28541563

Rethinking the Day of Silence

ERIC Educational Resources Information Center

Murphy, Adriana

2013-01-01

Back in 2006, 7th and 8th graders at Green Acres, the K-8 independent school where the author taught in suburban Maryland, participated in the Day of Silence. The Day of Silence is a national event: Students across the country take a one-day pledge of silence to show that they want to make schools safe for all students, regardless of their sexual…

Human Gamma Satellite Insulator Sequences to Prevent Gene Silencing | NCI Technology Transfer Center | TTC

Cancer.gov

This invention describes the use of chromatin insulators, or gamma satellite DNA, to inhibit gene silencing in a cell, which may have a significant impact on gene therapy across multiple diseases where gene silencing is the cause. Experimental data has demonstrated these gamma satellite DNAs overcome gene position effects and ultimately inhibit gene silencing.

The Discourse of Silence as Testimony in Jenn Díaz's "Es Un Decir"

ERIC Educational Resources Information Center

Riordan-Goncalves, Julia

2018-01-01

The explosion of interest in the recovery of historical memory in Spain seeks to address many decades of silence and forgetting during the years of the Franco dictatorship and afterwards. Working with trauma theory, Michel Foucault's understanding of silence as discourse, as well as queer theory's exploration of silence as strategy and power, this…

Ways to Promote the Classroom Participation of International Students by Understanding the Silence of Japanese University Students

ERIC Educational Resources Information Center

Kim, Soonhyang; Ates, Burcu; Grigsby, Yurimi; Kraker, Stefani; Micek, Timothy A.

2016-01-01

The authors explored the role of silence and deciphered its meaning and usefulness as a teaching and learning strategy for Japanese students through a survey of Japanese university students in their home country. This study has revealed that participant responses were evenly divided among comfortable with silence, uncomfortable with silence, and…

The Sound of Silence: The Case of Virtual Team Organising

ERIC Educational Resources Information Center

Panteli, N.; Fineman, S.

2005-01-01

In this paper we discuss the role of silence within a virtual organising context. The paper raises issues related to the construction of silence in the virtual team context and the implications it has on team interactions. By drawing upon existing studies on virtual teams, we argue that members' silence may not always have negative effects on team…

Light intensity affects RNA silencing of a transgene in Nicotiana benthamiana plants.

PubMed

Kotakis, Christos; Vrettos, Nicholas; Kotsis, Dimitrios; Tsagris, Mina; Kotzabasis, Kiriakos; Kalantidis, Kriton

2010-10-12

Expression of exogenous sequences in plants is often suppressed through one of the earliest described RNA silencing pathways, sense post-transcriptional gene silencing (S-PTGS). This type of suppression has made significant contributions to our knowledge of the biology of RNA silencing pathways and has important consequences in plant transgenesis applications. Although significant progress has been made in recent years, factors affecting the stability of transgene expression are still not well understood. It has been shown before that the efficiency of RNA silencing in plants is influenced by various environmental factors. Here we report that a major environmental factor, light intensity, significantly affects the induction and systemic spread of S-PTGS. Moreover, we show that photoadaptation to high or low light intensity conditions differentially affects mRNA levels of major components of the RNA silencing machinery. Light intensity is one of the previously unknown factors that affect transgene stability at the post-transcriptional level. Our findings demonstrate an example of how environmental conditions could affect RNA silencing.

Radical Conversations: Part Two--Cultivating Social-Constructivist Learning Methods in ABE Classrooms

ERIC Educational Resources Information Center

Muth, Bill; Kiser, Madeline

2008-01-01

In many U.S. prisons an overuse of individualized instruction silences literacy learners and reinforces oppressive notions about what knowledge is and whose knowledge counts. In these classrooms, methods that invite learners to tap their background knowledge, reflect on their worlds, and dialogue with others to construct meaning--commonplace in…

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations

PubMed Central

Gasparis, Sebastian; Kała, Maciej; Przyborowski, Mateusz; Orczyk, Waclaw; Nadolska-Orczyk, Anna

2017-01-01

Gene silencing by RNA interference is a particularly important tool in the study of gene function in polyploid cereal species for which the collections of natural or induced mutants are very limited. Previously we have been testing small interfering RNA-based approach of gene silencing in wheat and triticale. In this research, artificial microRNAs (amiRs) were studied in the same species and the same target genes to compare effectiveness of both gene silencing pathways. amiR cassettes were designed to silence Puroindoline a (Pina) and Puroindoline b (Pinb) hardness genes in wheat and their orthologues Secaloindoline a (Sina) and Secaloindoline b (Sinb) genes in triticale. Each of the two cassettes contained 21 nt microRNA (miR) precursor derived from conserved regions of Pina/Sina or Pinb/Sinb genes, respectively. Transgenic plants were obtained with high efficiency in two cultivars of wheat and one cultivar of triticale after using the Pinb-derived amiR vector for silencing of Pinb or Sinb, respectively. Lack of transgenic plants in wheat or very low transformation efficiency in triticale was observed using the Pina-derived amiR cassette, despite large numbers of embryos attempted. Silencing of Pinb in wheat and Sinb in triticale was highly efficient in the T1 generation. The transcript level of Pinb in wheat was reduced up to 92% and Sinb in triticale was reduced up to 98%. Moreover, intended silencing of Pinb/Sinb with Pinb-derived amiR cassette was highly correlated with simultaneous silencing of Pina/Sina in the same transgenic plants. High downregulation of Pinb/Pina genes in T1 plants of wheat and Sinb/Sina genes in T1 plants of triticale was associated with strong expression of Pinb-derived amiR. Silencing of the target genes correlated with increased grain hardness in both species. Total protein content in the grains of transgenic wheat was significantly lower. Although, the Pinb-derived amiR cassette was stably inherited in the T2 generation of wheat and triticale the silencing effect including strongly decreased expression of silenced genes as well as strong expression of Pinb-derived amiR was not transmitted. Advantages and disadvantages of posttranscriptional silencing of target genes by means of amiR and siRNA-based approaches in polyploid cereals are discussed. PMID:28119710

pHg/pSILBAγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA – triggering in the mycorrhizal fungus Laccaria bicolor

PubMed Central

Kemppainen, Minna J.; Pardo, Alejandro G.

2010-01-01

Summary pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy oriented two‐step PCR cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300‐based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAγ plasmid is used for Agrobacterium‐mediated transformation. The pHg/pSILBAγ system results in predominantly single integrations of RNA silencing triggering T‐DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAγ construct and two other pSILBA plasmid variants (pSILBA and pSILBAα) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA variants tested resulted in up to 65–76% of transformants with reduced growth on nitrate, pSILBAγ produced the highest number (65%) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T‐DNA integration in transcriptionally active genomic sites. pHg/pSILBAγ was shown to produce T‐DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAγ was further tested with Laccaria inositol‐1,4,5‐triphosphate 5‐phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAγ silencing system is optimized for L. bicolor but it should be highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing. PMID:21255319

The Initiation of Epigenetic Silencing of Active Transposable Elements Is Triggered by RDR6 and 21-22 Nucleotide Small Interfering RNAs1[W][OA

PubMed Central

Nuthikattu, Saivageethi; McCue, Andrea D.; Panda, Kaushik; Fultz, Dalen; DeFraia, Christopher; Thomas, Erica N.; Slotkin, R. Keith

2013-01-01

Transposable elements (TEs) are mobile fragments of DNA that are repressed in both plant and animal genomes through the epigenetic inheritance of repressed chromatin and expression states. The epigenetic silencing of TEs in plants is mediated by a process of RNA-directed DNA methylation (RdDM). Two pathways of RdDM have been identified: RNA Polymerase IV (Pol IV)-RdDM, which has been shown to be responsible for the de novo initiation, corrective reestablishment, and epigenetic maintenance of TE and/or transgene silencing; and RNA-dependent RNA Polymerase6 (RDR6)-RdDM, which was recently identified as necessary for maintaining repression for a few TEs. We have further characterized RDR6-RdDM using a genome-wide search to identify TEs that generate RDR6-dependent small interfering RNAs. We have determined that TEs only produce RDR6-dependent small interfering RNAs when transcriptionally active, and we have experimentally identified two TE subfamilies as direct targets of RDR6-RdDM. We used these TEs to test the function of RDR6-RdDM in assays for the de novo initiation, corrective reestablishment, and maintenance of TE silencing. We found that RDR6-RdDM plays no role in maintaining TE silencing. Rather, we found that RDR6 and Pol IV are two independent entry points into RdDM and epigenetic silencing that perform distinct functions in the silencing of TEs: Pol IV-RdDM functions to maintain TE silencing and to initiate silencing in an RNA Polymerase II expression-independent manner, while RDR6-RdDM functions to recognize active Polymerase II-derived TE mRNA transcripts to both trigger and correctively reestablish TE methylation and epigenetic silencing. PMID:23542151

The initiation of epigenetic silencing of active transposable elements is triggered by RDR6 and 21-22 nucleotide small interfering RNAs.

PubMed

Nuthikattu, Saivageethi; McCue, Andrea D; Panda, Kaushik; Fultz, Dalen; DeFraia, Christopher; Thomas, Erica N; Slotkin, R Keith

2013-05-01

Transposable elements (TEs) are mobile fragments of DNA that are repressed in both plant and animal genomes through the epigenetic inheritance of repressed chromatin and expression states. The epigenetic silencing of TEs in plants is mediated by a process of RNA-directed DNA methylation (RdDM). Two pathways of RdDM have been identified: RNA Polymerase IV (Pol IV)-RdDM, which has been shown to be responsible for the de novo initiation, corrective reestablishment, and epigenetic maintenance of TE and/or transgene silencing; and RNA-dependent RNA Polymerase6 (RDR6)-RdDM, which was recently identified as necessary for maintaining repression for a few TEs. We have further characterized RDR6-RdDM using a genome-wide search to identify TEs that generate RDR6-dependent small interfering RNAs. We have determined that TEs only produce RDR6-dependent small interfering RNAs when transcriptionally active, and we have experimentally identified two TE subfamilies as direct targets of RDR6-RdDM. We used these TEs to test the function of RDR6-RdDM in assays for the de novo initiation, corrective reestablishment, and maintenance of TE silencing. We found that RDR6-RdDM plays no role in maintaining TE silencing. Rather, we found that RDR6 and Pol IV are two independent entry points into RdDM and epigenetic silencing that perform distinct functions in the silencing of TEs: Pol IV-RdDM functions to maintain TE silencing and to initiate silencing in an RNA Polymerase II expression-independent manner, while RDR6-RdDM functions to recognize active Polymerase II-derived TE mRNA transcripts to both trigger and correctively reestablish TE methylation and epigenetic silencing.

The microenvironment induces collective migration in SDHB-silenced mouse pheochromocytoma spheroids.

PubMed

D'Antongiovanni, Vanessa; Martinelli, Serena; Richter, Susan; Canu, Letizia; Guasti, Daniele; Mello, Tommaso; Romagnoli, Paolo; Pacak, Karel; Eisenhofer, Graeme; Mannelli, Massimo; Rapizzi, Elena

2017-10-01

Pheochromocytomas (Pheos) and paragangliomas (PGLs) are neuroendocrine tumors. Approximately 30-40% of Pheos/PGLs are due to germline mutations in one of the susceptibility genes, including those encoding the succinate dehydrogenase subunits A-D ( SDHA-D ). Up to 2/3 of patients affected by SDHB mutated Pheo/PGL develop metastatic disease with no successful cure at present. Here, for the first time, we evaluated the effects of SDHB silencing in a three dimension (3D) culture using spheroids of a mouse Pheo cell line silenced or not (wild type/wt/control) for the SDHB subunit. We investigated the role of the microenvironment on spheroid growth and migration/invasion by co-culturing SDHB -silenced or wt spheroids with primary cancer-activated fibroblasts (CAFs). When spheroids were co-cultured with fibroblasts, SDHB -silenced cells showed a significant increase in matrigel invasion as demonstrated by the computation of the migratory areas ( P  < 0.001). Moreover, cells detaching from the SDHB -silenced spheroids moved collectively, unlike the cells of wt spheroids that moved individually. Additionally, SDHB- silenced spheroids developed long filamentous formations along which clusters of cells migrated far away from the spheroid, whereas these structures were not present in wt spheroids. We found that lactate, largely secreted by CAFs, plays a specific role in promoting migration only of SDHB -silenced cells. In this study, we demonstrated that SDHB silencing per se increases tumor cell migration/invasion and that microenvironment, as represented by CAFs, plays a pivotal role in enhancing collective migration/invasion in Pheo SDHB -silenced tumor cells, suggesting their role in increasing the tumor metastasizing potential. © 2017 Society for Endocrinology.

Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Curry, Merril C.; Peters, Amelia A.; Kenny, Paraic A.

Highlights: •Some clinical breast cancers are associated with MCU overexpression. •MCU silencing did not alter cell death initiated with the Bcl-2 inhibitor ABT-263. •MCU silencing potentiated caspase-independent cell death initiated by ionomycin. •MCU silencing promoted ionomycin-mediated cell death without changes in bulk Ca{sup 2+}. -- Abstract: The mitochondrial calcium uniporter (MCU) transports free ionic Ca{sup 2+} into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levelsmore » of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca{sup 2+} levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca{sup 2+} levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.« less

Cytosine methylation at CG and CNG sites is not a prerequisite for the initiation of transcriptional gene silencing in plants, but it is required for its maintenance.

PubMed

Diéguez, M J; Vaucheret, H; Paszkowski, J; Mittelsten Scheid, O

1998-08-01

Transgenes integrated into plant chromosomes, and/or endogenous plant genes, may be subjected to epigenetic silencing at the transcriptional or post-transcriptional level. Transcriptional inactivation is correlated with hypermethylation of CG/CNG sites at the silent loci. It is not known whether local hypermethylation is part of the inactivation process, or just an outcome of the silent state. To address this issue, we generated transgenic tobacco lines containing a selectable marker gene controlled by a derivative of the 35S promoter of the cauliflower mosaic virus (CaMV) devoid of CG and CNG methylation acceptor sites. Silencing was triggered by crossing to the silencer locus of tobacco line 271. This line contains inactive and methylated copies of the 35S promoter and is able to silence homologous promoter copies at ectopic chromosomal positions. The mutated promoter lacking CG/CNG methylation acceptor sites was as susceptible to Trans-silencing as the unmodified 35S promoter control. Thus, methylation at CG and CNG sites is not a prerequisite for the initiation of epigenetic gene inactivation. Interestingly, while methylation of the remaining cytosines is usually only slightly affected by silencing, it was significantly increased in the absence of CG/CNG sequences. Since this sequence preference is the same as that of known methyltransferases, this may imply that silencing is accompanied or directly followed by recruitment of methyltransferase, which, in the absence of cytosines in the optimal sequence context, modifies other C residues in the affected area. However, silencing without CG/CNG methylation was immediately relieved in the absence of the silencer. Thus, CG/CNG methylation is probably essential for the maintenance of previously established epigenetic states.

Development of antiviral gene therapy for Monodon baculovirus using dsRNA loaded chitosan-dextran sulfate nanocapsule delivery system in Penaeus monodon post-larvae.

PubMed

Ramesh Kumar, D; Elumalai, Rajasegaran; Raichur, Ashok M; Sanjuktha, M; Rajan, J J; Alavandi, S V; Vijayan, K K; Poornima, M; Santiago, T C

2016-07-01

In the present study, a suitable carrier system was developed for the delivery of dsRNA into Penaeus monodon (P. monodon) post larvae to silence the Monodon baculovirus (MBV) structural gene of p74. The carrier system was developed by layer by layer adsorption of oppositely charged chitosan-dextran sulfate, on charged silica nanoparticles. The silica template was removedto produce multilayered hollow nanocapsules (CS-DS) that were utilized for dsRNA loading at an alkaline pH. The capsule's surface was modified by conjugating with shrimp feed for enhanced cellular uptake. In vivo cellular uptake of CS-DS/FITC loaded nanocapsules conjugated with feed was studied after oral administration into post-larvae. The results revealed that the encapsulated FITC was effectively delivered and exhibited a sustained release into the cytoplasm of shrimp post-larvae. The MBV challenge study for structural gene p74was conducted after 3-25 days of post infection (dpi) with respective CS-DS/dsRNA coated with feed. The results showed a significant survival rate of 86.63% and effective gene silencing in P. monodon. Our findings indicated that the delivery of dsRNA using shrimp feed coatedCS-DSnanocapsules could be a novel approach to prevent viral infections in shrimp. Copyright © 2016 Elsevier B.V. All rights reserved.

Speech recognition in individuals with sensorineural hearing loss.

PubMed

de Andrade, Adriana Neves; Iorio, Maria Cecilia Martinelli; Gil, Daniela

2016-01-01

Hearing loss can negatively influence the communication performance of individuals, who should be evaluated with suitable material and in situations of listening close to those found in everyday life. To analyze and compare the performance of patients with mild-to-moderate sensorineural hearing loss in speech recognition tests carried out in silence and with noise, according to the variables ear (right and left) and type of stimulus presentation. The study included 19 right-handed individuals with mild-to-moderate symmetrical bilateral sensorineural hearing loss, submitted to the speech recognition test with words in different modalities and speech test with white noise and pictures. There was no significant difference between right and left ears in any of the tests. The mean number of correct responses in the speech recognition test with pictures, live voice, and recorded monosyllables was 97.1%, 85.9%, and 76.1%, respectively, whereas after the introduction of noise, the performance decreased to 72.6% accuracy. The best performances in the Speech Recognition Percentage Index were obtained using monosyllabic stimuli, represented by pictures presented in silence, with no significant differences between the right and left ears. After the introduction of competitive noise, there was a decrease in individuals' performance. Copyright © 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

Hypolipidaemic drug treatment: yesterday is not gone yet, today is challenging and tomorrow is coming soon; let us combine them all.

PubMed

Athyros, Vasilios G; Tziomalos, Konstantinos; Karagiannis, Asterios; Mikhailidis, Dimitri P

2014-01-01

Statins remain the cornerstone of hypolipidaemic drug treatment. The recent American College of Cardiology (ACC)/American Heart Association (AHA) lipid guidelines suggest using percent reductions of low density lipoprotein cholesterol (LDL-C), according to cardiovascular disease (CVD) risk, rather than specific LDL-C targets. These guidelines raised concerns and other Societies (US, International, European) have not endorsed them. The implementation of previous guidelines in clinical practice is suboptimal due to attitudes of physicians and restrictions in health care systems. Monoclonal antibodies that inhibit proprotein convertase subtilisin/ kexin type 9 (PCSK9), which degrades the LDL receptor, like alirocumab and evolocumab, are in phase 3 trials. These drugs are suitable for statin intolerant or resistant patients, heterozygous familial hypercholesterolaemia (HeFH) and some forms of homozygous FH (HoFH). Mipomersen (antisense oligonucleotide against apolipoprotein B) and lomitapide (microsomal triglyceride transfer protein blocker) have already been approved for HoFH. Eventually, silencing micro-RNA oligonucleotides may also become available. The repair or silencing of genes implicated in hyperlipidaemia and/or atherosclerosis is also on the horizon. If the new therapeutic options mentioned above prove to be effective and safe then by combining them with statins and/or ezetimibe we should be able to effectively control acquired or hereditary dyslipidaemias and substantially further reduce CVD morbidity and mortality.

Suppression of thymosin β10 increases cell migration and metastasis of cholangiocarcinoma

PubMed Central

2013-01-01

Background Thymosin β10 (Tβ10) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of Tβ10 in liver fluke-associated cholangiocarcinoma (CCA) are not fully understood. In this study, we investigated the expression of Tβ10 in CCA tumor tissues and cell lines as well as molecular mechanisms of Tβ10 in tumor metastasis of CCA cell lines. Methods Tβ10 expression was determined by real time RT-PCR or immunocytochemistry. Tβ10 silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell migration was assessed using modified Boyden chamber and wound healing assay. The effect of silencing Tβ10 on CCA tumor metastasis was determined in nude mice. Phosphorylation of ERK1/2 and the expression of EGR1, Snail and matrix metalloproteinases (MMPs) were studied. Results Ten pairs of CCA tissues (primary and metastatic tumors) and 5 CCA cell lines were studied. With real time RT-PCR and immunostaining analysis, Tβ10 was highly expressed in primary tumors of CCA; while it was relatively low in the metastatic tumors. Five CCA cell lines showed differential expression levels of Tβ10. Silence of Tβ10 significantly increased cell migration, invasion and wound healing of CCA cells in vitro; reversely, overexpression of Tβ10 reduced cell migration compared with control cells (P<0.05). In addition, silence of Tβ10 in CCA cells increased liver metastasis in a nude mouse model of CCA implantation into the spleen. Furthermore, silence of Tβ10 activated ERK1/2 and increased the expression of Snail and MMPs in CCA cell lines. Ras-GTPase inhibitor, FPT inhibitor III, effectively blocked Tβ10 silence-associated ERK1/2 activation, Snail expression and cell migration. Conclusions Low expression of Tβ10 is associated with metastatic phenotype of CCA in vitro and in vivo, which may be mediated by the activation of Ras, ERK1/2 and upregulation of Snail and MMPs. This study suggests a new molecular pathway of CCA pathogenesis and a novel strategy to treat or prevent CCA metastasis. PMID:24053380

A microRNA embedded AAV alpha-synuclein gene silencing vector for dopaminergic neurons

PubMed Central

Han, Ye; Khodr, Christina E.; Sapru, Mohan K.; Pedapati, Jyothi; Bohn, Martha C.

2011-01-01

Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons. PMID:21338582

Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1.

PubMed

Qu, Feng; Ye, Xiaohong; Morris, T Jack

2008-09-23

Plant RNA silencing machinery enlists four primary classes of proteins to achieve sequence-specific regulation of gene expression and mount an antiviral defense. These include Dicer-like ribonucleases (DCLs), Argonaute proteins (AGOs), dsRNA-binding proteins (DRBs), and RNA-dependent RNA polymerases (RDRs). Although at least four distinct endogenous RNA silencing pathways have been thoroughly characterized, a detailed understanding of the antiviral RNA silencing pathway is just emerging. In this report, we have examined the role of four DCLs, two AGOs, one DRB, and one RDR in controlling viral RNA accumulation in infected Arabidopsis plants by using a mutant virus lacking its silencing suppressor. Our results show that all four DCLs contribute to antiviral RNA silencing. We confirm previous reports implicating both DCL4 and DCL2 in this process and establish a minor role for DCL3. Surprisingly, we found that DCL1 represses antiviral RNA silencing through negatively regulating the expression of DCL4 and DCL3. We also implicate DRB4 in antiviral RNA silencing. Finally, we show that both AGO1 and AGO7 function to ensure efficient clearance of viral RNAs and establish that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 and RDR6 favor less structured RNA targets. Our results resolve several key steps in the antiviral RNA silencing pathway and provide a basis for further in-depth analysis.

The double-stranded RNA binding protein RDE-4 can act cell autonomously during feeding RNAi in C. elegans.

PubMed

Raman, Pravrutha; Zaghab, Soriayah M; Traver, Edward C; Jose, Antony M

2017-08-21

Long double-stranded RNA (dsRNA) can silence genes of matching sequence upon ingestion in many invertebrates and is therefore being developed as a pesticide. Such feeding RNA interference (RNAi) is best understood in the worm Caenorhabditis elegans, where the dsRNA-binding protein RDE-4 initiates silencing by recruiting an endonuclease to process long dsRNA into short dsRNA. These short dsRNAs are thought to move between cells because muscle-specific rescue of rde-4 using repetitive transgenes enables silencing in other tissues. Here, we extend this observation using additional promoters, report an inhibitory effect of repetitive transgenes, and discover conditions for cell-autonomous silencing in animals with tissue-specific rescue of rde-4. While expression of rde-4(+) in intestine, hypodermis, or neurons using a repetitive transgene can enable silencing also in unrescued tissues, silencing can be inhibited wihin tissues that express a repetitive transgene. Single-copy transgenes that express rde-4(+) in body-wall muscles or hypodermis, however, enable silencing selectively in the rescued tissue but not in other tissues. These results suggest that silencing by the movement of short dsRNA between cells is not an obligatory feature of feeding RNAi in C. elegans. We speculate that similar control of dsRNA movement could modulate tissue-specific silencing by feeding RNAi in other invertebrates. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe

PubMed Central

Tucker, James Franklin; Ohle, Corina; Schermann, Géza; Bendrin, Katja; Zhang, Wei; Fischer, Tamás; Zhang, Ke

2016-01-01

Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3’-5’ exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5’-3’ exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome. PMID:26889830

Tolerance of Sir1p/Origin Recognition Complex-Dependent Silencing for Enhanced Origin Firing at HMRa

PubMed Central

McConnell, Kristopher H.; Müller, Philipp; Fox, Catherine A.

2006-01-01

The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site. PMID:16479013

Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses

PubMed Central

Trubetskoy, Vladimir S.; Griffin, Jacob B.; Nicholas, Anthony L.; Nord, Eric M.; Xu, Zhao; Peterson, Ryan M.; Wooddell, Christine I.; Rozema, David B.; Wakefield, Darren H.; Lewis, David L.

2017-01-01

Abstract The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships. PMID:28180327

Gene silencing activity of siRNA polyplexes based on thiolated N,N,N-trimethylated chitosan.

PubMed

Varkouhi, Amir K; Verheul, Rolf J; Schiffelers, Raymond M; Lammers, Twan; Storm, Gert; Hennink, Wim E

2010-12-15

N,N,N-Trimethylated chitosan (TMC) is a biodegradable polymer emerging as a promising nonviral vector for nucleic acid and protein delivery. In the present study, we investigated whether the introduction of thiol groups in TMC enhances the extracellular stability of the complexes based on this polymer and promotes the intracellular release of siRNA. The gene silencing activity and the cellular cytotoxicity of polyplexes based on thiolated TMC were compared with those based on the nonthiolated counterpart and the regularly used lipidic transfection agent Lipofectamine. Incubation of H1299 human lung cancer cells expressing firefly luciferase with siRNA/thiolated TMC polyplexes resulted in 60-80% gene silencing activity, whereas complexes based on nonthiolated TMC showed less silencing (40%). The silencing activity of the complexes based on Lipofectamine 2000 was about 60-70%. Importantly, the TMC-SH polyplexes retained their silencing activity in the presence of hyaluronic acid, while nonthiolated TMC polyplexes hardly showed any silencing activity, demonstrating their stability against competing anionic macromolecules. Under the experimental conditions tested, the cytotoxicity of the thiolated and nonthiolated siRNA complexes was lower than those based on Lipofectamine. Given the good extracellular stability and good silencing activity, it is concluded that polyplexes based on TMC-SH are attractive systems for further in vivo evaluations.

The P0 protein encoded by cotton leafroll dwarf virus (CLRDV) inhibits local but not systemic RNA silencing.

PubMed

Delfosse, Verónica C; Agrofoglio, Yamila C; Casse, María F; Kresic, Iván Bonacic; Hopp, H Esteban; Ziegler-Graff, Véronique; Distéfano, Ana J

2014-02-13

Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Secondary RNA structure and its role in RNA interference to silence the respiratory syncytial virus fusion protein gene.

PubMed

Vig, Komal; Lewis, Nuruddeen; Moore, Eddie G; Pillai, Shreekumar; Dennis, Vida A; Singh, Shree R

2009-11-01

RNA interference (RNAi) is a post-transcriptional, gene silencing mechanism which uses small interfering RNA molecules (siRNA) for gene silencing. Respiratory Syncytial Virus (RSV) is an important respiratory pathogen of medical significance that causes high mortality in infants. The fusion (F) protein of RSV is a good target for therapeutic purposes as it is primarily responsible for penetration of the virus into host cells and subsequent syncytium formation during infection. In the present study, four siRNAs were designed and used individually as well as a mixture, to silence the RSV F gene. The relationship between siRNA design, target RNA structure, and their thermodynamics was also investigated. Silencing of F gene was observed using indirect immunofluorescence, western blot, reverse transcription PCR, and progeny viral titers. Our results show F gene silencing by all the four siRNAs individually and collectively. RT-PCR analysis revealed a decrease in mRNA level which corresponded to decreased F protein expression. siRNAs also inhibited RSV progeny as shown by viral titer estimation on infected HEp-2 cells. The present study demonstrates the silencing of the F gene using siRNA. Thermodynamic characteristics of the target RSV mRNA and siRNA seem to play an important role in siRNA gene silencing efficiency.

Is it just motion that silences awareness of other visual changes?

PubMed

Peirce, Jonathan W

2013-06-28

When an array of visual elements is changing color, size, or shape incoherently, the changes are typically quite visible even when the overall color, size, or shape statistics of the field may not have changed. When the dots also move, however, the changes become much less apparent; awareness of them is "silenced" (Suchow & Alvarez, 2011). This finding might indicate that the perception of motion is of particular importance to the visual system, such that it is given priority in processing over other forms of visual change. Here we test whether that is the case by examining the converse: whether awareness of motion signals can be silenced by potent coherent changes in color or size. We find that they can, and with very similar effects, indicating that motion is not critical for silencing. Suchow and Alvarez's dots always moved in the same direction with the same speed, causing them to be grouped as a single entity. We also tested whether this coherence was a necessary component of the silencing effect. It is not; when the dot speeds are randomly selected, such that no coherent motion is present, the silencing effect remains. It is clear that neither motion nor grouping is directly responsible for the silencing effect. Silencing can be generated from any potent visual change.

Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity.

PubMed

Clark, Josef P; Rahman, Reazur; Yang, Nachen; Yang, Linda H; Lau, Nelson C

2017-09-11

To test the directness of factors in initiating PIWI-directed gene silencing, we employed a Piwi-interacting RNA (piRNA)-targeted reporter assay in Drosophila ovary somatic sheet (OSS) cells [1]. This assay confirmed direct silencing roles for piRNA biogenesis factors and PIWI-associated factors [2-12] but suggested that chromatin-modifying proteins may act downstream of the initial silencing event. Our data also revealed that RNA-polymerase-II-associated proteins like PAF1 and RTF1 antagonize PIWI-directed silencing. PAF1 knockdown enhances PIWI silencing of reporters when piRNAs target the transcript region proximal to the promoter. Loss of PAF1 suppresses endogenous transposable element (TE) transcript maturation, whereas a subset of gene transcripts and long-non-coding RNAs adjacent to TE insertions are affected by PAF1 knockdown in a similar fashion to piRNA-targeted reporters. Additionally, transcription activation at specific TEs and TE-adjacent loci during PIWI knockdown is suppressed when PIWI and PAF1 levels are both reduced. Our study suggests a mechanistic conservation between fission yeast PAF1 repressing AGO1/small interfering RNA (siRNA)-directed silencing [13, 14] and Drosophila PAF1 opposing PIWI/piRNA-directed silencing. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extreme Metal Music and Anger Processing

PubMed Central

Sharman, Leah; Dingle, Genevieve A.

2015-01-01

The claim that listening to extreme music causes anger, and expressions of anger such as aggression and delinquency have yet to be substantiated using controlled experimental methods. In this study, 39 extreme music listeners aged 18–34 years were subjected to an anger induction, followed by random assignment to 10 min of listening to extreme music from their own playlist, or 10 min silence (control). Measures of emotion included heart rate and subjective ratings on the Positive and Negative Affect Scale (PANAS). Results showed that ratings of PANAS hostility, irritability, and stress increased during the anger induction, and decreased after the music or silence. Heart rate increased during the anger induction and was sustained (not increased) in the music condition, and decreased in the silence condition. PANAS active and inspired ratings increased during music listening, an effect that was not seen in controls. The findings indicate that extreme music did not make angry participants angrier; rather, it appeared to match their physiological arousal and result in an increase in positive emotions. Listening to extreme music may represent a healthy way of processing anger for these listeners. PMID:26052277

Human tRNA genes function as chromatin insulators

PubMed Central

Raab, Jesse R; Chiu, Jonathan; Zhu, Jingchun; Katzman, Sol; Kurukuti, Sreenivasulu; Wade, Paul A; Haussler, David; Kamakaka, Rohinton T

2012-01-01

Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes. PMID:22085927

Hyperactivation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2.

PubMed

Tchasovnikarova, Iva A; Timms, Richard T; Douse, Christopher H; Roberts, Rhys C; Dougan, Gordon; Kingston, Robert E; Modis, Yorgo; Lehner, Paul J

2017-07-01

Dominant mutations in the MORC2 gene have recently been shown to cause axonal Charcot-Marie-Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR-Cas9-mediated forward genetic screen, we identified MORC2 as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploited a new method, differential viral accessibility (DIVA), to show that loss of MORC2 results in chromatin decompaction at these target loci, which is concomitant with a loss of H3K9me3 deposition and transcriptional derepression. The ATPase activity of MORC2 is critical for HUSH-mediated silencing, and the most common alteration affecting the ATPase domain in CMT patients (p.Arg252Trp) hyperactivates HUSH-mediated repression in neuronal cells. These data define a critical role for MORC2 in epigenetic silencing by the HUSH complex and provide a mechanistic basis underpinning the role of MORC2 mutations in CMT disease.

Hyper-activation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2

PubMed Central

Douse, Christopher H.; Roberts, Rhys C.; Dougan, Gordon; Kingston, Robert E.; Modis, Yorgo; Lehner, Paul J.

2017-01-01

Dominant mutations in the MORC2 gene have recently been shown to cause axonal Charcot-Marie-Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR/Cas9-mediated forward genetic screen, we identify MORC2 as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploit a new method – Differential Viral Accessibility (DIVA) – to show that loss of MORC2 results in chromatin decompaction at these target loci, which is concomitant with a loss of H3K9me3 deposition and transcriptional derepression. The ATPase activity of MORC2 is critical for HUSH-mediated silencing, and the most common mutation affecting the ATPase domain found in CMT patients (R252W) hyper-activates HUSH-mediated repression in neuronal cells. These data define a critical role for MORC2 in epigenetic silencing by the HUSH complex and provide a mechanistic basis underpinning the role of MORC2 mutations in CMT disease. PMID:28581500

The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants.

PubMed

Poulet, Axel; Duc, Céline; Voisin, Maxime; Desset, Sophie; Tutois, Sylvie; Vanrobays, Emmanuel; Benoit, Matthias; Evans, David E; Probst, Aline V; Tatout, Christophe

2017-02-01

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its involvement in chromatin organisation has not been studied in plants. Here, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana in which heterochromatin clusters in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of the 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally, we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. © 2017. Published by The Company of Biologists Ltd.

“Real time” genetic manipulation: a new tool for ecological field studies

PubMed Central

Schäfer, Martin; Brütting, Christoph; Gase, Klaus; Reichelt, Michael; Baldwin, Ian; Meldau, Stefan

2014-01-01

Summary Field experiments with transgenic plants often reveal the functional significance of genetic traits important for plant performance in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene-related phenotypes in natural habitats. These methods do not allow sufficient control over gene expression to study ecological interactions in real-time, genetic traits playing essential roles in development, or dose-dependent effects. We applied the sensitive dexamethasone (DEX)-inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin-based DEX application method to facilitate ectopic gene expression and RNAi mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field-grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CK’s in both the glasshouse and field to understand resistance to the native herbivore Tupiocoris notatus, which attack plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, T. notatus damage increased, demonstrating CK’s role in this plant-herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature. PMID:23906159

Archaerhodopsin Selectively and Reversibly Silences Synaptic Transmission through Altered pH.

PubMed

El-Gaby, Mohamady; Zhang, Yu; Wolf, Konstantin; Schwiening, Christof J; Paulsen, Ole; Shipton, Olivia A

2016-08-23

Tools that allow acute and selective silencing of synaptic transmission in vivo would be invaluable for understanding the synaptic basis of specific behaviors. Here, we show that presynaptic expression of the proton pump archaerhodopsin enables robust, selective, and reversible optogenetic synaptic silencing with rapid onset and offset. Two-photon fluorescence imaging revealed that this effect is accompanied by a transient increase in pH restricted to archaerhodopsin-expressing boutons. Crucially, clamping intracellular pH abolished synaptic silencing without affecting the archaerhodopsin-mediated hyperpolarizing current, indicating that changes in pH mediate the synaptic silencing effect. To verify the utility of this technique, we used trial-limited, archaerhodopsin-mediated silencing to uncover a requirement for CA3-CA1 synapses whose afferents originate from the left CA3, but not those from the right CA3, for performance on a long-term memory task. These results highlight optogenetic, pH-mediated silencing of synaptic transmission as a spatiotemporally selective approach to dissecting synaptic function in behaving animals. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Sir- and silencer-independent disruption of silencing in Saccharomyces by Sas10p.

PubMed

Kamakaka, R T; Rine, J

1998-06-01

A promoter fusion library of Saccharomyces cerevisiae genes was used to exploit phenotypes associated with altered protein dosage. We identified a novel gene, SAS10, by the ability of Sas10p, when overproduced, to disrupt silencing. The predicted Sas10p was 70,200 kD and strikingly rich in charged amino acids. Sas10p was exclusively nuclear in all stages of the cell cycle. Overproduction of Sas10p caused derepression of mating type genes at both HML and HMR, as well as of URA3, TRP1, and ADE2 when inserted near a telomere or at HMR or the rDNA locus. Repressed genes not associated with silenced chromatin were unaffected. Sas10p was essential for viability, and the termination point following Sas10p depletion was as large budded cells. Remarkably, Sas10p overproduction disrupted silencing even under conditions that bypassed the requirement for Sir proteins, ORC, and Rap1p in silencing. These data implied that Sas10p function was intimately connected with the structure of silenced chromatin.

The dangerous role of silence in the relationship between trauma and violence: a group response.

PubMed

Phillips, Suzanne B

2015-01-01

This article considers that somewhere in the space between violence and trauma is dangerous silence. Silence intensifies the impact of trauma, and trauma that goes unspoken, un-witnessed, and unclaimed too often "outs itself" as more violence to self or others. Relevant empirical evidence on the impact of civilian interpersonal violence, combat trauma, school shootings, bullying, and domestic violence confirms this tragic cycle. Crucial to addressing the danger of silence in this cycle, the article examines the centrality of silence existentially, neuropsychologically, psychologically, developmentally, interpersonally, and culturally in relation to violence. The bridge to voicing and assimilating the unspeakable is empathic connection with others. Drawing upon two different types of group programs, the article demonstrates that group can serve as that bridge. Group process has the potential to undo the dangerous role of silence in the relationship of trauma and violence.

Epigenetic silencing of the DNA mismatch repair gene, MLH1, induced by hypoxic stress in a pathway dependent on the histone demethylase, LSD1

PubMed Central

Lu, Yuhong; Wajapeyee, Narendra; Turker, Mitchell S.; Glazer, Peter M.

2014-01-01

SUMMARY Silencing of the MLH1 gene is frequently seen in sporadic cancers. We report that hypoxia causes decreased H3K4 methylation at the MLH1 promoter via the H3K4 demethylases, LSD1 and PLU-1, and promotes long-term silencing of the promoter in a pathway that requires LSD1. Knockdown of LSD1 or its co-repressor, CoREST, also prevents the re-silencing (and cytosine DNA methylation) of the endogenous MLH1 promoter in RKO colon cancer cells following transient reactivation by the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC). The results demonstrate that hypoxia is a critical driving force for silencing of MLH1 through chromatin modification and indicate that the LSD1/CoREST complex is essential for MLH1 silencing. PMID:25043185

Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing.

PubMed

Chen, Chun-Kan; Blanco, Mario; Jackson, Constanza; Aznauryan, Erik; Ollikainen, Noah; Surka, Christine; Chow, Amy; Cerase, Andrea; McDonel, Patrick; Guttman, Mitchell

2016-10-28

The Xist long noncoding RNA orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the three-dimensional (3D) structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here, we show that Xist directly interacts with the Lamin B receptor, an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing by recruiting the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the 3D structure of DNA, enabling Xist and its silencing proteins to spread across the X to silence transcription. Copyright © 2016, American Association for the Advancement of Science.

Import routes and nuclear functions of Argonaute and other small RNA-silencing proteins.

PubMed

Schraivogel, Daniel; Meister, Gunter

2014-09-01

Small RNAs are important regulators of gene expression in many different organisms. Nuclear and cytoplasmic biogenesis enzymes generate functional small RNAs from double-stranded (ds) or single-stranded (ss) RNA precursors, and mature small RNAs are loaded into Argonaute proteins. In the cytoplasm, small RNAs guide Argonaute proteins to complementary RNAs leading to cleavage of these targets, translational silencing, or mRNA decay. In the nucleus Argonaute proteins engage in transcriptional silencing processes such as epigenetic silencing of repetitive elements at the chromatin level. During the past few years many novel functions of small RNA-guided gene silencing proteins in the nucleus have been reported. However, their specific import routes are largely unknown. In this review we summarize the current knowledge on nuclear transport routes that Argonaute and other RNA-silencing proteins take to carry out their various functions in the nucleus. Copyright © 2014 Elsevier Ltd. All rights reserved.

The RNA Silencing Pathway: The Bits and Pieces That Matter

PubMed Central

Groenenboom, Marian A. C; Marée, Athanasius F. M; Hogeweg, Paulien

2005-01-01

Cellular pathways are generally proposed on the basis of available experimental knowledge. The proposed pathways, however, may be inadequate to describe the phenomena they are supposed to explain. For instance, by means of concise mathematical models we are able to reveal shortcomings in the current description of the pathway of RNA silencing. The silencing pathway operates by cleaving siRNAs from dsRNA. siRNAs can associate with RISC, leading to the degradation of the target mRNA. We propose and analyze a few small extensions to the pathway: a siRNA degrading RNase, primed amplification of aberrant RNA pieces, and cooperation between aberrant RNA to trigger amplification. These extensions allow for a consistent explanation for various types of silencing phenomena, such as virus induced silencing, transgene and transposon induced silencing, and avoidance of self-reactivity, as well as for differences found between species groups. PMID:16110335

Workplace ostracism and employee silence in nursing: the mediating role of organizational identification.

PubMed

Gkorezis, Panagiotis; Panagiotou, Maria; Theodorou, Mamas

2016-10-01

The aim of this study was to examine the direct and indirect effect, through organizational identification, of workplace ostracism on nurses' silence towards patient safety. Employee silence in nursing has recently received attention in relation to its antecedents. Yet, very little is known about the role of workplace ostracism in generating nurses' silence. A cross-sectional survey was conducted in a public hospital in Cyprus. Data were collected from 157 nurses employed in a public hospital of Cyprus between November 2014-January 2015. To examine the present hypotheses bootstrapping analysis and Sobel test were conducted. Results demonstrated that workplace ostracism has an effect on nurses' silence towards patient safety. Moreover, this effect was partially mediated through organizational identification. Workplace ostracism among nurses significantly affects both nurses' attitude and behaviour namely organizational identification and employee silence. © 2016 John Wiley & Sons Ltd.

[Methods of hygromycin B phosphotransferase activity assay in transgenic plant].

PubMed

Zhuo, Qin; Yang, Xiaoguang

2004-07-01

Hygromycin B phosphotransferase (HPT) is a widely used selectable marker protein of transgenic plant. Detection of its activity is critical to studies on the development of various transgenic plants, silence of inserted gene, marker-free system development and safety assessment of transgenic food. In this paper, several methods for detecting the activity of this enzyme were reviewed.

The Art of Observation: Issues and Potential of Using Photo-Methods in Critical Ethnography with Adolescents

ERIC Educational Resources Information Center

Boucher, Michael L., Jr.

2017-01-01

The use of photographs in ethnographic education research is an emerging method that promises to enable scholars to collect deeper, more meaningful data from individuals who may otherwise be silenced. When used to empower participants, photo methodologies can remove what Foucault (1980) described as the analytical "gaze," allowing for…

RNA Interference Silencing of Glycogen Synthase Kinase 3β Inhibites Tau Phosphorylation in Mice with Alzheimer Disease.

PubMed

Bian, Hong; Bian, Wei; Lin, Xiaoying; Ma, Zhaoyin; Chen, Wen; Pu, Ying

2016-09-01

To explore the effect of glycogen synthase kinase 3β (GSK-3β) silencing on Tau-5 phosphorylation in mice suffering Alzheimer disease (AD). GSK-3β was firstly silenced in human neuroblastoma SH-SY5Y cells using special lentivirus (LV) and the content of Tau (A-12), p-Tau (Ser396) and p-Tau (PHF-6) proteins. GSK-3β was also silenced in APP/PS1 mouse model of AD mice, which were divided into three groups (n = 10): AD, vehicle, and LV group. Ten C57 mice were used as control. The memory ability of mice was tested by square water maze, and the morphological changes of hippocampus and neuron death were analyzed by haematoxylin-eosin staining. Moreover, the levels of Tau and phosphorylated Tau (p-Tau) were detected by western blotting and immunohistochemistry, respectively. The lentivirus-mediated GSK-3β silencing system was successfully developed and silencing GSK-3β at the cellular level reduced Tau phosphorylation obviously. Moreover, GSK-3β silence significantly improved the memory ability of AD mice in LV group compared with AD group (P < 0.05) according to the latency periods and error numbers. As for the hippocampus morphology and neuron death, no significant change was observed between LV group and normal control. Immunohistochemical detection and western blotting revealed that the levels of Tau and p-Tau were significantly down-regulated after GSK-3β silence. Silencing GSK-3β may have a positive effect on inhibiting the pathologic progression of AD through down-regulating the level of p-Tau.

Alfalfa dwarf cytorhabdovirus P protein is a local and systemic RNA silencing supressor which inhibits programmed RISC activity and prevents transitive amplification of RNA silencing.

PubMed

Bejerman, Nicolás; Mann, Krin S; Dietzgen, Ralf G

2016-09-15

Plants employ RNA silencing as an innate defense mechanism against viruses. As a counter-defense, plant viruses have evolved to express RNA silencing suppressor proteins (RSS), which target one or more steps of the silencing pathway. In this study, we show that the phosphoprotein (P) encoded by the negative-sense RNA virus alfalfa dwarf virus (ADV), a species of the genus Cytorhabdovirus, family Rhabdoviridae, is a suppressor of RNA silencing. ADV P has a relatively weak local RSS activity, and does not prevent siRNA accumulation. On the other hand, ADV P strongly suppresses systemic RNA silencing, but does not interfere with the short-distance spread of silencing, which is consistent with its lack of inhibition of siRNA accumulation. The mechanism of suppression appears to involve ADV P binding to RNA-induced silencing complex proteins AGO1 and AGO4 as shown in protein-protein interaction assays when ectopically expressed. In planta, we demonstrate that ADV P likely functions by inhibiting miRNA-guided AGO1 cleavage and prevents transitive amplification by repressing the production of secondary siRNAs. As recently described for lettuce necrotic yellows cytorhabdovirus P, but in contrast to other viral RSS known to disrupt AGO activity, ADV P sequence does not contain any recognizable GW/WG or F-box motifs, which suggests that cytorhabdovirus P proteins may use alternative motifs to bind to AGO proteins. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Silencing of CXCR4 Inhibits Tumor Cell Proliferation and Neural Invasion in Human Hilar Cholangiocarcinoma

PubMed Central

Tan, Xin-Yu; Chang, Shi; Liu, Wei

2014-01-01

Background/Aims To evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing. Methods An immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay. Results The expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (p<0.05). Silencing of CXCR4 in tumor cell lines by siRNA led to significantly decreased NI (p<0.05) and slightly decreased cell proliferation. Conclusions CXCR4 is likely correlated with clinical recurrence of hilar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA. PMID:24672662

Angiogenic effects of apigenin on endothelial cells after hypoxia-reoxygenation via the caveolin-1 pathway.

PubMed

Tu, Fengxia; Pang, Qiongyi; Chen, Xiang; Huang, Tingting; Liu, Meixia; Zhai, Qiongxiang

2017-12-01

In the present study, we aimed to elucidate whether apigenin contributes to the induction of angiogenesis and the related mechanisms in cell hypoxia-reoxygenation injury. The role of apigenin was examined in human umbilical vein endothelial cell (HUVEC) viability, migration and tube formation in vitro. To investigate the related mechanisms, we used caveolin-1 short interfering RNA. The viability of HUVECs was measured using Cell Counting Kit-8 assays, HUVEC migration was analyzed by crystal violet staining, and a tube formation assay was performed using the branch point method. Expression of caveolin-1, vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS) in HUVECs was examined by polymerase chain reaction and western blotting. Our data revealed that apigenin induced angiogenesis in vitro by increasing the tube formation ability of HUVECs, which was counteracted by caveolin-1 silencing. Compared to the NC group, Caveolin-1 and eNOS expression was upregulated by apigenin, whereas compared to the NC group, eNOS expression was increased upon caveolin-1 silencing. The expression of VEGF was increased by treatment with apigenin; however, compared to the NC group, caveolin-1 silencing did not affect VEGF expression, and apigenin did not increase VEGF expression in HUVECs after caveolin-1 silencing. These data suggest that apigenin may be a candidate therapeutic target for stroke recovery by promoting angiogenesis via the caveolin-1 signaling pathway.

RNA interference: a new strategy in the evolutionary arms race between human control strategies and insect pests.

PubMed

Machado, Vilmar; Rodríguez-García, María Juliana; Sánchez-García, Francisco Javier; Galan, Jose

2014-01-01

The relationship between humans and the insect pests of cultivated plants may be considered to be an indirect coevolutionary process, i.e., an arms race. Over time, humans have developed several strategies to minimize the negative impacts of insects on agricultural production. However, insects have made adaptive responses via the evolution of resistance to insecticides, and more recently against Bacillus thuriengiensis. Thus, we need to continuously invest resources in the development of new strategies for crop protection. Recent advances in genomics have demonstrated the possibility of a new weapon or strategy in this war, i.e., gene silencing, which involves blocking the expression of specific genes via mRNA inactivation. In the last decade, several studies have demonstrated the effectiveness of this strategy in the control of different species of insects. However, several technical difficulties need to be overcome to transform this potential into reality, such as the selection of target genes, the concentration of dsRNA, the nucleotide sequence of the dsRNA, the length of dsRNA, persistence in the insect body, and the life stage of the target species where gene silencing is most efficient. This study analyzes several aspects related to the use of gene silencing in pest control and it includes an overview of the inactivation process, as well as the problems that need to be resolved to transform gene silencing into an effective pest control method.

Carrying the burdens of poverty, parenting, and addiction: depression symptoms and self-silencing among ethnically diverse women.

PubMed

Grant, Therese M; Jack, Dana C; Fitzpatrick, Annette L; Ernst, Cara C

2011-02-01

Depression among women commonly co-occurs with substance abuse. We explore the association between women's depressive symptoms and self-silencing accounting for the effects of known childhood and adult risk indicators. Participants are 233 ethnically diverse, low-income women who abused alcohol/drugs prenatally. Depressive symptomatology was assessed using the Addiction Severity Index. Multivariate logistic regression models examined the association between self-silencing and the dependent depression variable. The full model indicated a 3% increased risk for depressive distress for each point increase in self-silencing score (OR = 1.03; P = .001). Differences in depressive symptomatology by ethnic groups were accounted for by their differences in self-silencing.

Lipocalin 2 Enhances Migration and Resistance against Cisplatin in Endometrial Carcinoma Cells

PubMed Central

Kashima, Hiroyasu; Yamada, Yasushi; Kobara, Hisanori; Asaka, Ryoichi; Ando, Hirofumi; Higuchi, Shotaro; Ida, Koichi; Mvunta, David Hamisi; Shiozawa, Tanri

2016-01-01

Purpose Lipocalin 2 (LCN2) is a secretory protein that is involved in various physiological processes including iron transport. We previously identified LCN2 as an up-regulated gene in endometrial carcinoma, and found that the overexpression of LCN2 and its receptor, SLC22A17, was associated with a poor prognosis. However, the functions and mechanism of action of LCN2 currently remain unclear. Methods The LCN2-overexpressing endometrial carcinoma cell lines, HHUA and RL95-2, and LCN2-low-expressing one, HEC1B, were used. The effects of LCN2 on cell migration, cell viability, and apoptosis under various stresses, including ultraviolet (UV) irradiation and cisplatin treatment, were examined using the scratch wound healing assay, WST-1 assay, and Apostrand assay, respectively. Results LCN2-silencing using shRNA method significantly reduced the migration ability of cells (p<0.05). Cytotoxic stresses significantly decreased the viability of LCN2-silenced cells more than that of control cells. In contrast, LCN2 overexpression was significantly increased cisplatin resistance. These effects were canceled by the addition of the iron chelator, deferoxamine. After UV irradiation, the expression of phosphorylated Akt (pAkt) was decreased in LCN2-silenced cells, and the PI3K inhibitor canceled the difference induced in UV sensitivity by LCN2. The cisplatin-induced expression of pAkt was not affected by LCN2; however, the expression of p53 and p21 was increased by LCN2-silencing. Conclusions These results indicated that LCN2 was involved in the migration and survival of endometrial carcinoma cells under various stresses in an iron-dependent manner. The survival function of LCN2 may be exerted through the PI3K pathway and suppression of the p53-p21 pathway. These functions of LCN2 may increase the malignant potential of endometrial carcinoma cells. PMID:27168162

Tomato Spotted Wilt Virus NSs Protein Supports Infection and Systemic Movement of a Potyvirus and Is a Symptom Determinant

PubMed Central

Gabriel Peralta, Sergio M.; Harte-Maxwell, Patricia A.

2018-01-01

Plant viruses are inducers and targets of antiviral RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressor proteins that interfere with antiviral RNA silencing. The NSs protein is an RNA silencing suppressor in orthotospoviruses, such as the tomato spotted wilt virus (TSWV). The mechanism of RNA silencing suppression by NSs and its role in virus infection and movement are poorly understood. Here, we cloned and tagged TSWV NSs and expressed it from a GFP-tagged turnip mosaic virus (TuMV-GFP) carrying either a wild-type or suppressor-deficient (AS9) helper component proteinase (HC-Pro). When expressed in cis, NSs restored pathogenicity and promoted systemic infection of suppressor-deficient TuMV-AS9-GFP in Nicotiana benthamiana and Arabidopsis thaliana. Inactivating mutations were introduced in NSs RNA-binding domain one. A genetic analysis with active and suppressor-deficient NSs, in combination with wild-type and mutant plants lacking essential components of the RNA silencing machinery, showed that the NSs insert is stable when expressed from a potyvirus. NSs can functionally replace potyviral HC-Pro, condition virus susceptibility, and promote systemic infection and symptom development by suppressing antiviral RNA silencing through a mechanism that partially overlaps that of potyviral HC-Pro. The results presented provide new insight into the mechanism of silencing suppression by NSs and its effect on virus infection. PMID:29538326

Tomato Spotted Wilt Virus NSs Protein Supports Infection and Systemic Movement of a Potyvirus and Is a Symptom Determinant.

PubMed

Garcia-Ruiz, Hernan; Gabriel Peralta, Sergio M; Harte-Maxwell, Patricia A

2018-03-14

Plant viruses are inducers and targets of antiviral RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressor proteins that interfere with antiviral RNA silencing. The NSs protein is an RNA silencing suppressor in orthotospoviruses, such as the tomato spotted wilt virus (TSWV). The mechanism of RNA silencing suppression by NSs and its role in virus infection and movement are poorly understood. Here, we cloned and tagged TSWV NSs and expressed it from a GFP-tagged turnip mosaic virus (TuMV-GFP) carrying either a wild-type or suppressor-deficient (AS9) helper component proteinase (HC-Pro). When expressed in cis, NSs restored pathogenicity and promoted systemic infection of suppressor-deficient TuMV-AS9-GFP in Nicotiana benthamiana and Arabidopsis thaliana . Inactivating mutations were introduced in NSs RNA-binding domain one. A genetic analysis with active and suppressor-deficient NSs, in combination with wild-type and mutant plants lacking essential components of the RNA silencing machinery, showed that the NSs insert is stable when expressed from a potyvirus. NSs can functionally replace potyviral HC-Pro, condition virus susceptibility, and promote systemic infection and symptom development by suppressing antiviral RNA silencing through a mechanism that partially overlaps that of potyviral HC-Pro. The results presented provide new insight into the mechanism of silencing suppression by NSs and its effect on virus infection.

The effect of eccentricity and spatiotemporal energy on motion silencing.

PubMed

Choi, Lark Kwon; Bovik, Alan C; Cormack, Lawrence K

2016-01-01

The now well-known motion-silencing illusion has shown that salient changes among a group of objects' luminances, colors, shapes, or sizes may appear to cease when objects move rapidly (Suchow & Alvarez, 2011). It has been proposed that silencing derives from dot spacing that causes crowding, coherent changes in object color or size, and flicker frequencies combined with dot spacing (Choi, Bovik, & Cormack, 2014; Peirce, 2013; Turi & Burr, 2013). Motion silencing is a peripheral effect that does not occur near the point of fixation. To better understand the effect of eccentricity on motion silencing, we measured the amount of motion silencing as a function of eccentricity in human observers using traditional psychophysics. Fifteen observers reported whether dots in any of four concentric rings changed in luminance over a series of rotational velocities. The results in the human experiments showed that the threshold velocity for motion silencing almost linearly decreases as a function of log eccentricity. Further, we modeled the response of a population of simulated V1 neurons to our stimuli. We found strong matches between the threshold velocities on motion silencing observed in the human experiment and those seen in the energy model of Adelson and Bergen (1985). We suggest the plausible explanation that as eccentricity increases, the combined motion-flicker signal falls outside the narrow spatiotemporal frequency response regions of the modeled receptive fields, thereby reducing flicker visibility.

Embracing Silence and the Emptiness between Unspoken Words

ERIC Educational Resources Information Center

VanSlyke-Briggs, Kjersti

2014-01-01

This article examines the use of silence as a constructive teaching tool in the classroom rather than as a punitive measure. The author offers suggestions for the inclusion of silence to benefit students specifically in a literature high school classroom.

PhOBF1, a petunia ocs element binding factor, plays an important role in antiviral RNA silencing.

PubMed

Sun, Daoyang; Li, Shaohua; Niu, Lixin; Reid, Michael S; Zhang, Yanlong; Jiang, Cai-Zhong

2017-02-01

Virus-induced gene silencing (VIGS) is a common reverse genetics strategy for characterizing the function of genes in plants. The detailed mechanism governing RNA silencing efficiency triggered by viruses is largely unclear. Here, we reveal that a petunia (Petunia hybrida) ocs element binding factor, PhOBF1, one of the basic leucine zipper (bZIP) transcription factors, was up-regulated by Tobacco rattle virus (TRV) infection. Simultaneous silencing of PhOBF1 and a reporter gene, phytoene desaturase (PDS) or chalcone synthase (CHS), by TRV-based VIGS led to a failure of the development of leaf photobleaching or the white-corollas phenotype. PhOBF1 silencing caused down-regulation of RNA silencing-related genes, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs). After inoculation with the TRV-PhPDS, PhOBF1-RNAi lines exhibited a substantially impaired PDS silencing efficiency, whereas overexpression of PhOBF1 resulted in a recovery of the silencing phenotype (photobleaching) in systemic leaves. A compromised resistance to TRV and Tobacco mosaic virus was found in PhOBF1-RNAi lines, while PhOBF1-overexpressing lines displayed an enhanced resistance to their infections. Compared with wild-type plants, PhOBF1-silenced plants accumulated lower levels of free salicylic acid (SA), salicylic acid glucoside, and phenylalanine, contrarily to higher levels of those in plants overexpressing PhOBF1. Furthermore, transcripts of a number of genes associated with the shikimate and phenylpropanoid pathways were decreased or increased in PhOBF1-RNAi or PhOBF1-overexpressing lines, respectively. Taken together, the data suggest that PhOBF1 regulates TRV-induced RNA silencing efficiency through modulation of RDRs, DCLs, and AGOs mediated by the SA biosynthesis pathway. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway*

PubMed Central

Foda, Bardees M.; Singh, Upinder

2015-01-01

RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5′-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

Octapartite negative-sense RNA genome of High Plains wheat mosaic virus encodes two suppressors of RNA silencing.

PubMed

Gupta, Adarsh K; Hein, Gary L; Graybosch, Robert A; Tatineni, Satyanarayana

2018-05-01

High Plains wheat mosaic virus (HPWMoV, genus Emaravirus; family Fimoviridae), transmitted by the wheat curl mite (Aceria tosichella Keifer), harbors a monocistronic octapartite single-stranded negative-sense RNA genome. In this study, putative proteins encoded by HPWMoV genomic RNAs 2-8 were screened for potential RNA silencing suppression activity by using a green fluorescent protein-based reporter agroinfiltration assay. We found that proteins encoded by RNAs 7 (P7) and 8 (P8) suppressed silencing induced by single- or double-stranded RNAs and efficiently suppressed the transitive pathway of RNA silencing. Additionally, a Wheat streak mosaic virus (WSMV, genus Tritimovirus; family Potyviridae) mutant lacking the suppressor of RNA silencing (ΔP1) but having either P7 or P8 from HPWMoV restored cell-to-cell and long-distance movement in wheat, thus indicating that P7 or P8 rescued silencing suppressor-deficient WSMV. Furthermore, HPWMoV P7 and P8 substantially enhanced the pathogenicity of Potato virus X in Nicotiana benthamiana. Collectively, these data demonstrate that the octapartite genome of HPWMoV encodes two suppressors of RNA silencing. Published by Elsevier Inc.

Titration and hysteresis in epigenetic chromatin silencing

NASA Astrophysics Data System (ADS)

Dayarian, Adel; Sengupta, Anirvan M.

2013-06-01

Epigenetic mechanisms of silencing via heritable chromatin modifications play a major role in gene regulation and cell fate specification. We consider a model of epigenetic chromatin silencing in budding yeast and study the bifurcation diagram and characterize the bistable and the monostable regimes. The main focus of this paper is to examine how the perturbations altering the activity of histone modifying enzymes affect the epigenetic states. We analyze the implications of having the total number of silencing proteins, given by the sum of proteins bound to the nucleosomes and the ones available in the ambient, to be constant. This constraint couples different regions of chromatin through the shared reservoir of ambient silencing proteins. We show that the response of the system to perturbations depends dramatically on the titration effect caused by the above constraint. In particular, for a certain range of overall abundance of silencing proteins, the hysteresis loop changes qualitatively with certain jump replaced by continuous merger of different states. In addition, we find a nonmonotonic dependence of gene expression on the rate of histone deacetylation activity of Sir2. We discuss how these qualitative predictions of our model could be compared with experimental studies of the yeast system under anti-silencing drugs.

Silencing of hygromycin phosphotransferase (hph) gene during sexual cycle and its reversible inactivation in heterokaryon of Neurospora crassa.

PubMed

Dev, Kamal; Maheshwari, Ramesh

2003-09-01

We transformed wild-type Neurospora crassa with hph gene encoding hygromycin phosphotransferase to obtain hygromycin-resistant (HygR) transformants and studied their behavior in the vegetative and sexual phases of growth. During vegetative growth in the absence of hygromycin, the hph gene was stable for at least three successive transfers with conidia. On the other hand, the behavior of the transformants in the sexual phase was different. The segregation of hph gene in the meiotic progeny was in accordance with the Mendelian ratio as inferred from PCR analysis. However, in spite of inheriting the hph gene, a proportion of the meiotic progeny failed to grow in the presence of hygromycin. This suggested that the hph gene is silenced in some progeny. The silencing effect was not confined to hph gene expression, since one-half of the meiotic progeny also showed poor conidiation. Genomic Southern analysis indicated deletions/rearrangements of the transgene in the progeny. A heterokaryon between silenced and non-silenced strains was able to grow on hygromycin-containing medium, showing that silencing was recessive. Silencing was reversed in homokaryotic nuclei extracted from such heterokaryon.

Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1.

PubMed

Csorba, Tibor; Lózsa, Rita; Hutvágner, György; Burgyán, József

2010-05-01

RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double-stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein-protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA-induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre-programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high-molecular-weight complex, suggesting the existence of a multi-protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA-RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co-expressed AGO1 in both the presence and absence of P0.

DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing.

PubMed

Saveliev, Alexander; Everett, Christopher; Sharpe, Tammy; Webster, Zoë; Festenstein, Richard

2003-04-24

Gene repression is crucial to the maintenance of differentiated cell types in multicellular organisms, whereas aberrant silencing can lead to disease. The organization of DNA into chromatin and heterochromatin is implicated in gene silencing. In chromatin, DNA wraps around histones, creating nucleosomes. Further condensation of chromatin, associated with large blocks of repetitive DNA sequences, is known as heterochromatin. Position effect variegation (PEV) occurs when a gene is located abnormally close to heterochromatin, silencing the affected gene in a proportion of cells. Here we show that the relatively short triplet-repeat expansions found in myotonic dystrophy and Friedreich's ataxia confer variegation of expression on a linked transgene in mice. Silencing was correlated with a decrease in promoter accessibility and was enhanced by the classical PEV modifier heterochromatin protein 1 (HP1). Notably, triplet-repeat-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective of chromosomal location. Because the phenomenon described here shares important features with PEV, the mechanisms underlying heterochromatin-mediated silencing might have a role in gene regulation at many sites throughout the mammalian genome and modulate the extent of gene silencing and hence severity in several triplet-repeat diseases.

The organisational silence of midwives and nurses: reasons and results.

PubMed

Yurdakul, Mine; Beşen, Meltem Aydin; Erdoğan, Semra

2016-07-01

The study was conducted to determine the issues about which nurses and midwives remain silent and the reasons for it and the perceived results of silence. Organisational silence is a vitally important issue in the health sector, due to the risks and mistakes that are not reported, and proposals for improvement that are not made. The sample of this descriptive survey, which investigated a cause and effect relationship, was 159 nurses and midwives. The data were collected using a questionnaire and the organisational silence scale. Of the study participants, 84.9% were nurses and 15.1% were midwives. Of all participants 88.7% were women. 8.8% of participants stated that they never remained silent about issues related to work and the workplace. Respondents most often remained silent about issues related to ethics and responsibility. 'Limited improvement and development' was frequently mentioned as a perceived result of organisational silence. Our study determined that organisational silence is quite common among nurses and midwives. Activities that raise the awareness of hospital administrations and employees about preventing the factors that cause and maintain silence in hospitals should be planned. © 2016 John Wiley & Sons Ltd.

Fluorescence-based high-throughput screening of dicer cleavage activity.

PubMed

Podolska, Katerina; Sedlak, David; Bartunek, Petr; Svoboda, Petr

2014-03-01

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

Phenotypic effects induced by knock-down of the period clock gene in Bombyx mori.

PubMed

Sandrelli, Federica; Cappellozza, Silvia; Benna, Clara; Saviane, Alessio; Mastella, Antonio; Mazzotta, Gabriella M; Moreau, Stephane; Pegoraro, Mirko; Piccin, Alberto; Zordan, Mauro A; Cappellozza, Luciano; Kyriacou, Charalambos P; Costa, Rodolfo

2007-04-01

The lepidopteran Bombyx mori is an insect of considerable scientific and economic importance. Recently, the B. mori circadian clock gene period has been molecularly characterized. We have transformed a B. mori strain with a construct encoding a period double-strand RNA in order to knock-down period gene expression. We observe that this post-transcriptional silencing produces a small but detectable disruption in the egg-hatching rhythm, as well as a reduction in egg-to-adult developmental time, without altering silk production parameters. Thus we show that both circadian and non-circadian phenotypes can be altered by changing per expression, and, at a practical level, these results suggest that per knock-down may provide a suitable strategy for improving the efficiency of rearing, without affecting silk productivity.

Going mobile: non-cell-autonomous small RNAs shape the genetic landscape of plants.

PubMed

Pyott, Douglas E; Molnar, Attila

2015-04-01

RNA silencing is a form of genetic regulation, which is conserved across eukaryotes and has wide ranging biological functions. Recently, there has been a growing appreciation for the importance of mobility in RNA silencing pathways, particularly in plants. Moreover, in addition to the importance for mobile RNA silencing in an evolutionary context, the potential for utilizing mobile short silencing RNAs in biotechnological applications is becoming apparent. This review aims to set current knowledge of this topic in a historical context and provides examples to illustrate the importance of mobile RNA silencing in both natural and artificially engineered systems in plants. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

PubMed Central

2014-01-01

Background Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene. Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. Results The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences. Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures. Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes. In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. Conclusions We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane. PMID:24708613

Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane.

PubMed

Chou, Ting-Chun; Moyle, Richard L

2014-04-08

Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene.Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences.Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures.Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes.In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.

A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.

Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the genemore » expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'- deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. In conclusion, the epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis.« less

A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

DOE PAGES

Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.; ...

2015-10-19

Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the genemore » expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'- deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. In conclusion, the epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis.« less

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants1[OPEN

PubMed Central

Liu, Na; Xie, Ke; Jia, Qi; Zhao, Jinping; Chen, Tianyuan; Li, Huangai; Wei, Xiang; Diao, Xianmin; Hong, Yiguo

2016-01-01

Virus-induced gene silencing (VIGS) is a powerful technique to study gene function in plants. However, very few VIGS vectors are available for monocot plants. Here we report that Foxtail mosaic virus (FoMV) can be engineered as an effective VIGS system to induce efficient silencing of endogenous genes in monocot plants including barley (Hordeum vulgare L.), wheat (Triticum aestivum) and foxtail millet (Setaria italica). This is evidenced by FoMV-based silencing of phytoene desaturase (PDS) and magnesium chelatase in barley, of PDS and Cloroplastos alterados1 in foxtail millet and wheat, and of an additional gene IspH in foxtail millet. Silencing of these genes resulted in photobleached or chlorosis phenotypes in barley, wheat, and foxtail millet. Furthermore, our FoMV-based gene silencing is the first VIGS system reported for foxtail millet, an important C4 model plant. It may provide an efficient toolbox for high-throughput functional genomics in economically important monocot crops. PMID:27225900

Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

PubMed

Amabile, Angelo; Migliara, Alessandro; Capasso, Paola; Biffi, Mauro; Cittaro, Davide; Naldini, Luigi; Lombardo, Angelo

2016-09-22

Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants.

PubMed

Liu, Na; Xie, Ke; Jia, Qi; Zhao, Jinping; Chen, Tianyuan; Li, Huangai; Wei, Xiang; Diao, Xianmin; Hong, Yiguo; Liu, Yule

2016-07-01

Virus-induced gene silencing (VIGS) is a powerful technique to study gene function in plants. However, very few VIGS vectors are available for monocot plants. Here we report that Foxtail mosaic virus (FoMV) can be engineered as an effective VIGS system to induce efficient silencing of endogenous genes in monocot plants including barley (Hordeum vulgare L.), wheat (Triticum aestivum) and foxtail millet (Setaria italica). This is evidenced by FoMV-based silencing of phytoene desaturase (PDS) and magnesium chelatase in barley, of PDS and Cloroplastos alterados1 in foxtail millet and wheat, and of an additional gene IspH in foxtail millet. Silencing of these genes resulted in photobleached or chlorosis phenotypes in barley, wheat, and foxtail millet. Furthermore, our FoMV-based gene silencing is the first VIGS system reported for foxtail millet, an important C4 model plant. It may provide an efficient toolbox for high-throughput functional genomics in economically important monocot crops. © 2016 American Society of Plant Biologists. All Rights Reserved.

F-box-like domain in the polerovirus protein P0 is required for silencing suppressor function

PubMed Central

Pazhouhandeh, Maghsoud; Dieterle, Monika; Marrocco, Katia; Lechner, Esther; Berry, Bassam; Brault, Véronique; Hemmer, Odile; Kretsch, Thomas; Richards, Kenneth E.; Genschik, Pascal; Ziegler-Graff, Véronique

2006-01-01

Plants employ small RNA-mediated posttranscriptional gene silencing as a virus defense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode of action of most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 of two Arabidopsis-infecting poleroviruses interacts by means of a conserved minimal F-box motif with Arabidopsis thaliana orthologs of S-phase kinase-related protein 1 (SKP1), a component of the SCF family of ubiquitin E3 ligases. Point mutations in the F-box-like motif abolished the P0–SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity of P0. Knockdown of expression of a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus infection. Together, the results support a model in which P0 acts as an F-box protein that targets an essential component of the host posttranscriptional gene silencing machinery. PMID:16446454

Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

PubMed Central

Pratt-Hyatt, Matthew; Pai, Dave A.; Haeusler, Rebecca A.; Wozniak, Glenn G.; Good, Paul D.; Miller, Erin L.; McLeod, Ian X.; Yates, John R.; Hopper, Anita K.; Engelke, David R.

2013-01-01

The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification. PMID:23898186

The Luteovirus P4 Movement Protein Is a Suppressor of Systemic RNA Silencing

PubMed Central

Fusaro, Adriana F.; Barton, Deborah A.; Nakasugi, Kenlee; Jackson, Craig; Kalischuk, Melanie L.; Kawchuk, Lawrence M.; Vaslin, Maite F. S.; Waterhouse, Peter M.

2017-01-01

The plant viral family Luteoviridae is divided into three genera: Luteovirus, Polerovirus and Enamovirus. Without assistance from another virus, members of the family are confined to the cells of the host plant’s vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant’s viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant’s silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant’s anti-viral defense. PMID:28994713

The Luteovirus P4 Movement Protein Is a Suppressor of Systemic RNA Silencing.

PubMed

Fusaro, Adriana F; Barton, Deborah A; Nakasugi, Kenlee; Jackson, Craig; Kalischuk, Melanie L; Kawchuk, Lawrence M; Vaslin, Maite F S; Correa, Regis L; Waterhouse, Peter M

2017-10-10

The plant viral family Luteoviridae is divided into three genera: Luteovirus , Polerovirus and Enamovirus . Without assistance from another virus, members of the family are confined to the cells of the host plant's vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant's viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant's silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant's anti-viral defense.

Two Components of the RNA-Directed DNA Methylation Pathway Associate with MORC6 and Silence Loci Targeted by MORC6 in Arabidopsis

PubMed Central

Liu, Zhang-Wei; Zhou, Jin-Xing; Huang, Huan-Wei; Li, Yong-Qiang; Shao, Chang-Rong; Li, Lin; Cai, Tao; Chen, She

2016-01-01

The SU(VAR)3-9 homolog SUVH9 and the double-stranded RNA-binding protein IDN2 were thought to be components of an RNA-directed DNA methylation (RdDM) pathway in Arabidopsis. We previously found that SUVH9 interacts with MORC6 but how the interaction contributes to transcriptional silencing remains elusive. Here, our genetic analysis indicates that SUVH2 and SUVH9 can either act in the same pathway as MORC6 or act synergistically with MORC6 to mediate transcriptional silencing. Moreover, we demonstrate that IDN2 interacts with MORC6 and mediates the silencing of a subset of MORC6 target loci. Like SUVH2, SUVH9, and IDN2, other RdDM components including Pol IV, Pol V, RDR2, and DRM2 are also required for transcriptional silencing at a subset of MORC6 target loci. MORC6 was previously shown to mediate transcriptional silencing through heterochromatin condensation. We demonstrate that the SWI/SNF chromatin-remodeling complex components SWI3B, SWI3C, and SWI3D interact with MORC6 as well as with SUVH9 and then mediate transcriptional silencing. These results suggest that the RdDM components are involved not only in DNA methylation but also in MORC6-mediated heterochromatin condensation. This study illustrates how DNA methylation is linked to heterochromatin condensation and thereby enhances transcriptional silencing at methylated genomic regions. PMID:27171427

Epistemologies of Silence

ERIC Educational Resources Information Center

Dénommé-Welch, Spy; Rowsell, Jennifer

2017-01-01

This paper engages some of the philosophical and epistemological underpinnings of silence, and its implications for teaching and learning both within and beyond educational settings. In this exploration, the authors draw on self-reflexive observations, woven throughout the paper as a series of vignettes, to explore questions of silence and its…

Virus-induced gene silencing in cultivated cotton (Gossypium spp.) using Tobacco rattle virus

USDA-ARS?s Scientific Manuscript database

The study described here has optimized the conditions for virus induced gene silencing (VIGS) in three cultivated cotton species (Gossypium hirsutum, G. arboreum and G. herbaceum) using a Tobacco rattle virus (TRV) vector. The system was used to silence the homolog of the Arabidopsis thaliana chloro...

Breaking the Silence (Teaching and Learning about Cultural Diversity).

ERIC Educational Resources Information Center

Miller, Howard M., Ed.

1997-01-01

Discusses the policy of silence (and its complex reasons) that often rules when it comes to teaching and learning about race, religion, ethnicity, and sexuality. Discusses briefly five books and articles that deal with breaking this silence, and offers observations about effective multiculturalism in the classroom. (SR)

Silence, an Eye of Knowledge

ERIC Educational Resources Information Center

Aghamohammadi, Mehdi

2017-01-01

One of the conspicuous features of the twentieth-century West was silence. This idea could be supported by examining reflections of Ludwig Wittgenstein, Fritz Mauthner, John Cage, Samuel Beckett, Ihab Hassan, Franz Kafka, Wassily Kandinsky, Jean-Paul Sartre, Virginia Woolf, Wolfgang Iser, Jacques Derrida, and Pierre Macherey. To me, silence is not…

Discourses that Silence: Teachers and Anti-Lesbian Harassment

ERIC Educational Resources Information Center

Ferfolja, Tania

2008-01-01

This paper examines the way lesbian identities are silenced in schools particularly through anti-lesbian harassment. Based on research with 30 self-identified lesbian teachers working across high schools in New South Wales, Australia, the discussion illustrates how various responses to anti-lesbian harassment silence the recognition of such…

Good Silences, Bad Silences, Unforgivable Silences

ERIC Educational Resources Information Center

Onwuachi-Willig, Angela

2012-01-01

For an untenured faculty member, perception is everything. For outsiders, such as women of color, the task of negotiating and performing identity can prove rather burdensome because of the need to counter negative stereotypes based on race, gender, and class. For many junior faculty members, a recurring conflict is the longstanding tension between…

Self-Silencing and Rejection Sensitivity in Adolescent Romantic Relationships

ERIC Educational Resources Information Center

Harper, Melinda S.; Dickson, Joseph W.; Welsh, Deborah P.

2006-01-01

This study examined the link between rejection sensitivity, self-silencing behaviors, and depressive symptomatology among adolescent dating couples. Self-silencing was hypothesized to be the process mediating the association between rejection sensitivity and depressive symptoms. Our sample included 211 couples between 14 and 21 who were dating at…

Organizational Silence in Sports Employees

ERIC Educational Resources Information Center

Bastug, Gulsum; Pala, Adem; Yilmaz, Taner; Duyan, Mehdi; Gunel, Ilker

2016-01-01

Organizational silence can be defined as a way of behaviour belonging to men and women employees in the organization exhibited without reflecting their feelings, ideas, concerns and suggestions related with their workplaces, works for which they are responsible or other activities of the organization. In the period of organizational silence,…

Listening for Silence in Text-Based, Online Encounters

ERIC Educational Resources Information Center

Zembylas, Michalinos; Vrasidas, Charalambos

2007-01-01

This article addresses the ways in which learners' silence plays out within asynchronous and synchronous text-based, online communication. Our study takes an ethnographic perspective in examining how learners and instructors in two online courses use and interpret silence. The ways in which those learners and instructors eventually integrated…

Switching on RNA Silencing Suppressor Activity by Restoring Argonaute Binding to a Viral Protein

PubMed Central

Szabó, Edit Z.; Manczinger, Máté; Göblös, Anikó; Kemény, Lajos

2012-01-01

We found that Sweet potato feathery mottle virus (SPFMV) P1, a close homologue of Sweet potato mild mottle virus P1, did not have any silencing suppressor activity. Remodeling the Argonaute (AGO) binding domain of SPFMV P1 by the introduction of two additional WG/GW motifs converted it to a silencing suppressor with AGO binding capacity. To our knowledge, this is the first instance of the transformation of a viral protein of unknown function to a functional silencing suppressor. PMID:22623784

Rice yellow stunt rhabdovirus protein 6 suppresses systemic RNA silencing by blocking RDR6-mediated secondary siRNA synthesis.

PubMed

Guo, Hongyan; Song, Xiaoguang; Xie, Chuanmiao; Huo, Yan; Zhang, Fujie; Chen, Xiaoying; Geng, Yunfeng; Fang, Rongxiang

2013-08-01

The P6 protein of Rice yellow stunt rhabdovirus (RYSV) is a virion structural protein that can be phosphorylated in vitro. However its exact function remains elusive. We found that P6 enhanced the virulence of Potato virus X (PVX) in Nicotiana benthamiana and N. tabacum plants, suggesting that it might function as a suppressor of RNA silencing. We examined the mechanism of P6-mediated silencing suppression by transiently expressing P6 in both N. benthamiana leaves and rice protoplasts. Our results showed that P6 could repress the production of secondary siRNAs and inhibit systemic green fluorescent protein RNA silencing but did not interfere with local RNA silencing in N. benthamiana plants or in rice protoplasts. Intriguingly, P6 and RDR6 had overlapping subcellular localization and P6 bound both rice and Arabidopsis RDR6 in vivo. Furthermore, transgenic rice plants expressing P6 showed enhanced susceptibility to infection by Rice stripe virus. Hence, we propose that P6 is part of the RYSV's counter-defense machinery against the plant RNA silencing system and plays a role mainly in affecting RDR6-mediated secondary siRNA synthesis. Our work provides a new perspective on how a plant-infecting nucleorhabdovirus may counteract host RNA silencing-mediated antiviral defense.

Virus-Induced Gene Silencing in Cultivated Cotton (Gossypium spp.) Using Tobacco Rattle Virus.

PubMed

Mustafa, Roma; Shafiq, Muhammad; Mansoor, Shahid; Briddon, Rob W; Scheffler, Brian E; Scheffler, Jodi; Amin, Imran

2016-01-01

The study described here has optimized the conditions for virus-induced gene silencing (VIGS) in three cultivated cotton species (Gossypium hirsutum, G. arboreum, and G. herbaceum) using a Tobacco rattle virus (TRV) vector. The system was used to silence the homolog of the Arabidopsis thaliana chloroplastos alterados 1 (AtCLA1) gene, involved in chloroplast development, in G. herbaceum, G. arboreum, and six commercial G. hirsutum cultivars. All plants inoculated with the TRV vector to silence CLA1 developed a typical albino phenotype indicative of silencing this gene. Although silencing in G. herbaceum and G. arboreum was complete, silencing efficiency differed for each G. hirsutum cultivar. Reverse transcriptase polymerase chain reaction (PCR) and real-time quantitative PCR showed a reduction in mRNA levels of the CLA1 homolog in all three species, with the highest efficiency (lowest CLA1 mRNA levels) in G. arboreum followed by G. herbaceum and G. hirsutum. The results indicate that TRV is a useful vector for VIGS in Gossypium species. However, selection of host cultivar is important. With the genome sequences of several cotton species recently becoming publicly available, this system has the potential to provide a very powerful tool for the rapid, large-scale reverse-genetic analysis of genes in Gossypium spp.

AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system.

PubMed

Schuck, Jana; Gursinsky, Torsten; Pantaleo, Vitantonio; Burgyán, Jozsef; Behrens, Sven-Erik

2013-05-01

AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.

AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system

PubMed Central

Schuck, Jana; Gursinsky, Torsten; Pantaleo, Vitantonio; Burgyán, Jozsef; Behrens, Sven-Erik

2013-01-01

AGO/RISC-mediated antiviral RNA silencing, an important component of the plant’s immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (−)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing. PMID:23535144

Function and diversity of P0 proteins among cotton leafroll dwarf virus isolates.

PubMed

Cascardo, Renan S; Arantes, Ighor L G; Silva, Tatiane F; Sachetto-Martins, Gilberto; Vaslin, Maité F S; Corrêa, Régis L

2015-08-12

The RNA silencing pathway is an important anti-viral defense mechanism in plants. As a counter defense, some members of the viral family Luteoviridae are able to evade host immunity by encoding the P0 RNA silencing suppressor protein. Here we explored the functional diversity of P0 proteins among eight cotton leafroll dwarf virus (CLRDV) isolates, a virus associated with a worldwide cotton disease known as cotton blue disease (CBD). CLRDV-infected cotton plants of different varieties were collected from five growing fields in Brazil and their P0 sequences compared to three previously obtained isolates. P0's silencing suppression activities were scored based on transient expression experiments in Nicotiana benthamiana leaves. High sequence diversity was observed among CLRDV P0 proteins, indicating that some isolates found in cotton varieties formerly resistant to CLRDV should be regarded as new genotypes within the species. All tested proteins were able to suppress local and systemic silencing, but with significantly variable degrees. All P0 proteins were able to mediate the decay of ARGONAUTE proteins, a key component of the RNA silencing machinery. The sequence diversity observed in CLRDV P0s is also reflected in their silencing suppression capabilities. However, the strength of local and systemic silencing suppression was not correlated for some proteins.

Sulfamethazine Suppresses Epigenetic Silencing in Arabidopsis by Impairing Folate Synthesis[W

PubMed Central

Zhang, Huiming; Deng, Xiangyang; Miki, Daisuke; Cutler, Sean; La, Honggui; Hou, Yueh-Ju; Oh, JeeEun; Zhu, Jian-Kang

2012-01-01

DNA methylation is a critical, dynamically regulated epigenetic mark. Small chemicals can be valuable tools in probing cellular processes, but the set of chemicals with broad effects on epigenetic regulation is very limited. Using the Arabidopsis thaliana repressor of silencing1 mutant, in which transgenes are transcriptionally silenced, we performed chemical genetic screens and found sulfamethazine (SMZ) as a chemical suppressor of epigenetic silencing. SMZ treatment released the silencing of transgenes as well as endogenous transposons and other repetitive elements. Plants treated with SMZ exhibit substantially reduced levels of DNA methylation and histone H3 Lys-9 dimethylation, but heterochromatic siRNA levels were not affected. SMZ is a structural analog and competitive antagonist to p-aminobenzoic acid (PABA), which is a precursor of folates. SMZ decreased the plant folate pool size and caused methyl deficiency, as demonstrated by reductions in S-adenosylmethionine levels and in global DNA methylation. Exogenous application of PABA or compounds downstream in the folate biosynthesis pathway restored transcriptional silencing in SMZ-treated plants. Together, our results revealed a novel type of chemical suppressor of epigenetic silencing, which may serve as a valuable tool for studying the roles and mechanisms of epigenetic regulation and underscores an important linkage between primary metabolism and epigenetic gene regulation. PMID:22447685

Provider interaction with the electronic health record: The effects on patient-centered communication in medical encounters

PubMed Central

Street, Richard L.; Liu, Lin; Farber, Neil J.; Chen, Yunan; Calvitti, Alan; Zuest, Danielle; Gabuzda, Mark T.; Bell, Kristin; Gray, Barbara; Rick, Steven; Ashfaq, Shazia; Agha, Zia

2015-01-01

Objective The computer with the electronic health record (EHR) is an additional ‘interactant’ in the medical consultation, as clinicians must simultaneously or in alternation engage patient and computer to provide medical care. Few studies have examined how clinicians' EHR workflow (e.g., gaze, keyboard activity, and silence) influences the quality of their communication, the patient's involvement in the encounter, and conversational control of the visit. Methods Twenty-three primary care providers (PCPs) from USA Veterans Administration (VA) primary care clinics participated in the study. Up to 6 patients per PCP were recruited. The proportion of time PCPs spent gazing at the computer was captured in real time via video-recording. Mouse click/scrolling activity was captured through Morae, a usability software that logs mouse clicks and scrolling activity. Conversational silence was coded as the proportion of time in the visit when PCP and patient were not talking. After the visit, patients completed patient satisfaction measures. Trained coders independently viewed videos of the interactions and rated the degree to which PCPs were patient-centered (informative, supportive, partnering) and patients were involved in the consultation. Conversational control was measured as the proportion of time the PCP held the floor compared to the patient. Results The final sample included 125 consultations. PCPs who spent more time in the consultation gazing at the computer and whose visits had more conversational silence were rated lower inpatient-centeredness. PCPs controlled more of the talk time in the visits that also had longer periods of mutual silence. Conclusions PCPs were rated as having less effective communication when they spent more time looking at the computer and when there was more periods of silence in the consultation. Because PCPs increasingly are using the EHR in their consultations, more research is needed to determine effective ways that they can verbally engage patients while simultaneously managing data in the EHR. Practice implications EHR activity consumes an increasing proportion of clinicians' time during consultations. To ensure effective communication with their patients, clinicians may benefit from using communication strategies that maintain the flow of conversation when working with the computer, as well as from learning EHR management skills that prevent extended periods of gaze at computer and long periods of silence. Next-generation EHR design must address better usability and clinical workflow integration, including facilitating patient-clinician communication. PMID:24882086

Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting

PubMed Central

De Paula, Daniel; Bentley, M. Vitória L.B.; Mahato, Ram I.

2007-01-01

RNA interference (RNAi) is an evolutionarily conserved process by which double-stranded small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing. Unlike other mRNA targeting strategies, RNAi takes advantage of the physiological gene silencing machinery. The potential use of siRNA as therapeutic agents has attracted great attention as a novel approach for treating severe and chronic diseases. RNAi can be achieved by either delivery of chemically synthesized siRNAs or endogenous expression of small hairpin RNA, siRNA, and microRNA (miRNA). However, the relatively high dose of siRNA required for gene silencing limits its therapeutic applications. This review discusses several strategies to improve therapeutic efficacy as well as to abrogate off-target effects and immunostimulation caused by siRNAs. There is an in-depth discussion on various issues related to the (1) mechanisms of RNAi, (2) methods of siRNA production, (3) barriers to RNAi-based therapies, (4) biodistribution, (5) design of siRNA molecules, (6) chemical modification and bioconjugation, (7) complex formation with lipids and polymers, (8) encapsulation into lipid particles, and (9) target specificity for enhanced therapeutic effectiveness. PMID:17329355

Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

PubMed Central

2012-01-01

Background We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns. Results The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively. Conclusions These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns. PMID:22853641

The Sign of Silence: Negotiating Musical Identities in an Improvising Ensemble

ERIC Educational Resources Information Center

Wilson, Graeme B.; MacDonald, Raymond A. R.

2012-01-01

Group musical improvisation, as a spontaneous process of collaborative creativity effected through non-verbal social interaction, is a unique psychological phenomenon and universal capacity. Existing studies focus on improvisation among professional jazz musicians, often using qualitative methods. However, improvisation transcends genres and…

The Evil Joker.

ERIC Educational Resources Information Center

Wagner, Michelle; Knudsen, Carla; Harper, Victoria

2000-01-01

A pumpkin decorating exercise gave California sixth-graders a chance to discuss a problematic school policy on gangs, using Pablo Freire's problem-posing method. Since the policy failed to understand student views, it officially sanctioned and promoted silence. Student journals revealed how gang activity was woven into their world. (MLH)

Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae).

PubMed

Yang, Chunxiao; Pan, Huipeng; Noland, Jeffrey Edward; Zhang, Deyong; Zhang, Zhanhong; Liu, Yong; Zhou, Xuguo

2015-12-10

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt method. In addition, RefFinder, a comprehensive platform integrating all the above-mentioned algorithms, ranked the overall stability of these candidate genes. As a result, various sets of suitable reference genes were recommended specifically for experiments involving different tissues, developmental stages, sex, and C. maculate larvae treated with dietary double stranded RNA. This study represents the critical first step to establish a standardized RT-qPCR protocol for the functional genomics research in a ladybeetle C. maculate. Furthermore, it lays the foundation for conducting ecological risk assessment of RNAi-based gene silencing biotechnologies on non-target organisms; in this case, a key predatory biological control agent.

Silencing of VEGF inhibits human osteosarcoma angiogenesis and promotes cell apoptosis via VEGF/PI3K/AKT signaling pathway

PubMed Central

Peng, Ningning; Gao, Shuming; Guo, Xu; Wang, Guangya; Cheng, Cai; Li, Min; Liu, Kehun

2016-01-01

Background: Osteosarcoma is a kind of highly malignant tumor and the growth and metastasis is closely related to angiogenesis. Vascular endothelial growth factor (VEGF) is an important angiogenesis-promoting factor. In the current study, we investigated the effects of suppressed VEGF on osteosarcoma and its molecular mechanism provided for a basis by targeting angiogenesis. Material/Methods: We established bearing human osteosarcoma Wistar rats model by subcutaneous inoculation of human SaOS-2 cells and the adenovirus vector Ad-VEGF-siRNA was constructed for further study. We assessed the efficiency of VEGF silencing and its influence on SaOS-2 cells. The expression of mRNA and protein were detected by RT-PCR and western blotting, respectively. Intratumoral microvessel density (MVD), VEGF and CD31 were evaluated by immunohistochemistry. We detected the cell apoptotic rates by flow cytometry. Results: Our results indicated that Ad-VEGF-siRNA could effectively suppressed the expression of VEGF expression, inhibited the proliferation capability and promoted apoptosis of SaOS-2 cells in vitro. Silencing of VEGF expression also suppress osteosarcoma tumor growth and reduce osteosarcoma angiogenesis in the Wistar rats model in vivo. Furthermore, We found that phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) activation were considerably reduced while inhibition VEGF expression in SaOS-2 cells. Conclusion: Our data demonstrated that VEGF silencing could suppress cells proliferation, promote cells apoptosis and reduce osteosarcoma angiogenesis through inactivation of VEGF/PI3K/AKT signaling pathway. PMID:27158386

Delivery of siRNA Silencing Runx2 Using a Multifunctional Polymer-Lipid Nanoparticle Inhibits Osteogenesis in a Cell Culture Model of Heterotopic Ossification

PubMed Central

Mishra, Swati; Vaughn, Asa D.; Devore, David I.

2015-01-01

Heterotopic ossification (HO) associated with traumatic neurological or musculoskeletal injuries remains a major clinical challenge. One approach to understanding better and potentially treating this condition is to silence one or more genes believed to be responsible for osteogenesis by small interfering RNA (siRNA) post-injury. Improved methods of delivering siRNA to myoprogenitor cells as well as relevant cell culture models of HO are needed to advance this approach. We utilize a model of HO featuring C2C12 myoprogenitor cells stimulated to the osteogenic phenotype by addition of BMP-2. For siRNA delivery, we utilize a nanocomposite consisting of DOTAP- based cationic liposomes coated with a graft copolymer of poly(propylacrylic acid) grafted with polyetheramine (Jeffamine), as this system has been shown previously to deliver antisense oligonucleotides safely into cells and out of endosomes for gene silencing in vitro and in vivo. Delivery of siRNA targeting Runx2, a transcription factor downstream of BMP-2, to stimulated C2C12 cells produced greater than 60% down-regulation of the Runx2 gene. This level of gene silencing was sufficient to inhibit alkaline phosphatase activity over the course of several days and calcium phosphate deposition over the course of 2 weeks. These results show the utility of the BMP-2/C2C12 model for capturing the cellular cell-fate decision in HO. Further, they suggest DOTAP/PPAA-g-Jeffamine as a promising delivery system for siRNA– based therapy for HO. PMID:23146945

Effects of siRNA-mediated suppression of HPV-11 L1 expression on the proliferation and apoptosis of vaginal epithelial cells

PubMed Central

Zeng, Juan; Yang, Shumei; Wang, Xiaorui; Gao, Yan; Zhang, Mei

2017-01-01

The aim of the present study was to investigate the effects of human papillomavirus (HPV) infection on the gynecological disease of vaginitis and to demonstrate how the small interfering RNA (siRNA) method may be used for HPV prevention in the clinic. Human vaginal epithelial cells were transfected with HPV-11 L1 expression vector and siRNA-HPV-11 L1 vectors and a control group was transfected with scrambled siRNA. Cell proliferation in each group was analyzed using the MTT assay and the expression of apoptosis-associated proteins was measured by western blot analysis. Compared with the control group, HPV-11 L1 mRNA and protein levels were significantly increased following transfection with the HPV-11 L1 expression vector in cells (P<0.05), but this result was significantly reversed by silencing of HPV-11 L1 (P<0.05). In addition, cell proliferation in the HPV-11 group was lower than that in the control group; however, cell proliferation was significantly increased in cells transfected with silenced L1 compared with that in the control group (P<0.05). Furthermore, silencing of HPV-11 L1 significantly decreased caspase-3 and caspase-9 expressions in cells, whereas the expression was increased in the HPV-11 L1 group (P<0.05). The present study suggested that siRNA-mediated silencing of HPV-11 L1 may have potential therapeutic applications for treating gynecological diseases associated with HPV-11 infection. PMID:28413509

Evolutionary Analysis of Heterochromatin Protein Compatibility by Interspecies Complementation in Saccharomyces

PubMed Central

Zill, Oliver A.; Scannell, Devin R.; Kuei, Jeffrey; Sadhu, Meru; Rine, Jasper

2012-01-01

The genetic bases for species-specific traits are widely sought, but reliable experimental methods with which to identify functionally divergent genes are lacking. In the Saccharomyces genus, interspecies complementation tests can be used to evaluate functional conservation and divergence of biological pathways or networks. Silent information regulator (SIR) proteins in S. bayanus provide an ideal test case for this approach because they show remarkable divergence in sequence and paralog number from those found in the closely related S. cerevisiae. We identified genes required for silencing in S. bayanus using a genetic screen for silencing-defective mutants. Complementation tests in interspecies hybrids identified an evolutionarily conserved Sir-protein-based silencing machinery, as defined by two interspecies complementation groups (SIR2 and SIR3). However, recessive mutations in S. bayanus SIR4 isolated from this screen could not be complemented by S. cerevisiae SIR4, revealing species-specific functional divergence in the Sir4 protein despite conservation of the overall function of the Sir2/3/4 complex. A cladistic complementation series localized the occurrence of functional changes in SIR4 to the S. cerevisiae and S. paradoxus branches of the Saccharomyces phylogeny. Most of this functional divergence mapped to sequence changes in the Sir4 PAD. Finally, a hemizygosity modifier screen in the interspecies hybrids identified additional genes involved in S. bayanus silencing. Thus, interspecies complementation tests can be used to identify (1) mutations in genetically underexplored organisms, (2) loci that have functionally diverged between species, and (3) evolutionary events of functional consequence within a genus. PMID:22923378

Fermitins, the Orthologs of Mammalian Kindlins, Regulate the Development of a Functional Cardiac Syncytium in Drosophila melanogaster

PubMed Central

Catterson, James H.; Heck, Margarete M. S.; Hartley, Paul S.

2013-01-01

The vertebrate Kindlins are an evolutionarily conserved family of proteins critical for integrin signalling and cell adhesion. Kindlin-2 (KIND2) is associated with intercalated discs in mice, suggesting a role in cardiac syncytium development; however, deficiency of Kind2 leads to embryonic lethality. Morpholino knock-down of Kind2 in zebrafish has a pleiotropic effect on development that includes the heart. It therefore remains unclear whether cardiomyocyte Kind2 expression is required for cardiomyocyte junction formation and the development of normal cardiac function. To address this question, the expression of Fermitin 1 and Fermitin 2 (Fit1, Fit2), the two Drosophila orthologs of Kind2, was silenced in Drosophila cardiomyocytes. Heart development was assessed in adult flies by immunological methods and videomicroscopy. Silencing both Fit1 and Fit2 led to a severe cardiomyopathy characterised by the failure of cardiomyocytes to develop as a functional syncytium and loss of synchrony between cardiomyocytes. A null allele of Fit1 was generated but this had no impact on the heart. Similarly, the silencing of Fit2 failed to affect heart function. In contrast, the silencing of Fit2 in the cardiomyocytes of Fit1 null flies disrupted syncytium development, leading to severe cardiomyopathy. The data definitively demonstrate a role for Fermitins in the development of a functional cardiac syncytium in Drosophila. The findings also show that the Fermitins can functionally compensate for each other in order to control syncytium development. These findings support the concept that abnormalities in cardiomyocyte KIND2 expression or function may contribute to cardiomyopathies in humans. PMID:23690969

Suppression of RNA Silencing by a Plant DNA Virus Satellite Requires a Host Calmodulin-Like Protein to Repress RDR6 Expression

PubMed Central

Li, Fangfang; Huang, Changjun; Li, Zhenghe; Zhou, Xueping

2014-01-01

In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection. PMID:24516387

Spread of X-chromosome inactivation into autosomal sequences: role for DNA elements, chromatin features and chromosomal domains

PubMed Central

Cotton, Allison M.; Chen, Chih-Yu; Lam, Lucia L.; Wasserman, Wyeth W.; Kobor, Michael S.; Brown, Carolyn J.

2014-01-01

X-chromosome inactivation results in dosage equivalence between the X chromosome in males and females; however, over 15% of human X-linked genes escape silencing and these genes are enriched on the evolutionarily younger short arm of the X chromosome. The spread of inactivation onto translocated autosomal material allows the study of inactivation without the confounding evolutionary history of the X chromosome. The heterogeneity and reduced extent of silencing on autosomes are evidence for the importance of DNA elements underlying the spread of silencing. We have assessed DNA methylation in six unbalanced X-autosome translocations using the Illumina Infinium HumanMethylation450 array. Two to 42% of translocated autosomal genes showed this mark of silencing, with the highest degree of inactivation observed for trisomic autosomal regions. Generally, the extent of silencing was greatest close to the translocation breakpoint; however, silencing was detected well over 100 kb into the autosomal DNA. Alu elements were found to be enriched at autosomal genes that escaped from inactivation while L1s were enriched at subject genes. In cells without the translocation, there was enrichment of heterochromatic features such as EZH2 and H3K27me3 for those genes that become silenced when translocated, suggesting that underlying chromatin structure predisposes genes towards silencing. Additionally, the analysis of topological domains indicated physical clustering of autosomal genes of common inactivation status. Overall, our analysis indicated a complex interaction between DNA sequence, chromatin features and the three-dimensional structure of the chromosome. PMID:24158853

RNA degradation and models for post-transcriptional gene-silencing.

PubMed

Meins, F

2000-06-01

Post-transcriptional gene silencing (PTGS) is a form of stable but potentially reversible epigenetic modification, which frequently occurs in transgenic plants. The interaction in trans of genes with similar transcribed sequences results in sequence-specific degradation of RNAs derived from the genes involved. Highly expressed single-copy loci, transcribed inverted repeats, and poorly transcribed complex loci can act as sources of signals that trigger PTGS. In some cases, mobile, sequence-specific silencing signals can move from cell to cell or even over long distances in the plant. Several current models hold that silencing signals are 'aberrant' RNAs (aRNA), which differ in some way from normal mRNAs. The most likely candidates are small antisense RNAs (asRNA) and double-stranded RNAs (dsRNA). Direct evidence that these or other aRNAs found in silent tissues can induce PTGS is still lacking. Most current models assume that silencing signals interact with target RNAs in a sequence-specific fashion. This results in degradation, usually in the cytoplasm, by exonucleolytic as well as endonucleolytic pathways, which are not necessarily PTGS-specific. Biochemical-switch models hold that the silent state is maintained by a positive auto-regulatory loop. One possibility is that concentrations of hypothetical silencing signals above a critical threshold trigger their own production by self-replication, by degradation of target RNAs, or by a combination of both mechanisms. These models can account for the stability, reversibility and multiplicity of silent states; the strong influence of transcription rate of target genes on the incidence and stability of silencing, and the amplification and systemic propagation of motile silencing signals.

Performing Silence: Gender, Violence, and Resistance in Women's Narratives from Lahaul, India

ERIC Educational Resources Information Center

Bhattacharya, Himika

2009-01-01

This article presents two different ways of understanding silence, through a discussion of women's narratives of violence from Lahaul, India. Here I illustrate how feminist ethnography works its way into re-conceptualizing silence as a tool women use to resist existing patriarchal discourses of honor, tribe and nation. (Contains 1 note.)

Silence as Right, Choice, Resistance and Strategy among Chinese "Me Generation" Students: Implications for Pedagogy

ERIC Educational Resources Information Center

Ha, Phan Le; Li, Binghui

2014-01-01

The topic of silence and "the Chinese learner" has been extensively studied often in relation to cross-cultural adjustment, intercultural issues, learning styles, language ability and differences of classroom expectations. These studies have often led to recommendations to understand silence and "the Chinese learner" in more…

Life Skills in Solitude and Silence in the School.

ERIC Educational Resources Information Center

Byrnes, Deborah A.

1983-01-01

Addresses the rationale for fostering the positive use of silent and alone time in school and notes how silence can be a useful setting for learning and thinking. Explains four sample activities to help acquaint children with the positive use of silence and solitude and suggests references for future reading. (SB)

Deriving Silence through Dependent Reference: Focus on Pronouns

ERIC Educational Resources Information Center

Livitz, Inna G.

2014-01-01

The starting point of this dissertation is the observation that pronouns that are obligatorily dependent on a sufficiently local antecedent are persistently silent. The classical hypothesis has been that silence is a lexical property of such elements. The central claim of this dissertation is that silence is instead a product of syntax--of the way…

Silences and Subtexts of Immigrant and Nonimmigrant Children.

ERIC Educational Resources Information Center

MacNaughton, Glenda

2001-01-01

Explored 4- and 5-year-old immigrant and nonimmigrant children's words and silences when they were asked about similarities and differences among 4 ethnic dolls. Found that skin color was salient in children's thought processes about appearance and identity. Recommended that educators listen to children's voices and silences to help children build…

PhOBF1, a petunia OCS element binding factor, plays an important role in antiviral RNA silencing

USDA-ARS?s Scientific Manuscript database

Virus-induced gene silencing (VIGS) is a common strategy of reverse genetics for characterizing function of genes in plant. The detailed mechanism governing RNA silencing efficiency triggered by virus is largely unclear. Here, we revealed that a petunia (Petunia hybrida) ocs element binding factor, ...

Self-Silencing, Emotional Awareness, and Eating Behaviors in College Women

ERIC Educational Resources Information Center

Shouse, Sarah H.; Nilsson, Johanna

2011-01-01

Self-silencing (or the suppression of expressing one's thoughts, feelings, and needs) can have a negative impact on the mental health of women, from depression to disordered eating behaviors. The authors examined the relationship between self-silencing and disordered eating as well as intuitive eating. The authors also explored whether emotional…

"Publicity Is Our Pride": The Passionate Grammar of Gertrude Stein.

ERIC Educational Resources Information Center

Cope, Karin M.

1992-01-01

Reexamines the associations between homosexuality and silence that have governed many gay and lesbian readings. Asks whether homosexuality is really a site of "silence" in Stein's texts, and by extension, whether to make such silences speak out is really what seems to be required of the reader by "queer texts." (RS)

Hearing the Silence: Acknowledging the Voice of My Latina Sisters

ERIC Educational Resources Information Center

Martinez-Vogt, Emily

2015-01-01

Latina community college students experience a number of challenges during their transition to college. Findings from a larger study indicated that Latina community college students experienced racism and stereotyping on campus responding with silence. Silence occurred in two ways: (1) Latinas were forced to be silent, and/or (2) Latinas chose to…

The Matter of Silence in Early Childhood Bilingual Education

ERIC Educational Resources Information Center

Martín-Bylund, Anna

2018-01-01

The relationship between silence as non-speech and bilingualism in early childhood education is intricate. This article maps this relationship with the help of diverse theoretical entrances to a video-recorded everyday episode from a bilingual (Spanish-Swedish) preschool in Sweden. Though this, three alternative readings of silence are produced.…

47 CFR 80.1125 - Search and rescue coordinating communications.

Code of Federal Regulations, 2011 CFR

2011-10-01

... station involved may impose silence on stations which interfere with that traffic. This instruction may be... “silence, m'aider”; (2) In narrow-band direct-printing telegraphy normally using forward-error correcting mode, the signal SILENCE MAYDAY. However, the ARQ mode may be used when it is advantageous to do so. (f...

Classroom Silence: Voices from Japanese EFL Learners

ERIC Educational Resources Information Center

Harumi, Seiko

2011-01-01

This article explores Japanese EFL learners' classroom silence in a Japanese EFL context. The existence of silence in second language learning contexts can be a source of conflict between students and teachers and even among students themselves. It can also be an obstacle to acquiring the target language. In order to tackle this problem and to…

Silence in the Second Language Classrooms of Japanese Universities

ERIC Educational Resources Information Center

King, Jim

2013-01-01

Japanese language learners' proclivity for silence has been alluded to by various writers (e.g. Anderson 1993; Korst 1997; Greer 2000) and is supported by plenty of anecdotal evidence, but large-scale, empirical studies aimed at measuring the extent of macro-level silence within Japanese university L2 classrooms are notably lacking. This article…

GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing.

PubMed

Sidorenko, Lyudmila V; Lee, Tzuu-Fen; Woosley, Aaron; Moskal, William A; Bevan, Scott A; Merlo, P Ann Owens; Walsh, Terence A; Wang, Xiujuan; Weaver, Staci; Glancy, Todd P; Wang, PoHao; Yang, Xiaozeng; Sriram, Shreedharan; Meyers, Blake C

2017-11-01

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Strategies to re-express epigenetically silenced p15(INK4b) and p21(WAF1) genes in acute myeloid leukemia.

PubMed

Geyer, C Ronald

2010-01-01

p15(INK4B) and p21(WAF1) are TGF-β targets that are silenced in leukemia by epigenetic mechanisms involving DNA methylation and/or histone modifications. Mechanisms for establishing and maintaining epigenetic silencing of p15(INK4B) and p21(WAF1) are not well established. The reversible nature of epigenetic modifications has lead to the development of drugs that target DNA methyltransferases, histone deacetylases, and histone methyltransferases, which have been used to re-express aberrantly silenced genes in leukemia. Recently, non-coding RNA, referred to as natural antisense transcripts (NATs), have been implicated in the regulation of epigenetic modifications. Here, we review epigenetic mechanisms for silencing p15(INK4B) and p21(WAF1) and the role of NATs in this process. We also review epigenetic drugs and drug combinations used to re-express p15(INK4B) and p21(WAF1). Lastly, we discuss the potential use of NATs to target the activity of epigenetic drugs to specific genes and to permanently re-express epigenetically silenced genes.

A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

PubMed

Tsai, Hsin-Yue; Chen, Chun-Chieh G; Conte, Darryl; Moresco, James J; Chaves, Daniel A; Mitani, Shohei; Yates, John R; Tsai, Ming-Daw; Mello, Craig C

2015-01-29

Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. Copyright © 2015 Elsevier Inc. All rights reserved.

A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi

PubMed Central

Tsai, Hsin-Yue; Chen, Chun-Chieh G.; Conte, Darryl; Moresco, James J.; Chaves, Daniel A.; Mitani, Shohei; Yates, John R.; Tsai, Ming-Daw; Mello, Craig C.

2015-01-01

SUMMARY Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering (si) RNAs are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3′ uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3’ uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. PMID:25635455

The RNA-induced transcriptional silencing complex targets chromatin exclusively via interacting with nascent transcripts.

PubMed

Shimada, Yukiko; Mohn, Fabio; Bühler, Marc

2016-12-01

Small RNAs regulate chromatin modification and transcriptional gene silencing across the eukaryotic kingdom. Although these processes have been well studied, fundamental mechanistic aspects remain obscure. Specifically, it is unclear exactly how small RNA-loaded Argonaute protein complexes target chromatin to mediate silencing. Here, using fission yeast, we demonstrate that transcription of the target locus is essential for RNA-directed formation of heterochromatin. However, high transcriptional activity is inhibitory; thus, a transcriptional window exists that is optimal for silencing. We further found that pre-mRNA splicing is compatible with RNA-directed heterochromatin formation. However, the kinetics of pre-mRNA processing is critical. Introns close to the 5' end of a transcript that are rapidly spliced result in a bistable response whereby the target either remains euchromatic or becomes fully silenced. Together, our results discount siRNA-DNA base pairing in RNA-mediated heterochromatin formation, and the mechanistic insights further reveal guiding paradigms for the design of small RNA-directed chromatin silencing studies in multicellular organisms. © 2016 Shimada et al.; Published by Cold Spring Harbor Laboratory Press.

The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription.

PubMed

Akay, Alper; Di Domenico, Tomas; Suen, Kin M; Nabih, Amena; Parada, Guillermo E; Larance, Mark; Medhi, Ragini; Berkyurek, Ahmet C; Zhang, Xinlian; Wedeles, Christopher J; Rudolph, Konrad L M; Engelhardt, Jan; Hemberg, Martin; Ma, Ping; Lamond, Angus I; Claycomb, Julie M; Miska, Eric A

2017-08-07

Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Negative core affect and employee silence: How differences in activation, cognitive rumination, and problem-solving demands matter.

PubMed

Madrid, Hector P; Patterson, Malcolm G; Leiva, Pedro I

2015-11-01

Employees can help to improve organizational performance by sharing ideas, suggestions, or concerns about practices, but sometimes they keep silent because of the experience of negative affect. Drawing and expanding on this stream of research, this article builds a theoretical rationale based on core affect and cognitive appraisal theories to describe how differences in affect activation and boundary conditions associated with cognitive rumination and cognitive problem-solving demands can explain employee silence. Results of a diary study conducted with professionals from diverse organizations indicated that within-person low-activated negative core affect increased employee silence when, as an invariant factor, cognitive rumination was high. Furthermore, within-person high-activated negative core affect decreased employee silence when, as an invariant factor, cognitive problem-solving demand was high. Thus, organizations should manage conditions to reduce experiences of low-activated negative core affect because these feelings increase silence in individuals high in rumination. In turn, effective management of experiences of high-activated negative core affect can reduce silence for individuals working under high problem-solving demand situations. (c) 2015 APA, all rights reserved).

Sound attenuation of a finite length dissipative flow duct silencer with internal mean flow in the absorbent

NASA Astrophysics Data System (ADS)

Cummings, A.; Chang, I.-J.

1988-11-01

Internal mean flow within the pores of a bulk-reacting porous acoustic absorbent, driven by mean static pressure gradients, is shown here to be an important feature of the acoustics of dissipative silencers in flow ducts, particularly in the case of internal combustion engine exhaust silencers. Theoretical treatments are presented here, both to describe the effect of internal flow on the bulk acoustic perties of the porous medium and to find the effect of the absorbent in situ, in the form of the sound transmission loss of the silencer. The measured transmission loss of an experimental silencer is compared to predicted data and good agreement between the two is obtained. The effects of mean fluid flow in the central passage and internal flow in the absorbent are separately demonstrated.

African American Faculty Expressing Concerns: Breaking the Silence at Predominantly White Research Oriented Universities

ERIC Educational Resources Information Center

Ross, Henry H.; Edwards, Willie J.

2016-01-01

A Delphi method was used with a panel of 24 African American faculty employed at 43 predominantly white doctoral extensive universities to arrive at a group consensus on a list of concerns that African American faculty in general experienced or held. Using the Delphi method a panel of African American faculty initially worked from a list of eight…

Light intensity and temperature affect systemic spread of silencing signal in transient agroinfiltration studies.

PubMed

Patil, Basavaprabhu L; Fauquet, Claude M

2015-06-01

RNA silencing is a sequence-specific post-transcriptional gene inactivation mechanism that operates in diverse organisms and that can extend beyond its site of initiation, owing to the movement of the silencing signal, called non-autonomous gene silencing. Previous studies have shown that several factors manifest the movement of the silencing signal, such as the size (21 or 24 nucleotides) of the secondary small interfering RNA (siRNA) produced, the steady-state concentration of siRNAs and their cognate messenger RNA (mRNA) or a change in the sink-source status of plant parts affecting phloem translocation. Our study shows that both light intensity and temperature have a significant impact on the systemic movement of the silencing signal in transient agroinfiltration studies in Nicotiana benthamiana. At higher light intensities (≥ 450 μE/m(2)/s) and higher temperatures (≥ 30 °C), gene silencing was localized to leaf tissue that was infiltrated, without any systemic spread. Interestingly, in these light and temperature conditions (≥ 450 μE/m(2) /s and ≥ 30 °C), the N. benthamiana plants showed recovery from the viral symptoms. However, the reduced systemic silencing and reduced viral symptom severity at higher light intensities were caused by a change in the sink-source status of the plant, ultimately affecting the phloem translocation of small RNAs or the viral genome. In contrast, at lower light intensities (<300 μE/m(2)/s) with a constant temperature of 25 °C, there was strong systemic movement of the silencing signal in the N. benthamiana plants and reduced recovery from virus infections. The accumulation of gene-specific siRNAs was reduced at higher temperature as a result of a reduction in the accumulation of transcript on transient agroinfiltration of RNA interference (RNAi) constructs, mostly because of poor T-DNA transfer activity of Agrobacterium, possibly also accompanied by reduced phloem translocation. © 2014 BSPP AND JOHN WILEY & SONS LTD.

10 CFR 963.16 - Postclosure suitability evaluation method.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 4 2014-01-01 2014-01-01 false Postclosure suitability evaluation method. 963.16 Section... Determination, Methods, and Criteria § 963.16 Postclosure suitability evaluation method. (a) DOE will evaluate postclosure suitability using the total system performance assessment method. DOE will conduct a total system...

10 CFR 963.16 - Postclosure suitability evaluation method.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 4 2011-01-01 2011-01-01 false Postclosure suitability evaluation method. 963.16 Section... Determination, Methods, and Criteria § 963.16 Postclosure suitability evaluation method. (a) DOE will evaluate postclosure suitability using the total system performance assessment method. DOE will conduct a total system...

10 CFR 963.16 - Postclosure suitability evaluation method.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 4 2012-01-01 2012-01-01 false Postclosure suitability evaluation method. 963.16 Section... Determination, Methods, and Criteria § 963.16 Postclosure suitability evaluation method. (a) DOE will evaluate postclosure suitability using the total system performance assessment method. DOE will conduct a total system...

Protection against common bean rust conferred by a gene silencing method

USDA-ARS?s Scientific Manuscript database

Rust disease of the dry bean plant, Phaseolus vulgaris, is caused by the fungus Uromyces appendiculatus. The fungus acquires its nutrients and energy from bean leaves using a specialized cell structure, the haustorium, through which it secretes effector proteins that contribute to pathogenicity by ...

Driving in unheard silence: Disability and the politics of shutting up.

PubMed

Lourens, Heidi

2018-03-01

The politics of silence is central to disability experience and the field of disability studies. In this analytical autoethnography, I write about my silences as a visually impaired woman. I explore and make sense of personal life stories through a theoretical perspective. The analysis of these personal experiences lead me to argue that disability-related silences are mostly created through the confluence of inaccessible physical and social environments and the psychological internalisation of these worlds. I also discuss the ways in which I am currently regaining my voice. Further research on resistance by disabled persons is recommended.

Genome-wide association study identified genetic variations and candidate genes for plant architecture component traits in Chinese upland cotton.

PubMed

Su, Junji; Li, Libei; Zhang, Chi; Wang, Caixiang; Gu, Lijiao; Wang, Hantao; Wei, Hengling; Liu, Qibao; Huang, Long; Yu, Shuxun

2018-06-01

Thirty significant associations between 22 SNPs and five plant architecture component traits in Chinese upland cotton were identified via GWAS. Four peak SNP loci located on chromosome D03 were simultaneously associated with more plant architecture component traits. A candidate gene, Gh_D03G0922, might be responsible for plant height in upland cotton. A compact plant architecture is increasingly required for mechanized harvesting processes in China. Therefore, cotton plant architecture is an important trait, and its components, such as plant height, fruit branch length and fruit branch angle, affect the suitability of a cultivar for mechanized harvesting. To determine the genetic basis of cotton plant architecture, a genome-wide association study (GWAS) was performed using a panel composed of 355 accessions and 93,250 single nucleotide polymorphisms (SNPs) identified using the specific-locus amplified fragment sequencing method. Thirty significant associations between 22 SNPs and five plant architecture component traits were identified via GWAS. Most importantly, four peak SNP loci located on chromosome D03 were simultaneously associated with more plant architecture component traits, and these SNPs were harbored in one linkage disequilibrium block. Furthermore, 21 candidate genes for plant architecture were predicted in a 0.95-Mb region including the four peak SNPs. One of these genes (Gh_D03G0922) was near the significant SNP D03_31584163 (8.40 kb), and its Arabidopsis homologs contain MADS-box domains that might be involved in plant growth and development. qRT-PCR showed that the expression of Gh_D03G0922 was upregulated in the apical buds and young leaves of the short and compact cotton varieties, and virus-induced gene silencing (VIGS) proved that the silenced plants exhibited increased PH. These results indicate that Gh_D03G0922 is likely the candidate gene for PH in cotton. The genetic variations and candidate genes identified in this study lay a foundation for cultivating moderately short and compact varieties in future Chinese cotton-breeding programs.

Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity

PubMed Central

Rand, Tim A.; Ginalski, Krzysztof; Grishin, Nick V.; Wang, Xiaodong

2004-01-01

RNA interference is carried out by the small double-stranded RNA-induced silencing complex (RISC). The RISC-bound small RNA guides the RISC complex to identify and cleave mRNAs with complementary sequences. The proteins that make up the RISC complex and cleave mRNA have not been unequivocally defined. Here, we report the biochemical purification of RISC activity to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 (Ago-2) is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, we found homology between the PIWI domain of Ago-2 and endonuclease V and identified potential active-site amino acid residues within the PIWI domain of Ago-2. PMID:15452342

Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity.

PubMed

Rand, Tim A; Ginalski, Krzysztof; Grishin, Nick V; Wang, Xiaodong

2004-10-05

RNA interference is carried out by the small double-stranded RNA-induced silencing complex (RISC). The RISC-bound small RNA guides the RISC complex to identify and cleave mRNAs with complementary sequences. The proteins that make up the RISC complex and cleave mRNA have not been unequivocally defined. Here, we report the biochemical purification of RISC activity to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 (Ago-2) is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, we found homology between the PIWI domain of Ago-2 and endonuclease V and identified potential active-site amino acid residues within the PIWI domain of Ago-2.

Choosing the Active X: The Human Version of X Inactivation.

PubMed

Migeon, Barbara R

2017-12-01

Humans and rodents differ in how they carry out X inactivation (XI), the mammalian method to compensate for the different number of X chromosomes in males and females. Evolutionary changes in staging embryogenesis and in mutations within the XI center alter the process among mammals. The mouse model of XI is predicated on X counting and subsequently choosing the X to 'inactivate'. However, new evidence suggests that humans initiate XI by protecting one X in both sexes from inactivation by XIST, the noncoding RNA that silences the inactive X. This opinion article explores the question of how the active X is protected from silencing by its own Xist locus, and the possibility of different solutions for mouse and human. Copyright © 2017 Elsevier Ltd. All rights reserved.

Paramutation phenomena in plants.

PubMed

Pilu, Roberto

2015-08-01

Paramutation is a particular epigenetic phenomenon discovered in Zea mays by Alexander Brink in the 1950s, and then also found in other plants and animals. Brink coined the term paramutation (from the Greek syllable "para" meaning beside, near, beyond, aside) in 1958, with the aim to differentiate paramutation from mutation. The peculiarity of paramutation with respect to other gene silencing phenomena consists in the ability of the silenced allele (named paramutagenic) to silence the other allele (paramutable) present in trans. The newly silenced (paramutated) allele remains stable in the next generations even after segregation from the paramutagenic allele and acquires paramutagenic ability itself. The inheritance behaviour of these epialleles permits a fast diffusion of a particular gene expression level/phenotype in a population even in the absence of other evolutionary influences, thus breaking the Hardy-Weinberg law. As with other gene silencing phenomena such as quelling in the fungus Neurospora crassa, transvection in Drosophila, co-suppression and virus-induced gene silencing (VIGS) described in transgenic plants and RNA interference (RNAi) in the nematode Caenorhabditis elegans, paramutation occurs without changes in the DNA sequence. So far the molecular basis of paramutation remains not fully understood, although many studies point to the involvement of RNA causing changes in DNA methylation and chromatin structure of the silenced genes. In this review I summarize all paramutation phenomena described in plants, focusing on the similarities and differences between them. Copyright © 2015 Elsevier Ltd. All rights reserved.

The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways.

PubMed

Gómez-Muñoz, Neus; Velázquez, Karelia; Vives, María Carmen; Ruiz-Ruiz, Susana; Pina, José Antonio; Flores, Ricardo; Moreno, Pedro; Guerri, José

2017-12-01

Citrus tristeza virus (CTV) induces in the field the decline and death of citrus varieties grafted on sour orange (SO) rootstock, which has forced the use of alternative decline-tolerant rootstocks in affected countries, despite the highly desirable agronomic features of the SO rootstock. Declining citrus plants display phloem necrosis below the bud union. In addition, SO is minimally susceptible to CTV compared with other citrus varieties, suggesting partial resistance of SO to CTV. Here, by silencing different citrus genes with a Citrus leaf blotch virus-based vector, we have examined the implication of the RNA silencing and salicylic acid (SA) defence pathways in the resistance of SO to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, associated with these defence pathways, enhanced virus spread and accumulation in SO plants in comparison with non-silenced controls, whereas silencing of the genes NPR3/NPR4, associated with the hypersensitive response, produced a slight decrease in CTV accumulation and reduced stunting of SO grafted on CTV-infected rough lemon plants. We also found that the CTV RNA silencing suppressors p20 and p23 also suppress the SA signalling defence, with the suppressor activity being higher in the most virulent isolates. © 2016 BSPP AND JOHN WILEY & SONS LTD.

The molecular basis for stability of heterochromatin-mediated silencing in mammals.

PubMed

Hiragami-Hamada, Kyoko; Xie, Sheila Q; Saveliev, Alexander; Uribe-Lewis, Santiago; Pombo, Ana; Festenstein, Richard

2009-11-04

The archetypal epigenetic phenomenon of position effect variegation (PEV) in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3), DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac) and lysine 4 di or tri methylation (H3K4me2/3) are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo.

Dihydroartemisinin induces apoptosis preferentially via a Bim-mediated intrinsic pathway in hepatocarcinoma cells.

PubMed

Qin, Guiqi; Zhao, ChuBiao; Zhang, Lili; Liu, Hongyu; Quan, Yingyao; Chai, Liuying; Wu, Shengnan; Wang, Xiaoping; Chen, Tongsheng

2015-08-01

This report is designed to dissect the detail molecular mechanism by which dihydroartemisinin (DHA), a derivative of artemisinin, induces apoptosis in human hepatocellular carcinoma (HCC) cells. DHA induced a loss of the mitochondrial transmemberane potential (ΔΨm), release of cytochrome c, activation of caspases, and externalization of phosphatidylserine indicative of apoptosis induction. Compared with the modest inhibitory effects of silencing Bax, silencing Bak largely prevented DHA-induced ΔΨm collapse and apoptosis though DHA induced a commensurable activation of Bax and Bak, demonstrating a key role of the Bak-mediated intrinsic apoptosis pathway. DHA did not induce Bid cleavage and translocation from cytoplasm to mitochondria and had little effects on the expressions of Puma and Noxa, but did increase Bim and Bak expressions and decrease Mcl-1 expression. Furthermore, the cytotoxicity of DHA was remarkably reduced by silencing Bim, and modestly but significantly reduced by silencing Puma or Noxa. Silencing Bim or Noxa preferentially reduced DHA-induced Bak activation, while silencing Puma preferentially reduced DHA-induced Bax activation, demonstrating that Bim and to a lesser extent Noxa act as upstream mediators to trigger the Bak-mediated intrinsic apoptosis pathway. In addition, silencing Mcl-1 enhanced DHA-induced Bak activation and apoptosis. Taken together, our data demonstrate a crucial role of Bim in preferentially regulating the Bak/Mcl-1 rheostat to mediate DHA-induced apoptosis in HCC cells.

LINE-1 silencing by retinoblastoma proteins is effected through the nucleosomal and remodeling deacetylase multiprotein complex.

PubMed

Montoya-Durango, Diego E; Ramos, Kenneth A; Bojang, Pasano; Ruiz, Lorell; Ramos, Irma N; Ramos, Kenneth S

2016-01-25

Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1. Chromatin immunoprecipitation and silencing RNA technology were used to identify the repressor complex that silences L1 in human and murine cells. Components of the Nucleosomal and Remodeling Deacetylase (NuRD) multiprotein complex specifically enriched the L1 5'-untranslated DNA sequence in human and murine cells. Genetic ablation of RB proteins in murine cells destabilized interactions within the NuRD macromolecular complex and mediated nuclear rearrangement of Mi2-β, an ATP-dependent helicase subunit with nucleosome remodeling activity. Depletion of Mi2-β, RbAP46 and HDAC2 reduced the repressor activity of the NuRD complex and reactivated a synthetic L1 reporter in human cells. Epigenetic reactivation of L1 in RB-null cells by DNA damage was markedly enhanced compared to wild type cells. RB proteins stabilize interactions of the NuRD corepressor complex within the L1 promoter to effect L1 silencing. L1 retroelements may serve as a scaffold on which RB builds heterochromatic regions that regulate chromatin function.

Pnc1p-Mediated Nicotinamide Clearance Modifies the Epigenetic Properties of rDNA Silencing in Saccharomyces cerevisiae

PubMed Central

McClure, Julie M.; Gallo, Christopher M.; Smith, Daniel L.; Matecic, Mirela; Hontz, Robert D.; Buck, Stephen W.; Racette, Frances G.; Smith, Jeffrey S.

2008-01-01

The histone deacetylase activity of Sir2p is dependent on NAD+ and inhibited by nicotinamide (NAM). As a result, Sir2p-regulated processes in Saccharomyces cerevisiae such as silencing and replicative aging are susceptible to alterations in cellular NAD+ and NAM levels. We have determined that high concentrations of NAM in the growth medium elevate the intracellular NAD+ concentration through a mechanism that is partially dependent on NPT1, an important gene in the Preiss–Handler NAD+ salvage pathway. Overexpression of the nicotinamidase, Pnc1p, prevents inhibition of Sir2p by the excess NAM while maintaining the elevated NAD+ concentration. This growth condition alters the epigenetics of rDNA silencing, such that repression of a URA3 reporter gene located at the rDNA induces growth on media that either lacks uracil or contains 5-fluoroorotic acid (5-FOA), an unusual dual phenotype that is reminiscent of telomeric silencing (TPE) of URA3. Despite the similarities to TPE, the modified rDNA silencing phenotype does not require the SIR complex. Instead, it retains key characteristics of typical rDNA silencing, including RENT and Pol I dependence, as well as a requirement for the Preiss–Handler NAD+ salvage pathway. Exogenous nicotinamide can therefore have negative or positive impacts on rDNA silencing, depending on the PNC1 expression level. PMID:18780747

A Study of Pragmatic Functions of Silence in Colloquial Jordanian Arabic

ERIC Educational Resources Information Center

Al-Harahsheh, Ahmad Mohammad Ahmad

2013-01-01

The pragmatic study of silence has not got much concern in the Arab world in general and in Jordanian Arabic in particular. The purpose of the current study is to investigate the perception and practice of silence in casual conversation in Jordanian society from a pragmatic point of view. This study adapts Volosinov's notion of…

In a Silent Way: Student Perceptions of Silence in Community

ERIC Educational Resources Information Center

Wood, Margaret; Tribe, Robert

2016-01-01

This paper explores young people's perceptions of the role and value of shared "gathered" silence in the corporate life of a school community. It draws on a small-scale qualitative investigation in a Quaker school setting. There may be particular things to learn about the practice of stillness and silence inherent in the ethos of a…

The Phenomenon of Intrusive Thoughts in Yejide Kilanko's "Daughters Who Walk This Path"

ERIC Educational Resources Information Center

Dodhy, Shamaila

2017-01-01

Hegemonic masculinity, with patriarchal supremacy and female subservience are the norms of many African societies. Suffering in silence goes along with the traditional place of woman in African societies as they observe sexist hierarchy of power. Physical attack and its wounds flourish in an atmosphere of secrecy and silence. Silence to such…

The Educators' Guide to the Day of Silence

ERIC Educational Resources Information Center

Gay, Lesbian and Straight Education Network (GLSEN), 2010

2010-01-01

The Day of Silence is the largest single student-led action towards creating safer schools for all, regardless of sexual orientation, gender identity or gender expression. From the first-ever Day of Silence at the University of Virginia in 1996, to the organizing efforts in over 8,000 middle schools, high schools, colleges and universities across…

Silent Pedagogy and Rethinking Classroom Practice: Structuring Teaching through Silence Rather than Talk

ERIC Educational Resources Information Center

Ollin, Ros

2008-01-01

Classroom observations are an important source of information about teaching and about the practice of particular teachers. The paper considers the value placed on talk as opposed to silence in this context and suggests that a cultural bias towards talk means that silence is commonly perceived negatively. The paper is based on a qualitative…

Rethinking Classroom Participation: Listening to Silent Voices

ERIC Educational Resources Information Center

Schultz, Katherine

2009-01-01

Many educators understand how to gauge learning by paying close attention to student talk. Few know how to interpret and attend to student silence as a form of participation. In her new book, Katherine Schultz examines the complex role student silence can play in teaching and learning. Urging teachers to listen to student silence in new ways, this…

Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors.

PubMed

Huang, Ting-Kuo; Falk, Bryce W; Dandekar, Abhaya M; McDonald, Karen A

2018-05-24

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter ( Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium -mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24.

Calcium signalling silencing in atrial fibrillation.

PubMed

Greiser, Maura

2017-06-15

Subcellular calcium signalling silencing is a novel and distinct cellular and molecular adaptive response to rapid cardiac activation. Calcium signalling silencing develops during short-term sustained rapid atrial activation as seen clinically during paroxysmal atrial fibrillation (AF). It is the first 'anti-arrhythmic' adaptive response in the setting of AF and appears to counteract the maladaptive changes that lead to intracellular Ca 2+ signalling instability and Ca 2+ -based arrhythmogenicity. Calcium signalling silencing results in a failed propagation of the [Ca 2+ ] i signal to the myocyte centre both in patients with AF and in a rabbit model. This adaptive mechanism leads to a substantial reduction in the expression levels of calcium release channels (ryanodine receptors, RyR2) in the sarcoplasmic reticulum, and the frequency of Ca 2+ sparks and arrhythmogenic Ca 2+ waves remains low. Less Ca 2+ release per [Ca 2+ ] i transient, increased fast Ca 2+ buffering strength, shortened action potentials and reduced L-type Ca 2+ current contribute to a substantial reduction of intracellular [Na + ]. These features of Ca 2+ signalling silencing are distinct and in contrast to the changes attributed to Ca 2+ -based arrhythmogenicity. Some features of Ca 2+ signalling silencing prevail in human AF suggesting that the Ca 2+ signalling 'phenotype' in AF is a sum of Ca 2+ stabilizing (Ca 2+ signalling silencing) and Ca 2+ destabilizing (arrhythmogenic unstable Ca 2+ signalling) factors. Calcium signalling silencing is a part of the mechanisms that contribute to the natural progression of AF and may limit the role of Ca 2+ -based arrhythmogenicity after the onset of AF. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Tomato yellow leaf curl virus infection of a resistant tomato line with a silenced sucrose transporter gene LeHT1 results in inhibition of growth, enhanced virus spread, and necrosis.

PubMed

Eybishtz, Assaf; Peretz, Yuval; Sade, Dagan; Gorovits, Rena; Czosnek, Henryk

2010-02-01

To identify genes involved in resistance of tomato to Tomato yellow leaf curl virus (TYLCV), cDNA libraries from lines resistant (R) and susceptible (S) to the virus were compared. The hexose transporter LeHT1 was found to be expressed preferentially in R tomato plants. The role of LeHT1 in the establishment of TYLCV resistance was studied in R plants where LeHT1 has been silenced using Tobacco rattle virus-induced gene silencing (TRV VIGS). Following TYLCV inoculation, LeHT1-silenced R plants showed inhibition of growth and enhanced virus accumulation and spread. In addition, a necrotic response was observed along the stem and petioles of infected LeHT1-silenced R plants, but not on infected not-silenced R plants. This response was specific of R plants since it was absent in infected LeHT1-silenced S plants. Necrosis had several characteristics of programmed cell death (PCD): DNA from necrotic tissues presented a PCD-characteristic ladder pattern, the amount of a JNK analogue increased, and production of reactive oxygen was identified by DAB staining. A similar necrotic reaction along stem and petioles was observed in LeHT1-silenced R plants infected with the DNA virus Bean dwarf mosaic virus and the RNA viruses Cucumber mosaic virus and Tobacco mosaic virus. These results constitute the first evidence for a necrotic response backing natural resistance to TYLCV in tomato, confirming that plant defense is organized in multiple layers. They demonstrate that the hexose transporter LeHT1 is essential for the expression of natural resistance against TYLCV and its expression correlates with inhibition of virus replication and movement.

P19-dependent and P19-independent reversion of F1-V gene silencing in tomato.

PubMed

Alvarez, M Lucrecia; Pinyerd, Heidi L; Topal, Emel; Cardineau, Guy A

2008-09-01

As a part of a project to develop a plant-made plague vaccine, we expressed the Yersinia pestis F1-V antigen fusion protein in tomato. We discovered that in some of these plants the expression of the f1-v gene was undetectable in leaves and fruit by ELISA, even though they had multiple copies of f1-v according to Southern-blot analysis. A likely explanation of these results is the phenomenon of RNA silencing, a group of RNA-based processes that produces sequence-specific inhibition of gene expression and may result in transgene silencing in plants. Here we report the reversion of the f1-v gene silencing in transgenic tomato plants through two different mechanisms. In the P19-dependent Reversion or Type I, the viral suppressor of gene silencing, P19, induces the reversion of gene silencing. In the P19-independent Reversion or Type II, the f1-v gene expression is restored after the substantial loss of gene copies as a consequence of transgene segregation in the progeny. The transient and stable expression of the p19 gene driven by a constitutive promoter as well as an ethanol inducible promoter induced a P19-dependent reversion of f1-v gene silencing. In particular, the second generation plant 3D1.6 had the highest P19 protein levels and correlated with the highest F1-V protein accumulation, almost a three-fold increase of F1-V protein levels in fruit than that previously reported for the non-silenced F1-V elite tomato lines. These results confirm the potential exploitation of P19 to substantially increase the expression of value-added proteins in plants.

Folate Polyglutamylation Is Involved in Chromatin Silencing by Maintaining Global DNA Methylation and Histone H3K9 Dimethylation in Arabidopsis[C][W

PubMed Central

Zhou, Hao-Ran; Zhang, Fang-Fang; Ma, Ze-Yang; Huang, Huan-Wei; Jiang, Ling; Cai, Tao; Zhu, Jian-Kang; Zhang, Chuyi; He, Xin-Jian

2013-01-01

DNA methylation and repressive histone Histone3 Lysine9 (H3K9) dimethylation correlate with chromatin silencing in plants and mammals. To identify factors required for DNA methylation and H3K9 dimethylation, we screened for suppressors of the repressor of silencing1 (ros1) mutation, which causes silencing of the expression of the RD29A (RESPONSE TO DESSICATION 29A) promoter-driven luciferase transgene (RD29A-LUC) and the 35S promoter-driven NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) transgene (35S-NPTII). We identified the folylpolyglutamate synthetase FPGS1 and the known factor DECREASED DNA METHYLATION1 (DDM1). The fpgs1 and ddm1 mutations release the silencing of both RD29A-LUC and 35S-NPTII. Genome-wide analysis indicated that the fpgs1 mutation reduces DNA methylation and releases chromatin silencing at a genome-wide scale. The effect of fpgs1 on chromatin silencing is correlated with reduced levels of DNA methylation and H3K9 dimethylation. Supplementation of fpgs1 mutants with 5-formyltetrahydrofolate, a stable form of folate, rescues the defects in DNA methylation, histone H3K9 dimethylation, and chromatin silencing. The competitive inhibitor of methyltransferases, S-adenosylhomocysteine, is markedly upregulated in fpgs1, by which fpgs1 reduces S-adenosylmethionine accessibility to methyltransferases and accordingly affects DNA and histone methylation. These results suggest that FPGS1-mediated folate polyglutamylation is required for DNA methylation and H3K9 dimethylation through its function in one-carbon metabolism. Our study makes an important contribution to understanding the complex interplay among metabolism, development, and epigenetic regulation. PMID:23881414

Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs

PubMed Central

García-Chapa, Meritxell; López-Moya, Juan José; Burgyán, József

2010-01-01

RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC. PMID:20657820

Characterization of the RNA silencing suppression activity of the Ebola virus VP35 protein in plants and mammalian cells.

PubMed

Zhu, Yali; Cherukuri, Nil Celebi; Jackel, Jamie N; Wu, Zetang; Crary, Monica; Buckley, Kenneth J; Bisaro, David M; Parris, Deborah S

2012-03-01

Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.

Sex-specific silencing of X-linked genes by Xist RNA

PubMed Central

Gayen, Srimonta; Maclary, Emily; Hinten, Michael; Kalantry, Sundeep

2016-01-01

X-inactive specific transcript (Xist) long noncoding RNA (lncRNA) is thought to catalyze silencing of X-linked genes in cis during X-chromosome inactivation, which equalizes X-linked gene dosage between male and female mammals. To test the impact of Xist RNA on X-linked gene silencing, we ectopically induced endogenous Xist by ablating the antisense repressor Tsix in mice. We find that ectopic Xist RNA induction and subsequent X-linked gene silencing is sex specific in embryos and in differentiating embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). A higher frequency of XΔTsixY male cells displayed ectopic Xist RNA coating compared with XΔTsixX female cells. This increase reflected the inability of XΔTsixY cells to efficiently silence X-linked genes compared with XΔTsixX cells, despite equivalent Xist RNA induction and coating. Silencing of genes on both Xs resulted in significantly reduced proliferation and increased cell death in XΔTsixX female cells relative to XΔTsixY male cells. Thus, whereas Xist RNA can inactivate the X chromosome in females it may not do so in males. We further found comparable silencing in differentiating XΔTsixY and 39,XΔTsix (XΔTsixO) ESCs, excluding the Y chromosome and instead implicating the X-chromosome dose as the source of the sex-specific differences. Because XΔTsixX female embryonic epiblast cells and EpiSCs harbor an inactivated X chromosome prior to ectopic inactivation of the active XΔTsix X chromosome, we propose that the increased expression of one or more X-inactivation escapees activates Xist and, separately, helps trigger X-linked gene silencing. PMID:26739568

The role of mutation in the new cancer paradigm.

PubMed

Prehn, Richmond T

2005-04-26

The almost universal belief that cancer is caused by mutation may gradually be giving way to the belief that cancer begins as a cellular adaptation that involves the local epigenetic silencing of various genes. In my own interpretation of the new epigenetic paradigm, the genes epigenetically suppressed are genes that normally serve in post-embryonic life to suppress and keep suppressed those other genes upon which embryonic development depends. Those other genes, if not silenced or suppressed in the post-embryonic animal, become, I suggest, the oncogenes that are the basis of neoplasia.Mutations that occur in silenced genes supposedly go unrepaired and are, therefore, postulated to accumulate, but such mutations probably play little or no causative role in neoplasia because they occur in already epigenetically silenced genes. These mutations probably often serve to make the silencing, and therefore the cancer, epigenetically irreversible.

The role of mutation in the new cancer paradigm

PubMed Central

Prehn, Richmond T

2005-01-01

The almost universal belief that cancer is caused by mutation may gradually be giving way to the belief that cancer begins as a cellular adaptation that involves the local epigenetic silencing of various genes. In my own interpretation of the new epigenetic paradigm, the genes epigenetically suppressed are genes that normally serve in post-embryonic life to suppress and keep suppressed those other genes upon which embryonic development depends. Those other genes, if not silenced or suppressed in the post-embryonic animal, become, I suggest, the oncogenes that are the basis of neoplasia. Mutations that occur in silenced genes supposedly go unrepaired and are, therefore, postulated to accumulate, but such mutations probably play little or no causative role in neoplasia because they occur in already epigenetically silenced genes. These mutations probably often serve to make the silencing, and therefore the cancer, epigenetically irreversible. PMID:15854226

Stability of miRNA 5′terminal and seed regions is correlated with experimentally observed miRNA-mediated silencing efficacy

PubMed Central

Hibio, Naoki; Hino, Kimihiro; Shimizu, Eigo; Nagata, Yoshiro; Ui-Tei, Kumiko

2012-01-01

MicroRNAs (miRNAs) are key regulators of sequence-specific gene silencing. However, crucial factors that determine the efficacy of miRNA-mediated target gene silencing are poorly understood. Here we mathematized base-pairing stability and showed that miRNAs with an unstable 5′ terminal duplex and stable seed-target duplex exhibit strong silencing activity. The results are consistent with the previous findings that an RNA strand with unstable 5′ terminal in miRNA duplex easily loads onto the RNA-induced silencing complex (RISC), and miRNA recognizes target mRNAs with seed-complementary sequences to direct posttranscriptional repression. Our results suggested that both the unwinding and target recognition processes of miRNAs could be proficiently controlled by the thermodynamics of base-pairing in protein-free condition. Interestingly, such thermodynamic parameters might be evolutionarily well adapted to the body temperatures of various species. PMID:23251782

Gold Nanobeacons for Tracking Gene Silencing in Zebrafish

PubMed Central

Cordeiro, Milton; Carvalho, Lara; Silva, Joana; Saúde, Leonor; Fernandes, Alexandra R.; Baptista, Pedro V.

2017-01-01

The use of gold nanoparticles for effective gene silencing has demonstrated its potential as a tool for gene expression experiments and for the treatment of several diseases. Here, we used a gold nanobeacon designed to specifically silence the enhanced green fluorescence protein (EGFP) mRNA in embryos of a fli-EGFP transgenic zebrafish line, while simultaneously allowing the tracking and localization of the silencing events via the beacon’s emission. Fluorescence imaging measurements demonstrated a decrease of the EGFP emission with a concomitant increase in the fluorescence of the Au-nanobeacon. Furthermore, microinjection of the Au-nanobeacon led to a negligible difference in mortality and malformations in comparison to the free oligonucleotide, indicating that this system is a biocompatible platform for the administration of gene silencing moieties. Together, these data illustrate the potential of Au-nanobeacons as tools for in vivo zebrafish gene modulation with low toxicity which may be used towards any gene of interest. PMID:28336844

Two suppressors of RNA silencing encoded by cereal-infecting members of the family Luteoviridae.

PubMed

Liu, Yan; Zhai, Hao; Zhao, Kun; Wu, Beilei; Wang, Xifeng

2012-08-01

Several members of the family Luteoviridae are important pathogens of cultivated plant species of the family Gramineae. In this study, we explored RNA-silencing suppressors (RSSs) encoded by two cereal-infecting luteoviruses: barley yellow dwarf virus and wheat yellow dwarf virus (BYDV and WYDV, respectively). The P0 protein of WYDV-GPV (P0(GPV)) and the P6 protein of BYDV-GAV (P6(GAV)) displayed RSS activities when expressed in agro-infiltrated leaves of Nicotiana benthamiana, by their local ability to inhibit post-transcriptional gene silencing of GFP. Analysis of GFP, mRNA and GFP-specific small interfering RNA indicated that both P0(GPV) and P6(GAV) are suppressors of silencing that can restrain not only local but also systemic gene silencing. This is the first report of RSS activity of the P6 protein in a member of the genus Luteovirus.

Mutations in Ran system affected telomere silencing in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayashi, Naoyuki; Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-0934; Kobayashi, Masahiko

The Ran GTPase system regulates the direction and timing of several cellular events, such as nuclear-cytosolic transport, centrosome formation, and nuclear envelope assembly in telophase. To gain insight into the Ran system's involvement in chromatin formation, we investigated gene silencing at the telomere in several mutants of the budding yeast Saccharomyces cerevisiae, which had defects in genes involved in the Ran system. A mutation of the RanGAP gene, rna1-1, caused reduced silencing at the telomere, and partial disruption of the nuclear Ran binding factor, yrb2-{delta}2, increased this silencing. The reduced telomere silencing in rna1-1 cells was suppressed by a highmore » dosage of the SIR3 gene or the SIT4 gene. Furthermore, hyperphosphorylated Sir3 protein accumulated in the rna1-1 mutant. These results suggest that RanGAP is required for the heterochromatin structure at the telomere in budding yeast.« less

A Loud Silence: Working with Research-Based Theatre and A/R/Tography

ERIC Educational Resources Information Center

Lea, Graham W.; Belliveau, George; Wager, Amanda; Beck, Jaime L.

2011-01-01

Arts-based approaches to research have emerged as an integral component of current scholarship in the social sciences, education, health research, and humanities. Integrating arts-based methods and methodologies with research generates possibilities for fresh approaches for creating, translating, and exchanging knowledge (Barone & Eisner, 1997;…

Agrobacterium rhizogenes - based transformation of soybean roots to form composite plants

USDA-ARS?s Scientific Manuscript database

Composite plants are a powerful tool to rapidly analyze the effects of gene overexpression, gene silencing, and examine test promoter expression in transgenic roots. No sterile tissue culture is needed. This avoids loss of valuable material due to contamination of sterile cultures. This method uses ...

Popular Media, Critical Pedagogy, and Inner City Youth

ERIC Educational Resources Information Center

Leard, Diane Wishart; Lashua, Brett

2006-01-01

In this article, we explored ways youth, traditionally silenced, engaged with popular culture to voice experiences and challenge dominant narratives of public schools and daily lives. We also considered how educators use popular culture as critical pedagogy with inner city youth. Through ethnographic bricolage and case study methods, and drawing…

The Relationship between Organizational Silence and Burnout among Academicians: A Research on Universities in Turkey

ERIC Educational Resources Information Center

Akin, Ugur; Ulusoy, Tarik

2016-01-01

The aim of this research is to analyze the relationship between organizational silence and burnout levels of academicians. The study group consisted of 190 academicians, who work in 17 state universities that are located in 15 different provinces of Turkey. Data were collected through Causes of Faculty Members' Silence Scale and Maslach Burnout…

The Study of Relationship between Women Teachers' Career Barriers and Organizational Silence: Viewpoint of Women and Men Teachers

ERIC Educational Resources Information Center

Inandi, Yusuf; Gün, Meryem Evsen; Giliç, Fahrettin

2017-01-01

The purpose of the study is to reveal the relationship between women teachers' career barriers and their organizational silence, and also whether career barriers predict their silence.* Study group is comprised of 522 teachers working at elementary and high schools in central districts of Mersin. In this descriptive study, data were collected…

Self-Serving Biases in Perceiving the Opinions of Others: Implications for the Spiral of Silence (Review Essay).

ERIC Educational Resources Information Center

Kennamer, J. David

1990-01-01

Synthesizes research supporting the assertion that people are not very accurate perceivers of the opinions of others. Assesses the impact of these findings on E. Noelle-Neumann's "spiral of silence" theory. Suggests an alternative research strategy drawn from social psychology to test this aspect of the spiral of silence process. (SG)

The Role of Silence in Teaching and Learning

ERIC Educational Resources Information Center

Schultz, Katherine

2013-01-01

The author's first teaching position was as a 4th and 5th grade teacher at a school in Philadelphia. There, she learned the Quaker value of adding silence and periods of reflection to her teaching to provide a wider range of students with the opportunity to participate in classroom discussions. Later, a focus on silence as a teaching strategy led…

Chromatin and RNAi factors protect the C. elegans germline against repetitive sequences

PubMed Central

Robert, Valérie J.P.; Sijen, Titia; van Wolfswinkel, Josien; Plasterk, Ronald H.A.

2005-01-01

Protection of genomes against invasion by repetitive sequences, such as transposons, viruses, and repetitive transgenes, involves strong and selective silencing of these sequences. During silencing of repetitive transgenes, a trans effect (“cosuppression”) occurs that results in silencing of cognate endogenous genes. Here we report RNA interference (RNAi) screens performed to catalog genes required for cosuppression in the Caenorhabditis elegans germline. We find factors with a putative role in chromatin remodeling and factors involved in RNAi. Together with molecular data also presented in this study, these results suggest that in C. elegans repetitive sequences trigger transcriptional gene silencing using RNAi and chromatin factors. PMID:15774721

Role of the tomato TAGL1 gene in regulating fruit metabolites elucidated using RNA sequence and metabolomics analyses.

PubMed

Zhao, Xiaodan; Yuan, Xinyu; Chen, Sha; Meng, Lanhuan; Fu, Daqi

2018-01-01

Fruit ripening is a complex biological process affecting fruit quality. In tomato the fruit ripening process is delicately regulated by transcription factors (TFs). Among these, the TOMATO AGAMOUS-LIKE 1 (TAGL1) gene plays an important role in both the development and ripening of fruit. In this study, the TAGL1 gene was successfully silenced by virus-induced gene silencing technology (VIGS), and the global gene expression and metabolites profiles of TAGL1-silenced fruits were analyzed by RNA-sequence analysis (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS/MS). The TAGL1-silenced fruits phenotypically displayed an orange pericarp, which was in accordance with the results expected from the down-regulation of genes associated with carotenoid synthesis. Levels of several amino acids and organic acids were lower in the TAGL1-silenced fruits than in the wild-type fruits, whereas, α-tomatine content was greatly increased (more than 10-fold) in the TAGL1-silenced fruits compared to wild-type fruits. The findings of this study showed that TAGL1 not only regulates the ripening of tomato fruits, but also affects the synthesis and levels of nutrients in the fruit.

Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair.

PubMed

Zhu, Kai; Lai, Hao; Guo, Changfa; Li, Jun; Wang, Yulin; Wang, Lingyan; Wang, Chunsheng

2014-01-01

Mesenchymal stem cell (MSC) transplantation has attracted much attention in myocardial infarction therapy. One of the limitations is the poor survival of grafted cells in the ischemic microenvironment. Small interfering RNA-mediated prolyl hydroxylase domain protein 2 (PHD2) silencing in MSCs holds tremendous potential to enhance their survival and paracrine effect after transplantation. However, an efficient and biocompatible PHD2 silencing system for clinical application is lacking. Herein, we developed a novel PHD2 silencing system based on arginine-terminated generation 4 poly(amidoamine) (Arg-G4) nanoparticles. The system exhibited effective and biocompatible small interfering RNA delivery and PHD2 silencing in MSCs in vitro. After genetically modified MSC transplantation in myocardial infarction models, MSC survival and paracrine function of IGF-1 were enhanced significantly in vivo. As a result, we observed decreased cardiomyocyte apoptosis, scar size, and interstitial fibrosis, and increased angiogenesis in the diseased myocardium, which ultimately attenuated ventricular remodeling and improved heart function. This work demonstrated that an Arg-G4 nanovector-based PHD2 silencing system could enhance the efficiency of MSC transplantation for infarcted myocardium repair.

ADP-glucose pyrophosphorylase gene plays a key role in the quality of corm and yield of cormels in gladiolus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seng, Shanshan, E-mail: seshsh108@126.com; Wu, Jian; Sui, Juanjuan

Starch is the main storage compound in underground organs like corms. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in storage organs and is likely one of the most important determinant of sink strength. Here, we identify an AGPase gene (GhAGPS1) from gladiolus. The highest transcriptional levels of GhAGPS1 were observed in cormels and corms. Transformation of GhAGPS1 into Arabidopsis rescued the phenotype of aps1 mutant. Silencing GhAGPS1 in gladiolus corms by virus-induced gene silencing (VIGS) decreased the transcriptional levels of two genes and starch content. Transmission electron microscopy analyses of leaf and corm sections confirmed thatmore » starch biosynthesis was inhibited. Corm weight and cormel number reduced significantly in the silenced plants. Taken together, these results indicate that inhibiting the expression of AGPase gene could impair starch synthesis, which results in the lowered corm quality and cormel yield in gladiolus. -- Highlights: •Cormel quantity was reduced significantly in silenced Gladiolus plants. •Corm quality was declined significantly in silenced Gladiolus plants. •Starch synthesis was inhibited in silenced Gladiolus plants.« less

Trans-Kingdom RNA Silencing in Plant-Fungal Pathogen Interactions.

PubMed

Hua, Chenlei; Zhao, Jian-Hua; Guo, Hui-Shan

2018-02-05

Fungal pathogens represent a major group of plant invaders that are the causative agents of many notorious plant diseases. Large quantities of RNAs, especially small RNAs involved in gene silencing, have been found to transmit bidirectionally between fungal pathogens and their hosts. Although host-induced gene silencing (HIGS) technology has been developed and applied to protect crops from fungal infections, the mechanisms of RNA transmission, especially small RNAs regulating trans-kingdom RNA silencing in plant immunity, are largely unknown. In this review, we summarize and discuss recent important findings regarding trans-kingdom sRNAs and RNA silencing in plant-fungal pathogen interactions compared with the well-known RNAi mechanisms in plants and fungi. We focus on the interactions between plant and fungal pathogens with broad hosts, represented by the vascular pathogen Verticillium dahliae and non-vascular pathogen Botrytis cinerea, and discuss the known instances of natural RNAi transmission between fungal pathogens and host plants. Given that HIGS has been developed and recently applied in controlling Verticillium wilt diseases, we propose an ideal research system exploiting plant vasculature-Verticillium interaction to further study trans-kingdom RNA silencing. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

A non-canonical site reveals the cooperative mechanisms of microRNA-mediated silencing.

PubMed

Flamand, Mathieu N; Gan, Hin Hark; Mayya, Vinay K; Gunsalus, Kristin C; Duchaine, Thomas F

2017-07-07

Although strong evidence supports the importance of their cooperative interactions, microRNA (miRNA)-binding sites are still largely investigated as functionally independent regulatory units. Here, a survey of alternative 3΄UTR isoforms implicates a non-canonical seedless site in cooperative miRNA-mediated silencing. While required for target mRNA deadenylation and silencing, this site is not sufficient on its own to physically recruit miRISC. Instead, it relies on facilitating interactions with a nearby canonical seed-pairing site to recruit the Argonaute complexes. We further show that cooperation between miRNA target sites is necessary for silencing in vivo in the C. elegans embryo, and for the recruitment of the Ccr4-Not effector complex. Using a structural model of cooperating miRISCs, we identified allosteric determinants of cooperative miRNA-mediated silencing that are required for both embryonic and larval miRNA functions. Our results delineate multiple cooperative mechanisms in miRNA-mediated silencing and further support the consideration of target site cooperation as a fundamental characteristic of miRNA function. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Analysis of absorption and reflection mechanisms in a three-dimensional plate silencer

NASA Astrophysics Data System (ADS)

Wang, Chunqi; Huang, Lixi

2008-06-01

When a segment of a rigid duct is replaced by a plate backed by a hard-walled cavity, grazing incident sound waves induce plate vibration, hence sound reflection. Based on this mechanism, a broadband plate silencer, which works effectively from low-to-medium frequencies have been developed recently. A typical plate silencer consists of an expansion chamber with two side-branch cavities covered by light but extremely stiff plates. Such a configuration is two-dimensional in nature. In this paper, numerical study is extended to three-dimensional configurations to investigate the potential improvement in sound reflection. Finite element simulation shows that the three-dimensional configurations perform better than the corresponding two-dimensional design, especially in the relatively high frequency region. Further analysis shows that the three-dimensional design gives better plate response at higher axial modes than the simple two-dimensional design. Sound absorption mechanism is also introduced to the plate silencer by adding two dissipative chambers on the two lateral sides of a two-cavity wave reflector, hence a hybrid silencer. Numerical simulation shows that the proposed hybrid silencer is able to achieve a good moderate bandwidth with much reduced total length in comparison with pure absorption design.

Post-transcriptional gene silencing of the gene encoding aldolase from soybean cyst nematode by transformed soybean roots.

PubMed

Youssef, Reham M; Kim, Kyung-Hwan; Haroon, Sanaa A; Matthews, Benjamin F

2013-06-01

Plant parasitic nematodes cause approximately 157 billion US dollars in losses worldwide annually. The soybean cyst nematode (SCN), Heterodera glycines, is responsible for an estimated one billion dollars in losses to the US farmer each year. A promising new approach for control of plant parasitic nematode control is gene silencing. We tested this approach by silencing the SCN gene HgALD, encoding fructose-1,6-diphosphate aldolase. This enzyme is important in the conversion of glucose into energy and may be especially important in actin-based motility during parasite invasion of its host. An RNAi construct targeted to silence HgALD was transformed into soybean roots of composite plants to examine its efficacy to reduce the development of females formed by SCN. The number of mature females on roots transformed with the RNAi construct designed to silence the HgALD gene was reduced by 58%. These results indicate that silencing the aldolase gene of SCN +can greatly decrease the number of female SCN reaching maturity, and it is a promising step towards broadening resistance of plants against plant-parasitic nematodes. Published by Elsevier Inc.

Raman microscopy of bladder cancer cells expressing green fluorescent protein

NASA Astrophysics Data System (ADS)

Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

2016-11-01

Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

RNA Pol IV and V in Gene Silencing: Rebel Polymerases Evolving Away From Pol II’s Rules

PubMed Central

Zhou, Ming; Law, Julie A.

2015-01-01

Noncoding RNAs regulate gene expression at both the transcriptional and post-transcriptional levels, and play critical roles in development, imprinting and the maintenance of genome integrity in eukaryotic organisms [1–3]. Therefore, it is important to understand how the production of such RNAs are controlled. In addition to the three canonical DNA dependent RNA polymerases (Pol) Pol I, II and III, two non-redundant plant-specific RNA polymerases, Pol IV and Pol V, have been identified and shown to generate noncoding RNAs that are required for transcriptional gene silencing via the RNA-directed DNA methylation (RdDM) pathway. Thus, somewhat paradoxically, transcription is required for gene silencing. This paradox extends beyond plants, as silencing pathways in yeast, fungi, flies, worms, and mammals also require transcriptional machinery [4,5]. As plants have evolved specialized RNA polymerases to carry out gene silencing in a manner that is separate from the essential roles of Pol II, their characterization offers unique insight into how RNA polymerases facilitate gene silencing. In this review, we focus on the mechanisms of Pol IV and Pol V function, including their compositions, their transcripts, and their modes of recruitment to chromatin. PMID:26344361

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

PubMed Central

Gartenberg, Marc R.; Smith, Jeffrey S.

2016-01-01

Transcriptional silencing in Saccharomyces cerevisiae occurs at several genomic sites including the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA) tandem array. Epigenetic silencing at each of these domains is characterized by the absence of nearly all histone modifications, including most prominently the lack of histone H4 lysine 16 acetylation. In all cases, silencing requires Sir2, a highly-conserved NAD+-dependent histone deacetylase. At locations other than the rDNA, silencing also requires additional Sir proteins, Sir1, Sir3, and Sir4 that together form a repressive heterochromatin-like structure termed silent chromatin. The mechanisms of silent chromatin establishment, maintenance, and inheritance have been investigated extensively over the last 25 years, and these studies have revealed numerous paradigms for transcriptional repression, chromatin organization, and epigenetic gene regulation. Studies of Sir2-dependent silencing at the rDNA have also contributed to understanding the mechanisms for maintaining the stability of repetitive DNA and regulating replicative cell aging. The goal of this comprehensive review is to distill a wide array of biochemical, molecular genetic, cell biological, and genomics studies down to the “nuts and bolts” of silent chromatin and the processes that yield transcriptional silencing. PMID:27516616

Epigenetic chromatin silencing: bistability and front propagation

NASA Astrophysics Data System (ADS)

Sedighi, Mohammad; Sengupta, Anirvan M.

2007-12-01

The role of post-translational modification of histones in eukaryotic gene regulation is well recognized. Epigenetic silencing of genes via heritable chromatin modifications plays a major role in cell fate specification in higher organisms. We formulate a coarse-grained model of chromatin silencing in yeast and study the conditions under which the system becomes bistable, allowing for different epigenetic states. We also study the dynamics of the boundary between the two locally stable states of chromatin: silenced and unsilenced. The model could be of use in guiding the discussion on chromatin silencing in general. In the context of silencing in budding yeast, it helps us understand the phenotype of various mutants, some of which may be non-trivial to see without the help of a mathematical model. One such example is a mutation that reduces the rate of background acetylation of particular histone side chains that competes with the deacetylation by Sir2p. The resulting negative feedback due to a Sir protein depletion effect gives rise to interesting counter-intuitive consequences. Our mathematical analysis brings forth the different dynamical behaviors possible within the same molecular model and guides the formulation of more refined hypotheses that could be addressed experimentally.

Expression of cancer stem markers could be influenced by silencing of p16 gene in HeLa cervical carcinoma cells.

PubMed

Wu, H; Zhang, J; Shi, H

2016-01-01

Effect of the tumor suppression gene p16 on the biological characteristics of HeLa cervical carcinoma cells was explored. The expression of p16 protein was increased in HeLa tumor sphere cells, and no significant difference in tumor spheres from the first to the fourth passages. Compared with those of parental HeLa cells, the proportion of CD44+/CD24- and ABCG2+ cells increased significantly in tumor spheres. However after the cells were silenced by the p16-sh289 vector, expression of P16 protein and the cell number of CD44+/CD24- and ABCG2+ decreased. Moreover, HeLa cells with p16 gene silencing showed decreased abilities of sphere formation and matrigel invasion. More HeLa cells with p16 gene silence were needed for tumor formation in nude mice. Tumor size and weight in mouse model established with p16 gene silenced HeLa cells were less than those with HeLa parental cell model. The present results indicate that silencing of the p16 gene inhibits expression of cancer stem cell markers and tumorigenic ability of HeLa cells.

Deep sequencing uncovers commonality in small RNA profiles between transgene-induced and naturally occurring RNA silencing of chalcone synthase-A gene in petunia.

PubMed

Kasai, Megumi; Matsumura, Hideo; Yoshida, Kentaro; Terauchi, Ryohei; Taneda, Akito; Kanazawa, Akira

2013-01-30

Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs. We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved. The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these silencing systems especially in the biosynthetic processes of siRNAs including cleavage of CHS-A transcripts and subsequent production of secondary siRNAs in exon 2. The data also suggest that these events occurred at multiple sites, which can be a feature of these silencing phenomena.

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo

PubMed Central

Coles, Andrew H.; Osborn, Maire F.; Alterman, Julia F.; Turanov, Anton A.; Godinho, Bruno M.D.C.; Kennington, Lori; Chase, Kathryn; Aronin, Neil

2016-01-01

Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene® branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra- and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo. PMID:26595721

Annexin II-Dependent Mechanism of Breast Cancer Progression

DTIC Science & Technology

2008-06-01

and migratory capacities of the annexin II-suppressed cells. Methods: We used antisense RNA technology to silence the annexin II gene in MDA...gene in mDA-MB231 cells using polymerase chain reaction-based short hairpin RNA (1–7 months) b) Characterize the proliferative, invasive, and...MB231 cells according to methods described by Li et al. (24). Briefly, three different diothionated antisense nucleotides (ODN) were synthesized

Silencing of X-Linked MicroRNAs by Meiotic Sex Chromosome Inactivation

PubMed Central

Royo, Hélène; Seitz, Hervé; ElInati, Elias; Peters, Antoine H. F. M.; Stadler, Michael B.; Turner, James M. A.

2015-01-01

During the pachytene stage of meiosis in male mammals, the X and Y chromosomes are transcriptionally silenced by Meiotic Sex Chromosome Inactivation (MSCI). MSCI is conserved in therian mammals and is essential for normal male fertility. Transcriptomics approaches have demonstrated that in mice, most or all protein-coding genes on the X chromosome are subject to MSCI. However, it is unclear whether X-linked non-coding RNAs behave in a similar manner. The X chromosome is enriched in microRNA (miRNA) genes, with many exhibiting testis-biased expression. Importantly, high expression levels of X-linked miRNAs (X-miRNAs) have been reported in pachytene spermatocytes, indicating that these genes may escape MSCI, and perhaps play a role in the XY-silencing process. Here we use RNA FISH to examine X-miRNA expression in the male germ line. We find that, like protein-coding X-genes, X-miRNAs are expressed prior to prophase I and are thereafter silenced during pachynema. X-miRNA silencing does not occur in mouse models with defective MSCI. Furthermore, X-miRNAs are expressed at pachynema when present as autosomally integrated transgenes. Thus, we conclude that silencing of X-miRNAs during pachynema in wild type males is MSCI-dependent. Importantly, misexpression of X-miRNAs during pachynema causes spermatogenic defects. We propose that MSCI represents a chromosomal mechanism by which X-miRNAs, and other potential X-encoded repressors, can be silenced, thereby regulating genes with critical late spermatogenic functions. PMID:26509798

A Violaxanthin Deepoxidase Interacts with a Viral Suppressor of RNA Silencing to Inhibit Virus Amplification1[OPEN

PubMed Central

Chen, Ling; Yan, Zhaoling; Cheng, Yuqin; Jiao, Zhiyuan; Sun, Biao

2017-01-01

RNA silencing plays a critical role against viral infection. To counteract this antiviral silencing, viruses have evolved various RNA silencing suppressors. Meanwhile, plants have evolved counter-counter defense strategies against RNA silencing suppression (RSS). In this study, the violaxanthin deepoxidase protein of maize (Zea mays), ZmVDE, was shown to interact specifically with the helper component-proteinase (HC-Pro; a viral RNA silencing suppressor) of Sugarcane mosaic virus (SCMV) via its mature protein region by yeast two-hybrid assay, which was confirmed by coimmunoprecipitation in Nicotiana benthamiana cells. It was demonstrated that amino acids 101 to 460 in HC-Pro and the amino acid glutamine-292 in ZmVDE mature protein were essential for this interaction. The mRNA levels of ZmVDE were down-regulated 75% to 65% at an early stage of SCMV infection. Interestingly, ZmVDE, which normally localized in the chloroplasts and cytoplasm, could relocalize to HC-Pro-containing aggregate bodies in the presence of HC-Pro alone or SCMV infection. In addition, ZmVDE could attenuate the RSS activity of HC-Pro in a specific protein interaction-dependent manner. Subsequently, transient silencing of the ZmVDE gene facilitated SCMV RNA and coat protein accumulation. Taken together, our results suggest that ZmVDE interacts with SCMV HC-Pro and attenuates its RSS activity, contributing to decreased SCMV accumulation. This study demonstrates that a host factor can be involved in secondary defense responses against viral infection in monocot plants. PMID:29021224

Resilience among women with HIV: Impact of silencing the self and socioeconomic factors

PubMed Central

Dale, Sannisha K.; Cohen, Mardge H.; Kelso, Gwendolyn A.; Cruise, Ruth C.; Weber, Kathleen M.; Watson, Cheryl; Burke-Miller, Jane K.; Brody, Leslie R.

2014-01-01

In the U.S., women account for over a quarter of the approximately 50,000 annual new HIV diagnoses and face intersecting and ubiquitous adversities including gender inequities, sexism, poverty, violence, and limited access to quality education and employment. Women are also subjected to prescribed gender roles such as silencing their needs in interpersonal relationships, which may lessen their ability to be resilient and function adaptively following adversity. Previous studies have often highlighted the struggles encountered by women with HIV without focusing on their strengths. The present cross-sectional study investigated the relationships of silencing the self and socioeconomic factors (education, employment, and income) with resilience in a sample of women with HIV. The sample consisted of 85 women with HIV, diverse ethnic/racial groups, aged 24 – 65 enrolled at the Chicago site of the Women’s Interagency HIV Study in the midwestern region of the United States. Measures included the Connor-Davidson Resilience Scale -10 item and the Silencing the Self Scale (STSS). Participants showed high levels of resilience. Women with lower scores on the STSS (lower self-silencing) reported significantly higher resilience compared to women with higher STSS scores. Although employment significantly related to higher resilience, silencing the self tended to predict resilience over and above the contributions of employment, income, and education. Results suggest that intervention and prevention efforts aimed at decreasing silencing the self and increasing employment opportunities may improve resilience. PMID:24932061

The molecular basis for stability of heterochromatin-mediated silencing in mammals

PubMed Central

2009-01-01

The archetypal epigenetic phenomenon of position effect variegation (PEV) in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3), DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac) and lysine 4 di or tri methylation (H3K4me2/3) are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo. PMID:19889207

Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.].

PubMed

Trusov, Yuri; Botella, José Ramón

2006-01-01

Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering 'natural flowering' in mature pineapples under commercial field conditions.

Transcriptome and proteome analyses and the role of atypical calpain protein and autophagy in the spliced leader silencing pathway in Trypanosoma brucei.

PubMed

Hope, Ronen; Egarmina, Katarina; Voloshin, Konstantin; Waldman Ben-Asher, Hiba; Carmi, Shai; Eliaz, Dror; Drori, Yaron; Michaeli, Shulamit

2016-10-01

Under persistent ER stress, Trypanosoma brucei parasites induce the spliced leader silencing (SLS) pathway. In SLS, transcription of the SL RNA gene, the SL donor to all mRNAs, is extinguished, arresting trans-splicing and leading to programmed cell death (PCD). In this study, we investigated the transcriptome following silencing of SEC63, a factor essential for protein translocation across the ER membrane, and whose silencing induces SLS. The proteome of SEC63-silenced cells was analyzed with an emphasis on SLS-specific alterations in protein expression, and modifications that do not directly result from perturbations in trans-splicing. One such protein identified is an atypical calpain SKCRP7.1/7.2. Co-silencing of SKCRP7.1/7.2 and SEC63 eliminated SLS induction due its role in translocating the PK3 kinase. This kinase initiates SLS by migrating to the nucleus and phosphorylating TRF4 leading to shut-off of SL RNA transcription. Thus, SKCRP7.1 is involved in SLS signaling and the accompanying PCD. The role of autophagy in SLS was also investigated; eliminating autophagy through VPS34 or ATG7 silencing demonstrated that autophagy is not essential for SLS induction, but is associated with PCD. Thus, this study identified factors that are used by the parasite to cope with ER stress and to induce SLS and PCD. © 2016 John Wiley & Sons Ltd.

Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata.

PubMed

Gherbi, Hassen; Nambiar-Veetil, Mathish; Zhong, Chonglu; Félix, Jessy; Autran, Daphné; Girardin, Raphaël; Vaissayre, Virginie; Auguy, Florence; Bogusz, Didier; Franche, Claudine

2008-05-01

In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the beta-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP(+) hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.

RNA-mediated gene silencing in Candida albicans: inhibition of hyphae formation by use of RNAi technology.

PubMed

Moazeni, Maryam; Khoramizadeh, Mohammad Reza; Kordbacheh, Parivash; Sepehrizadeh, Zargham; Zeraati, Hojat; Noorbakhsh, Fatemeh; Teimoori-Toolabi, Ladan; Rezaie, Sassan

2012-09-01

The introduction of RNA silencing machinery in fungi has led to the promising application of RNAi methodology to knock down essential vital factor or virulence factor genes in the microorganisms. Efg1p is required for development of a true hyphal growth form which is known to be essential for interactions with human host cells and for the yeast's pathogenesis. In this paper, we describe the development of a system for presenting and studying the RNAi function on the EFG1 gene in C. albicans. The 19-nucleotide siRNA was designed on the basis of the cDNA sequence of the EFG1 gene in C. albicans and transfection was performed by use of a modified-PEG/LiAc method. To investigate EFG1 gene silencing in siRNA-treated cells, the yeasts were grown in human serum; to induce germ tubes a solid medium was used with the serum. Quantitative changes in expression of the EFG1 gene were analyzed by measuring the cognate EFG1 mRNA level by use of a quantitative real-time RT-PCR assay. Compared with the positive control, true hyphae formation was significantly reduced by siRNA at concentrations of 1 μM, 500 nM, and 100 nM (P < 0.05). In addition, siRNA at a concentration of 1 μM was revealed to inhibit expression of the EFG1 gene effectively (P < 0.05). On the basis of the potential of post-transcriptional gene silencing to control the expression of specific genes, these techniques may be regarded as promising means of drug discovery, with applications in biomedicine and functional genomics analysis.

Small-Interfering RNA–Eluting Surfaces as a Novel Concept for Intravascular Local Gene Silencing

PubMed Central

Nolte, Andrea; Walker, Tobias; Schneider, Martina; Kray, Oya; Avci-Adali, Meltem; Ziemer, Gerhard; Wendel, Hans Peter

2011-01-01

New drug-eluting stent (DES) methods have recently been demonstrated to improve outcomes of intravascular interventions. A novel technique is the design of gene-silencing stents that elute specific small-interfering RNAs (siRNAs) for better vascular wall regeneration. Although siRNAs used to alter gene expression have surpassed expectations in in vitro experiments, the functional and local delivery of siRNAs is still the major obstacle for the in vivo application of RNA interference. In this preliminary in vitro study we investigated a surface-immobilized siRNA delivery technique that would be readily adaptable for local intravascular applications in vivo. The transfection potency of gelatin coatings consisting of a specific siRNA complexed with polyethylenimine (PEI) was examined in primary human endothelial cells by flow cytometry and quantitative real-time polymerase chain reaction. Several media conditions, such as the presence or absence of serum during cultivation, were investigated. Furthermore, different siRNA and PEI amounts, as well as nitrogen/phosphate ratios, were tested for their transfection efficiency. Gelatin coatings consisting of PEI and siRNA against an exemplary endothelial adhesion molecule receptor achieved a significant knockdown of around 70%. The transfection efficiency of the coatings was not influenced by the presence of serum. The results of this preliminary study support the expectation that this novel coating may be favorable for local in vivo gene silencing (for example, when immobilized on stents or balloons for percutanous transluminal coronary angioplasty). However, further animal experiments are needed to confirm the translation into clinical practice. This intriguing technology leads the way to more sophisticated and individualized coatings for the post-DES era, toward silencing of genes involved in the pathway of intimal hyperplasia. PMID:21792480

Virus-Induced Gene Silencing Identifies an Important Role of the TaRSR1 Transcription Factor in Starch Synthesis in Bread Wheat.

PubMed

Liu, Guoyu; Wu, Yufang; Xu, Mengjun; Gao, Tian; Wang, Pengfei; Wang, Lina; Guo, Tiancai; Kang, Guozhang

2016-09-23

The function of a wheat starch regulator 1 (TaRSR1) in regulating the synthesis of grain storage starch was determined using the barley stripe mosaic virus-virus induced gene-silencing (BSMV-VIGS) method in field experiments. Chlorotic stripes appeared on the wheat spikes infected with barley stripe mosaic virus-virus induced gene-silencing- wheat starch regulator 1 (BSMV-VIGS-TaRSR1) at 15 days after anthesis, at which time the transcription levels of the TaRSR1 gene significantly decreased. Quantitative real-time PCR was also used to measure the transcription levels of 26 starch synthesis-related enzyme genes in the grains of BSMV-VIGS-TaRSR1-silenced wheat plants at 20, 27, and 31 days after anthesis. The results showed that the transcription levels of some starch synthesis-related enzyme genes were markedly induced at different sampling time points: TaSSI, TaSSIV, TaBEIII, TaISA1, TaISA3, TaPHOL, and TaDPE1 genes were induced at each of the three sampling time points and TaAGPS1-b, TaAGPL1, TaAGPL2, TaSSIIb, TaSSIIc, TaSSIIIb, TaBEI, TaBEIIa, TaBEIIb, TaISA2, TaPHOH, and TaDPE2 genes were induced at one sampling time point. Moreover, both the grain starch contents, one thousand kernel weights, grain length and width of BSMV-VIGS-TaRSR1-infected wheat plants significantly increased. These results suggest that TaRSR1 acts as a negative regulator and plays an important role in starch synthesis in wheat grains by temporally regulating the expression of specific starch synthesis-related enzyme genes.

Virus-Induced Gene Silencing Identifies an Important Role of the TaRSR1 Transcription Factor in Starch Synthesis in Bread Wheat

PubMed Central

Liu, Guoyu; Wu, Yufang; Xu, Mengjun; Gao, Tian; Wang, Pengfei; Wang, Lina; Guo, Tiancai; Kang, Guozhang

2016-01-01

The function of a wheat starch regulator 1 (TaRSR1) in regulating the synthesis of grain storage starch was determined using the barley stripe mosaic virus—virus induced gene-silencing (BSMV-VIGS) method in field experiments. Chlorotic stripes appeared on the wheat spikes infected with barley stripe mosaic virus-virus induced gene-silencing- wheat starch regulator 1 (BSMV-VIGS-TaRSR1) at 15 days after anthesis, at which time the transcription levels of the TaRSR1 gene significantly decreased. Quantitative real-time PCR was also used to measure the transcription levels of 26 starch synthesis-related enzyme genes in the grains of BSMV-VIGS-TaRSR1-silenced wheat plants at 20, 27, and 31 days after anthesis. The results showed that the transcription levels of some starch synthesis-related enzyme genes were markedly induced at different sampling time points: TaSSI, TaSSIV, TaBEIII, TaISA1, TaISA3, TaPHOL, and TaDPE1 genes were induced at each of the three sampling time points and TaAGPS1-b, TaAGPL1, TaAGPL2, TaSSIIb, TaSSIIc, TaSSIIIb, TaBEI, TaBEIIa, TaBEIIb, TaISA2, TaPHOH, and TaDPE2 genes were induced at one sampling time point. Moreover, both the grain starch contents, one thousand kernel weights, grain length and width of BSMV-VIGS-TaRSR1-infected wheat plants significantly increased. These results suggest that TaRSR1 acts as a negative regulator and plays an important role in starch synthesis in wheat grains by temporally regulating the expression of specific starch synthesis-related enzyme genes. PMID:27669224

RNAi-mediated endogene silencing in strawberry fruit: detection of primary and secondary siRNAs by deep sequencing.

PubMed

Härtl, Katja; Kalinowski, Gregor; Hoffmann, Thomas; Preuss, Anja; Schwab, Wilfried

2017-05-01

RNA interference (RNAi) has been exploited as a reverse genetic tool for functional genomics in the nonmodel species strawberry (Fragaria × ananassa) since 2006. Here, we analysed for the first time different but overlapping nucleotide sections (>200 nt) of two endogenous genes, FaCHS (chalcone synthase) and FaOMT (O-methyltransferase), as inducer sequences and a transitive vector system to compare their gene silencing efficiencies. In total, ten vectors were assembled each containing the nucleotide sequence of one fragment in sense and corresponding antisense orientation separated by an intron (inverted hairpin construct, ihp). All sequence fragments along the full lengths of both target genes resulted in a significant down-regulation of the respective gene expression and related metabolite levels. Quantitative PCR data and successful application of a transitive vector system coinciding with a phenotypic change suggested propagation of the silencing signal. The spreading of the signal in strawberry fruit in the 3' direction was shown for the first time by the detection of secondary small interfering RNAs (siRNAs) outside of the primary targets by deep sequencing. Down-regulation of endogenes by the transitive method was less effective than silencing by ihp constructs probably because the numbers of primary siRNAs exceeded the quantity of secondary siRNAs by three orders of magnitude. Besides, we observed consistent hotspots of primary and secondary siRNA formation along the target sequence which fall within a distance of less than 200 nt. Thus, ihp vectors seem to be superior over the transitive vector system for functional genomics in strawberry fruit. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Silencing of miR156 confers enhanced resistance to brown planthopper in rice.

PubMed

Ge, Yafei; Han, Junyou; Zhou, Guoxin; Xu, Yunmin; Ding, Yue; Shi, Min; Guo, Changkui; Wu, Gang

2018-06-22

Silencing of miR156 in rice confers enhanced resistance to brown planthopper through reducing JA and JA-Ile biosynthesis. Rice brown planthopper (BPH, Nilaparvata lugens Stål) threatens the sustainability of rice production and global food security. Due to the rapid adaptation of BPH to current germplasms in rice, development of novel types of resistant germplasms becomes increasingly important. Plant ontogenetic defense against pathogen and herbivores offers a broad spectrum and durable resistance, and has been experimentally tested in many plants; however, the underlying molecular mechanism remains unclear. miR156 is the master regulator of ontogeny in plants; modulation of miR156 is, therefore, expected to cause corresponding changes in BPH resistance. To test this hypothesis, we silenced miR156 using a target mimicry method in rice, and analyzed the resistance of miR156-silenced plants (MIM156) to BPH. MIM156 plants exhibited enhanced resistance to BPH based on analyses of honeydew excretion, nymph survival, fecundity of BPH, and the survival ratio of rice plants after BPH infestation. Molecular analysis indicated that the expression of MPK3, MPK6, and WRKY70, three genes involved in BPH resistance and jasmonic acid (JA) signaling, was altered in MIM156 plants. The JA and bioactive jasmonoyl-isoleucine levels and the expression of genes involved in JA biosynthesis were significantly reduced in MIM156 plants. Restoration of JA level by exogenous application increased the number of BPH feeding on MIM156 plants and reduced its resistance to BPH. Our findings suggest that miR156 negatively regulates BPH resistance by increasing JA level in rice; therefore, modulation of miR156-SPLs' pathway may offer a promising way to breed rice varieties with enhanced resistance against BPH and elite agronomically important traits.

A Pre- and Co-Knockdown of RNAseT Enzyme, Eri-1, Enhances the Efficiency of RNAi Induced Gene Silencing in Caenorhabditis elegans

PubMed Central

Jadiya, Pooja; Nazir, Aamir

2014-01-01

Background The approach of RNAi mediated gene knockdown, employing exogenous dsRNA, is being beneficially exploited in various fields of functional genomics. The immense utility of the approach came to fore from studies with model system C. elegans, but quickly became applicable with varied research models ranging from in vitro to various in vivo systems. Previously, there have been reports on the refractoriness of the neuronal cells to RNAi mediated gene silencing following which several modulators like eri-1 and lin-15 were described in C. elegans which, when present, would negatively impact the gene knockdown. Methodology/Principal Findings Taking a clue from these findings, we went on to screen hypothesis-driven- methodologies towards exploring the efficiency in the process of RNAi under various experimental conditions, wherein these genes would be knocked down preceding to, or concurrently with, the knocking down of a gene of interest. For determining the efficiency of gene knockdown, we chose to study visually stark phenotypes of uncoordinated movement, dumpy body morphology and blistered cuticle obtained by knocking down of genes unc-73, dpy-9 and bli-3 respectively, employing the RNAi-by-feeding protocol in model system C. elegans. Conclusions/Significance Our studies led to a very interesting outcome as the results reveal that amongst various methods tested, pre-incubation with eri-1 dsRNA synthesizing bacteria followed by co-incubation with eri-1 and gene-of-interest dsRNA synthesizing bacteria leads to the most efficient gene silencing as observed by the analysis of marker phenotypes. This provides an approach for effectively employing RNAi induced gene silencing while working with different genetic backgrounds including transgenic and mutant strains. PMID:24475317

Mechanism of Action of 2-Aminobenzamide HDAC Inhibitors in Reversing Gene Silencing in Friedreich’s Ataxia

PubMed Central

Soragni, Elisabetta; Chou, C. James; Rusche, James R.; Gottesfeld, Joel M.

2015-01-01

The genetic defect in Friedreich’s ataxia (FRDA) is the hyperexpansion of a GAA•TTC triplet in the first intron of the FXN gene, encoding the essential mitochondrial protein frataxin. Histone post-translational modifications near the expanded repeats are consistent with heterochromatin formation and consequent FXN gene silencing. Using a newly developed human neuronal cell model, derived from patient-induced pluripotent stem cells, we find that 2-aminobenzamide histone deacetylase (HDAC) inhibitors increase FXN mRNA levels and frataxin protein in FRDA neuronal cells. However, only compounds targeting the class I HDACs 1 and 3 are active in increasing FXN mRNA in these cells. Structural analogs of the active HDAC inhibitors that selectively target either HDAC1 or HDAC3 do not show similar increases in FXN mRNA levels. To understand the mechanism of action of these compounds, we probed the kinetic properties of the active and inactive inhibitors, and found that only compounds that target HDACs 1 and 3 exhibited a slow-on/slow-off mechanism of action for the HDAC enzymes. HDAC1- and HDAC3-selective compounds did not show this activity. Using siRNA methods in the FRDA neuronal cells, we show increases in FXN mRNA upon silencing of either HDACs 1 or 3, suggesting the possibility that inhibition of each of these class I HDACs is necessary for activation of FXN mRNA synthesis, as there appears to be redundancy in the silencing mechanism caused by the GAA•TTC repeats. Moreover, inhibitors must have a long residence time on their target enzymes for this activity. By interrogating microarray data from neuronal cells treated with inhibitors of different specificity, we selected two genes encoding histone macroH2A (H2AFY2) and Polycomb group ring finger 2 (PCGF2) that were specifically down-regulated by the inhibitors targeting HDACs1 and 3 versus the more selective inhibitors for further investigation. Both genes are involved in transcriptional repression and we speculate their involvement in FXN gene silencing. Our results shed light on the mechanism whereby HDAC inhibitors increase FXN mRNA levels in FRDA neuronal cells. PMID:25798128

Silencing or inhibition of endoplasmic reticulum aminopeptidase 1 (ERAP1) suppresses free heavy chain expression and Th17 responses in ankylosing spondylitis

PubMed Central

Chen, Liye; Ridley, Anna; Hammitzsch, Ariane; Al-Mossawi, Mohammad Hussein; Bunting, Helen; Georgiadis, Dimitris; Chan, Antoni; Kollnberger, Simon; Bowness, Paul

2016-01-01

Objective Human leucocyte antigen (HLA)-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly associated with ankylosing spondylitis (AS). ERAP1 is a key aminopeptidase in HLA class I presentation and can potentially alter surface expression of HLA-B27 free heavy chains (FHCs). We studied the effects of ERAP1 silencing/inhibition/variations on HLA-B27 FHC expression and Th17 responses in AS. Methods Flow cytometry was used to measure surface expression of HLA class I in peripheral blood mononuclear cells (PBMCs) from patients with AS carrying different ERAP1 genotypes (rs2287987, rs30187 and rs27044) and in ERAP1-silenced/inhibited/mutated HLA-B27-expressing antigen presenting cells (APCs). ERAP1-silenced/inhibited APCs were cocultured with KIR3DL2CD3ε-reporter cells or AS CD4+ T cells. Th17 responses of AS CD4+ T cells were measured by interleukin (IL)-17A ELISA and Th17 intracellular cytokine staining. FHC cell surface expression and Th17 responses were also measured in AS PBMCs following ERAP1 inhibition. Results The AS-protective ERAP1 variants, K528R and Q730E, were associated with reduced surface FHC expression by monocytes from patients with AS and HLA-B27-expressing APCs. ERAP1 silencing or inhibition in APCs downregulated HLA-B27 FHC surface expression, reduced IL-2 production by KIR3DL2CD3ε-reporter cells and suppressed the Th17 expansion and IL-17A secretion by AS CD4+ T cells. ERAP1 inhibition of AS PBMCs reduced HLA class I FHC surface expression by monocytes and B cells, and suppressed Th17 expansion. Conclusions ERAP1 activity determines surface expression of HLA-B27 FHCs and potentially promotes Th17 responses in AS through binding of HLA-B27 FHCs to KIR3DL2. Our data suggest that ERAP1 inhibition has potential for AS treatment. PMID:26130142

Silencing of grapevine pectate lyase-like genes VvPLL2 and VvPLL3 confers resistance against Erysiphe necator and differentially modulates gene expression

USDA-ARS?s Scientific Manuscript database

Broad-spectrum resistance against powdery mildew (PM) has been reported by silencing susceptibility genes in the model plant Arabidopsis. Here we used artificial microRNA constructs in PM-susceptible Vitis vinifera cv. Chardonnay to stably silence two pectate lyase-like orthologs (VvPLL2 and VvPLL3)...

Riding in silence: a little snowboarding, a lot of small RNAs

PubMed Central

2010-01-01

The recent symposium, RNA silencing: Mechanism, Biology and Applications, organized by Phillip D. Zamore (University of Massachusetts Medical School) and Beverly Davidson (University of Iowa), and held in Keystone, Colorado, brought together scientists working on diverse aspects of RNA silencing, a field that comprises a multitude of gene regulatory pathways guided by microRNAs, small interfering RNAs and PIWI-interacting RNAs. PMID:20230614

Conversion of a Replication Origin to a Silencer through a Pathway Shared by a Forkhead Transcription Factor and an S Phase Cyclin

PubMed Central

Casey, Laurieann; Patterson, Erin E.; Müller, Ulrika

2008-01-01

Silencing of the mating-type locus HMR in Saccharomyces cerevisiae requires DNA elements called silencers. To establish HMR silencing, the origin recognition complex binds the HMR-E silencer and recruits the silent information regulator (Sir)1 protein. Sir1 in turn helps establish silencing by stabilizing binding of the other Sir proteins, Sir2–4. However, silencing is semistable even in sir1Δ cells, indicating that SIR1-independent establishment mechanisms exist. Furthermore, the requirement for SIR1 in silencing a sensitized version of HMR can be bypassed by high-copy expression of FKH1 (FKH1hc), a conserved forkhead transcription factor, or by deletion of the S phase cyclin CLB5 (clb5Δ). FKH1hc caused only a modest increase in Fkh1 levels but effectively reestablished Sir2–4 chromatin at HMR as determined by Sir3-directed chromatin immunoprecipitation. In addition, FKH1hc prolonged the cell cycle in a manner distinct from deletion of its close paralogue FKH2, and it created a cell cycle phenotype more reminiscent to that caused by a clb5Δ. Unexpectedly, and in contrast to SIR1, both FKH1hc and clb5Δ established silencing at HMR using the replication origins, ARS1 or ARSH4, as complete substitutes for HMR-E (HMRΔE::ARS). HMRΔE::ARS1 was a robust origin in CLB5 cells. However, initiation by HMRΔE::ARS1 was reduced by clb5Δ or FKH1hc, whereas ARS1 at its native locus was unaffected. The CLB5-sensitivity of HMRΔE::ARS1 did not result from formation of Sir2–4 chromatin because sir2Δ did not rescue origin firing in clb5Δ cells. These and other data supported a model in which FKH1 and CLB5 modulated Sir2–4 chromatin and late-origin firing through opposing regulation of a common pathway. PMID:18045995

Interspecific RNA Interference of SHOOT MERISTEMLESS-Like Disrupts Cuscuta pentagona Plant Parasitism[C][W][OA

PubMed Central

Alakonya, Amos; Kumar, Ravi; Koenig, Daniel; Kimura, Seisuke; Townsley, Brad; Runo, Steven; Garces, Helena M.; Kang, Julie; Yanez, Andrea; David-Schwartz, Rakefet; Machuka, Jesse; Sinha, Neelima

2012-01-01

Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA–mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection. PMID:22822208

Interspecific RNA interference of SHOOT MERISTEMLESS-like disrupts Cuscuta pentagona plant parasitism.

PubMed

Alakonya, Amos; Kumar, Ravi; Koenig, Daniel; Kimura, Seisuke; Townsley, Brad; Runo, Steven; Garces, Helena M; Kang, Julie; Yanez, Andrea; David-Schwartz, Rakefet; Machuka, Jesse; Sinha, Neelima

2012-07-01

Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA-mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.

CpG island methylator phenotype in adenocarcinomas from the digestive tract: Methods, conclusions, and controversies

PubMed Central

Sánchez-Vega, Francisco; Gotea, Valer; Chen, Yun-Ching; Elnitski, Laura

2017-01-01

Over the last two decades, cancer-related alterations in DNA methylation that regulate transcription have been reported for a variety of tumors of the gastrointestinal tract. Due to its relevance for translational research, great emphasis has been placed on the analysis and molecular characterization of the CpG island methylator phenotype (CIMP), defined as widespread hypermethylation of CpG islands in clinically distinct subsets of cancer patients. Here, we present an overview of previous work in this field and also explore some open questions using cross-platform data for esophageal, gastric, and colorectal adenocarcinomas from The Cancer Genome Atlas. We provide a data-driven, pan-gastrointestinal stratification of individual samples based on CIMP status and we investigate correlations with oncogenic alterations, including somatic mutations and epigenetic silencing of tumor suppressor genes. Besides known events in CIMP such as BRAF V600E mutation, CDKN2A silencing or MLH1 inactivation, we discuss the potential role of emerging actors such as Wnt pathway deregulation through truncating mutations in RNF43 and epigenetic silencing of WIF1. Our results highlight the existence of molecular similarities that are superimposed over a larger backbone of tissue-specific features and can be exploited to reduce heterogeneity of response in clinical trials. PMID:28344746

Key enzymes and proteins of crop insects as candidate for RNAi based gene silencing

PubMed Central

Kola, Vijaya Sudhakara Rao; Renuka, P.; Madhav, Maganti Sheshu; Mangrauthia, Satendra K.

2015-01-01

RNA interference (RNAi) is a mechanism of homology dependent gene silencing present in plants and animals. It operates through 21–24 nucleotides small RNAs which are processed through a set of core enzymatic machinery that involves Dicer and Argonaute proteins. In recent past, the technology has been well appreciated toward the control of plant pathogens and insects through suppression of key genes/proteins of infecting organisms. The genes encoding key enzymes/proteins with the great potential for developing an effective insect control by RNAi approach are actylcholinesterase, cytochrome P450 enzymes, amino peptidase N, allatostatin, allatotropin, tryptophan oxygenase, arginine kinase, vacuolar ATPase, chitin synthase, glutathione-S-transferase, catalase, trehalose phosphate synthase, vitellogenin, hydroxy-3-methylglutaryl coenzyme A reductase, and hormone receptor genes. Through various studies, it is demonstrated that RNAi is a reliable molecular tool which offers great promises in meeting the challenges imposed by crop insects with careful selection of key enzymes/proteins. Utilization of RNAi tool to target some of these key proteins of crop insects through various approaches is described here. The major challenges of RNAi based insect control such as identifying potential targets, delivery methods of silencing trigger, off target effects, and complexity of insect biology are very well illustrated. Further, required efforts to address these challenges are also discussed. PMID:25954206

Ferritin heavy chain is a negative regulator of ovarian cancer stem cell expansion and epithelial to mesenchymal transition

PubMed Central

Pisanu, Maria Elena; Faniello, Maria Concetta; Jakopin, Žiga; Chiarella, Emanuela; Giovannone, Emilia Dora; Mancini, Rita; Ciliberto, Gennaro

2016-01-01

Objectives Ferritin is the major intracellular iron storage protein essential for maintaining the cellular redox status. In recent years ferritin heavy chain (FHC) has been shown to be involved also in the control of cancer cell growth. Analysis of public microarray databases in ovarian cancer revealed a correlation between low FHC expression levels and shorter survival. To better understand the role of FHC in cancer, we have silenced the FHC gene in SKOV3 cells. Results FHC-KO significantly enhanced cell viability and induced a more aggressive behaviour. FHC-silenced cells showed increased ability to form 3D spheroids and enhanced expression of NANOG, OCT4, ALDH and Vimentin. These features were accompanied by augmented expression of SCD1, a major lipid metabolism enzyme. FHC apparently orchestrates part of these changes by regulating a network of miRNAs. Methods FHC-silenced and control shScr SKOV3 cells were monitored for changes in proliferation, migration, ability to propagate as 3D spheroids and for the expression of stem cell and epithelial-to-mesenchymal-transition (EMT) markers. The expression of three miRNAs relevant to spheroid formation or EMT was assessed by q-PCR. Conclusions In this paper we uncover a new function of FHC in the control of cancer stem cells. PMID:27566559

Investigating Gene Function in Cereal Rust Fungi by Plant-Mediated Virus-Induced Gene Silencing.

PubMed

Panwar, Vinay; Bakkeren, Guus

2017-01-01

Cereal rust fungi are destructive pathogens, threatening grain production worldwide. Targeted breeding for resistance utilizing host resistance genes has been effective. However, breakdown of resistance occurs frequently and continued efforts are needed to understand how these fungi overcome resistance and to expand the range of available resistance genes. Whole genome sequencing, transcriptomic and proteomic studies followed by genome-wide computational and comparative analyses have identified large repertoire of genes in rust fungi among which are candidates predicted to code for pathogenicity and virulence factors. Some of these genes represent defence triggering avirulence effectors. However, functions of most genes still needs to be assessed to understand the biology of these obligate biotrophic pathogens. Since genetic manipulations such as gene deletion and genetic transformation are not yet feasible in rust fungi, performing functional gene studies is challenging. Recently, Host-induced gene silencing (HIGS) has emerged as a useful tool to characterize gene function in rust fungi while infecting and growing in host plants. We utilized Barley stripe mosaic virus-mediated virus induced gene silencing (BSMV-VIGS) to induce HIGS of candidate rust fungal genes in the wheat host to determine their role in plant-fungal interactions. Here, we describe the methods for using BSMV-VIGS in wheat for functional genomics study in cereal rust fungi.

Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance

PubMed Central

Jeyapalan, J N; Noor, D A Mohamed; Lee, S-H; Tan, C L; Appleby, V A; Kilday, J P; Palmer, R D; Schwalbe, E C; Clifford, S C; Walker, D A; Murray, M J; Coleman, N; Nicholson, J C; Scotting, P J

2011-01-01

Background: Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. Methods: A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. Results: Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a ‘methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. Conclusion: Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy. PMID:21712824

Combined lentiviral and RNAi technologies for the delivery and permanent silencing of the hsp25 gene.

PubMed

Kaur, Punit; Nagaraja, Ganachari M; Asea, Alexzander

2011-01-01

Elevated heat shock protein 27 (Hsp27) expression has been found in a number of tumors, including breast, prostate, gastric, uterine, ovarian, head and neck, and tumor arising from the nervous system and urinary system, and determined to be a predictor of poor clinical outcome. Although the mechanism of action of Hsp27 has been well documented, there are currently no available inhibitors of Hsp27 in clinical trials. RNA interference (RNAi) has the potential to offer more specificity and flexibility than traditional drugs to silence gene expression. Not surprisingly, RNAi has become a major focus for biotechnology and pharmaceutical companies, which are now in the early stages of developing RNAi therapeutics, mostly based on short interfering RNA (siRNAs), to target viral infection, cancer, hypercholesterolemia, cardiovascular disease, macular degeneration, and neurodegenerative diseases. However, the critical issues associated with RNAi as a therapeutic are delivery, specificity, and stability of the RNAi reagents. To date, the delivery is currently considered the biggest hurdle, as the introduction of siRNAs systemically into body fluids can result in their degradation, off-target effects, and immune detection. In this chapter, we discuss a method of combined lentiviral and RNAi-based technology for the delivery and permanent silencing of the hsp25 gene.

A Novel High Throughput Assay for Anthelmintic Drug Screening and Resistance Diagnosis by Real-Time Monitoring of Parasite Motility

PubMed Central

Smout, Michael J.; Kotze, Andrew C.; McCarthy, James S.; Loukas, Alex

2010-01-01

Background Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. Methodology/Principal Findings Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus. Conclusions/Significance Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing. PMID:21103363

Experience and Perspectives of Caregivers of Spouse with Mild Cognitive Impairment

PubMed Central

Lu, Yueh-Feng Yvonne; Haase, Joan E.

2010-01-01

The purpose of this paper is to describe commonalities of the lived experience of being a spouse caregiver of a person with mild cognitive impairment (MCI). The Colaizzi method of empirical phenomenology was used for inter-viewing and analyzing data obtained from 10 spouse caregivers of persons with MCI. Four major themes were found and labeled: (a) Putting the Puzzle Pieces Together-There Really is Something Wrong; (b) A Downward Spiral into a World of Silence; (c) Consequences to Caregivers of Living in a World of Silence; (d) Taking Charge of Care. The findings of this study provided rich data to guide interventions to help caregivers to improve their awareness of MCI, gain new information and skills to deal more effectively with and adjust to the caregiving of their spouse with MCI over the long-term. PMID:19689238

Analysis of Variability in HIV-1 Subtype A Strains in Russia Suggests a Combination of Deep Sequencing and Multitarget RNA Interference for Silencing of the Virus.

PubMed

Kretova, Olga V; Chechetkin, Vladimir R; Fedoseeva, Daria M; Kravatsky, Yuri V; Sosin, Dmitri V; Alembekov, Ildar R; Gorbacheva, Maria A; Gashnikova, Natalya M; Tchurikov, Nickolai A

2017-02-01

Any method for silencing the activity of the HIV-1 retrovirus should tackle the extremely high variability of HIV-1 sequences and mutational escape. We studied sequence variability in the vicinity of selected RNA interference (RNAi) targets from isolates of HIV-1 subtype A in Russia, and we propose that using artificial RNAi is a potential alternative to traditional antiretroviral therapy. We prove that using multiple RNAi targets overcomes the variability in HIV-1 isolates. The optimal number of targets critically depends on the conservation of the target sequences. The total number of targets that are conserved with a probability of 0.7-0.8 should exceed at least 2. Combining deep sequencing and multitarget RNAi may provide an efficient approach to cure HIV/AIDS.

A role for the nucleoporin Nup170p in chromatin structure and gene silencing

PubMed Central

Van de Vosse, David W.; Wan, Yakun; Lapetina, Diego L.; Chen, Wei-Ming; Chiang, Jung-Hsien; Aitchison, John D.; Wozniak, Richard W.

2013-01-01

Embedded in the nuclear envelope, nuclear pore complexes (NPCs) not only regulate nuclear transport, but also interface with transcriptionally active euchromatin, largely silenced heterochromatin, as well as the boundaries between these regions. It is unclear what functional role NPCs play in establishing or maintaining these distinct chromatin domains. We report that the yeast NPC protein Nup170p interacts with regions of the genome containing ribosomal protein and subtelomeric genes. Here, it functions in nucleosome positioning and as a repressor of transcription. We show that the role of Nup170p in subtelomeric gene silencing is linked to its association with the RSC chromatin-remodeling complex and the silencing factor Sir4p, and that the binding of Nup170p and Sir4p to subtelomeric chromatin is cooperative and necessary for the association of telomeres with the nuclear envelope. Our results establish the NPC as an active participant in silencing and the formation of peripheral heterochromatin. PMID:23452847

Cytorhabdovirus phosphoprotein shows RNA silencing suppressor activity in plants, but not in insect cells.

PubMed

Mann, Krin S; Johnson, Karyn N; Dietzgen, Ralf G

2015-02-01

RNA silencing in plants and insects provides an antiviral defense and as a countermeasure most viruses encode RNA silencing suppressors (RSS). For the family Rhabdoviridae, no detailed functional RSS studies have been reported in plant hosts and insect vectors. In agroinfiltrated Nicotiana benthamiana leaves we show for the first time for a cytorhabdovirus, lettuce necrotic yellows virus (LNYV), that one of the nucleocapsid core proteins, phosphoprotein (P) has relatively weak local RSS activity and delays systemic silencing of a GFP reporter. Analysis of GFP small RNAs indicated that the P protein did not prevent siRNA accumulation. To explore RSS activity in insects, we used a Flock House virus replicon system in Drosophila S2 cells. In contrast to the plant host, LNYV P protein did not exhibit RSS activity in the insect cells. Taken together our results suggest that P protein may target plant-specific components of RNA silencing post siRNA biogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

Sharp wave ripples during learning stabilize hippocampal spatial map

PubMed Central

Roux, Lisa; Hu, Bo; Eichler, Ronny; Stark, Eran; Buzsáki, György

2017-01-01

Cognitive representation of the environment requires a stable hippocampal map but the mechanisms maintaining map representation are unknown. Because sharp wave-ripples (SPW-R) orchestrate both retrospective and prospective spatial information, we hypothesized that disrupting neuronal activity during SPW-Rs affects spatial representation. Mice learned daily a new set of three goal locations on a multi-well maze. We used closed-loop SPW-R detection at goal locations to trigger optogenetic silencing of a subset of CA1 pyramidal neurons. Control place cells (non-silenced or silenced outside SPW-Rs) largely maintained the location of their place fields after learning and showed increased spatial information content. In contrast, the place fields of SPW-R-silenced place cells remapped, and their spatial information remained unaltered. SPW-R silencing did not impact the firing rates or the proportions of place cells. These results suggest that interference with SPW-R-associated activity during learning prevents the stabilization and refinement of the hippocampal map. PMID:28394323

Two classes of silencing RNAs move between C. elegans tissues

PubMed Central

Jose, Antony M; Garcia, Giancarlo A; Hunter, Craig P

2011-01-01

Summary Organism-wide RNA interference (RNAi) is due to the transport of mobile silencing RNA throughout the organism but the identities of these mobile RNA species in animals are unknown. Here we present genetic evidence that both the initial double-stranded RNA (dsRNA), which triggers RNAi, and at least one dsRNA intermediate produced during RNAi can act as or generate mobile silencing RNA in Caenorhabditis elegans. This dsRNA intermediate requires the long dsRNA-binding protein RDE-4, the endonuclease DCR-1, which cleaves long dsRNA into double-stranded short-interfering RNA (ds-siRNA), and the putative nucleotidyltransferase MUT-2 (RDE-3). However, single-stranded siRNA and downstream secondary siRNA produced upon amplification by the RNA-dependent RNA Polymerase RRF-1 do not generate mobile silencing RNA. Restricting inter-tissue transport to long dsRNA and directly processed siRNA intermediates rather than amplified siRNA may serve to modulate the extent of systemic silencing in proportion to available dsRNA. PMID:21984186

Organizational Silence and Hidden Threats to Patient Safety

PubMed Central

Henriksen, Kerm; Dayton, Elizabeth

2006-01-01

Organizational silence refers to a collective-level phenomenon of saying or doing very little in response to significant problems that face an organization. The paper focuses on some of the less obvious factors contributing to organizational silence that can serve as threats to patient safety. Converging areas of research from the cognitive, social, and organizational sciences and the study of sociotechnical systems help to identify some of the underlying factors that serve to shape and sustain organizational silence. These factors have been organized under three levels of analysis: (1) individual factors, including the availability heuristic, self-serving bias, and the status quo trap; (2) social factors, including conformity, diffusion of responsibility, and microclimates of distrust; and (3) organizational factors, including unchallenged beliefs, the good provider fallacy, and neglect of the interdependencies. Finally, a new role for health care leaders and managers is envisioned. It is one that places high value on understanding system complexity and does not take comfort in organizational silence. PMID:16898978

Organizational silence and hidden threats to patient safety.

PubMed

Henriksen, Kerm; Dayton, Elizabeth

2006-08-01

Organizational silence refers to a collective-level phenomenon of saying or doing very little in response to significant problems that face an organization. The paper focuses on some of the less obvious factors contributing to organizational silence that can serve as threats to patient safety. Converging areas of research from the cognitive, social, and organizational sciences and the study of sociotechnical systems help to identify some of the underlying factors that serve to shape and sustain organizational silence. These factors have been organized under three levels of analysis: (1) individual factors, including the availability heuristic, self-serving bias, and the status quo trap; (2) social factors, including conformity, diffusion of responsibility, and microclimates of distrust; and (3) organizational factors, including unchallenged beliefs, the good provider fallacy, and neglect of the interdependencies. Finally, a new role for health care leaders and managers is envisioned. It is one that places high value on understanding system complexity and does not take comfort in organizational silence.

E(y)2/Sus1 is required for blocking PRE silencing by the Wari insulator in Drosophila melanogaster.

PubMed

Erokhin, Maksim; Parshikov, Alexander; Georgiev, Pavel; Chetverina, Darya

2010-06-01

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers to the spread of repressive chromatin. Recently, we have found an insulator, named Wari, located on the 3' side of the white gene. Here, we show that the previously identified 368-bp core of this insulator is sufficient for blocking Polycomb response element-mediated silencing. Although Wari does not contain binding sites for known insulator proteins, the E(y)2 and CP190 proteins bind to Wari as well as to the Su(Hw)-containing insulators in vivo. It may well be that these proteins are recruited to the insulator by as yet unidentified DNA-binding protein. Partial inactivation of E(y)2 in a weak e(y)2 ( u1 ) mutation impairs only the anti-silencing but not the enhancer-blocking activity of the Wari insulator. Thus, the E(y)2 protein in different Drosophila insulators serves to protect gene expression from silencing.

Music Enhances Autobiographical Memory in Mild Alzheimer's Disease

ERIC Educational Resources Information Center

El Haj, Mohamad; Postal, Virginie; Allain, Philippe

2012-01-01

Studies have shown that the "Four Seasons" music may enhance the autobiographical performance of Alzheimer's disease (AD) patients. We used a repeated measures design in which autobiographical recall of 12 mild AD patients was assessed using a free narrative method under three conditions: (a) in "Silence," (b) after being exposed to the opus "Four…

Leading Higher Education Professionals: Local, Connected, and Artful

ERIC Educational Resources Information Center

Bathurst, Ralph; van Gelderen, Marco

2014-01-01

In this paper, we explore the work life of a scholar new to academia in her methods of leading informally. We discuss five vignettes that offer moments of learning and reflection, including reducing performance anxiety, privileging financial accountability, the rise of the virtual world, the impact of silence, and the potential for confrontation…

Listening to the Voices: The Experiences of African American Female Student Athletes

ERIC Educational Resources Information Center

Bruening, Jennifer E.; Armstrong, Ketra L.; Pastore, Donna L.

2005-01-01

This study examined the sport participation patterns of 12 African American female collegiate student athletes using qualitative methods. Data were collected at a large midwestern university during the 1998-99 academic year. An emergent theme was the effect of silencing by the media, athletic administrators, coaches, and other student athletes on…

The Aesthetic and the Spiritual Attitude in Learning: Lessons from Simone Weil

ERIC Educational Resources Information Center

Caranfa, Angelo

2010-01-01

This essay attempts to set out the fundamental notions--attention, detachment, silence, solitude, prayer, and apprenticeship--of Weil's educational method, claiming that such a pedagogy expresses a vision of learning in which the moral, the aesthetic, and the spiritual life are harmoniously balanced. Simone Weil's approach to education is not only…

Emancipatory Education without Enlightenment? Thais, Americans, and the "Pedagogy of the Oppressed."

ERIC Educational Resources Information Center

Matzen, Richard N., Jr.

If the educational methods of Paulo Freire are imposed on Thais and other Asians, the outcome may not be the reinforcing of, but instead the losing of, their cultural identities. Freire reveals what are vital cultural assumptions for his pedagogy when defining "freedom, silence, confrontation, communication, and self" in "Pedagogy…

Gender Inequity in the Workshop: Methods Which Silence Women Writers.

ERIC Educational Resources Information Center

McCabe, Nancy

In a creative writing workshop of seven male and nine female graduate students, two female students became increasingly aware of a sexual bias. Though the male instructor made an effort to create a non-hierarchical, student-centered environment, informal hierarchies developed among students that granted the male instructor and the male students…

Identification of Potential Therapeutic Mechanisms for HIP1 Inhibition in Breast Cancer

DTIC Science & Technology

2006-05-01

repressing transcription when knocked down using a silencing RNA approach and activating tran- scription when overexpressed. We have also identified a...HIP1, huntingtin interacting protein 1; PSA, prostate-specific antigen; siRNA, silencing RNA . The online version of this article includes supplemental...We examined whether endogenous HIP1 was also required to sustain AR transcriptional activity by taking a silencing RNA (siRNA) approach to knockdown

A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants

PubMed Central

Binder, Andreas; Lambert, Jayne; Morbitzer, Robert; Popp, Claudia; Ott, Thomas; Lahaye, Thomas; Parniske, Martin

2014-01-01

The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct. PMID:24551083

Restriction of Retrotransposon Mobilization in Schizosaccharomyces pombe by Transcriptional Silencing and Higher-Order Chromatin Organization

PubMed Central

Murton, Heather E.; Grady, Patrick J. R.; Chan, Tsun Ho; Cam, Hugh P.; Whitehall, Simon K.

2016-01-01

Uncontrolled propagation of retrotransposons is potentially detrimental to host genome integrity. Therefore, cells have evolved surveillance mechanisms to restrict the mobility of these elements. In Schizosaccharomyces pombe the Tf2 LTR retrotransposons are transcriptionally silenced and are also clustered in the nucleus into structures termed Tf bodies. Here we describe the impact of silencing and clustering on the mobility of an endogenous Tf2 element. Deletion of genes such as set1+ (histone H3 lysine 4 methyltransferase) or abp1+ (CENP-B homolog) that both alleviate silencing and clustering, result in a corresponding increase in mobilization. Furthermore, expression of constitutively active Sre1, a transcriptional activator of Tf2 elements, also alleviates clustering and induces mobilization. In contrast, clustering is not disrupted by loss of the HIRA histone chaperone, despite high levels of expression, and in this background, mobilization frequency is only marginally increased. Thus, mutations that compromise transcriptional silencing but not Tf bodies are insufficient to drive mobilization. Furthermore, analyses of mutant alleles that separate the transcriptional repression and clustering functions of Set1 are consistent with control of Tf2 propagation via a combination of silencing and spatial organization. Our results indicate that host surveillance mechanisms operate at multiple levels to restrict Tf2 retrotransposon mobilization. PMID:27343236

[Silence pact from the perspective of caretakers of palliative care patients].

PubMed

Cejudo López, Ángela; López López, Begoña; Duarte Rodríguez, Miguel; Crespo Serván, María Pilar; Coronado Illescas, Concepción; de la Fuente Rodríguez, Carlota

2015-01-01

To determine why terminally-ill patients, family caregivers and health care providers make a pact of silence about the terminal status of the patient, and to identify the attitudes, experiences and opinions of family caregivers concerning the conspiracy of silence in palliative care. A qualitative phenomenological study based on an interpretive analysis, conducted in Primary Health Care, Seville, Spain. Study dimensions: knowledge of the diagnosis and prognosis of the condition; disclosure of information; reaction to information received, feelings and approach to death; information disclosed to the patient and reasons behind partial disclosure; communication between patients, families, and health care providers. Family caregivers of patients on palliative care suspicious about a pact of silence. Family caregivers hamper professional-patient communication; use of deceit to conceal the truth; suspicion that the patient knows the truth; the clinician conceals the truth; paternalist attitudes; feelings of sadness, grief, resignation, acceptance of the disease. The pact of silence has negative effects on coping with death, quality of life in the last days of life, and mourning. Communication between patients, health care providers, and families should be improved to prevent the pact of silence, and help patients cope with death. Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

Endomembrane-associated RSD-3 is important for RNAi induced by extracellular silencing RNA in both somatic and germ cells of Caenorhabditis elegans

PubMed Central

Imae, Rieko; Dejima, Katsufumi; Kage-Nakadai, Eriko; Arai, Hiroyuki; Mitani, Shohei

2016-01-01

RNA silencing signals in C. elegans spread among cells, leading to RNAi throughout the body. During systemic spread of RNAi, membrane trafficking is thought to play important roles. Here, we show that RNAi Spreading Defective-3 (rsd-3), which encodes a homolog of epsinR, a conserved ENTH (epsin N-terminal homology) domain protein, generally participates in cellular uptake of silencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only in germ cells, but we isolated several deletion alleles of rsd-3, and found that these mutants are defective in the spread of silencing RNA not only into germ cells but also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularly localized to the trans-Golgi network (TGN) and endosomes. Tissue-specific rescue experiments indicate that RSD-3 is required for importing silencing RNA into cells rather than exporting from cells. Structure/function analysis showed that the ENTH domain alone is sufficient, and membrane association of the ENTH domain is required, for RSD-3 function in systemic RNAi. Our results suggest that endomembrane trafficking through the TGN and endosomes generally plays an important role in cellular uptake of silencing RNA. PMID:27306325

Endomembrane-associated RSD-3 is important for RNAi induced by extracellular silencing RNA in both somatic and germ cells of Caenorhabditis elegans.

PubMed

Imae, Rieko; Dejima, Katsufumi; Kage-Nakadai, Eriko; Arai, Hiroyuki; Mitani, Shohei

2016-06-16

RNA silencing signals in C. elegans spread among cells, leading to RNAi throughout the body. During systemic spread of RNAi, membrane trafficking is thought to play important roles. Here, we show that RNAi Spreading Defective-3 (rsd-3), which encodes a homolog of epsinR, a conserved ENTH (epsin N-terminal homology) domain protein, generally participates in cellular uptake of silencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only in germ cells, but we isolated several deletion alleles of rsd-3, and found that these mutants are defective in the spread of silencing RNA not only into germ cells but also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularly localized to the trans-Golgi network (TGN) and endosomes. Tissue-specific rescue experiments indicate that RSD-3 is required for importing silencing RNA into cells rather than exporting from cells. Structure/function analysis showed that the ENTH domain alone is sufficient, and membrane association of the ENTH domain is required, for RSD-3 function in systemic RNAi. Our results suggest that endomembrane trafficking through the TGN and endosomes generally plays an important role in cellular uptake of silencing RNA.

Integration of promoters, inverted repeat sequences and proteomic data into a model for high silencing efficiency of coeliac disease related gliadins in bread wheat

PubMed Central

2013-01-01

Background Wheat gluten has unique nutritional and technological characteristics, but is also a major trigger of allergies and intolerances. One of the most severe diseases caused by gluten is coeliac disease. The peptides produced in the digestive tract by the incomplete digestion of gluten proteins trigger the disease. The majority of the epitopes responsible reside in the gliadin fraction of gluten. The location of the multiple gliadin genes in blocks has to date complicated their elimination by classical breeding techniques or by the use of biotechnological tools. As an approach to silence multiple gliadin genes we have produced 38 transgenic lines of bread wheat containing combinations of two endosperm-specific promoters and three different inverted repeat sequences to silence three fractions of gliadins by RNA interference. Results The effects of the RNA interference constructs on the content of the gluten proteins, total protein and starch, thousand seed weights and SDSS quality tests of flour were analyzed in these transgenic lines in two consecutive years. The characteristics of the inverted repeat sequences were the main factor that determined the efficiency of silencing. The promoter used had less influence on silencing, although a synergy in silencing efficiency was observed when the two promoters were used simultaneously. Genotype and the environment also influenced silencing efficiency. Conclusions We conclude that to obtain wheat lines with an optimum reduction of toxic gluten epitopes one needs to take into account the factors of inverted repeat sequences design, promoter choice and also the wheat background used. PMID:24044767

Cannabinoid Type 1 Receptors Transiently Silence Glutamatergic Nerve Terminals of Cultured Cerebellar Granule Cells

PubMed Central

Ramírez-Franco, Jorge; Bartolomé-Martín, David; Alonso, Beatris; Torres, Magdalena; Sánchez-Prieto, José

2014-01-01

Cannabinoid receptors are the most abundant G protein-coupled receptors in the brain and they mediate retrograde short-term inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at many excitatory synapses. The induction of presynaptically silent synapses is a means of modulating synaptic strength, which is important for synaptic plasticity. Persistent activation of cannabinoid type 1 receptors (CB1Rs) mutes GABAergic terminals, although it is unclear if CB1Rs can also induce silencing at glutamatergic synapses. Cerebellar granule cells were transfected with VGLUT1-pHluorin to visualise the exo-endocytotic cycle. We found that prolonged stimulation (10 min) of cannabinoid receptors with the agonist HU-210 induces the silencing of previously active synapses. However, the presynaptic silencing induced by HU-210 is transient as it reverses after 20 min. cAMP with forskolin prevented CB1R-induced synaptic silencing, via activation of the Exchange Protein directly Activated by cAMP (Epac). Furthermore, Epac activation accelerated awakening of already silent boutons. Electron microscopy revealed that silencing was associated with synaptic vesicle (SV) redistribution within the nerve terminal, which diminished the number of vesicles close to the active zone of the plasma membrane. Finally, by combining functional and immunocytochemical approaches, we observed a strong correlation between the release capacity of the nerve terminals and RIM1α protein content, but not that of Munc13-1 protein. These results suggest that prolonged stimulation of cannabinoid receptors can transiently silence glutamatergic nerve terminals. PMID:24533119

Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity.

PubMed

Huang, Chung-Hao; Hsiao, Weng-Rong; Huang, Ching-Wen; Chen, Kuan-Chun; Lin, Shih-Shun; Chen, Tsung-Chi; Raja, Joseph A J; Wu, Hui-Wen; Yeh, Shyi-Dong

2015-01-01

The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) ((109)KFTMHNQ(117)), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal β-sheet motif ((397)IYFL(400)) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.

Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

PubMed Central

Huang, Chung-Hao; Hsiao, Weng-Rong; Huang, Ching-Wen; Chen, Kuan-Chun; Lin, Shih-Shun; Chen, Tsung-Chi; Raja, Joseph A. J.; Wu, Hui-Wen; Yeh, Shyi-Dong

2015-01-01

The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) (109KFTMHNQ117), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal β-sheet motif (397IYFL400) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism. PMID:25993336

Optically measured NADH concentrations are unaffected by propofol induced EEG silence during transient cerebral hypoperfusion in anesthetized rabbits☆

PubMed Central

Wang, Mei; Agarwal, Sachin; Mayevsky, Avraham; Joshi, Shailendra

2014-01-01

The neuroprotective benefit of intra-operative anesthetics is widely described and routinely aimed to invoke electroencephalographic (EEG) silence in anticipation of transient cerebral ischemia. Previous rat survival studies have questioned an additional benefit from achieving EEG silence during transient global cerebral hypoperfusion. Surgical preparation on twelve New Zealand white rabbits under ketamine–propofol anesthesia, included placement of skull screws for bilateral EEG monitoring, skull shaving for laser Doppler probes, and a 5 mm diameter right temporal craniotomy for the NADH probe. Transient global cerebral hypoperfusion was achieved with bilateral internal carotid artery occlusion and pharmacologically induced systemic hypotension. All animals acted as controls, and had cerebral hypoperfusion under baseline propofol anesthesia with an active EEG. Thereafter, animals were randomized to receive bolus injection of intracarotid (3–5 mg) or intravenous (10–20 mg) 1% propofol to create EEG silence for 1–2 min. The data collected at baseline, peak hypoperfusion, and 5 and 10 min post hypoperfusion was analyzed by repeated measures ANOVA with post hoc Bonferroni–Dunn test. Eleven of the twelve rabbits completed the protocol. Hemodynamics and cerebral blood flow changes were comparable in all the animals. Compared to controls, the increase in NADH during ischemia was unaffected by EEG silence with either intravenous or intraarterial propofol. We failed to observe any significant additional attenuation of the elevation in NADH levels with propofol induced EEG silence during transient global cerebral hypoperfusion. This is consistent with previous rat survival studies showing that EEG silence was not required for full neuroprotective effects of pentothal anesthesia. PMID:21570061

CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene

PubMed Central

Liu, Yu-Chin; Chang, Pu-Yuan; Chao, Chuck C.-K.

2015-01-01

In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy. PMID:26384430

Silencing of E2F3 suppresses tumor growth of Her2+ breast cancer cells by restricting mitosis.

PubMed

Lee, Miyoung; Oprea-Ilies, Gabriela; Saavedra, Harold I

2015-11-10

The E2F transcriptional activators E2F1, E2F2 and E2F3a regulate many important cellular processes, including DNA replication, apoptosis and centrosome duplication. Previously, we demonstrated that silencing E2F1 or E2F3 suppresses centrosome amplification (CA) and chromosome instability (CIN) in Her2+ breast cancer cells without markedly altering proliferation. However, it is unknown whether and how silencing a single E2F activator, E2F3, affects malignancy of human breast cancer cells. Thus, we injected HCC1954 Her2+ breast cancer cells silenced for E2F3 into mammary fat pads of immunodeficient mice and demonstrated that loss of E2F3 retards tumor growth. Surprisingly, silencing of E2F3 led to significant reductions in mitotic indices relative to vector controls, while the percentage of cells undergoing S phase were not affected. Nek2 is a mitotic kinase commonly upregulated in breast cancers and a critical regulator of Cdk4- or E2F-mediated CA. In this report, we found that Nek2 overexpression rescued back the CA caused by silencing of shE2F3. However, the effects of Nek2 overexpression in affecting tumor growth rates of shE2F3 and shE2F3; GFP cells were inconclusive. Taken together, our results indicate that E2F3 silencing decreases mammary tumor growth by reducing percentage of cells undergoing mitosis.

Modeling Interactions between Transposable Elements and the Plant Epigenetic Response: A Surprising Reliance on Element Retention.

PubMed

Roessler, Kyria; Bousios, Alexandros; Meca, Esteban; Gaut, Brandon S

2018-03-01

Transposable elements (TEs) compose the majority of angiosperm DNA. Plants counteract TE activity by silencing them epigenetically. One form of epigenetic silencing requires 21-22 nt small interfering RNAs that act to degrade TE mRNA and may also trigger DNA methylation. DNA methylation is reinforced by a second mechanism, the RNA-dependent DNA methylation (RdDM) pathway. RdDM relies on 24 nt small interfering RNAs and ultimately establishes TEs in a quiescent state. These host factors interact at a systems level, but there have been no system level analyses of their interactions. Here, we define a deterministic model that represents the propagation of active TEs, aspects of the host response and the accumulation of silenced TEs. We describe general properties of the model and also fit it to biological data in order to explore two questions. The first is why two overlapping pathways are maintained, given that both are likely energetically expensive. Under our model, RdDM silenced TEs effectively even when the initiation of silencing was weak. This relationship implies that only a small amount of RNAi is needed to initiate TE silencing, but reinforcement by RdDM is necessary to efficiently counter TE propagation. Second, we investigated the reliance of the host response on rates of TE deletion. The model predicted that low levels of deletion lead to few active TEs, suggesting that silencing is most efficient when methylated TEs are retained in the genome, thereby providing one explanation for the large size of plant genomes.

Arabidopsis homologs of retinoblastoma-associated protein 46/48 associate with a histone deacetylase to act redundantly in chromatin silencing.

PubMed

Gu, Xiaofeng; Jiang, Danhua; Yang, Wannian; Jacob, Yannick; Michaels, Scott D; He, Yuehui

2011-11-01

RNA molecules such as small-interfering RNAs (siRNAs) and antisense RNAs (asRNAs) trigger chromatin silencing of target loci. In the model plant Arabidopsis, RNA-triggered chromatin silencing involves repressive histone modifications such as histone deacetylation, histone H3 lysine-9 methylation, and H3 lysine-27 monomethylation. Here, we report that two Arabidopsis homologs of the human histone-binding proteins Retinoblastoma-Associated Protein 46/48 (RbAp46/48), known as MSI4 (or FVE) and MSI5, function in partial redundancy in chromatin silencing of various loci targeted by siRNAs or asRNAs. We show that MSI5 acts in partial redundancy with FVE to silence FLOWERING LOCUS C (FLC), which is a crucial floral repressor subject to asRNA-mediated silencing, FLC homologs, and other loci including transposable and repetitive elements which are targets of siRNA-directed DNA Methylation (RdDM). Both FVE and MSI5 associate with HISTONE DEACETYLASE 6 (HDA6) to form complexes and directly interact with the target loci, leading to histone deacetylation and transcriptional silencing. In addition, these two genes function in de novo CHH (H = A, T, or C) methylation and maintenance of symmetric cytosine methylation (mainly CHG methylation) at endogenous RdDM target loci, and they are also required for establishment of cytosine methylation in the previously unmethylated sequences directed by the RdDM pathway. This reveals an important functional divergence of the plant RbAp46/48 relatives from animal counterparts.

Sound therapies for tinnitus management.

PubMed

Jastreboff, Margaret M

2007-01-01

Many people with bothersome (suffering) tinnitus notice that their tinnitus changes in different acoustical surroundings, it is more intrusive in silence and less profound in the sound enriched environments. This observation led to the development of treatment methods for tinnitus utilizing sound. Many of these methods are still under investigation in respect to their specific protocol and effectiveness and only some have been objectively evaluated in clinical trials. This chapter will review therapies for tinnitus using sound stimulation.

HSP27 is required for invasion and metastasis triggered by hepatocyte growth factor.

PubMed

Pavan, Simona; Musiani, Daniele; Torchiaro, Erica; Migliardi, Giorgia; Gai, Marta; Di Cunto, Ferdinando; Erriquez, Jessica; Olivero, Martina; Di Renzo, Maria Flavia

2014-03-15

The hepatocyte growth factor (HGF) also known as scatter factor activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27 kDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the remodeling of actin filaments induced by HGF and that motility in vitro depended on the p38MAPK-MK2 axis. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired the ability of circulating ovarian cancer cells to home to the lungs and to form experimental hematogenous metastases and the capability of cancer cells to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the proinvasive and prometastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy. © 2013 UICC.

Gene signature associated with benign neurofibroma transformation to malignant peripheral nerve sheath tumors

PubMed Central

Sorzano, Carlos O. S.; Pascual-Montano, Alberto; Carazo, Jose M.

2017-01-01

Benign neurofibromas, the main phenotypic manifestations of the rare neurological disorder neurofibromatosis type 1, degenerate to malignant tumors associated to poor prognosis in about 10% of patients. Despite efforts in the field of (epi)genomics, the lack of prognostic biomarkers with which to predict disease evolution frustrates the adoption of appropriate early therapeutic measures. To identify potential biomarkers of malignant neurofibroma transformation, we integrated four human experimental studies and one for mouse, using a gene score-based meta-analysis method, from which we obtained a score-ranked signature of 579 genes. Genes with the highest absolute scores were classified as promising disease biomarkers. By grouping genes with similar neurofibromatosis-related profiles, we derived panels of potential biomarkers. The addition of promoter methylation data to gene profiles indicated a panel of genes probably silenced by hypermethylation. To identify possible therapeutic treatments, we used the gene signature to query drug expression databases. Trichostatin A and other histone deacetylase inhibitors, as well as cantharidin and tamoxifen, were retrieved as putative therapeutic means to reverse the aberrant regulation that drives to malignant cell proliferation and metastasis. This in silico prediction corroborated reported experimental results that suggested the inclusion of these compounds in clinical trials. This experimental validation supported the suitability of the meta-analysis method used to integrate several sources of public genomic information, and the reliability of the gene signature associated to the malignant evolution of neurofibromas to generate working hypotheses for prognostic and drug-responsive biomarkers or therapeutic measures, thus showing the potential of this in silico approach for biomarker discovery. PMID:28542306

Targeting Quiescence in Prostate Cancer

DTIC Science & Technology

2017-10-01

CRISPR /Cas9 to generate cell lines where the reporters are integrated endogenously into 5 essential cell cycle genes to avoid epigenetic silencing. In...Developed and began an improved CRISPR /Cas9-based strategy to target reporters to endogenous gene loci in PC3 and C4-2B cells to prevent silencing...serum. An improved CRISPR /Cas9-based strategy to avoid cell cycle reporter silencing and incorporate a constitutive nuclear marker As described

The ethics of silence: Does conflict of interest explain employee silence?

PubMed

Anderson, James

2018-03-01

Employee silence constitutes a significant threat to organizational success. This article argues that silence is a by-product of a structural Conflict of Interest (COI) between employees and their employers. This argument turns on the claim, also defended here, that employees are in a privileged position vis-à-vis knowledge of their work and that leaders-whether they recognize it or not-are dependent on their employees for reliable information about the work they are doing. Employee voice, therefore, is an organizational necessity. It is also a moral achievement as it involves risking one's personal interests for the sake of the organization. Leaders must take steps to mitigate COI and encourage employee voice; this article provides several strategies for doing exactly that.

Soybean Homologs of MPK4 Negatively Regulate Defense Responses and Positively Regulate Growth and Development1[W][OA

PubMed Central

Liu, Jian-Zhong; Horstman, Heidi D.; Braun, Edward; Graham, Michelle A.; Zhang, Chunquan; Navarre, Duroy; Qiu, Wen-Li; Lee, Yeunsook; Nettleton, Dan; Hill, John H.; Whitham, Steven A.

2011-01-01

Mitogen-activated protein kinase (MAPK) cascades play important roles in disease resistance in model plant species such as Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum). However, the importance of MAPK signaling pathways in the disease resistance of crops is still largely uninvestigated. To better understand the role of MAPK signaling pathways in disease resistance in soybean (Glycine max), 13, nine, and 10 genes encoding distinct MAPKs, MAPKKs, and MAPKKKs, respectively, were silenced using virus-induced gene silencing mediated by Bean pod mottle virus. Among the plants silenced for various MAPKs, MAPKKs, and MAPKKKs, those in which GmMAPK4 homologs (GmMPK4s) were silenced displayed strong phenotypes including stunted stature and spontaneous cell death on the leaves and stems, the characteristic hallmarks of activated defense responses. Microarray analysis showed that genes involved in defense responses, such as those in salicylic acid (SA) signaling pathways, were significantly up-regulated in GmMPK4-silenced plants, whereas genes involved in growth and development, such as those in auxin signaling pathways and in cell cycle and proliferation, were significantly down-regulated. As expected, SA and hydrogen peroxide accumulation was significantly increased in GmMPK4-silenced plants. Accordingly, GmMPK4-silenced plants were more resistant to downy mildew and Soybean mosaic virus compared with vector control plants. Using bimolecular fluorescence complementation analysis and in vitro kinase assays, we determined that GmMKK1 and GmMKK2 might function upstream of GmMPK4. Taken together, our results indicate that GmMPK4s negatively regulate SA accumulation and defense response but positively regulate plant growth and development, and their functions are conserved across plant species. PMID:21878550

Recovery of Nicotiana benthamiana plants from a necrotic response induced by a nepovirus is associated with RNA silencing but not with reduced virus titer.

PubMed

Jovel, Juan; Walker, Melanie; Sanfaçon, Hélène

2007-11-01

Recovery of plants from virus-induced symptoms is often described as a consequence of RNA silencing, an antiviral defense mechanism. For example, recovery of Nicotiana clevelandii from a nepovirus (tomato black ring virus) is associated with a decreased viral RNA concentration and sequence-specific resistance to further virus infection. In this study, we have characterized the interaction of another nepovirus, tomato ringspot virus (ToRSV), with host defense responses during symptom induction and subsequent recovery. Early in infection, ToRSV induced a necrotic phenotype in Nicotiana benthamiana that showed characteristics typical of a hypersensitive response. RNA silencing was also activated during ToRSV infection, as evidenced by the presence of ToRSV-derived small interfering RNAs (siRNAs) that could direct degradation of ToRSV sequences introduced into sensor constructs. Surprisingly, disappearance of symptoms was not accompanied by a commensurate reduction in viral RNA levels. The stability of ToRSV RNA after recovery was also observed in N. clevelandii and Cucumis sativus and in N. benthamiana plants carrying a functional RNA-dependent RNA polymerase 1 ortholog from Medicago truncatula. In experiments with a reporter transgene (green fluorescent protein), ToRSV did not suppress the initiation or maintenance of transgene silencing, although the movement of the silencing signal was partially hindered. Our results demonstrate that although RNA silencing is active during recovery, reduction of virus titer is not required for the initiation of this phenotype. This scenario adds an unforeseen layer of complexity to the interaction of nepoviruses with the host RNA silencing machinery. The possibility that viral proteins, viral RNAs, and/or virus-derived siRNAs inactivate host defense responses is discussed.

Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells

PubMed Central

Molenaar, Jan J.; Ebus, Marli E.; Geerts, Dirk; Koster, Jan; Lamers, Fieke; Valentijn, Linda J.; Westerhout, Ellen M.; Versteeg, Rogier; Caron, Huib N.

2009-01-01

Two genes have a synthetically lethal relationship when the silencing or inhibiting of 1 gene is only lethal in the context of a mutation or activation of the second gene. This situation offers an attractive therapeutic strategy, as inhibition of such a gene will only trigger cell death in tumor cells with an activated second oncogene but spare normal cells without activation of the second oncogene. Here we present evidence that CDK2 is synthetically lethal to neuroblastoma cells with MYCN amplification and over-expression. Neuroblastomas are childhood tumors with an often lethal outcome. Twenty percent of the tumors have MYCN amplification, and these tumors are ultimately refractory to any therapy. Targeted silencing of CDK2 by 3 RNA interference techniques induced apoptosis in MYCN-amplified neuroblastoma cell lines, but not in MYCN single copy cells. Silencing of MYCN abrogated this apoptotic response in MYCN-amplified cells. Inversely, silencing of CDK2 in MYCN single copy cells did not trigger apoptosis, unless a MYCN transgene was activated. The MYCN induced apoptosis after CDK2 silencing was accompanied by nuclear stabilization of P53, and mRNA profiling showed up-regulation of P53 target genes. Silencing of P53 rescued the cells from MYCN-driven apoptosis. The synthetic lethality of CDK2 silencing in MYCN activated neuroblastoma cells can also be triggered by inhibition of CDK2 with a small molecule drug. Treatment of neuroblastoma cells with roscovitine, a CDK inhibitor, at clinically achievable concentrations induced MYCN-dependent apoptosis. The synthetically lethal relationship between CDK2 and MYCN indicates CDK2 inhibitors as potential MYCN-selective cancer therapeutics. PMID:19525400

Mycoviruses as Triggers and Targets of RNA Silencing in White Mold Fungus Sclerotinia sclerotiorum.

PubMed

Mochama, Pauline; Jadhav, Prajakta; Neupane, Achal; Lee Marzano, Shin-Yi

2018-04-22

This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum by infecting wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a DNA virus. Key silencing-related genes were disrupted to dissect the RNA silencing pathway. Specifically, dicer genes ( dcl-1, dcl-2 , and both dcl-1 / dcl-2 ) were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, and susceptibility to virus infections. Wild-type and mutant strains were transfected with a single-stranded RNA virus, SsHV2-L, and copies of a single-stranded DNA mycovirus, SsHADV-1, as a synthetic virus constructed in this study. Disruption of dcl-1 or dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum ; however, the double dicer mutant strain exhibited significantly slower growth. Furthermore, the Δdcl-1/dcl-2 double mutant, which was slow growing without virus infection, exhibited much more severe debilitation following virus infections including phenotypic changes such as slower growth, reduced pigmentation, and delayed sclerotial formation. These phenotypic changes were absent in the single mutants, Δdcl-1 and Δdcl-2 . Complementation of a single dicer in the double disruption mutant reversed viral susceptibility to the wild-type state. Virus-derived small RNAs were accumulated from virus-infected wild-type strains with strand bias towards the negative sense. The findings of these studies indicate that S. sclerotiorum has robust RNA silencing mechanisms that process both DNA and RNA mycoviruses and that, when both dicers are silenced, invasive nucleic acids can greatly debilitate the virulence of this fungus.

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis.

PubMed

Almeida Garcia, Rayssa; Lima Pepino Macedo, Leonardo; Cabral do Nascimento, Danila; Gillet, François-Xavier; Moreira-Pinto, Clidia Eduarda; Faheem, Muhammad; Moreschi Basso, Angelina Maria; Mattar Silva, Maria Cristina; Grossi-de-Sa, Maria Fatima

2017-01-01

RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis

PubMed Central

Almeida Garcia, Rayssa; Lima Pepino Macedo, Leonardo; Cabral do Nascimento, Danila; Gillet, François-Xavier; Moreira-Pinto, Clidia Eduarda; Faheem, Muhammad; Moreschi Basso, Angelina Maria; Mattar Silva, Maria Cristina

2017-01-01

RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests. PMID:29261729

A Genetic Network for Systemic RNA Silencing in Plants1[OPEN

PubMed Central

Chen, Weiwei; Zhang, Xian; Fan, Yaya; Li, Bin; Shi, Nongnong; Zhao, Mei; Qin, Cheng; Zheng, Qianqian; Zhang, Pengcheng; Wang, Huizhong; Jackson, Stephen; Cheng, Qi

2018-01-01

Non-cell autonomous RNA silencing can spread from cell to cell and over long distances in animals and plants. However, the genetic requirements and signals involved in plant mobile gene silencing are poorly understood. Here, we identified a DICER-LIKE2 (DCL2)-dependent mechanism for systemic spread of posttranscriptional RNA silencing, also known as posttranscriptional gene silencing (PTGS), in Nicotiana benthamiana. Using a suite of transgenic DCL RNAi lines coupled with a GFP reporter, we demonstrated that N. benthamiana DCL1, DCL2, DCL3, and DCL4 are required to produce microRNAs and 22, 24, and 21nt small interfering RNAs (siRNAs), respectively. All investigated siRNAs produced in local incipient cells were present at low levels in distal tissues. Inhibition of DCL2 expression reduced the spread of gene silencing, while suppression of DCL3 or DCL4 expression enhanced systemic PTGS. In contrast to DCL4 RNAi lines, DCL2-DCL4 double-RNAi lines developed systemic PTGS similar to that observed in DCL2 RNAi. We further showed that the 21 or 24 nt local siRNAs produced by DCL4 or DCL3 were not involved in long-distance gene silencing. Grafting experiments demonstrated that DCL2 was required in the scion to respond to the signal, but not in the rootstock to produce/send the signal. These results suggest a coordinated DCL genetic pathway in which DCL2 plays an essential role in systemic PTGS in N. benthamiana, while both DCL4 and DCL3 attenuate systemic PTGS. We discuss the potential role of 21, 22, and 24 nt siRNAs in systemic PTGS. PMID:29439213

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

PubMed

Yamanaka, Yuki; Winardhi, Ricksen S; Yamauchi, Erika; Nishiyama, So-Ichiro; Sowa, Yoshiyuki; Yan, Jie; Kawagishi, Ikuro; Ishihama, Akira; Yamamoto, Kaneyoshi

2018-06-15

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Recovery of Nicotiana benthamiana Plants from a Necrotic Response Induced by a Nepovirus Is Associated with RNA Silencing but Not with Reduced Virus Titer▿

PubMed Central

Jovel, Juan; Walker, Melanie; Sanfaçon, Hélène

2007-01-01

Recovery of plants from virus-induced symptoms is often described as a consequence of RNA silencing, an antiviral defense mechanism. For example, recovery of Nicotiana clevelandii from a nepovirus (tomato black ring virus) is associated with a decreased viral RNA concentration and sequence-specific resistance to further virus infection. In this study, we have characterized the interaction of another nepovirus, tomato ringspot virus (ToRSV), with host defense responses during symptom induction and subsequent recovery. Early in infection, ToRSV induced a necrotic phenotype in Nicotiana benthamiana that showed characteristics typical of a hypersensitive response. RNA silencing was also activated during ToRSV infection, as evidenced by the presence of ToRSV-derived small interfering RNAs (siRNAs) that could direct degradation of ToRSV sequences introduced into sensor constructs. Surprisingly, disappearance of symptoms was not accompanied by a commensurate reduction in viral RNA levels. The stability of ToRSV RNA after recovery was also observed in N. clevelandii and Cucumis sativus and in N. benthamiana plants carrying a functional RNA-dependent RNA polymerase 1 ortholog from Medicago truncatula. In experiments with a reporter transgene (green fluorescent protein), ToRSV did not suppress the initiation or maintenance of transgene silencing, although the movement of the silencing signal was partially hindered. Our results demonstrate that although RNA silencing is active during recovery, reduction of virus titer is not required for the initiation of this phenotype. This scenario adds an unforeseen layer of complexity to the interaction of nepoviruses with the host RNA silencing machinery. The possibility that viral proteins, viral RNAs, and/or virus-derived siRNAs inactivate host defense responses is discussed. PMID:17728227

Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines

PubMed Central

Noor, Dzul Azri Mohamed; Jeyapalan, Jennie N; Alhazmi, Safiah; Carr, Matthew; Squibb, Benjamin; Wallace, Claire; Tan, Christopher; Cusack, Martin; Hughes, Jaime; Reader, Tom; Shipley, Janet; Sheer, Denise; Scotting, Paul J

2016-01-01

Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome-wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours’ biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription–quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes. PMID:29263807

Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines.

PubMed

Noor, Dzul Azri Mohamed; Jeyapalan, Jennie N; Alhazmi, Safiah; Carr, Matthew; Squibb, Benjamin; Wallace, Claire; Tan, Christopher; Cusack, Martin; Hughes, Jaime; Reader, Tom; Shipley, Janet; Sheer, Denise; Scotting, Paul J

2016-01-01

Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome-wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours' biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription-quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes.

An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene.

PubMed

Cui, Hongguang; Wang, Aiming

2017-03-01

RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus-induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense-orientated target gene sequence of 100-200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV-based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E-knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector-mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Environmentally Induced Gene Silencing in Breast Cancer

DTIC Science & Technology

2007-07-01

fibrosarcoma cell line (HTD114), and a human breast cancer cell line (MCF7). The MLH1 promoter was only tested in the MCG7 cells. The control TRE-Luc...TRE- Luc MLH1 - Luc step in silencing is quite unstable. Nonetheless, cells that exhibit stable silencing of the HPRT construct can arise in...mechanism (i.e., gene repression). Finally, during the last year we have isolated or acquired functional promoters for the BRCA-1, MLH1 , and E

Temporal Progression of Visual Injury from Blast Exposure

DTIC Science & Technology

2013-09-01

included the design and construction of a silencer and dump tank. The final design is shown in Figure 8. A steel barrel lined with 2” of acoustic foam...was selected as the dump tank. It surrounds a rubber barrel lined with foam composite. The steel barrel is allowed to recoil on a cart, absorbing...test. Figure 8. (Left) Inner silencer assembly completed during Q4 of Year 1. (Right) Final silencer assembly with the outer steel drum

Developing Gene Silencing for the Study and Treatment of Dystonia

DTIC Science & Technology

2017-12-01

1 AWARD NUMBER: W81XWH-14-1-0282 TITLE: Developing Gene Silencing for the Study and Treatment of Dystonia PRINCIPAL INVESTIGATOR: Pedro...30/2014-9/29/2017 4. TITLE AND SUBTITLE Developing Gene Silencing for the Study and Treatment of Dystonia 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c...function. More importantly, we will check if no side effects or toxicity occurs. Successful completion of our studies will move us a step closer to

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263.

PubMed

Curry, Merril; Roberts-Thomson, Sarah J; Monteith, Gregory R

2016-09-30

PMCA2 overexpression in some breast cancers suggests that this calcium pump isoform may play a role in breast pathophysiology. To investigate PMCA2 as a potential drug target for breast cancer therapy, we assessed the functional consequence of PMCA2 silencing on cell death pathways and calcium signals in the basal-like MDA-MB-231 breast cancer cell line. Silencing PMCA2 expression alone has no effect on MDA-MB-231 cell viability, however, PMCA2 silencing promotes calcium-induced cell death initiated with the calcium ionophore ionomycin. Assessment of cytoplasmic calcium responses generated with various agents including ionomycin demonstrates that in MDA-MB-231 cells, PMCA2 does not play a major role in shaping global calcium signals. We also examined the ability of PMCA2 silencing to modulate caspase-dependent cell death triggered by a Bcl-2 inhibitor that is in clinical development for the treatment of various cancers, ABT-263 (Navitoclax). Despite the lack of effect on global calcium responses, PMCA2 silencing augmented Bcl-2 inhibitor (ABT-263)-mediated MDA-MB-231 breast cancer cell death. These studies provide evidence that PMCA2 inhibitors could sensitize PMCA2-positive breast cancers to cell death initiators that work through mechanisms involving the Bcl-2 survival pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Lettuce chlorosis virus P23 Suppresses RNA Silencing and Induces Local Necrosis with Increased Severity at Raised Temperatures.

PubMed

Kubota, Kenji; Ng, James C K

2016-06-01

RNA silencing functions as an antivirus defense strategy in plants, one that plant viruses counter by producing viral suppressors of RNA silencing (VSRs). VSRs have been identified in three members of the genus Crinivirus but they do not all share identical suppression mechanisms. Here, we used Agrobacterium co-infiltration assays to investigate the suppressor activity of proteins encoded by Lettuce chlorosis virus (LCV). Of 7 LCV proteins (1b, P23, HSP70 homolog, P60, CP, CPm, and P27) tested for the suppression of silencing of green fluorescent protein (GFP) expression in wild-type Nicotiana benthamiana plants, only P23 suppressed the onset of local silencing. Small-interfering (si)RNA accumulation was reduced in leaves co-infiltrated with P23, suggesting that P23 inhibited the accumulation or enhanced the degradation of siRNA. P23 also inhibited the cell-to-cell and systemic movement of RNA silencing in GFP-expressing transgenic N. benthamiana plants. Expression of P23 via agroinfiltration of N. benthamiana leaves induced local necrosis that increased in severity at elevated temperatures, a novelty given that a direct temperature effect on necrosis severity has not been reported for the other crinivirus VSRs. These results further affirm the sophistication of crinivirus VSRs in mediating the evasion of host's antiviral defenses and in symptom modulation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong Junmei; Wei Na; Chalk, Alistair

RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5' end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests thatmore » the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5' end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.« less

Development of an Efficient Virus Induced Gene Silencing Strategy in the Non-Model Wild Ginger-Zingiber zerumbet and Investigation of Associated Proteome Changes

PubMed Central

Mahadevan, Chidambareswaren; Jaleel, Abdul; Deb, Lokesh; Thomas, George; Sakuntala, Manjula

2015-01-01

Zingiber zerumbet (Zingiberaceae) is a wild, tropical medicinal herb that shows a high degree of resistance to diseases affecting cultivated ginger. Barley stripe mosaic virus (BSMV) silencing vectors containing an endogenous phytoene desaturase (PDS) gene fragment were agroinfiltrated into young leaves of Z. zerumbet under controlled growth conditions to effect virus-induced gene silencing (VIGS). Infiltrated leaves as well as newly emerged leaves and tillers showed visual signs of PDS silencing after 30 days. Replication and systemic movement of the viral vectors in silenced plants were confirmed by RT-PCR. Real-time quantitative PCR analysis verified significant down-regulation of PDS transcripts in the silenced tissues. Label-free proteomic analysis was conducted in leaves with established PDS transcript down regulation and buffer-infiltrated (mock) leaves. A total of 474 proteins were obtained, which were up-regulated, down-regulated or modulated de novo during VIGS. Most of these proteins were localized to the chloroplast, as revealed by UniprotKB analysis, and among the up-regulated proteins there were abiotic stress responsive, photosynthetic, metabolic and membrane proteins. Moreover, the demonstration of viral proteins together with host proteins proved successful viral infection. We report for the first time the establishment of a high-throughput gene functional analysis platform using BSMV-mediated VIGS in Z. zerumbet, as well as proteomic changes associated with VIGS. PMID:25918840

Silencing the Girdin gene enhances radio-sensitivity of hepatocellular carcinoma via suppression of glycolytic metabolism.

PubMed

Yu, Li; Sun, Yifan; Li, Jingjing; Wang, Yan; Zhu, Yuxing; Shi, Yong; Fan, Xiaojun; Zhou, Jianda; Bao, Ying; Xiao, Jie; Cao, Ke; Cao, Peiguo

2017-08-15

Radiotherapy has been used increasingly to treat primary hepatocellular carcinoma. Clinically, the main cause of radiotherapy failure is cellular radioresistance, conferred via glycolytic metabolism. Our previous study demonstrated that Girdin is upregulated in primary hepatocellular carcinoma and promotes the invasion and metastasis of tumor cells. However, whether Girdin underlies the radio-sensitivity of hepatocellular carcinoma remains unclear. A short hairpin RNA (shRNA) was used to silence CCDC88A (encoding Girdin), and real-time PCR was performed to determine CCDC88A mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of Girdin silencing on cellular radiosensitivity. Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the signaling pathway downstream of Girdin. Finally, animal experiments were performed to demonstrate the effect of CCDC88A silencing on the radiosensitivity of hepatoma in vivo. shRNA-induced Girdin silencing suppressed glycolysis and enhanced the radio-sensitivity of hepatic cell lines, HepG2 and Huh-7. Furthermore, silencing of Girdin inhibited the PI3K/AKT/HIF-1α signaling pathway, which is a central regulator of glycolysis. Girdin can regulate glycolysis in hepatocellular carcinoma cells through the PI3K/AKT/HIF-1α signaling pathway, which decreases the sensitivity of tumor cells to radiotherapy.

Focusing on RISC assembly in mammalian cells.

PubMed

Hong, Junmei; Wei, Na; Chalk, Alistair; Wang, Jue; Song, Yutong; Yi, Fan; Qiao, Ren-Ping; Sonnhammer, Erik L L; Wahlestedt, Claes; Liang, Zicai; Du, Quan

2008-04-11

RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5' end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5' end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.

Shine-Dalgarno sequence enhances the efficiency of lacZ repression by artificial anti-lac antisense RNAs in Escherichia coli.

PubMed

Stefan, Alessandra; Schwarz, Flavio; Bressanin, Daniela; Hochkoeppler, Alejandro

2010-11-01

Silencing of the lacZ gene in Escherichia coli was attempted by means of the expression of antisense RNAs (asRNAs) in vivo. A short fragment of lacZ was cloned into the pBAD expression vector, in reverse orientation, using the EcoRI and PstI restriction sites. This construct (pBAD-Zcal1) was used to transform E. coli cells, and the antisense transcription was induced simply by adding arabinose to the culture medium. We demonstrated that the Zcal1 asRNA effectively silenced lacZ using β-galactosidase activity determinations, SDS-PAGE, and Western blotting. Because the concentration of the lac mRNA was always high in cells that expressed Zcal1, we hypothesize that this antisense acts by inhibiting messenger translation. Similar analyses, performed with a series of site-specific Zcal1 mutants, showed that the Shine-Dalgarno sequence, which is conferred by the pBAD vector, is an essential requisite for silencing competence. Indeed, the presence of the intact Shine-Dalgarno sequence positively affects asRNA stability and, hence, silencing effectiveness. Our observations will contribute to the understanding of the main determinants of silencing as exerted by asRNAs as well as provide useful support for the design of robust and efficient prokaryotic gene silencers. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Local gene silencing in plants via synthetic dsRNA and carrier peptide.

PubMed

Numata, Keiji; Ohtani, Misato; Yoshizumi, Takeshi; Demura, Taku; Kodama, Yutaka

2014-10-01

Quick and facile transient RNA interference (RNAi) is one of the most valuable plant biotechnologies for analysing plant gene functions. To establish a novel double-strand RNA (dsRNA) delivery system for plants, we developed an ionic complex of synthetic dsRNA with a carrier peptide in which a cell-penetrating peptide is fused with a polycation sequence as a gene carrier. The dsRNA-peptide complex is 100-300 nm in diameter and positively charged. Infiltration of the complex into intact leaf cells of Arabidopsis thaliana successfully induced rapid and efficient down-regulation of exogenous and endogenous genes such as yellow fluorescent protein and chalcone synthase. The present method realizes quick and local gene silencing in specific tissues and/or organs in plants. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Virus-induced gene silencing offers a functional genomics platform for studying plant cell wall formation.

PubMed

Zhu, Xiaohong; Pattathil, Sivakumar; Mazumder, Koushik; Brehm, Amanda; Hahn, Michael G; Dinesh-Kumar, S P; Joshi, Chandrashekhar P

2010-09-01

Virus-induced gene silencing (VIGS) is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus (TRV)-based VIGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post-VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VIGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.

Oxime ether lipids containing hydroxylated head groups are more superior siRNA delivery agents than their nonhydroxylated counterparts

PubMed Central

Gupta, Kshitij; Mattingly, Stephanie J; Knipp, Ralph J; Afonin, Kirill A; Viard, Mathias; Bergman, Joseph T; Stepler, Marissa; Nantz, Michael H; Puri, Anu; Shapiro, Bruce A

2015-01-01

Aim: To evaluate the structure–activity relationship of oxime ether lipids (OELs) containing modifications in the hydrophobic domains (chain length, degree of unsaturation) and hydrophilic head groups (polar domain hydroxyl groups) toward complex formation with siRNA molecules and siRNA delivery efficiency of resulting complexes to a human breast cancer cell line (MDA-MB-231). Materials & methods: Ability of lipoplex formation between oxime ether lipids with nucleic acids were examined using biophysical techniques. The potential of OELs to deliver nucleic acids and silence green fluorescent protein (GFP) gene was analyzed using MDA-MB-231 and MDA-MB-231/GFP cells, respectively. Results & conclusion: Introduction of hydroxyl groups to the polar domain of the OELs and unsaturation into the hydrophobic domain favor higher transfection and gene silencing in a cell culture system. PMID:26107486

A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics.

PubMed

Alterman, Julia F; Coles, Andrew H; Hall, Lauren M; Aronin, Neil; Khvorova, Anastasia; Didiot, Marie-Cécile

2017-08-20

Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo . We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, and accurately quantify the mRNA silencing of hundreds of hsiRNAs, we use the QuantiGene 2.0 quantitative gene expression assay. This high-throughput, 96-well plate-based assay can quantify mRNA levels directly from sample lysate. Here, we describe a method to prepare short-term cultures of mouse primary cortical neurons in a 96-well plate format for high-throughput testing of oligonucleotide therapeutics. This method supports the testing of hsiRNA libraries and the identification of potential therapeutics within just two weeks. We detail methodologies of our high throughput assay workflow from primary neuron preparation to data analysis. This method can help identify oligonucleotide therapeutics for treatment of various neurological diseases.

Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis

PubMed Central

Blevins, Todd; Pontes, Olga; Pikaard, Craig S.; Meins, Frederick

2009-01-01

5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays. PMID:19529764

Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

PubMed

Hedil, Marcio; Kormelink, Richard

2016-07-23

The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins

PubMed Central

Hedil, Marcio; Kormelink, Richard

2016-01-01

The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far. PMID:27455310

Insights on ornithine decarboxylase silencing as a potential strategy for targeting retinoblastoma.

PubMed

Muthukumaran, Sivashanmugam; Bhuvanasundar, Renganathan; Umashankar, Vetrivel; Sulochana, K N

2018-02-01

Ornithine Decarboxylase (ODC) is a key enzyme involved in polyamine synthesis and is reported to be up regulated in several cancers. However, the effect of ODC gene silencing in retinoblastoma is to be understood for utilization in therapeutic applications. Hence, in this study, a novel siRNA (small interference RNA) targeting ODC was designed and validated in Human Y79 retinoblastoma cells for its effects on intracellular polyamine levels, Matrix Metalloproteinase 2 & 9 activity and Cell cycle. The designed siRNA showed efficient silencing of ODC mRNA expression and protein levels in Y79 cells. It also showed significant reduction of intracellular polyamine levels and altered levels of oncogenic LIN28b expression. By this study, a regulatory loop is proposed, wherein, ODC silencing in Y79 cells to result in decreased polyamine levels, thereby, leading to altered protein levels of Lin28b, MMP-2 and MMP-9, which falls in line with earlier studies in neuroblastoma. Thus, by this study, we propose ODC silencing as a prospective strategy for targeting retinoblastoma. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Suffering in silence: Investigating the role of fear in the relationship between abusive supervision and defensive silence.

PubMed

Kiewitz, Christian; Restubog, Simon Lloyd D; Shoss, Mindy K; Garcia, Patrick Raymund James M; Tang, Robert L

2016-05-01

Drawing from an approach-avoidance perspective, we examine the relationships between subordinates' perceptions of abusive supervision, fear, defensive silence, and ultimately abusive supervision at a later time point. We also account for the effects of subordinates' assertiveness and individual perceptions of a climate of fear on these predicted mediated relationships. We test this moderated mediation model with data from three studies involving different sources collected across various measurement periods. Results corroborated our predictions by showing (a) a significant association between abusive supervision and subordinates' fear, (b) second-stage moderation effects of subordinates' assertiveness and their individual perceptions of a climate of fear in the abusive supervision-fear-defensive silence relationship (with lower assertiveness and higher levels of climate-of-fear perceptions exacerbating the detrimental effects of fear resulting from abusive supervision), and (c) first-stage moderation effects of subordinates' assertiveness and climate-of-fear perceptions in a model linking fear to defensive silence and abusive supervision at a later time. Theoretical and practical implications are discussed. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA.

PubMed

Samuel, Glady Hazitha; Wiley, Michael R; Badawi, Atif; Adelman, Zach N; Myles, Kevin M

2016-11-29

Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito's RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular "arms race" between vector and pathogen underlies the continued existence of flaviviruses in nature.

Interplays between Soil-Borne Plant Viruses and RNA Silencing-Mediated Antiviral Defense in Roots

PubMed Central

Andika, Ida Bagus; Kondo, Hideki; Sun, Liying

2016-01-01

Although the majority of plant viruses are transmitted by arthropod vectors and invade the host plants through the aerial parts, there is a considerable number of plant viruses that infect roots via soil-inhabiting vectors such as plasmodiophorids, chytrids, and nematodes. These soil-borne viruses belong to diverse families, and many of them cause serious diseases in major crop plants. Thus, roots are important organs for the life cycle of many viruses. Compared to shoots, roots have a distinct metabolism and particular physiological characteristics due to the differences in development, cell composition, gene expression patterns, and surrounding environmental conditions. RNA silencing is an important innate defense mechanism to combat virus infection in plants, but the specific information on the activities and molecular mechanism of RNA silencing-mediated viral defense in root tissue is still limited. In this review, we summarize and discuss the current knowledge regarding RNA silencing aspects of the interactions between soil-borne viruses and host plants. Overall, research evidence suggests that soil-borne viruses have evolved to adapt to the distinct mechanism of antiviral RNA silencing in roots. PMID:27695446

Localized epigenetic silencing of a damage-activated WNT enhancer limits regeneration in mature Drosophila imaginal discs

PubMed Central

Harris, Robin E; Setiawan, Linda; Saul, Josh; Hariharan, Iswar K

2016-01-01

Many organisms lose the capacity to regenerate damaged tissues as they mature. Damaged Drosophila imaginal discs regenerate efficiently early in the third larval instar (L3) but progressively lose this ability. This correlates with reduced damage-responsive expression of multiple genes, including the WNT genes wingless (wg) and Wnt6. We demonstrate that damage-responsive expression of both genes requires a bipartite enhancer whose activity declines during L3. Within this enhancer, a damage-responsive module stays active throughout L3, while an adjacent silencing element nucleates increasing levels of epigenetic silencing restricted to this enhancer. Cas9-mediated deletion of the silencing element alleviates WNT repression, but is, in itself, insufficient to promote regeneration. However, directing Myc expression to the blastema overcomes repression of multiple genes, including wg, and restores cellular responses necessary for regeneration. Localized epigenetic silencing of damage-responsive enhancers can therefore restrict regenerative capacity in maturing organisms without compromising gene functions regulated by developmental signals. DOI: http://dx.doi.org/10.7554/eLife.11588.001 PMID:26840050

RNAi-mediated gene silencing as a principle of action of venoms and poisons.

PubMed

Pereira, Tiago Campos; Lopes-Cendes, Iscia

2008-01-01

RNA interference (RNAi) is a natural phenomenon in which double-stranded RNA molecules (dsRNAs) promote silencing of genes with similar sequence. It is noteworthy that in some instances the effects of gene silencing are similar to those caused by venoms and natural poisons (e.g., hemorrhage and low blood pressure). This observation raises the possibility that venomous/poisonous species in fact produce dsRNAs in their venoms/poisons and leading to the deleterious effects in the victim by RNAi-mediated gene silencing. Two approaches could be used to test this hypothesis, first, the neutralization of the dsRNAs and comparing to a non-treated venom sample; and second, to identify the dsRNA present in the venom and attempt to artificially reproduce its effects in the laboratory. In addition, we present three innovative treatment strategies for accidental interactions with venomous or poisonous species. RNAi has several roles in biological systems: gene regulation, antiviral defense, transposon silencing and heterochromatin formation. The hypothesis presented here provides a new role: a natural attack mechanism.

Sustained Local Delivery of siRNA from an Injectable Scaffold

PubMed Central

Nelson, Christopher E.; Gupta, Mukesh K.; Adolph, Elizabeth J.; Shannon, Joshua M.; Guelcher, Scott A.; Duvall, Craig L.

2011-01-01

Controlled gene silencing technologies have significant, unrealized potential for use in tissue regeneration applications. The design described herein provides a means to package and protect siRNA within pH-responsive, endosomolytic micellar nanoparticles (si-NPs) that can be incorporated into nontoxic, biodegradable, and injectable polyurethane (PUR) tissue scaffolds. The si-NPs were homogeneously incorporated throughout the porous PUR scaffolds, and they were shown to be released via a diffusion-based mechanism for over three weeks. The siRNA-loaded micelles were larger but retained nano particulate morphology of approximately 100 nm diameter following incorporation into and release from the scaffolds. PUR scaffold releasate collected in vitro in PBS at 37°C for 1–4 days was able to achieve dose-dependent siRNA-mediated silencing with approximately 50% silencing achieved of the model gene GAPDH in NIH3T3 mouse fibroblasts. This promising platform technology provides both a research tool capable of probing the effects of local gene silencing and a potentially high-impact therapeutic approach for sustained, local silencing of deleterious genes within tissue defects. PMID:22061489

Optogenetic silencing of locus coeruleus activity in mice impairs cognitive flexibility in an attentional set-shifting task

PubMed Central

Janitzky, Kathrin; Lippert, Michael T.; Engelhorn, Achim; Tegtmeier, Jennifer; Goldschmidt, Jürgen; Heinze, Hans-Jochen; Ohl, Frank W.

2015-01-01

The locus coeruleus (LC) is the sole source of noradrenergic projections to the cortex and essential for attention-dependent cognitive processes. In this study we used unilateral optogenetic silencing of the LC in an attentional set-shifting task (ASST) to evaluate the influence of the LC on prefrontal cortex-dependent functions in mice. We expressed the halorhodopsin eNpHR 3.0 to reversibly silence LC activity during task performance, and found that silencing selectively impaired learning of those parts of the ASST that most strongly rely on cognitive flexibility. In particular, extra-dimensional set-shifting (EDS) and reversal learning was impaired, suggesting an involvement of the medial prefrontal cortex (mPFC) and the orbitofrontal cortex. In contrast, those parts of the task that are less dependent on cognitive flexibility, i.e., compound discrimination (CD) and the intra-dimensional shifts (IDS) were not affected. Furthermore, attentional set formation was unaffected by LC silencing. Our results therefore suggest a modulatory influence of the LC on cognitive flexibility, mediated by different frontal networks. PMID:26582980

The Sounds of Silence: Language, Cognition, and Anxiety in Selective Mutism

ERIC Educational Resources Information Center

Manassis, Katharina; Tannock, Rosemary; Garland, E. Jane; Minde, Klaus; McInnes, Alison; Clark, Sandra

2007-01-01

Objectives: To determine whether oral language, working memory, and social anxiety differentiate children with selective mutism (SM), children with anxiety disorders (ANX), and normal controls (NCs) and explore predictors of mutism severity. Method: Children ages 6 to 10 years with SM (n = 44) were compared with children with ANX (n = 28) and NCs…

Utilization of a tobacco rattle virus vector to clone an Nicotiana benthamiana cDNA library for VIGS

USDA-ARS?s Scientific Manuscript database

Virus-induced gene silencing (VIGS) is an efficient and rapid method to identify plant gene functions. One of the most widely used VIGS vectors is Tobacco rattle virus (TRV) which has been used successfully for RNA interference (RNAi) in N. benthamiana and tomato. We previously modified a TRV VIGS v...

Teacher Technology Narratives: Native Hawaiian Views on Education and Change

ERIC Educational Resources Information Center

Yong, D. Lilinoe; Hoffman, Ellen S.

2014-01-01

Narrative inquiry is a method by which "silenced voices" may be heard. In this study, eight Native Hawaiian teachers share their experiences of the Hawaiian Language Immersion Program (HLIP), or Papahana Kaiapuni, within the Hawai'i public school system. The teachers describe change over time in HLIP with a focus on technology and…

RNAi-induced silencing of embryonic tryptophan oxygenase in the Pyralid moth, Plodia interpunctella

PubMed Central

Fabrick, Jeffrey A.; Kanost, Michael R.; Baker, James E.

2004-01-01

Gene silencing through the introduction of double-stranded RNA (RNA interference, RNAi) provides a powerful tool for the elucidation of gene function in many systems, including those where genomics and proteomics are incomplete. The use of RNAi technology for gene silencing in Lepidoptera has lacked significant attention compared to other systems. To demonstrate that RNAi can be utilized in the lepidopteran, Plodia interpunctella, we cloned a cDNA for tryptophan oxygenase, and showed that silencing of tryptophan oxygenase through RNAi during embryonic development resulted in loss of eye-color pigmentation. The complete amino acid sequence of Plodia tryptophan oxygenase can be accessed through NCBI Protein Database under NCBI Accession # AY427951. Abbreviation RNAi RNA interference PCR polymerase chain reaction RT-PCR reverse transcription-PCR PMID:15861231

An intronic microRNA silences genes that are functionally antagonistic to its host gene.

PubMed

Barik, Sailen

2008-09-01

MicroRNAs (miRNAs) are short noncoding RNAs that down-regulate gene expression by silencing specific target mRNAs. While many miRNAs are transcribed from their own genes, nearly half map within introns of 'host' genes, the significance of which remains unclear. We report that transcriptional activation of apoptosis-associated tyrosine kinase (AATK), essential for neuronal differentiation, also generates miR-338 from an AATK gene intron that silences a family of mRNAs whose protein products are negative regulators of neuronal differentiation. We conclude that an intronic miRNA, transcribed together with the host gene mRNA, may serve the interest of its host gene by silencing a cohort of genes that are functionally antagonistic to the host gene itself.

The role of green fluorescent protein (GFP) in transgenic plants to reduce gene silencing phenomena.

PubMed

El-Shemy, Hany A; Khalafalla, Mutasim M; Ishimoto, Masao

2009-01-01

The green fluorescent protein (GFP) of jellyfish (Aequorea victoria) has significant advantages over other reporter genes, because expression can be detected in living cells without any substrates. Recently, epigenetic phenomena are important to consider in plant biotechnology experiments for elucidate unknown mechanism. Therefore, soybean immature cotyledons were generated embryogenesis cells and engineered with two different gene constructs (pHV and pHVS) using gene gun method. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. However, sGFP(S65T) as a reporter gene was used only in pHVS as a reporter gene for study the relation between using sGFP(S65T) and gene silencing phenomena. Fluorescence microscopic was used for screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Protein analysis was used to detect gene expression overall seeds using SDS-PAGE. Percentage of gene down regulation was highly in pHV construct compared with pHVS. Thus, sGFP(S65T ) as a reporter gene in vector system may be play useful role for transgenic evaluation and avoid gene silencing in plants for the benefit of plant transformation system.

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

PubMed

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

2014-04-20

A transient expression vector based on Citrus tristeza virus (CTV) is unusually stable. Because of its stability it is being considered for use in the field to control Huanglongbing (HLB), which is caused by Candidatus Liberibacter asiaticus (CLas) and vectored by Asian citrus psyllid, Diaphorina citri. In the absence of effective control strategies for CLas, emphasis has been on control of D. citri. Coincident cohabitation in phloem tissue by CLas, D. citri and CTV was exploited to develop a novel method to mitigate HLB through RNA interference (RNAi). Since CTV has three RNA silencing suppressors, it was not known if CTV-based vector could induce RNAi in citrus. Yet, expression of sequences targeting citrus phytoene desaturase gene by CTV-RNAi resulted in photo-bleaching phenotype. CTV-RNAi vector, engineered with truncated abnormal wing disc (Awd) gene of D. citri, induced altered Awd expression when silencing triggers ingested by feeding D. citri nymphs. Decreased Awd in nymphs resulted in malformed-wing phenotype in adults and increased adult mortality. This impaired ability of D. citri to fly would potentially limit the successful vectoring of CLas bacteria between citrus trees in the grove. CTV-RNAi vector would be relevant for fast-track screening of candidate sequences for RNAi-mediated pest control. Copyright © 2014. Published by Elsevier B.V.

Silencing Prx1 and/or Prx5 sensitizes human esophageal cancer cells to ionizing radiation and increases apoptosis via intracellular ROS accumulation

PubMed Central

Gao, Mai-cang; Jia, Xiao-di; Wu, Qi-fei; Cheng, Yan; Chen, Fen-rong; Zhang, Jun

2011-01-01

Aim: To investigate whether down-regulation of peroxiredoxin 1 (Prx1) and/or peroxiredoxin 5 (Prx5) sensitizes human esophageal cancer cells to ionizing radiation (IR). Methods: Human esophageal carcinoma cell lines Eca-109 and TE-1 were used. Prx mRNA expression profiles in Eca-109 and TE-1 cells were determined using RT-PCR. Two highly expressed isoforms of Prxs, Prx1 and Prx5, were silenced by RNA interference (RNAi). Following IR, intracellular reactive oxygen species (ROS) and apoptosis were measured using flow cytometry, the activities of catalase, superoxide dismutase and glutathione peroxidase were measured, and the radiosensitizing effect of RNAi was observed. Tumor xenograft model was also used to examine the radiosensitizing effect of RNAi in vivo. Results: Down-regulation of Prx1 and/or Prx5 by RNAi does not alter the activities of catalase, superoxide dismutase and glutathione peroxidase, but made human tumor cells more sensitive to IR-induced apoptosis both in vitro and in vivo. When the two isoforms were decreased simultaneously, intracellular ROS and apoptosis significantly increased after IR. Conclusion: Silencing Prx1 and/or Prx5 by RNAi sensitizes human Eca-109 and TE-1 cells to IR, and the intracellular ROS accumulation may contribute to the radiosensitizing effect of the RNAi. PMID:21468086

Impact of Paracoccin Gene Silencing on Paracoccidioides brasiliensis Virulence.

PubMed

Fernandes, Fabrício F; Oliveira, Aline F; Landgraf, Taise N; Cunha, Cristina; Carvalho, Agostinho; Vendruscolo, Patrícia E; Gonçales, Relber A; Almeida, Fausto; da Silva, Thiago A; Rodrigues, Fernando; Roque-Barreira, Maria Cristina

2017-07-18

Among the endemic deep mycoses in Latin America, paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a major cause of morbidity. Disease development and its manifestations are associated with both host and fungal factors. Concerning the latter, several recent studies have employed the methodology of gene modulation in P. brasiliensis using antisense RNA (AsRNA) and Agrobacterium tumefaciens -mediated transformation (ATMT) to identify proteins that influence fungus virulence. Our previous observations suggested that paracoccin (PCN), a multidomain fungal protein with both lectin and enzymatic activities, may be a potential P. brasiliensis virulence factor. To explore this, we used AsRNA and ATMT methodology to obtain three independent PCN-silenced P. brasiliensis yeast strains (As PCN1 , As PCN2 , and As PCN3 ) and characterized them with regard to P. brasiliensis biology and pathogenicity. As PCN1 , As PCN2 , and As PCN3 showed relative PCN expression levels that were 60%, 40%, and 60% of that of the wild-type (WT) strain, respectively. PCN silencing led to the aggregation of fungal cells, blocked the morphological yeast-to-mycelium transition, and rendered the yeast less resistant to macrophage fungicidal activity. In addition, mice infected with As PCN1 , As PCN2 , and As PCN3 showed a reduction in fungal burden of approximately 96% compared with those inoculated with the WT strain, which displayed a more extensive destruction of lung tissue. Finally, mice infected with the PCN-silenced yeast strains had lower mortality than those infected with the WT strain. These data demonstrate that PCN acts as a P. brasiliensis contributory virulence factor directly affecting fungal pathogenesis. IMPORTANCE The nonexistence of efficient genetic transformation systems has hampered studies in the dimorphic fungus Paracoccidioides brasiliensis , the etiological agent of the most frequent systemic mycosis in Latin America. The recent development of a method for gene expression knockdown by antisense RNA technology, associated with an Agrobacterium tumefaciens -mediated transformation system, provides new strategies for studying P. brasiliensis Through this technology, we generated yeasts that were silenced for paracoccin (PCN), a P. brasiliensis component that has lectin and enzymatic properties. By comparing the phenotypes of PCN-silenced and wild-type strains of P. brasiliensis , we identified PCN as a virulence factor whose absence renders the yeasts unable to undergo the transition to mycelium and causes a milder pulmonary disease in mice, with a lower mortality rate. Our report highlights the importance of the technology used for P. brasiliensis transformation and demonstrates that paracoccin is a virulence factor acting on fungal biology and pathogenesis. Copyright © 2017 Fernandes et al.

Sustained delivery of siRNA/mesoporous silica nanoparticle (siRNA/MSN) complexes from nanofiber scaffolds for long-term gene silencing.

PubMed

Pinese, Coline; Lin, Junquan; Milbreta, Ulla; Li, Mingqiang; Wang, Yucai; Leong, Kam W; Chew, Sing Yian

2018-06-08

A low toxicity and efficient delivery system is needed to deliver small interfering RNAs (siRNA) in vitro and in vivo. The use of mesoporous silica nanoparticles (MSN) is becoming increasingly common due to its biocompatibility, tunable pore size and customizable properties. However, bolus delivery of siRNA/MSN complexes remains suboptimal, especially when a sustained and long-term administration is required. Here, we utilized electrospun scaffolds for sustained delivery of siRNA/MSN-PEI through surface adsorption and nanofiber encapsulation. As a proof-of-concept, we targeted collagen type I expression to modulate fibrous capsule formation. Surface adsorption of siRNA/MSN-PEI provided sustained availability of siRNA for at least 30 days in vitro. As compared to conventional bolus delivery, such scaffold-mediated transfection provided more effective gene silencing (p < 0.05). On the contrary, a longer sustained release was attained (at least 5 months) when siRNA/MSN-PEI complexes were encapsulated within the electrospun fibers. In vivo subcutaneous implantation and biodistribution analysis of these scaffolds revealed that siRNA remained localized up to ∼290 μm from the implants. Finally, a fibrous capsule reduction of ∼45.8 % was observed after 4 weeks in vivo as compared to negative scrambled siRNA treatment. Taken together, these results demonstrate the efficacy of scaffold-mediated sustained delivery of siRNA/MSN-PEI for long-term non-viral gene silencing applications. The bolus delivery of siRNA/ Mesoporous Silica Nanoparticles (MSN) complexes shows high efficiency to silence protein agonists of tumoral processes as cancer treatments. However, in tissue engineering area, scaffold mediated delivery is desired to achieve a local and sustained release of therapeutics. We showed the feasibility and the efficacy of siRNA/MSN delivered from electrospun scaffolds through surface adsorption and nanofiber encapsulation. We showed that this method enhances siRNA transfection efficiency and sustained targeted proteins silencing in vitro and in vivo. As a proof of concept, in this study, we targeted collagen type I expression to modulate fibrous capsule formation. However this platform can be applied to the release and transfection of siRNA or miRNA in cancer and tissue engineering applications. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Transgenerational inheritance of an acquired small RNA-based antiviral response in C.elegans

PubMed Central

Rechavi, Oded; Minevich, Gregory; Hobert, Oliver

2011-01-01

Induced expression of the Flock House virus in the soma of C. elegans results in the RNAi-dependent production of virus-derived, small interfering RNAs (viRNAs), which in turn silence the viral genome. We show here that the viRNA-mediated viral silencing effect is transmitted in a non-Mendelian manner to many ensuing generations. We show that the viral silencing agents, viRNAs, are transgenerationally transmitted in a template-independent manner and work in trans to silence viral genomes present in animals that are deficient in producing their own viRNAs. These results provide evidence for the transgenerational inheritance of an acquired trait, induced by the exposure of animals to a specific, biologically relevant physiological challenge. The ability to inherit such extragenic information may provide adaptive benefits to an animal. PMID:22119442

Biomimetic RNA-silencing nanocomplexes: overcoming multidrug resistance in cancer cells.

PubMed

Wang, Zhongliang; Wang, Zhe; Liu, Dingbin; Yan, Xuefeng; Wang, Fu; Niu, Gang; Yang, Min; Chen, Xiaoyuan

2014-02-10

RNA interference (RNAi) is an RNA-dependent gene silencing approach controlled by an RNA-induced silencing complex (RISC). Herein, we present a synthetic RISC-mimic nanocomplex, which can actively cleave its target RNA in a sequence-specific manner. With high enzymatic stability and efficient self-delivery to target cells, the designed nanocomplex can selectively and potently induce gene silencing without cytokine activation. These nanocomplexes, which target multidrug resistance, are not only able to bypass the P-glycoprotein (Pgp) transporter, due to their nano-size effect, but also effectively suppress Pgp expression, thus resulting in successful restoration of drug sensitivity of OVCAR8/ADR cells to Pgp-transportable cytotoxic agents. This nanocomplex approach has the potential for both functional genomics and cancer therapy. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Silencing of the tomato phosphatidylinositol-phospholipase C2 (SlPLC2) reduces plant susceptibility to Botrytis cinerea.

PubMed

Gonorazky, Gabriela; Guzzo, María Carla; Abd-El-Haliem, Ahmed M; Joosten, Matthieu H A J; Laxalt, Ana María

2016-12-01

The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated on pathogen attack. We have previously shown that the fungal elicitor xylanase induces a raise of SlPLC2 and SlPLC5 transcripts and that SlPLC2, but not SlPLC5, is required for xylanase-induced expression of defense-related genes. In this work we studied the role of SlPLC2 in the interaction between tomato and the necrotrophic fungus Botrytis cinerea. Inoculation of tomato leaves with B. cinerea increases SlPLC2 transcript levels. We knocked-down the expression of SlPLC2 by virus-induced gene silencing and plant defense responses were analyzed upon B. cinerea inoculation. SlPLC2 silenced plants developed smaller necrotic lesions concomitantly with less proliferation of the fungus. Silencing of SlPLC2 resulted as well in a reduced production of reactive oxygen species. Upon B. cinerea inoculation, transcript levels of the salicylic acid (SA)-defense pathway marker gene SlPR1a were diminished in SlPLC2 silenced plants compared to non-silenced infected plants, while transcripts of the jasmonic acid (JA)-defense gene markers Proteinase Inhibitor I and II (SlPI-I and SlPI-II) were increased. This implies that SlPLC2 participates in plant susceptibility to B. cinerea. © 2016 BSPP and John Wiley & Sons Ltd.

Breaking down the wall of silence around children affected by AIDS in Thailand to support their psychosocial health.

PubMed

Ishikawa, Naoko; Pridmore, Pat; Carr-Hill, Roy; Chaimuangdee, Kreangkrai

2010-03-01

This study examines the psychosocial needs of the children affected by AIDS. Eight primary school children aged 10-13 years who lost parents to AIDS or whose parents were living with HIV were closely followed for a period of one year and qualitative data on the psychosocial challenges they faced were collected using semi-structured interviews, observation, drawings and diaries. Data were also collected from their caregivers and classroom teachers using semi-structured interviews, as well as data from their classmates using a self-completion questionnaire. The findings strongly suggested that adults were creating a "wall of silence" around children affected by AIDS by hiding the parents' HIV status from them and avoid talking to them about HIV and AIDS. The silence was intended to protect the children from sadness, embarrassment, bullying and discrimination. In reality, however, the silence was found to have isolated them and increased their psychosocial vulnerability by blocking open communication with family members, peers and teachers, and left them to cope with their problems on their own. It is argued that to support the psychosocial health of these children, it is necessary for the adults to recognise the negative impact of silence and for the families and the school to be involved in a process of participatory learning and action to find culturally appropriate ways to break down the wall of silence, and promote more open communication.

The Silencing of Carotenoid β-Hydroxylases by RNA Interference in Different Maize Genetic Backgrounds Increases the β-Carotene Content of the Endosperm

PubMed Central

Berman, Judit; Zorrilla-López, Uxue; Sandmann, Gerhard; Capell, Teresa; Christou, Paul

2017-01-01

Maize (Zea mays L.) is a staple food in many parts of Africa, but the endosperm generally contains low levels of the pro-vitamin A carotenoid β-carotene, leading to vitamin A deficiency disease in populations relying on cereal-based diets. However, maize endosperm does accumulate high levels of other carotenoids, including zeaxanthin, which is derived from β-carotene via two hydroxylation reactions. Blocking these reactions could therefore improve the endosperm β-carotene content. Accordingly, we used RNA interference (RNAi) to silence the endogenous ZmBCH1 and ZmBCH2 genes, which encode two non-heme di-iron carotenoid β-hydroxylases. The genes were silenced in a range of maize genetic backgrounds by introgressing the RNAi cassette, allowing us to determine the impact of ZmBCH1/ZmBCH2 silencing in diverse hybrids. The β-carotene content of the endosperm increased substantially in all hybrids in which ZmBCH2 was silenced, regardless of whether or not ZmBCH1 was silenced simultaneously. However, the β-carotene content did not change significantly in C17 hybrids (M7 × C17 and M13 × C17) compared to C17 alone, because ZmBCH2 is already expressed at negligible levels in the C17 parent. Our data indicate that ZmBCH2 is primarily responsible for the conversion of β-carotene to zeaxanthin in maize endosperm. PMID:29186806

On the robustness of SAC silencing in closed mitosis

NASA Astrophysics Data System (ADS)

Ruth, Donovan; Liu, Jian

Mitosis equally partitions sister chromatids to two daughter cells. This is achieved by properly attaching these chromatids via their kinetochores to microtubules that emanate from the spindle poles. Once the last kinetochore is properly attached, the spindle microtubules pull the sister chromatids apart. Due to the dynamic nature of microtubules, however, kinetochore-microtubule attachment often goes wrong. When this erroneous attachment occurs, it locally activates an ensemble of proteins, called the spindle assembly checkpoint proteins (SAC), which halts the mitotic progression until all the kinetochores are properly attached by spindle microtubules. The timing of SAC silencing thus determines the fidelity of chromosome segregation. We previously established a spatiotemporal model that addresses the robustness of SAC silencing in open mitosis for the first time. Here, we focus on closed mitosis by examining yeast mitosis as a model system. Though much experimental work has been done to study the SAC in cells undergoing closed mitosis, the processes responsible are not well understood. We leverage and extend our previous model to study SAC silencing mechanism in closed mitosis. We show that a robust signal of the SAC protein accumulation at the spindle pole body can be achieved. This signal is a nonlinear increasing function of number of kinetochore-microtubule attachments, and can thus serve as a robust trigger to time the SAC silencing. Together, our mechanism provides a unified framework across species that ensures robust SAC silencing and fidelity of chromosome segregation in mitosis. Intramural research program in NHLBI at NIH.

Lack of sex chromosome specific meiotic silencing in platypus reveals origin of MSCI in therian mammals.

PubMed

Daish, Tasman J; Casey, Aaron E; Grutzner, Frank

2015-12-10

In therian mammals heteromorphic sex chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during meiotic prophase I while the autosomes maintain transcriptional activity. The evolution of this sex chromosome silencing is thought to result in retroposition of genes required in spermatogenesis from the sex chromosomes to autosomes. In birds sex chromosome specific silencing appears to be absent and global transcriptional reductions occur through pachytene and sex chromosome-derived autosomal retrogenes are lacking. Egg laying monotremes are the most basal mammalian lineage, feature a complex and highly differentiated XY sex chromosome system with homology to the avian sex chromosomes, and also lack autosomal retrogenes. In order to delineate the point of origin of sex chromosome specific silencing in mammals we investigated whether MSCI exists in platypus. Our results show that platypus sex chromosomes display only partial or transient colocalisation with a repressive histone variant linked to therian sex chromosome silencing and surprisingly lack a hallmark MSCI epigenetic signature present in other mammals. Remarkably, platypus instead feature an avian like period of general low level transcription through prophase I with the sex chromosomes and the future mammalian X maintaining association with a nucleolus-like structure. Our work demonstrates for the first time that in mammals meiotic silencing of sex chromosomes evolved after the divergence of monotremes presumably as a result of the differentiation of the therian XY sex chromosomes. We provide a novel evolutionary scenario on how the future therian X chromosome commenced the trajectory toward MSCI.

Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae.

PubMed

Oling, David; Masoom, Rehan; Kvint, Kristian

2014-06-15

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes. © 2014 Öling 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).

TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats.

PubMed

Sun, Xiaoyan; Song, Ming; Wang, Hui; Zhou, Huimin; Wang, Feng; Li, Ya; Zhang, Yun; Zhang, Wei; Zhong, Ming; Ti, Yun

2017-06-17

Our previous study had suggested Tribbles homolog 3 (TRB3) might be involved in metabolic syndrome via adipose tissue. Given prior studies, we sought to determine whether TRB3 plays a major role in adipocytes and adipose tissue with beneficial metabolic effects in obese and diabetic rats. Fully differentiated 3T3-L1 adipocytes were incubated to induce insulin resistant adipocytes. Forty male Sprague-Dawley rats were all fed high-fat (HF) diet. Type 2 diabetic rat model was induced by high-fat diet and low-dose streptozotocin (STZ). Compared with control group, in insulin resistant adipocytes, protein levels of insulin receptor substrate-1(IRS-1), glucose transporter 4(GLUT4) and phosphorylated-AMP-activated protein kinase (p-AMPK)were reduced, TRB3 protein level and triglyceride level were significantly increased, glucose uptake was markedly decreased. TRB3 silencing alleviated adipocytes insulin resistance. With TRB3 gene silencing, protein levels of IRS-1, GLUT4 and p-AMPK were significantly increased in adipocytes. TRB3 gene silencing decreased blood glucose, ameliorated insulin sensitivity and adipose tissue remodeling in diabetic rats. TRB3 silencing decreased triglyceride, increased glycogen simultaneously in diabetic epididymal and brown adipose tissues (BAT). Consistently, p-AMPK levels were increased in diabetic epididymal adipose tissue, and BAT after TRB3-siRNA treatment. TRB3silencing increased phosphorylation of Akt in liver, and improved liver insulin resistance. Copyright © 2017. Published by Elsevier Inc.

Gene Silencing in Insect Cells Using RNAi.

PubMed

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

2016-01-01

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

Silenced Memories: An Examination of the Sociocultural Knowledge on Race and Racial Violence in Official School Curriculum

ERIC Educational Resources Information Center

Brown, Keffrelyn D.; Brown, Anthony L.

2010-01-01

Drawing from the theoretical lenses of cultural memory and critical race theory, we examined how elementary level and middle school level social studies textbooks represent the history of racial violence directed toward African Americans and resistance to this violence in the U.S. Using a literary analysis method, we found that textbooks often…

Child Development Theory as a Mediator of Novice Teachers' Ethnotheories to Increase Learning and Justice in the Classroom

ERIC Educational Resources Information Center

Cardwell, Nancy Michele

2014-01-01

Many urban public schools use teaching methods that isolate and silence children to compel compliance (Schwebel, 2004; Saltman & Gabbard, 2003; Baumrind, 1991). In these contexts, black and brown children are disciplined more often and harshly than white, sent through the court system 70% of the time (Alexander, 2012). Novice teachers,…

Problem-Posing in a Primary Grade Classroom: Utilizing Freire's Methods to Break the Culture of Silence

ERIC Educational Resources Information Center

Reed, Judith; Saunders, Karen; Pfadenhauer-Simonds, Susan

2015-01-01

Using Paulo Freire's, idea that the aim of education must always be to transform the world so as to liberate oppressed peoples. The authors: Reed, Saunders, and Pfadenhauer-Simonds address this issue by noting that state-backed education systems in the United States, as elsewhere, arguably can be seen as supporting repressive forces that keep…

Expression of cytosolic NADP(+)-dependent isocitrate dehydrogenase in melanocytes and its role as an antioxidant.

PubMed

Kim, Ji Young; Shin, Jae Yong; Kim, Miri; Hann, Seung-Kyung; Oh, Sang Ho

2012-02-01

Cytosolic NADP(+)-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione. To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant. The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked. The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H(2)O(2) compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H(2)O(2). This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes. Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Lipocalin 2 Enhances Migration and Resistance against Cisplatin in Endometrial Carcinoma Cells.

PubMed

Miyamoto, Tsutomu; Kashima, Hiroyasu; Yamada, Yasushi; Kobara, Hisanori; Asaka, Ryoichi; Ando, Hirofumi; Higuchi, Shotaro; Ida, Koichi; Mvunta, David Hamisi; Shiozawa, Tanri

2016-01-01

Lipocalin 2 (LCN2) is a secretory protein that is involved in various physiological processes including iron transport. We previously identified LCN2 as an up-regulated gene in endometrial carcinoma, and found that the overexpression of LCN2 and its receptor, SLC22A17, was associated with a poor prognosis. However, the functions and mechanism of action of LCN2 currently remain unclear. The LCN2-overexpressing endometrial carcinoma cell lines, HHUA and RL95-2, and LCN2-low-expressing one, HEC1B, were used. The effects of LCN2 on cell migration, cell viability, and apoptosis under various stresses, including ultraviolet (UV) irradiation and cisplatin treatment, were examined using the scratch wound healing assay, WST-1 assay, and Apostrand assay, respectively. LCN2-silencing using shRNA method significantly reduced the migration ability of cells (p<0.05). Cytotoxic stresses significantly decreased the viability of LCN2-silenced cells more than that of control cells. In contrast, LCN2 overexpression was significantly increased cisplatin resistance. These effects were canceled by the addition of the iron chelator, deferoxamine. After UV irradiation, the expression of phosphorylated Akt (pAkt) was decreased in LCN2-silenced cells, and the PI3K inhibitor canceled the difference induced in UV sensitivity by LCN2. The cisplatin-induced expression of pAkt was not affected by LCN2; however, the expression of p53 and p21 was increased by LCN2-silencing. These results indicated that LCN2 was involved in the migration and survival of endometrial carcinoma cells under various stresses in an iron-dependent manner. The survival function of LCN2 may be exerted through the PI3K pathway and suppression of the p53-p21 pathway. These functions of LCN2 may increase the malignant potential of endometrial carcinoma cells.

Enhanced Host-Parasite Resistance Based on Down-Regulation of Phelipanche aegyptiaca Target Genes Is Likely by Mobile Small RNA

PubMed Central

Dubey, Neeraj K.; Eizenberg, Hanan; Leibman, Diana; Wolf, Dalia; Edelstein, Menahem; Abu-Nassar, Jackline; Marzouk, Sally; Gal-On, Amit; Aly, Radi

2017-01-01

RNA silencing refers to diverse mechanisms that control gene expression at transcriptional and post-transcriptional levels which can also be used in parasitic pathogens of plants that Broomrapes (Orobanche/Phelipanche spp.) are holoparasitic plants that subsist on the roots of a variety of agricultural crops and cause severe negative effects on the yield and yield quality of those crops. Effective methods for controlling parasitic weeds are scarce, with only a few known cases of genetic resistance. In the current study, we suggest an improved strategy for the control of parasitic weeds based on trans-specific gene-silencing of three parasite genes at once. We used two strategies to express dsRNA containing selected sequences of three Phelipanche aegyptiaca genes PaACS, PaM6PR, and PaPrx1 (pma): transient expression using Tobacco rattle virus (TRV:pma) as a virus-induced gene-silencing vector and stable expression in transgenic tomato Solanum lycopersicum (Mill.) plants harboring a hairpin construct (pBINPLUS35:pma). siRNA-mediated transgene-silencing (20–24 nt) was detected in the host plants. Our results demonstrate that the quantities of PaACS and PaM6PR transcripts from P. aegyptiaca tubercles grown on transgenic tomato or on TRV-infected Nicotiana benthamiana plants were significantly reduced. However, only partial reductions in the quantity of PaPrx1 transcripts were observed in the parasite tubercles grown on tomato and on N. benthamiana plants. Concomitant with the suppression of the target genes, there were significant decreases in the number and weight of the parasite tubercles that grew on the host plants, in both the transient and the stable experimental systems. The results of the work carried out using both strategies point to the movement of mobile exogenous siRNA from the host to the parasite, leading to the impaired expression of essential parasite target genes. PMID:28955363

MSH3-Deficiency Initiates EMAST without Oncogenic Transformation of Human Colon Epithelial Cells

PubMed Central

Campregher, Christoph; Schmid, Gerald; Ferk, Franziska; Knasmüller, Siegfried; Khare, Vineeta; Kortüm, Benedikt; Dammann, Kyle; Lang, Michaela; Scharl, Theresa; Spittler, Andreas; Roig, Andres I.; Shay, Jerry W.; Gerner, Christopher; Gasche, Christoph

2012-01-01

Background/Aim Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency. Methods HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay. Results Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10−4) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold. Conclusions MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis. PMID:23209772

Silencing of Tuberin Enhances Photoreceptor Survival and Function in a Preclinical Model of Retinitis Pigmentosa (An American Ophthalmological Society Thesis)

PubMed Central

Tsang, Stephen H.; Chan, Lawrence; Tsai, Yi-Ting; Wu, Wen-Hsuan; Hsu, Chun-Wei; Yang, Jin; Tosi, Joaquin; Wert, Katherine J.; Davis, Richard J.; Mahajan, Vinit B.

2014-01-01

Purpose: To assess the functional consequences of silencing of tuberin, an inhibitor of the mTOR signaling pathway, in a preclinical model of retinitis pigmentosa (RP) in order to test the hypothesis that insufficient induction of the protein kinase B (PKB)-regulated tuberin/mTOR self-survival pathway initiates apoptosis. Methods: In an unbiased genome-scale approach, kinase peptide substrate arrays were used to analyze self-survival pathways at the onset of photoreceptor degeneration. The mutant Pde6bH620Q/Pde6bH620Q at P14 and P18 photoreceptor outer segment (OS) lysates were labeled with P-ATP and hybridized to an array of 1,164 different synthetic peptide substrates. At this stage, OS of Pde6bH620Q/Pde6bH620Q rods are morphologically normal. In vitro kinase assays and immunohistochemistry were used to validate phosphorylation. Short hairpin RNA (shRNA) gene silencing was used to validate tuberin’s role in regulating survival. Results: At the onset of degeneration, 162 peptides were differentially phosphorylated. Protein kinases A, G, C (AGC kinases), and B exhibited increased activity in both peptide array and in vitro kinase assays. Immunohistochemical data confirmed altered phosphorylation patterns for phosphoinositide-dependent kinase-1 (PDK1), ribosomal protein S6 (RPS6), and tuberin. Tuberin gene silencing rescued photoreceptors from degeneration. Conclusions: Phosphorylation of tuberin and RPS6 is due to the upregulated activity of PKB. PKB/tuberin cell growth/survival signaling is activated before the onset of degeneration. Substrates of the AGC kinases in the PKB/tuberin pathway are phosphorylated to promote cell survival. Knockdown of tuberin, the inhibitor of the mTOR pathway, increased photoreceptor survival and function in a preclinical model of RP. PMID:25646031

Comparison of anti-EGFR-Fab’ conjugated immunoliposomes modified with two different conjugation linkers for siRNa delivery in SMMC-7721 cells

PubMed Central

Deng, Li; Zhang, Yingying; Ma, Lulu; Jing, Xiaolong; Ke, Xingfa; Lian, Jianhao; Zhao, Qiang; Yan, Bo; Zhang, Jinfeng; Yao, Jianzhong; Chen, Jianming

2013-01-01

Background Targeted liposome-polycation-DNA complex (LPD), mainly conjugated with antibodies using functionalized PEG derivatives, is an effective nanovector for systemic delivery of small interference RNA (siRNA). However, there are few studies reporting the effect of different conjugation linkers on LPD for gene silencing. To clarify the influence of antibody conjugation linkers on LPD, we prepared two different immunoliposomes to deliver siRNA in which DSPE-PEG-COOH and DSPE-PEG-MAL, the commonly used PEG derivative linkers, were used to conjugate anti-EGFR Fab’ with the liposome. Methods First, 600 μg of anti-EGFR Fab’ was conjugated with 28.35 μL of a micelle solution containing DSPE-PEG-MAL or DSPE-PEG-COOH, and then post inserted into the prepared LPD. Various liposome parameters, including particle size, zeta potential, stability, and encapsulation efficiency were evaluated, and the targeting ability and gene silencing activity of TLPD-FPC (DSPE-PEG-COOH conjugated with Fab’) was compared with that of TLPD-FPM (DSPE-PEG-MAL conjugated with Fab’) in SMMC-7721 hepatocellular carcinoma cells. Results There was no significant difference in particle size between the two TLPDs, but the zeta potential was significantly different. Further, although there was no significant difference in siRNA encapsulation efficiency, cell viability, or serum stability between TLPD-FPM and TLPD-FPC, cellular uptake of TLPD-FPM was significantly greater than that of TLPD-FPC in EGFR-overexpressing SMMC-7721 cells. The luciferase gene silencing efficiency of TLPD-FPM was approximately three-fold high than that of TLPD-FPC. Conclusion Different conjugation linkers whereby antibodies are conjugated with LPD can affect the physicochemical properties of LPD and antibody conjugation efficiency, thus directly affecting the gene silencing effect of TLPD. Immunoliposomes prepared by DSPE-PEG-MAL conjugation with anti-EGFR Fab’ are more effective than TLPD containing DSPE-PEG-COOH in targeting hepatocellular carcinoma cells for siRNA delivery. PMID:24023515

Artificial small RNA for sequence specific cleavage of target RNA through RNase III endonuclease Dicer

PubMed Central

Liu, Yali; Liu, Li; Zhan, Yonghao; Zhuang, Chengle; Lin, Junhao; Chen, Mingwei; Li, Jianfa; Cai, Zhiming; Huang, Weiren; Zhang, Yong

2016-01-01

CRISPR-Cas9 system uses a guide RNA which functions in conjunction with Cas9 proteins to target a DNA and cleaves double-strand DNA. This phenomenon raises a question whether an artificial small RNA (asRNA), composed of a Dicer–binding RNA element and an antisense RNA, could also be used to induce Dicer to process and degrade a specific RNA. If so, we could develop a new method which is named DICERi for gene silencing or RNA editing. To prove the feasibility of asRNA, we selected MALAT-1 as target and used Hela and MDA-MB-231 cells as experimental models. The results of qRT-PCR showed that the introduction of asRNA decreased the relative expression level of target gene significantly. Next, we analyzed cell proliferation using CCK-8 and EdU staining assays, and then cell migration using wound scratch and Transwell invasion assays. We found that cell proliferation and cell migration were both suppressed remarkably after asRNA was expressed in Hela and MDA-MB-231 cells. Cell apoptosis was also detected through Hoechst staining and ELISA assays and the data indicated that he numbers of apoptotic cell in experimental groups significantly increased compared with negative controls. In order to prove that the gene silencing effects were caused by Dicer, we co-transfected shRNA silencing Dicer and asRNA. The relative expression levels of Dicer and MALAT-1 were both detected and the results indicated that when the cleavage role of Dicer was silenced, the relative expression level of MALAT-1 was not affected after the introduction of asRNA. All the above results demonstrated that these devices directed by Dicer effectively excised target RNA and repressed the target genes, thus causing phenotypic changes. Our works adds a new dimension to gene regulating technologies and may have broad applications in construction of gene circuits. PMID:27231846