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  1. SOCS3 Silencing Attenuates Eosinophil Functions in Asthma Patients

    PubMed Central

    Zafra, Mª Paz; Cañas, Jose A.; Mazzeo, Carla; Gámez, Cristina; Sanz, Veronica; Fernández-Nieto, Mar; Quirce, Santiago; Barranco, Pilar; Ruiz-Hornillos, Javier; Sastre, Joaquín; del Pozo, Victoria

    2015-01-01

    Eosinophils are one of the key inflammatory cells in asthma. Eosinophils can exert a wide variety of actions through expression and secretion of multiple molecules. Previously, we have demonstrated that eosinophils purified from peripheral blood from asthma patients express high levels of suppressor of cytokine signaling 3 (SOCS3). In this article, SOCS3 gene silencing in eosinophils from asthmatics has been carried out to achieve a better understanding of the suppressor function in eosinophils. SOCS3 siRNA treatment drastically reduced SOCS3 expression in eosinophils, leading to an inhibition of the regulatory transcription factors GATA-3 and FoxP3, also interleukin (IL)-10; in turn, an increased STAT3 phosphorilation was observed. Moreover, SOCS3 abrogation in eosinophils produced impaired migration, adhesion and degranulation. Therefore, SOCS3 might be regarded as an important regulator implicated in eosinophil mobilization from the bone marrow to the lungs during the asthmatic process. PMID:25764157

  2. SOCS3 silencing attenuates eosinophil functions in asthma patients.

    PubMed

    Zafra, Ma Paz; Cañas, Jose A; Mazzeo, Carla; Gámez, Cristina; Sanz, Veronica; Fernández-Nieto, Mar; Quirce, Santiago; Barranco, Pilar; Ruiz-Hornillos, Javier; Sastre, Joaquín; del Pozo, Victoria

    2015-01-01

    Eosinophils are one of the key inflammatory cells in asthma. Eosinophils can exert a wide variety of actions through expression and secretion of multiple molecules. Previously, we have demonstrated that eosinophils purified from peripheral blood from asthma patients express high levels of suppressor of cytokine signaling 3 (SOCS3). In this article, SOCS3 gene silencing in eosinophils from asthmatics has been carried out to achieve a better understanding of the suppressor function in eosinophils. SOCS3 siRNA treatment drastically reduced SOCS3 expression in eosinophils, leading to an inhibition of the regulatory transcription factors GATA-3 and FoxP3, also interleukin (IL)-10; in turn, an increased STAT3 phosphorilation was observed. Moreover, SOCS3 abrogation in eosinophils produced impaired migration, adhesion and degranulation. Therefore, SOCS3 might be regarded as an important regulator implicated in eosinophil mobilization from the bone marrow to the lungs during the asthmatic process. PMID:25764157

  3. Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa.

    PubMed

    Kumar, Lokender; Chhibber, Sanjay; Kumar, Rajnish; Kumar, Manoj; Harjai, Kusum

    2015-04-01

    Quorum sensing in Pseudomonas aeruginosa plays an imperative role in virulence factor, biofilm formation and antimicrobial resistance. Blocking quorum sensing pathways are viewed as viable anti-virulent therapy in association with traditional antimicrobial therapy. Anti-quorum sensing dietary phytochemicals with may prove to be a safe and viable choice as anti-virulent drug candidates. Previously, our lab proved zingerone as potent anti-biofilm agent hence; further its anti-virulent and anti-quorum activities were evaluated. Zingerone, besides decreasing swimming, swarming and twitching phenotypes of P. aeruginosa PAO1, reduced biofilm forming capacity and production of virulence factors including rhamnolipid, elastase, protease, pyocyanin, cell free and cell bound hemolysin (p<0.001) indicating anti-virulent property attributing towards attenuation of virulence of P. aeruginosa. Further zingerone not only had marked effect on the production of quorum sensing signal molecules by clinical isolates of P. aeruginosa but also showed significant interference with the activation of QS reporter strains. To study the mechanism of blocking quorum sensing cascade, in silico analysis was carried out. Anti-QS activity was attributed to interference with the ligand receptor interaction of zingerone with QS receptors (TraR, LasR, RhlR and PqsR). Zingerone showed a good comparative docking score to respective autoinducer molecules which was even higher than that of vanillin, a proven anti-quorum sensing phytochemical. The results of the present study revealed the anti-quorum sensing activity of zingerone targeting ligand-receptor interaction, hence proposing zingerone as a suitable anti-virulent drug candidate against P. aeruginosa infections. PMID:25704369

  4. RNA-induced silencing attenuates G protein-mediated calcium signals.

    PubMed

    Philip, Finly; Sahu, Shriya; Golebiewska, Urszula; Scarlata, Suzanne

    2016-05-01

    Phospholipase Cβ (PLCβ) is activated by G protein subunits in response to environmental stimuli to increase intracellular calcium. In cells, a significant portion of PLCβ is cytosolic, where it binds a protein complex required for efficient RNA-induced silencing called C3PO (component 3 promoter of RISC). Binding between C3PO and PLCβ raises the possibility that RNA silencing activity can affect the ability of PLCβ to mediate calcium signals. By use of human and rat neuronal cell lines (SK-N-SH and PC12), we show that overexpression of one of the main components of C3PO diminishes Ca(2+) release in response to Gαq/PLCβ stimulation by 30 to 40%. In untransfected SK-N-SH or PC12 cells, the introduction of siRNA(GAPDH) [small interfering RNA(glyceraldehyde 3-phosphate dehydrogenase)] reduces PLCβ-mediated calcium signals by ∼30%, but addition of siRNA(Hsp90) (heat shock protein 90) had little effect. Fluorescence imaging studies suggest an increase in PLCβ-C3PO association in cells treated with siRNA(GAPDH) but not siRNA(Hsp90). Taken together, our studies raise the possibility that Ca(2+) responses to extracellular stimuli can be modulated by components of the RNA silencing machinery.-Philip, F., Sahu, S., Golebiewska, U., Scarlata, S. RNA-induced silencing attenuates G protein-mediated calcium signals. PMID:26862135

  5. Silencing MR-1 attenuates inflammatory damage in mice heart induced by AngII

    SciTech Connect

    Dai, Wenjian; Chen, Haiyang; Jiang, Jiandong; Kong, Weijia; Wang, Yiguang

    2010-01-15

    Myofibrillogenesis regulator-1(MR-1) can aggravate cardiac hypertrophy induced by angiotensin(Ang) II in mice through activation of NF-{kappa}B signaling pathway, and nuclear transcription factor (NF)-{kappa}B and activator protein-1(AP-1) regulate inflammatory and immune responses by increasing the expression of specific inflammatory genes in various tissues including heart. Whether inhibition of MR-1 expression will attenuate AngII-induced inflammatory injury in mice heart has not been explored. Herein, we monitored the activation of NF-{kappa}B and AP-1, together with expression of pro-inflammatory of interleukin(IL)-6, tumor necrosis factor(TNF)-{alpha}, vascular-cell adhesion molecule (VCAM)-1, platelet endothelial cell adhesion molecule (PECAM), and inflammatory cell infiltration in heart of mice which are induced firstly by AngII (PBS),then received MR-1-siRNA or control-siRNA injecting. We found that the activation of NF-{kappa}B and AP-1 was inhibited significantly, together with the decreased expression of IL-6, TNF-{alpha}, VCAM-1, and PECAM in AngII-induced mice myocardium in MR-1-siRNA injection groups compared with control-siRNA injecting groups. However, the expression level of MR-1 was not an apparent change in PBS-infused groups than in unoperation groups, and MR-1-siRNA do not affect the expression of MR-1 in PBS-infused mice. Our findings suggest that silencing MR-1 protected mice myocardium against inflammatory injury induced by AngII by suppression of pro-inflammatory transcription factors NF-{kappa}B and AP-1 signaling pathway.

  6. Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells.

    PubMed

    Dushpanova, Anar; Agostini, Silvia; Ciofini, Enrica; Cabiati, Manuela; Casieri, Valentina; Matteucci, Marco; Del Ry, Silvia; Clerico, Aldo; Berti, Sergio; Lionetti, Vincenzo

    2016-01-01

    Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2-) levels, known triggers of ET-1 expression. Moreover, no increase in O2- or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2- production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction. PMID:27443965

  7. Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells

    PubMed Central

    Dushpanova, Anar; Agostini, Silvia; Ciofini, Enrica; Cabiati, Manuela; Casieri, Valentina; Matteucci, Marco; Del Ry, Silvia; Clerico, Aldo; Berti, Sergio; Lionetti, Vincenzo

    2016-01-01

    Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2−) levels, known triggers of ET-1 expression. Moreover, no increase in O2− or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2− production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction. PMID:27443965

  8. Attenuation of Histone Methyltransferase KRYPTONITE-mediated transcriptional gene silencing by Geminivirus

    PubMed Central

    Sun, Yan-Wei; Tee, Chuan-Sia; Ma, Yong-Huan; Wang, Gang; Yao, Xiang-Mei; Ye, Jian

    2015-01-01

    Although histone H3K9 methylation has been intensively studied in animals and a model plant Arabidopsis thaliana, little is known about the evolution of the histone methyltransferase and its roles in plant biotic stress response. Here we identified a Nicotiana benthamiana homolog of H3K9 histone methyltransferase KRYPTONITE (NbKYP) and demonstrated its fundamental roles on methylation of plant and virus, beside of leading to the suppression of endogenous gene expression and virus replication. NbKYP and another gene encoding DNA methyltransferase CHROMOMETHYLTRANSFERASE 3 (NbCMT3-1) were further identified as the key components of maintenance of transcriptional gene silencing, a DNA methylation involved anti-virus machinery. All three types of DNA methylations (asymmetric CHH and symmetric CHG/CG) were severely affected in NbKYP-silenced plants, but only severe reduction of CHG methylation found in NbCMT3-1-silenced plants. Attesting to the importance of plant histone H3K9 methylation immunity to virus, the virulence of geminiviruses requires virus-encoded trans-activator AC2 which inhibits the expression of KYP via activation of an EAR-motif-containing transcription repressor RAV2 (RELATED TO ABI3 and VP1). The reduction of KYP was correlated with virulence of various similar geminiviruses. These findings provide a novel mechanism of how virus trans-activates a plant endogenous anti-silencing machinery to gain high virulence. PMID:26602265

  9. Silencing of ILK attenuates the abnormal proliferation and migration of human Tenon's capsule fibroblasts induced by TGF-β2

    PubMed Central

    Xing, Yao; Cui, Lijun; Kang, Qianyan

    2016-01-01

    The cytokine, transforming growth factor-β (TGF-β), plays a key role in wound healing and tissue repair. Integrin-linked kinase (ILK) is a downstream factor of the TGF-β signaling pathway. Research on ILK has mainly focused on its role in the invasion and metastasis of cancer cells. However, little has been reported on the effects of ILK in human Tenon's capsule fibroblasts (HTFs). In this study, we investigated the role of ILK in the proliferation and migration of HTFs exposed to TGF-β2. A lentiviral vector targeting ILK was screened from three candidates and the experimental result indicated that RNA interference can be used to inhibit ILK expression at both the mRNA and protein level. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to assess ILK mRNA expression. Cell proliferation was quantified by MTT assay and cell cycle progression was detected by flow cytometric analysis. Migration was measured by wound healing assay. It was observed that the silencing of ILK suppressed the TGF-β2-induced proliferation of HTFs and led to G1 phase cell cycle arrest and the significant downregulation of cyclin D1 expression. The migration ability of the HTFs decreased following the silencing of ILK, while the downregulation of α-smooth muscle actin expression and the upregulation of E-cadherin expression were observed. The findings of our study indicate that the silencing of ILK attenuates the abnormal proliferation and migration of HTFs induced by TGF-β2, which reveals the therapeutic potential of ILK inhibition in the prevention of scarring following glaucoma filtration surgery. PMID:27315599

  10. Silencing of ILK attenuates the abnormal proliferation and migration of human Tenon's capsule fibroblasts induced by TGF-β2.

    PubMed

    Xing, Yao; Cui, Lijun; Kang, Qianyan

    2016-08-01

    The cytokine, transforming growth factor-β (TGF‑β), plays a key role in wound healing and tissue repair. Integrin‑linked kinase (ILK) is a downstream factor of the TGF-β signaling pathway. Research on ILK has mainly focused on its role in the invasion and metastasis of cancer cells. However, little has been reported on the effects of ILK in human Tenon's capsule fibroblasts (HTFs). In this study, we investigated the role of ILK in the proliferation and migration of HTFs exposed to TGF-β2. A lentiviral vector targeting ILK was screened from three candidates and the experimental result indicated that RNA interference can be used to inhibit ILK expression at both the mRNA and protein level. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to assess ILK mRNA expression. Cell proliferation was quantified by MTT assay and cell cycle progression was detected by flow cytometric analysis. Migration was measured by wound healing assay. It was observed that the silencing of ILK suppressed the TGF-β2-induced proliferation of HTFs and led to G1 phase cell cycle arrest and the significant downregulation of cyclin D1 expression. The migration ability of the HTFs decreased following the silencing of ILK, while the downregulation of α-smooth muscle actin expression and the upregulation of E-cadherin expression were observed. The findings of our study indicate that the silencing of ILK attenuates the abnormal proliferation and migration of HTFs induced by TGF-β2, which reveals the therapeutic potential of ILK inhibition in the prevention of scarring following glaucoma filtration surgery. PMID:27315599

  11. BSCTV C2 attenuates the degradation of SAMDC1 to suppress DNA methylation-mediated gene silencing in Arabidopsis.

    PubMed

    Zhang, Zhonghui; Chen, Hao; Huang, Xiahe; Xia, Ran; Zhao, Qingzhen; Lai, Jianbin; Teng, Kunling; Li, Yin; Liang, Liming; Du, Quansheng; Zhou, Xueping; Guo, Huishan; Xie, Qi

    2011-01-01

    Plant viruses are excellent tools for studying microbial-plant interactions as well as the complexities of host activities. Our study focuses on the role of C2 encoded by Beet severe curly top virus (BSCTV) in the virus-plant interaction. Using BSCTV C2 as bait in a yeast two-hybrid screen, a C2-interacting protein, S-adenosyl-methionine decarboxylase 1 (SAMDC1), was identified from an Arabidopsis thaliana cDNA library. The interaction was confirmed by an in vitro pull-down assay and a firefly luciferase complemention imaging assay in planta. Biochemical analysis further showed that the degradation of the SAMDC1 protein was inhibited by MG132, a 26S proteasome inhibitor, and that C2 could attenuate the degradation of the SAMDC1 protein. Genetic analysis showed that loss of function of SAMDC1 resulted in reduced susceptibility to BSCTV infection and reduced viral DNA accumulation, similar to the effect of BSCTV C2 deficiency. Bisulfite sequencing analysis further showed that C2 deficiency caused enhanced DNA methylation of the viral genome in infected plants. We also showed that C2 can suppress de novo methylation in the FWA transgenic assay in the C2 transgene background. Overexpression of SAMDC1 can mimic the suppressive activity of C2 against green fluorescent protein-directed silencing. These results suggest that C2 interferes with the host defense mechanism of DNA methylation-mediated gene silencing by attenuating the 26S proteasome-mediated degradation of SAMDC1. PMID:21245466

  12. CA3 Synaptic Silencing Attenuates Kainic Acid-Induced Seizures and Hippocampal Network Oscillations123

    PubMed Central

    Yu, Lily M. Y.; Wintzer, Marie E.

    2016-01-01

    Abstract Epilepsy is a neurological disorder defined by the presence of seizure activity, manifest both behaviorally and as abnormal activity in neuronal networks. An established model to study the disorder in rodents is the systemic injection of kainic acid, an excitatory neurotoxin that at low doses quickly induces behavioral and electrophysiological seizures. Although the CA3 region of the hippocampus has been suggested to be crucial for kainic acid-induced seizure, because of its strong expression of kainate glutamate receptors and its high degree of recurrent connectivity, the precise role of excitatory transmission in CA3 in the generation of seizure and the accompanying increase in neuronal oscillations remains largely untested. Here we use transgenic mice in which CA3 pyramidal cell synaptic transmission can be inducibly silenced in the adult to demonstrate CA3 excitatory output is required for both the generation of epileptiform oscillatory activity and the progression of behavioral seizures. PMID:27022627

  13. Silencing of miR-34a Attenuates Cardiac Dysfunction in a Setting of Moderate, but Not Severe, Hypertrophic Cardiomyopathy

    PubMed Central

    Bernardo, Bianca C.; Gao, Xiao-Ming; Tham, Yow Keat; Kiriazis, Helen; Winbanks, Catherine E.; Ooi, Jenny Y. Y.; Boey, Esther J. H.; Obad, Susanna; Kauppinen, Sakari; Gregorevic, Paul; Du, Xiao-Jun; Lin, Ruby C. Y.; McMullen, Julie R.

    2014-01-01

    Therapeutic inhibition of the miR-34 family (miR-34a,-b,-c), or miR-34a alone, have emerged as promising strategies for the treatment of cardiac pathology. However, before advancing these approaches further for potential entry into the clinic, a more comprehensive assessment of the therapeutic potential of inhibiting miR-34a is required for two key reasons. First, miR-34a has ∼40% fewer predicted targets than the miR-34 family. Hence, in cardiac stress settings in which inhibition of miR-34a provides adequate protection, this approach is likely to result in less potential off-target effects. Secondly, silencing of miR-34a alone may be insufficient in settings of established cardiac pathology. We recently demonstrated that inhibition of the miR-34 family, but not miR-34a alone, provided benefit in a chronic model of myocardial infarction. Inhibition of miR-34 also attenuated cardiac remodeling and improved heart function following pressure overload, however, silencing of miR-34a alone was not examined. The aim of this study was to assess whether inhibition of miR-34a could attenuate cardiac remodeling in a mouse model with pre-existing pathological hypertrophy. Mice were subjected to pressure overload via constriction of the transverse aorta for four weeks and echocardiography was performed to confirm left ventricular hypertrophy and systolic dysfunction. After four weeks of pressure overload (before treatment), two distinct groups of animals became apparent: (1) mice with moderate pathology (fractional shortening decreased ∼20%) and (2) mice with severe pathology (fractional shortening decreased ∼37%). Mice were administered locked nucleic acid (LNA)-antimiR-34a or LNA-control with an eight week follow-up. Inhibition of miR-34a in mice with moderate cardiac pathology attenuated atrial enlargement and maintained cardiac function, but had no significant effect on fetal gene expression or cardiac fibrosis. Inhibition of miR-34a in mice with severe pathology

  14. Load-induced inattentional deafness.

    PubMed

    Raveh, Dana; Lavie, Nilli

    2015-02-01

    High perceptual load in a task is known to reduce the visual perception of unattended items (e.g., Lavie, Beck, & Konstantinou, 2014). However, it remains an open question whether perceptual load in one modality (e.g., vision) can affect the detection of stimuli in another modality (e.g., hearing). We report four experiments that establish that high visual perceptual load leads to reduced detection sensitivity in hearing. Participants were requested to detect a tone that was presented during performance of a visual search task of either low or high perceptual load (varied through item similarity). The findings revealed that auditory detection sensitivity was consistently reduced with higher load, and that this effect persisted even when the auditory detection response was made first (before the search response) and when the auditory stimulus was highly expected (50 % present). These findings demonstrate a phenomenon of load-induced deafness and provide evidence for shared attentional capacity across vision and hearing. PMID:25287617

  15. Caspase-12 silencing attenuates inhibitory effects of cigarette smoke extract on NOD1 signaling and hBDs expression in human oral mucosal epithelial cells.

    PubMed

    Wang, Xiang; Qian, Ya-jie; Zhou, Qian; Ye, Pei; Duan, Ning; Huang, Xiao-feng; Zhu, Ya-nan; Li, Jing-jing; Hu, Li-ping; Zhang, Wei-yun; Han, Xiao-dong; Wang, Wen-mei

    2014-01-01

    Cigarette smoke exposure is associated with increased risk of various diseases. Epithelial cells-mediated innate immune responses to infectious pathogens are compromised by cigarette smoke. Although many studies have established that cigarette smoke exposure affects the expression of Toll-liked receptor (TLR), it remains unknown whether the nucleotide-binding oligomerization domain-containing protein 1 (NOD1) expression is affected by cigarette smoke exposure. In the study, we investigated effects of cigarette smoke extract (CSE) on NOD1 signaling in an immortalized human oral mucosal epithelial (Leuk-1) cell line. We first found that CSE inhibited NOD1 expression in a dose-dependent manner. Moreover, CSE modulated the expression of other crucial molecules in NOD1 signaling and human β defensin (hBD) 1, 2 and 3. We found that RNA interference-induced Caspase-12 silencing increased NOD1 and phospho-NF-κB (p-NF-κB) expression and down-regulated RIP2 expression. The inhibitory effects of CSE on NOD1 signaling can be attenuated partially through Caspase-12 silencing. Intriguingly, Caspase-12 silencing abrogated inhibitory effects of CSE on hBD1, 3 expression and augmented induced effect of CSE on hBD2 expression. Caspase-12 could play a vital role in the inhibitory effects of cigarette smoke on NOD1 signaling and hBDs expression in oral mucosal epithelial cells. PMID:25503380

  16. Caspase-12 Silencing Attenuates Inhibitory Effects of Cigarette Smoke Extract on NOD1 Signaling and hBDs Expression in Human Oral Mucosal Epithelial Cells

    PubMed Central

    Wang, Xiang; Qian, Ya-jie; Zhou, Qian; Ye, Pei; Duan, Ning; Huang, Xiao-feng; Zhu, Ya-nan; Li, Jing-jing; Hu, Li-ping; Zhang, Wei-yun; Han, Xiao-dong; Wang, Wen-mei

    2014-01-01

    Cigarette smoke exposure is associated with increased risk of various diseases. Epithelial cells-mediated innate immune responses to infectious pathogens are compromised by cigarette smoke. Although many studies have established that cigarette smoke exposure affects the expression of Toll-liked receptor (TLR), it remains unknown whether the nucleotide-binding oligomerization domain-containing protein 1 (NOD1) expression is affected by cigarette smoke exposure. In the study, we investigated effects of cigarette smoke extract (CSE) on NOD1 signaling in an immortalized human oral mucosal epithelial (Leuk-1) cell line. We first found that CSE inhibited NOD1 expression in a dose-dependent manner. Moreover, CSE modulated the expression of other crucial molecules in NOD1 signaling and human β defensin (hBD) 1, 2 and 3. We found that RNA interference-induced Caspase-12 silencing increased NOD1 and phospho-NF-κB (p-NF-κB) expression and down-regulated RIP2 expression. The inhibitory effects of CSE on NOD1 signaling can be attenuated partially through Caspase-12 silencing. Intriguingly, Caspase-12 silencing abrogated inhibitory effects of CSE on hBD1, 3 expression and augmented induced effect of CSE on hBD2 expression. Caspase-12 could play a vital role in the inhibitory effects of cigarette smoke on NOD1 signaling and hBDs expression in oral mucosal epithelial cells. PMID:25503380

  17. Silencing MicroRNA-155 Attenuates Cardiac Injury and Dysfunction in Viral Myocarditis via Promotion of M2 Phenotype Polarization of Macrophages.

    PubMed

    Zhang, Yingying; Zhang, Mengying; Li, Xueqin; Tang, Zongsheng; Wang, Xiangmin; Zhong, Min; Suo, Qifeng; Zhang, Yao; Lv, Kun

    2016-01-01

    Macrophage infiltration is a hallmark feature of viral myocarditis. As studies have shown that microRNA-155 regulates the differentiation of macrophages, we aimed to investigate the role of microRNA-155 in VM. We report that silencing microRNA-155 protects mice from coxsackievirus B3 induced myocarditis. We found that microRNA-155 expression was upregulated and localized primarily in heart-infiltrating macrophages and CD4(+) T lymphocytes during acute myocarditis. In contrast with wildtype (WT) mice, microRNA-155(-/-) mice developed attenuated viral myocarditis, which was characterized by decreased cardiac inflammation and decreased intracardiac CD45(+) leukocytes. Hearts of microRNA-155(-/-) mice expressed decreased levels of the IFN-γ and increased levels of the cytokines IL-4 and IL-13. Although total CD4(+) and regulatory T cells were unchanged in miR-155(-/-) spleen proportionally, the activation of T cells and CD4(+) T cell proliferation in miR-155(-/-) mice were significantly decreased. Beyond the acute phase, microRNA-15(5-/-) mice had reduced mortality and improved cardiac function during 5 weeks of follow-up. Moreover, silencing microRNA-155 led to increased levels of alternatively-activated macrophages (M2) and decreased levels of classically-activated macrophages (M1) in the heart. Combined, our studies suggest that microRNA-155 confers susceptibility to viral myocarditis by affecting macrophage polarization, and thus may be a potential therapeutic target for viral myocarditis. PMID:26931072

  18. Silencing MicroRNA-155 Attenuates Cardiac Injury and Dysfunction in Viral Myocarditis via Promotion of M2 Phenotype Polarization of Macrophages

    PubMed Central

    Zhang, Yingying; Zhang, Mengying; Li, Xueqin; Tang, Zongsheng; Wang, Xiangmin; Zhong, Min; Suo, Qifeng; Zhang, Yao; Lv, Kun

    2016-01-01

    Macrophage infiltration is a hallmark feature of viral myocarditis. As studies have shown that microRNA-155 regulates the differentiation of macrophages, we aimed to investigate the role of microRNA-155 in VM. We report that silencing microRNA-155 protects mice from coxsackievirus B3 induced myocarditis. We found that microRNA-155 expression was upregulated and localized primarily in heart-infiltrating macrophages and CD4+ T lymphocytes during acute myocarditis. In contrast with wildtype (WT) mice, microRNA-155−/− mice developed attenuated viral myocarditis, which was characterized by decreased cardiac inflammation and decreased intracardiac CD45+ leukocytes. Hearts of microRNA-155−/− mice expressed decreased levels of the IFN-γ and increased levels of the cytokines IL-4 and IL-13. Although total CD4+ and regulatory T cells were unchanged in miR-155−/− spleen proportionally, the activation of T cells and CD4+ T cell proliferation in miR-155−/− mice were significantly decreased. Beyond the acute phase, microRNA-155−/− mice had reduced mortality and improved cardiac function during 5 weeks of follow-up. Moreover, silencing microRNA-155 led to increased levels of alternatively-activated macrophages (M2) and decreased levels of classically-activated macrophages (M1) in the heart. Combined, our studies suggest that microRNA-155 confers susceptibility to viral myocarditis by affecting macrophage polarization, and thus may be a potential therapeutic target for viral myocarditis. PMID:26931072

  19. Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities

    PubMed Central

    Iyer, Swathi V.; Parrales, Alejandro; Begani, Priya; Narkar, Akshay; Adhikari, Amit S.; Martinez, Luis A.; Iwakuma, Tomoo

    2016-01-01

    Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies. PMID:26700961

  20. Cystathionine-γ-lyase gene silencing with siRNA in monocytes/ macrophages attenuates inflammation in cecal ligation and puncture-induced sepsis in the mouse.

    PubMed

    Badiei, A; Chambers, S T; Gaddam, R R; Bhatia, M

    2016-03-01

    Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H2S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture (CLP)-induced sepsis is characterized by increased levels of myeloperoxidase (MPO) activity, morphological changes in liver and pro-inflammatory cytokines and chemokines in the liver and lung. SiRNA treatment attenuated inflammation in the liver and lungs of mice following CLP-induced sepsis. Liver MPO activity increased in CLP-induced sepsis and treatment with siRNA significantly reduced this. Similarly, lung MPO activity increased following induction of sepsis with CLP while siRNA treatment significantly reduced MPO activity. Liver and lung cytokine and chemokine levels in CLP-induced sepsis reduced following treatment with siRNA. These findings show a crucial pro-inflammatory role for H2S synthesized by CSE in macrophages in sepsis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition. PMID:26949091

  1. Attenuation of atherosclerotic lesions in diabetic apolipoprotein E-deficient mice using gene silencing of macrophage migration inhibitory factor

    PubMed Central

    Sun, Hui; Zhang, XianJun; Zhao, Lei; Zhen, Xi; Huang, ShanYing; Wang, ShaSha; He, Hong; Liu, ZiMo; Xu, NaNa; Yang, FaLin; Qu, ZhongHua; Ma, ZhiYong; Zhang, Cheng; Zhang, Yun; Hu, Qin

    2015-01-01

    Macrophage migration inhibitory factor (MIF) involves the pathogenesis of atherosclerosis (AS) and increased plasma MIF levels in diabetes mellitus (DM) patients are associated with AS. Here, we have been suggested that MIF could be a critical contributor for the pathological process of diabetes-associated AS by using adenovirus-mediated RNA interference. First, streptozotocin (STZ)-induced diabetic animal model was constructed in 114 apolipoprotein E-deficient mice (apoE−/− mice) fed on a regular chow diet. Then, the animals were randomly divided into three groups: Adenovirus-mediated MIF interference (Ad-MIFi), Ad-enhanced green fluorescent protein (EGFP) and normal saline (NS) group (n ≈ 33/group). Non-diabetic apoE−/− mice (n = 35) were served as controls. Ad-MIFi, Ad-EGFP and NS were, respectively, injected into the tail vein of mice from Ad-MIFi, Ad-EGFP and NS group, which were injected repeatedly 4 weeks later. Physical, biochemical, morphological and molecular parameters were measured. The results showed that diabetic apoE−/− mice had significantly aggravated atherosclerotic lesions. MIF gene interference attenuated atherosclerotic lesions and stabilized atheromatous plaque, accompanied by the decreased macrophages and lipids deposition and inflammatory cytokines production, improved glucose intolerance and plasma cholesterol level, the decreased ratio of matrix matalloproteinase-2/tissue inhibitor of metalloproteinase-1 and plaque instability index. An increased expression of MIF and its ligand CD74 was also detected in the diabetic patients with coronary artery disease. The results suggest that MIF gene interference is able to inhibit atherosclerotic lesions and increase plaque stability in diabetic apoE−/−mice. MIF inhibition could be a novel and promising approach to the treatment of DM-associated AS. PMID:25661015

  2. Transient silencing of Npr3 gene expression improved the circulatory levels of atrial natriuretic peptides and attenuated β-adrenoceptor activation- induced cardiac hypertrophic growth in experimental rats.

    PubMed

    Venkatesan, Balaji; Tumala, Anusha; Subramanian, Vimala; Vellaichamy, Elangovan

    2016-07-01

    Natriuretic peptide receptor-C (NPR-C) is considered as a clearance receptor that maintains the circulatory levels of natriuretic peptides. It has been suggested that augmented expression of NPR-C as a cause for the diminished anti-hypertrophic action of natriuretic peptides in the failing heart. Hence, we sought to determine the level of Npr3 gene (coding for NPR-C) expression in the Isoproterenol (ISO) treated Wistar rats. In addition, we studied the effect of Npr3 gene silencing on the hypertrophic growth. A significant increase in heart weight-to-body weight ratio (HW/BW-24%,P<0.01), an indicator of cardiac hypertrophic growth was observed in the ISO (10mg/kg BW/day,i.p for 7 days) treated rats. As expected, the cardiac NPR-C protein expression was significantly increased by 4 fold as compared to control rats. In parallel, the circulatory atrial natriuretic peptide (ANP) level was significantly decreased (2 fold) in ISO treated rats. Upon treatment with siRNA-Npr3, a significant decrease in the cardiac NPR-C protein expression (70%,P<0.01), HW/BW ratio (70%,P<0.01) and hypertrophic marker genes (α-Sk, β-MHC, c-fos, P<0.01, respectively) mRNA expression were observed. Interestingly, the circulatory ANP level was increased by 1.5 fold in the siRNA-Npr3 treated rats as compared to ISO treated rats. Moreover, the cardiac collagen content, matrixmetalloprotinases-2 (MMP-2) and enzymatic antioxidant status (P<0.01, respectively) were found to be restored back to near normal upon siRNA-Npr3 treatment. Taken together, the results of this study indicates that specific down-regulation of Npr3 gene improves the circulatory levels of ANP and antioxidant system and there by attenuates the β-adrenoceptor over-activation mediated cardiac hypertrophic growth in experimental rats. PMID:27108789

  3. Tissue transglutaminase is involved in mechanical load-induced osteogenic differentiation of human ligamentum flavum cells.

    PubMed

    Chao, Yuan-Hung; Huang, Shih-Yung; Yang, Ruei-Cheng; Sun, Jui-Sheng

    2016-07-01

    Mechanical load-induced osteogenic differentiation might be the key cellular event in the calcification and ossification of ligamentum flavum. The aim of this study was to investigate the influence of tissue transglutaminase (TGM2) on mechanical load-induced osteogenesis of ligamentum flavum cells. Human ligamentum flavum cells were obtained from 12 patients undergoing lumbar spine surgery. Osteogenic phenotypes of ligamentum flavum cells, such as alkaline phosphatase (ALP), Alizarin red-S stain, and gene expression of osteogenic makers were evaluated following the administration of mechanical load and BMP-2 treatment. The expression of TGM2 was evaluated by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) analysis. Our results showed that mechanical load in combination with BMP-2 enhanced calcium deposition and ALP activity. Mechanical load significantly increased ALP and OC gene expression on day 3, whereas BMP-2 significantly increased ALP, OPN, and Runx2 on day 7. Mechanical load significantly induced TGM2 gene expression and enzyme activity in human ligamentum flavum cells. Exogenous TGM2 increased ALP and OC gene expression; while, inhibited TG activity significantly attenuated mechanical load-induced and TGM2-induced ALP activity. In summary, mechanical load-induced TGM2 expression and enzyme activity is involved in the progression of the calcification of ligamentum flavum. PMID:27115725

  4. AUTOPHAGY IN LOAD-INDUCED HEART DISEASE

    PubMed Central

    Zhu, Hongxin; Rothermel, Beverly A.; Hill, Joseph A.

    2009-01-01

    The heart is a highly plastic organ capable of remodeling in response to changes in physiological or pathological demand. When workload increases, the heart compensates through hypertrophic growth of individual cardiomyocytes to increase cardiac output. However, sustained stress, such as occurs with hypertension or following myocardial infarction, triggers changes in sarcomeric protein composition and energy metabolism, loss of cardiomyocytes, ventricular dilation, reduced pump function, and ultimately heart failure. It has been known for some time that autophagy is active in cardiomyocytes, occurring at increased levels in disease. Yet the potential contribution of cardiomyocyte autophagy to ventricular remodeling and disease pathogenesis has only recently been explored. This latter fact stems largely from the recent emergence of tools to probe molecular mechanisms governing cardiac plasticity and to define the role of autophagic flux in the context of heart disease. In this chapter, we briefly review prominent mouse models useful in the study of load-induced heart disease and standard techniques used to assess whether a molecular or cellular event is adaptive or maladaptive. We then outline methods available for monitoring autophagic activity in the heart, providing detailed protocols for several techniques unique to working with heart and other striated muscles. PMID:19216915

  5. Silencing Glycogen Synthase Kinase-3β Inhibits Acetaminophen Hepatotoxicity and Attenuates JNK Activation and Loss of Glutamate Cysteine Ligase and Myeloid Cell Leukemia Sequence 1*

    PubMed Central

    Shinohara, Mie; Ybanez, Maria D.; Win, Sanda; Than, Tin Aung; Jain, Shilpa; Gaarde, William A.; Han, Derick; Kaplowitz, Neil

    2010-01-01

    Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3β (GSK-3β) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury (∼1 h). The silencing of GSK-3β, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3α (GSK-3α), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3β affected several key pathways important in conferring protection against APAP-induced liver injury. APAP treatment was observed to promote the loss of glutamate cysteine ligase (GCL, rate-limiting enzyme in GSH synthesis) in liver. The silencing of GSK-3β decreased the loss of hepatic GCL, and promoted greater GSH recovery in liver following APAP treatment. Silencing JNK1 and -2 also prevented the loss of GCL. APAP treatment also resulted in GSK-3β translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3β reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3β also resulted in an inhibition of the early phase (0–2 h), and blunted the late phase (after 4 h) of JNK activation and translocation to mitochondria in liver following APAP treatment. Taken together our results suggest that activation of GSK-3β is a key mediator of the initial phase of APAP-induced liver injury through modulating GCL and Mcl-1 degradation, as well as JNK activation in liver. PMID:20061376

  6. RuBPCase activase mediates growth-defense tradeoffs: Silencing RCA redirects JA flux from JA-Ile to MeJA to attenuate induced defense responses in Nicotiana attenuata

    PubMed Central

    Mitra, Sirsha; Baldwin, Ian T.

    2014-01-01

    Summary RuBPCase activase (RCA), an abundant photosynthetic protein is strongly down-regulated in response to Manduca sexta’s oral secretion (OS) in Nicotiana attenuata. RCA-silenced plants are impaired not only in photosynthetic capacity and growth, but also in jasmonic acid (JA)-isoleucine (Ile) signaling, and herbivore resistance mediated by JA-Ile dependent defense traits. These responses are consistent with a resource-based growth-defense trade-off. Since JA+Ile-supplementation of OS restored WT levels of JA-Ile, defenses and resistance to M. sexta, but OS supplemented individually with JA- or Ile did not, the JA-Ile deficiency of RCA-silenced plants could not be attributed to lower JA or Ile pools or JAR4/6 conjugating activity. Similar levels of JA-Ile derivatives after OS elicitation indicated unaltered JA-Ile turnover and lower levels of other JA-conjugates ruled out competition from other conjugation reactions. RCA-silenced plants accumulated more methyl jasmonate (MeJA) after OS elicitation, which corresponded with increased jasmonate methyltransferase (JMT) activity. RCA-silencing phenocopies JMT over-expression, wherein elevated JMT activity redirects OS-elicited JA flux towards inactive MeJA, creating a JA sink which depletes JA-Ile and its associated defense responses. Hence RCA plays an additional non-photosynthetic role in attenuating JA-mediated defenses and their associated costs potentially allowing plants to anticipate resource-based constraints on growth before they actually occur. PMID:24491116

  7. Rescuing Loading Induced Bone Formation at Senescence

    PubMed Central

    Srinivasan, Sundar; Ausk, Brandon J.; Prasad, Jitendra; Threet, Dewayne; Bain, Steven D.; Richardson, Thomas S.; Gross, Ted S.

    2010-01-01

    The increasing incidence of osteoporosis worldwide requires anabolic treatments that are safe, effective, and, critically, inexpensive given the prevailing overburdened health care systems. While vigorous skeletal loading is anabolic and holds promise, deficits in mechanotransduction accrued with age markedly diminish the efficacy of readily complied, exercise-based strategies to combat osteoporosis in the elderly. Our approach to explore and counteract these age-related deficits was guided by cellular signaling patterns across hierarchical scales and by the insight that cell responses initiated during transient, rare events hold potential to exert high-fidelity control over temporally and spatially distant tissue adaptation. Here, we present an agent-based model of real-time Ca2+/NFAT signaling amongst bone cells that fully described periosteal bone formation induced by a wide variety of loading stimuli in young and aged animals. The model predicted age-related pathway alterations underlying the diminished bone formation at senescence, and hence identified critical deficits that were promising targets for therapy. Based upon model predictions, we implemented an in vivo intervention and show for the first time that supplementing mechanical stimuli with low-dose Cyclosporin A can completely rescue loading induced bone formation in the senescent skeleton. These pre-clinical data provide the rationale to consider this approved pharmaceutical alongside mild physical exercise as an inexpensive, yet potent therapy to augment bone mass in the elderly. Our analyses suggested that real-time cellular signaling strongly influences downstream bone adaptation to mechanical stimuli, and quantification of these otherwise inaccessible, transient events in silico yielded a novel intervention with clinical potential. PMID:20838577

  8. The silencing of Pokemon attenuates the proliferation of hepatocellular carcinoma cells in vitro and in vivo by inhibiting the PI3K/Akt pathway.

    PubMed

    Lin, Chan-Chan; Zhou, Jing-Ping; Liu, Yun-Peng; Liu, Jing-Jing; Yang, Xiao-Ning; Jazag, Amarsanaa; Zhang, Zhi-Ping; Guleng, Bayasi; Ren, Jian-Lin

    2012-01-01

    Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon assumes a critical function in cellular differentiation and oncogenesis. Our study identified an oncogenic role for Pokemon in human hepatocellular carcinoma (HCC). We successfully established human HepG2 and Huh-7 cell lines in which Pokemon was stably knocked down. We demonstrated that Pokemon silencing inhibited cell proliferation and migration. Pokemon knockdown inhibited the PI3K/Akt and c-Raf/MEK/ERK pathways and modulated the expression of various cell cycle regulators in HepG2 and Huh-7 cells. Therefore, Pokemon may also be involved in cell cycle progression in these cells. We confirmed that Pokemon silencing suppresses hepatocellular carcinoma growth in tumor xenograft mice. These results suggest that Pokemon promotes cell proliferation and migration in hepatocellular carcinoma and accelerates tumor development in an Akt- and ERK-signaling-dependent manner. PMID:23300578

  9. Adenylyl cyclase 6 mediates loading-induced bone adaptation in vivo

    PubMed Central

    Lee, Kristen L.; Hoey, David A.; Spasic, Milos; Tang, Tong; Hammond, H. Kirk; Jacobs, Christopher R.

    2014-01-01

    Primary cilia are single, nonmotile, antenna-like structures extending from the apical membrane of most mammalian cells. They may mediate mechanotransduction, the conversion of external mechanical stimuli into biochemical intracellular signals. Previously we demonstrated that adenylyl cyclase 6 (AC6), a membrane-bound enzyme enriched in primary cilia of MLO-Y4 osteocyte-like cells, may play a role in a primary cilium-dependent mechanism of osteocyte mechanotransduction in vitro. In this study, we determined whether AC6 deletion impairs loading-induced bone formation in vivo. Skeletally mature mice with a global knockout of AC6 exhibited normal bone morphology and responded to osteogenic chemical stimuli similar to wild-type mice. Following ulnar loading over 3 consecutive days, bone formation parameters were assessed using dynamic histomorphometry. Mice lacking AC6 formed significantly less bone than control animals (41% lower bone formation rate). Furthermore, there was an attenuated flow-induced increase in COX-2 mRNA expression levels in primary bone cells isolated from AC6 knockout mice compared to controls (1.3±0.1- vs. 2.6±0.2-fold increase). Collectively, these data indicate that AC6 plays a role in loading-induced bone adaptation, and these findings are consistent with our previous studies implicating primary cilia and AC6 in a novel mechanism of osteocyte mechanotransduction.—Lee, K. L., Hoey, D. A., Spasic, M., Tang, T., Hammond, H. K., Jacobs, C. R. Adenylyl cyclase 6 mediates loading-induced bone adaptation in vivo. PMID:24277577

  10. Adenylyl cyclase 6 mediates loading-induced bone adaptation in vivo.

    PubMed

    Lee, Kristen L; Hoey, David A; Spasic, Milos; Tang, Tong; Hammond, H Kirk; Jacobs, Christopher R

    2014-03-01

    Primary cilia are single, nonmotile, antenna-like structures extending from the apical membrane of most mammalian cells. They may mediate mechanotransduction, the conversion of external mechanical stimuli into biochemical intracellular signals. Previously we demonstrated that adenylyl cyclase 6 (AC6), a membrane-bound enzyme enriched in primary cilia of MLO-Y4 osteocyte-like cells, may play a role in a primary cilium-dependent mechanism of osteocyte mechanotransduction in vitro. In this study, we determined whether AC6 deletion impairs loading-induced bone formation in vivo. Skeletally mature mice with a global knockout of AC6 exhibited normal bone morphology and responded to osteogenic chemical stimuli similar to wild-type mice. Following ulnar loading over 3 consecutive days, bone formation parameters were assessed using dynamic histomorphometry. Mice lacking AC6 formed significantly less bone than control animals (41% lower bone formation rate). Furthermore, there was an attenuated flow-induced increase in COX-2 mRNA expression levels in primary bone cells isolated from AC6 knockout mice compared to controls (1.3±0.1- vs. 2.6±0.2-fold increase). Collectively, these data indicate that AC6 plays a role in loading-induced bone adaptation, and these findings are consistent with our previous studies implicating primary cilia and AC6 in a novel mechanism of osteocyte mechanotransduction. PMID:24277577

  11. The caspase-1 inhibitor AC-YVAD-CMK attenuates acute gastric injury in mice: involvement of silencing NLRP3 inflammasome activities

    PubMed Central

    Zhang, Fang; Wang, Liang; Wang, Jun-jie; Luo, Peng-fei; Wang, Xing-tong; Xia, Zhao-fan

    2016-01-01

    This study evaluated the protective effects of inhibiting caspase-1 activity or gastric acid secretion on acute gastric injury in mice. AC-YVAD-CMK, omeprazole, or vehicle were administered to mice before cold-restraint stress- or ethanol-induced gastric injury. Survival rates and histological evidence of gastric injury of mice pretreated with AC-YVAD-CMK or omeprazole, and exposed to cold-restraint stress, improved significantly relative to the vehicle group. The increased levels of tumour necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-18 following cold-stress injury were decreased by AC-YVAD-CMK, but not omeprazole, pretreatment. The increased expression of CD68 in gastric tissues was inhibited significantly by AC-YVAD-CMK pretreatment. Inhibiting caspase-1 activity in the NLRP3 inflammasome decreased gastric cell apoptosis, and the expression of Bax and cleaved caspase-3. AC-YVAD-CMK pretreatment significantly inhibited cold-restraint stress-induced increases in the expression of phosphorylated IκB-alpha and P38. General anatomy and histological results showed the protective effect of AC-YVAD-CMK on ethanol-induced acute gastric injury. Overall, our results showed that the caspase-1 inhibitor AC-YVAD-CMK protected against acute gastric injury in mice by affecting the NLRP3 inflammasome and attenuating inflammatory processes and apoptosis. This was similar to the mechanism associated with NF-κB and P38 mitogen-activated protein kinase signalling pathways. PMID:27053298

  12. The Molecular Basis for Load-Induced Skeletal Muscle Hypertrophy

    PubMed Central

    Marcotte, George R.; West, Daniel W.D.; Baar, Keith

    2016-01-01

    In a mature (weight neutral) animal, an increase in muscle mass only occurs when the muscle is loaded sufficiently to cause an increase in myofibrillar protein balance. A tight relationship between muscle hypertrophy, acute increases in protein balance, and the activity of the mechanistic target of rapamycin complex 1 (mTORC1) was demonstrated 15 years ago. Since then, our understanding of the signals that regulate load-induced hypertrophy has evolved considerably. For example, we now know that mechanical load activates mTORC1 in the same way as growth factors, by moving TSC2 (a primary inhibitor of mTORC1) away from its target (the mTORC activator) Rheb. However, the kinase that phosphorylates and moves TSC2 is different in the two processes. Similarly, we have learned that a distinct pathway exists whereby amino acids activate mTORC1 by moving it to Rheb. While mTORC1 remains at the forefront of load-induced hypertrophy, the importance of other pathways that regulate muscle mass are becoming clearer. Myostatin, is best known for its control of developmental muscle size. However, new mechanisms to explain how loading regulates this process are suggesting that it could play an important role in hypertrophic muscle growth as well. Lastly, new mechanisms are highlighted for how β2 receptor agonists could be involved in load-induced muscle growth and why these agents are being developed as non-exercise-based therapies for muscle atrophy. Overall, the results highlight how studying the mechanism of load-induced skeletal muscle mass is leading the development of pharmaceutical interventions to promote muscle growth in those unwilling or unable to perform resistance exercise. PMID:25359125

  13. 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.…

  14. Load-Induced Confinement Activates Diamond Lubrication by Water

    NASA Astrophysics Data System (ADS)

    Zilibotti, G.; Corni, S.; Righi, M. C.

    2013-10-01

    Tribochemical reactions are chemical processes, usually involving lubricant or environment molecules, activated at the interface between two solids in relative motion. They are difficult to be monitored in situ, which leaves a gap in the atomistic understanding required for their control. Here we report the real-time atomistic description of the tribochemical reactions occurring at the interface between two diamond films in relative motion, by means of large scale ab initio molecular dynamics. We show that the load-induced confinement is able to catalyze diamond passivation by water dissociative adsorption. Such passivation decreases the energy of the contacting surfaces and increases their electronic repulsion. At sufficiently high coverages, the latter prevents surface sealing, thus lowering friction. Our findings elucidate effects of the nanoscale confinement on reaction kinetics and surface thermodynamics, which are important for the design of new lubricants.

  15. Noise suppression by flexible fan silencers

    SciTech Connect

    Partyka, J.; Kelly, T.R.J.

    1995-12-31

    This paper presents the results on noise testing of a fan only, as well as the results of a steel silencer and of flexible silencers that were connected directly to a fan. On-site facilities and free-field method set by the British Standards Institution were used to measure and then compare the fan only and different practical silencer configuration setups. In order to determine the fan-silencer combination that would give the maximum noise attenuation, total noise intensity, noise contributed to by the fan motor only, as well as aerodynamical noise created through air interacting with the fan parts were considered to obtain decibel readings for the octave bands. Subsequently, the optimal configuration found was the setup with flexible silencers on the fan inlet and the fan outlet. If only one silencer is used, it should be installed on the fan inlet. The aerodynamic noise affects the low frequencies. The flow noise is then overtaken at 1 kHz by the mechanical noise.

  16. RNA silencing: an antiviral mechanism.

    PubMed

    Csorba, T; Pantaleo, V; Burgyán, J

    2009-01-01

    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 which can counteract the host silencing-based antiviral process. After the discovery of virus-encoded silencing suppressors, it was shown that these viral proteins can target one or more key points in the silencing machinery. Here we review recent progress in our understanding of the mechanism and function of antiviral RNA silencing in plants, and on the virus's counterattack by expression of silencing-suppressor proteins. We also discuss emerging evidence that RNA silencing and expression of viral silencing-suppressor proteins are tools forged as a consequence of virus-host coevolution for fine-tuning host-pathogen coexistence. PMID:20109663

  17. Analysis and design of pod silencers

    NASA Astrophysics Data System (ADS)

    Munjal, M. L.

    2003-05-01

    Parallel baffle mufflers or split silencers are used extensively in heating, ventilation and air conditioning systems for increased attenuation of noise within a short or given length. Acoustic analysis of rectangular parallel baffle mufflers runs on the same lines as that of a rectangular duct lined on two sides. This simplification would not hold for circular configurations. Often, a cylindrical pod is inserted into a circular lined duct to increase its attenuation (or transmission loss), thereby making the flow passage annular and providing an additional absorptive layer on the inner side of this annular passage. This configuration, called a pod silencer, is analyzed here for the four-pole parameters as well as transmission loss, making use of the bulk reaction model. The effect of thin protective film or a highly perforated metallic plate is duly incorporated by means of a grazing-flow impedance. Use of appropriate boundary conditions leads to a set of linear homogeneous equations which in turn lead to a transcendental frequency equation in the unknown complex axial wave number. This is solved by means of the Newton-Raphson method, and the axial wave number is then used in the expressions for transmission loss as well as the transfer matrix parameters. Finally, results of a parametric study are reported to help the designer in optimization of a pod silencer configuration within a given overall size for minimal cost.

  18. The Fruit of Silence

    ERIC Educational Resources Information Center

    Nelson, Marilyn

    2006-01-01

    This presentation explores how contemplative practices, especially those anchored in an active listening to silence, are integrated into creative writing courses. It pays particular attention to a course taught at the United States Military Academy at West Point and to a course on the poetry of war and peace taught at the University of…

  19. The Gift of Silence

    ERIC Educational Resources Information Center

    Haskins, Cathleen

    2011-01-01

    Slowing down, quieting the mind and body, and experiencing silence nourishes the spirit. Montessori educators are mandated to cultivate not just the intellect but the whole child. They recognize that nurturing the spirit of the child is part of what makes this form of education work so well. This article discusses the benefits of stillness and…

  20. The Conspiracy Of Silence

    PubMed Central

    Cohen, Saul

    1980-01-01

    The issue of the impaired physician is compounded by not only mass denial of the problem, but also a 'conspiracy of silence' among many groups associated with the physician. The conspirators—including the physician himself, his family, community, professional colleagues and nurses as well as hospital boards and administration—are unable to reconcile deteriorating performance due to alcohol or drugs with an otherwise gifted professional who should know the dangers of substance abuse. They may also fear the effects of labels such as 'alcoholism', 'drug abuse' and 'psychiatric illness', and they may not know how to handle the problem. A Saskatchewan Medical Association committee was formed in 1976 to penetrate the shroud of silence by identifying and rehabilitating impaired physicians. However, the committee's experience since that time has been largely frustrating because its function has been viewed as more punitive than therapeutic. Imagesp849-a PMID:21293652

  1. Small interference RNA-mediated gene silencing of human biliverdin reductase, but not that of heme oxygenase-1, attenuates arsenite-mediated induction of the oxygenase and increases apoptosis in 293A kidney cells.

    PubMed

    Miralem, Tihomir; Hu, Zhenbo; Torno, Michael D; Lelli, Katherine M; Maines, Mahin D

    2005-04-29

    BVR reduces biliverdin, the HO-1 and HO-2 product, to bilirubin. Human biliverdin (BVR) is a serine/threonine kinase activated by free radicals. It is a leucine zipper (bZip) DNA-binding protein and a regulatory factor for 8/7-bp AP-1-regulated genes, including HO-1 and ATF-2/CREB. Presently, small interference (si) RNA constructs were used to investigate the role of human BVR in sodium arsenite (As)-mediated induction of HO-1 and in cytoprotection against apoptosis. Activation of BVR involved increased serine/threonine phosphorylation but not its protein or transcript levels. The peak activity at 1 h (4-5-fold) after treatment of 293A cells with 5 mum As preceded induction of HO-1 expression by 3 h. The following suggests BVR involvement in regulating oxidative stress response of HO-1: siBVR attenuated As-mediated increase in HO-1 expression; siBVR, but not siHO-1, inhibited As-dependent increased c-jun promoter activity; treatment of cells with As increased AP-1 binding of nuclear proteins; BVR was identified in the DNA-protein complex; and AP-1 binding of the in vitro translated BVR was phosphorylation-dependent and was attenuated by biliverdin. Most unexpectedly, cells transfected with siBVR, but not siHO-1, displayed a 4-fold increase in apoptotic cells when treated with 10 mum As as detected by flow cytometry. The presence of BVR small interference RNA augmented the effect of As on levels of cytochrome c, TRAIL, and DR-5 mRNA and cleavage of poly(ADP-ribose) polymerase. The findings describe the function of BVR in HO-1 oxidative response and, demonstrate, for the first time, not only that BVR advances the role of HO-1 in cytoprotection but also affords cytoprotection independent of heme degradation. PMID:15741166

  2. Hybrid silencers with micro-perforated panels and internal partitions.

    PubMed

    Yu, Xiang; Cheng, Li; You, Xiangyu

    2015-02-01

    A sub-structuring approach, along with a unit cell treatment, is proposed to model expansion chamber silencers with internal partitions and micro-perforated panels (MPPs) in the absence of internal flow. The side-branch of the silencer is treated as a combination of unit cells connected in series. It is shown that, by connecting multiple unit cells with varying parameters, the noise attenuation bandwidth can be enlarged. With MPPs, the hybrid noise attenuation mechanism of the silencer is revealed. Depending on the size of the perforation hole, noise attenuation can be dominated by dissipative, reactive, or combined effects together. For a broadband sound absorption, the hole size, together with the perforation ratio and other parameters, can be optimized to strike a balance between the dissipative and reactive effect, for ultimately achieving the desired noise attenuation performance within a prescribed frequency region. The modular nature of the proposed formulation allows doing this in a flexible, accurate, and cost effective manner. The accuracy of the proposed approach is validated through comparisons with finite element method and experiments. PMID:25698027

  3. 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…

  4. Epigenetic silencing in transgenic plants

    PubMed Central

    Rajeevkumar, Sarma; Anunanthini, Pushpanathan; Sathishkumar, Ramalingam

    2015-01-01

    Epigenetic silencing is a natural phenomenon in which the expression of genes is regulated through modifications of DNA, RNA, or histone proteins. It is a mechanism for defending host genomes against the effects of transposable elements and viral infection, and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was the discovery of silencing in transgenic tobacco plants due to the interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, that is, transcriptional gene silencing, which is associated with heavy methylation of promoter regions and blocks the transcription of transgenes, and post-transcriptional gene silencing (PTGS), the basic mechanism is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of major concern in the transgenic technologies used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes is required. The current status of epigenetic silencing in transgenic technology is discussed and summarized in this mini-review. PMID:26442010

  5. Edwin Hubble's Silence

    NASA Astrophysics Data System (ADS)

    Lago, D.

    2013-04-01

    In late 1928 Edwin Hubble was right in the middle of using V. M. Slipher's redshift data to prove that the universe is expanding, when Hubble's boss, George Hale, directed him to drop everything and rush to the Grand Canyon and test it as a possible site for Hale's planned 200-inch telescope. On his way, Hubble stopped at Lowell Observatory and met with V. M. Slipher. The letters both men wrote about this visit suggest that Hubble never said a word about his being in the middle of using Slipher's research to transform the universe. At the least, this silence is symbolic of the silence with which astronomical history has often treated Slipher's work. A survey of the historical literature suggests several reasons for this. Theorists and observers in astronomy (and other sciences) have long had different perspectives about how science works, and those who place more importance on theory have tended to credit the idea of the expanding universe to the theorists. Also, many sources indicate that Edwin Hubble was not a modest man or generous about sharing credit.

  6. The Search for Strategic Silence.

    ERIC Educational Resources Information Center

    Lentz, Richard

    Media content analysts seldom observe the principle that editorial omissions are as telling as what is published or broadcast; hence, the purpose of this paper is to explore, and thus stimulate debate about, editorial omissions or "strategic silence." It is observed that as a concept, strategic silence embraces both tact and strategy--the former…

  7. The eerie silence

    NASA Astrophysics Data System (ADS)

    Davies, Paul

    2010-03-01

    Whether or not we are alone in the universe is one of the great outstanding questions of existence. For thousands of years it was restricted to the realm of philosophy and theology, but 50 years ago it became part of science. In April 1960 a young US astronomer, Frank Drake, began using a radio telescope to investigate whether signals from an extraterrestrial community might be coming our way. Known as the Search for Extraterrestrial Intelligence, or SETI, it has grown into a major international enterprise, involving scientific institutions in several countries. Apart from a few oddities, however, all that the radio astronomers have encountered is an eerie silence. So is humankind the only technological civilization in the universe after all? Or might we be looking for the wrong thing in the wrong place at the wrong time?

  8. The rest is silence.

    PubMed Central

    Bernstein, E; Denli, A M; Hannon, G J

    2001-01-01

    Over the past several years, RNAi and its related phenomena have emerged not only as a powerful experimental tool but also as a new mode of gene regulation. Through a combination of genetic and biochemical approaches we have learned much about the mechanisms underlying dsRNA responses. However, many of the most intriguing aspects of dsRNA-induced gene silencing have yet to be illuminated. What has become abundantly clear is that the complex and highly conserved biology underlying RNA interference is critical both for genome maintenance and for the development of complex organisms. However, it seems probable that we have only begun to reveal the diversity of biological roles played by RNAi-related processes. PMID:11720281

  9. Silence Amenity Engineering

    NASA Astrophysics Data System (ADS)

    Fujita, Hajime

    Engineering civilization brought convenient and comfortable life to us. However, some environmental problems such as various pollutions have also been developed with it. Acoustical noise is one of the major problems in modern life. Noise is generated from a noise source and propagates through transmitting medium such as the air and eventually reaches a receiver, usually a human being. The noise problem can be avoided, therefore, if one of those three elements in the noise problem is removed completely. In actual case, engineers are looking for most efficient way combining the controls for these three elements. In this article, basic characteristics of noise is reviewed briefly at first, then sound field analysis to predict sound transmission is discussed Aerodynamic noise is one of the major problems in silence amenity engineering today. Basic concept of the aerodynamic noise generation mechanism is discussed in detail with applications to turbo-machinery and high speed train noise control technology.

  10. Probabilistic evaluation of initiation time in RC bridge beams with load-induced cracks exposed to de-icing salts

    SciTech Connect

    Lu Zhaohui; Zhao Yangang; Yu Zhiwu; Ding Faxing

    2011-03-15

    In this study, a reliability-based method for predicting the initiation time of reinforced concrete bridge beams with load-induced cracks exposed to de-icing salts is presented. A practical model for predicting the diffusion coefficient of chloride ingress into load-induced cracked concrete is proposed. Probabilistic information about uncertainties related to the surface chloride content and the threshold chloride concentration has been estimated from a wide review of previous experimental or statistical studies. Probabilistic analysis to estimate the time to corrosion initiation with/without considering the effect of the load-induced cracks on the chloride ingress into concrete has been carried out. Results of the analysis demonstrate the importance of considering the effect of the load-induced cracks for correct prediction of corrosion initiation in RC bridge beams exposed to chlorides.

  11. 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)

  12. RNA silencing movement in plants.

    PubMed

    Mermigka, Glykeria; Verret, Frédéric; Kalantidis, Kriton

    2016-04-01

    Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant, respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review. PMID:26297506

  13. Traffic into silence: endomembranes and post-transcriptional RNA silencing

    PubMed Central

    Kim, Yun Ju; Maizel, Alexis; Chen, Xuemei

    2014-01-01

    microRNAs (miRNAs) and small interfering RNAs (siRNAs) are small RNAs that repress gene expression at the post-transcriptional level in plants and animals. Small RNAs guide Argonaute-containing RNA-induced silencing complexes to target RNAs in a sequence-specific manner, resulting in mRNA deadenylation followed by exonucleolytic decay, mRNA endonucleolytic cleavage, or translational inhibition. Although our knowledge of small RNA biogenesis, turnover, and mechanisms of action has dramatically expanded in the past decade, the subcellular location of small RNA-mediated RNA silencing still needs to be defined. In contrast to the prevalent presumption that RNA silencing occurs in the cytosol, emerging evidence reveals connections between the endomembrane system and small RNA activities in plants and animals. Here, we summarize the work that uncovered this link between small RNAs and endomembrane compartments and present an overview of the involvement of the endomembrane system in various aspects of RNA silencing. We propose that the endomembrane system is an integral component of RNA silencing that has been long overlooked and predict that a marriage between cell biology and RNA biology holds the key to a full understanding of post-transcriptional gene regulation by small RNAs. PMID:24668229

  14. RNA Polymerase I and Fob1 contributions to transcriptional silencing at the yeast rDNA locus.

    PubMed

    Buck, Stephen W; Maqani, Nazif; Matecic, Mirela; Hontz, Robert D; Fine, Ryan D; Li, Mingguang; Smith, Jeffrey S

    2016-07-27

    RNA polymerase II (Pol II)-transcribed genes embedded within the yeast rDNA locus are repressed through a Sir2-dependent process called 'rDNA silencing'. Sir2 is recruited to the rDNA promoter through interactions with RNA polymerase I (Pol I), and to a pair of DNA replication fork block sites (Ter1 and Ter2) through interaction with Fob1. We utilized a reporter gene (mURA3) integrated adjacent to the leftmost rDNA gene to investigate localized Pol I and Fob1 functions in silencing. Silencing was attenuated by loss of Pol I subunits or insertion of an ectopic Pol I terminator within the adjacent rDNA gene. Silencing left of the rDNA array is naturally attenuated by the presence of only one intact Fob1 binding site (Ter2). Repair of the 2nd Fob1 binding site (Ter1) dramatically strengthens silencing such that it is no longer impacted by local Pol I transcription defects. Global loss of Pol I activity, however, negatively affects Fob1 association with the rDNA. Loss of Ter2 almost completely eliminates localized silencing, but is restored by artificially targeting Fob1 or Sir2 as Gal4 DNA binding domain fusions. We conclude that Fob1 and Pol I make independent contributions to establishment of silencing, though Pol I also reinforces Fob1-dependent silencing. PMID:27060141

  15. Ice-load induced tectonics controlled tunnel valley evolution - instances from the southwestern Baltic Sea

    NASA Astrophysics Data System (ADS)

    Al Hseinat, M.; Hübscher, C.

    2014-08-01

    Advancing ice sheets have a strong impact on the earth's topography. For example, they leave behind an erosional unconformity, bulldozer the underlying strata and form tunnel valleys, primarily by subglacial melt-water erosion and secondarily by direct glacial erosion. The conceptual models of the reactivation of faults within the upper crust, due to the ice sheets' load, are also established. However, this phenomenon is also rather under-explored. Here, we propose a causal link between ice-load induced tectonics, the generation of near-vertical faults in the upper crust above an inherited deep-rooted fault and the evolution of tunnel valleys. The Kossau tunnel valley in the southeastern Bay of Kiel has been surveyed by means of high-resolution multi-channel seismic and echosounder data. It strikes almost south to north and can be mapped over a distance of ca 50 km. It is 1200-8000 m wide with a valley of up to 200 m deep. Quaternary deposits fill the valley and cover the adjacent glaciogenic unconformity. A near-vertical fault system with an apparent dip angle of >80°, which reaches from the top Zechstein upwards into the Quaternary, underlies the valley. The fault partially pierces the seafloor and growth is observed within the uppermost Quaternary strata only. Consequently, the fault evolved in the Late Quaternary. The fault is associated with an anticline that is between 700 and 3000 m wide and about 20-40 m high. The fault-anticline assemblage neither resembles any typical extensional, compressional or strike-slip deformation pattern, nor is it related to salt tectonics. Based on the observed position and deformation pattern of the fault-anticline assemblage, we suggest that these structures formed as a consequence of the differential ice-load induced tectonics above an inherited deep-rooted sub-salt fault related to the Glückstadt Graben. Lateral variations in the ice-load during the ice sheet's advance caused differential subsidence, thus rejuvenating the

  16. Communicative Silences: Forms and Functions

    ERIC Educational Resources Information Center

    Bruneau, Thomas J.

    1973-01-01

    The nature of silence is discussed as an imposition of mind, as an interdependent signification ground for speech signs, as a relationship to mental time (as opposed to artificial time), and as it relates to sensation, perception and metaphorical movement. (Author)

  17. Silence as the Foundation of Learning

    ERIC Educational Resources Information Center

    Caranfa, Angelo

    2004-01-01

    Past and present discussions on education all too frequently neglect the role that silence plays in learning. In this article I set out to demonstrate that silence is the very foundation of learning. My claim is that we must find ways of freeing silence in our pedagogical practices so that our discourse does not denigrate into mere empty words,…

  18. Teaching Note: Gaining Voice through Silence

    ERIC Educational Resources Information Center

    Kaufman, Peter

    2008-01-01

    For educators striving to create an egalitarian classroom based on open, reflexive, and honest dialogue, silence can be the ultimate obstacle. Not only does silence stifle individual expression; more importantly, silence also prevents the collective production of knowledge, understanding, compassion, and empathy. When learners and teachers feel…

  19. Is silence killing your company?

    PubMed

    Perlow, Leslie; Williams, Stephanie

    2003-05-01

    Many times, often with the best of intentions, people at work decide it's more productive to remain silent about their differences than to air them. There's no time, they think, or no point in going against what the boss says. But as new research by the authors shows, silencing doesn't smooth things over or make people more productive. It merely pushes differences beneath the surface and can set in motion powerfully destructive forces. When people stay silent about important disagreements, they can begin to fill with anxiety, anger, and resentment. As long as the conflict is unresolved, their repressed feelings remain potent, making them increasingly distrustful, self-protective, and all the more fearful that if they speak up they will be embarrassed or rejected. Their sense of insecurity grows, leading to further acts of silence, more defensiveness, and more distrust, thereby setting into motion a destructive "spiral of silence." Sooner or later, they mentally opt out--sometimes merely doing what they're told but contributing nothing of their own, sometimes spreading discontent and frustration throughout the workplace that can lead them, and others, to leave without thinking it through. These vicious spirals of silence can be replaced with virtuous spirals of communication, but that requires individuals to find the courage to act differently and executives to create the conditions in which people will value the expression of differences. All too often, behind failed products, broken processes, and mistaken career decisions are people who chose to hold their tongues. Breaking the silence can bring an outpouring of fresh ideas from all levels of an organization--ideas that might just raise the organization's performance to a whole new level. PMID:12747162

  20. Load-induced debonding of FRP composites applied to reinforced concrete

    NASA Astrophysics Data System (ADS)

    Blok, Joel; Brown, Jeff

    2009-05-01

    Fiber-reinforced polymer (FRP) composites are widely used to increase the flexural and shear capacity of reinforced concrete (RC) elements. One potential disadvantage is that strengthened surfaces are no longer visible and cracks or delaminations that result from excessive loading or fatigue may go undetected. This research investigated thermal imaging techniques for monitoring and evaluating load-induced delamination of FRP composites applied to small scale RC beams. Two beams (3.5 in x 4.5 in x 58 in) were loaded monotonically to failure. Infrared thermography (IRT) inspections were performed at various load levels through failure using a composite phase imaging technique. Two similar beams were tested in fatigue and periodic IRT inspections were performed at 50,000-cycle intervals. Individual phase values for each pixel were designated as "well-bonded", "suspect" or "unbonded" to indicate the quality of FRP bond. Suspect areas included regions of excess thickened-epoxy tack-coat and smaller installation defects in the unloaded specimens. The long-term objective of this research is to develop a practical framework for conducting quantitative IRT inspections of FRP composites applied to RC and incorporating these results into acceptance criteria for new installations and predictions for the remaining service life of in-service FRP systems. This method may also offer insight into the necessity for repairs to in-service systems.

  1. Loading-Induced Heat-Shock Response in Bovine Intervertebral Disc Organ Culture.

    PubMed

    Chooi, Wai Hon; Chan, Samantha Chun Wai; Gantenbein, Benjamin; Chan, Barbara Pui

    2016-01-01

    Mechanical loading has been shown to affect cell viability and matrix maintenance in the intervertebral disc (IVD) but there is no investigation on how cells survive mechanical stress and whether the IVD cells perceive mechanical loading as stress and respond by expression of heat shock proteins. This study investigates the stress response in the IVD in response to compressive loading. Bovine caudal disc organ culture was used to study the effect of physiological range static loading and dynamic loading. Cell activity, gene expression and immunofluorescence staining were used to analyze the cell response. Cell activity and cytoskeleton of the cells did not change significantly after loading. In gene expression analysis, significant up-regulation of heat shock protein-70 (HSP70) was observed in nucleus pulposus after two hours of loading. However, the expression of the matrix remodeling genes did not change significantly after loading. Similarly, expressions of stress response and matrix remodeling genes changed with application and removal of the dynamic loading. The results suggest that stress response was induced by physiological range loading without significantly changing cell activity and upregulating matrix remodeling. This study provides direct evidence on loading induced stress response in IVD cells and contributes to our understanding in the mechanoregulation of intervertebral disc cells. PMID:27580124

  2. Transcriptional gene silencing in humans.

    PubMed

    Weinberg, Marc S; Morris, Kevin V

    2016-08-19

    It has been over a decade since the first observation that small non-coding RNAs can functionally modulate epigenetic states in human cells to achieve functional transcriptional gene silencing (TGS). TGS is mechanistically distinct from the RNA interference (RNAi) gene-silencing pathway. TGS can result in long-term stable epigenetic modifications to gene expression that can be passed on to daughter cells during cell division, whereas RNAi does not. Early studies of TGS have been largely overlooked, overshadowed by subsequent discoveries of small RNA-directed post-TGS and RNAi. A reappraisal of early work has been brought about by recent findings in human cells where endogenous long non-coding RNAs function to regulate the epigenome. There are distinct and common overlaps between the proteins involved in small and long non-coding RNA transcriptional regulatory mechanisms, suggesting that the early studies using small non-coding RNAs to modulate transcription were making use of a previously unrecognized endogenous mechanism of RNA-directed gene regulation. Here we review how non-coding RNA plays a role in regulation of transcription and epigenetic gene silencing in human cells by revisiting these earlier studies and the mechanistic insights gained to date. We also provide a list of mammalian genes that have been shown to be transcriptionally regulated by non-coding RNAs. Lastly, we explore how TGS may serve as the basis for development of future therapeutic agents. PMID:27060137

  3. [A gun silencer of a special kind].

    PubMed

    Schyma, C; Schyma, P; Milbradt, H

    2000-01-01

    The authors report about a small bore pistol with silencer. In addition, the silencer can be sealed up by a rubber plate. This leads to a false estimation of the shooting distance. Also at close range shots the shot with the silencer and the rubber seal leaves hardly gunshot residues. The bullet wipe persists but microradiography shows his changed morphological composition. The use of the rubber seal leads predominantly to atypical bullet holes. PMID:10829239

  4. [E. M. Jellinek's silenced and silencing transgenerational story].

    PubMed

    Kelemen, Gábor; Márk, Mónika

    2013-01-01

    Jellinek is a kind of archetypal character for future generations in the field of addiction studies. His implosion in the arena of alcoholism around the age of 50 was an unexpected challenge to medical science. We know very little about his own role models giving an intellectual and moral compass to his pragmatic creativity. More than 30 years has passed since Jellinek's death when an American sociologist Ron Roizen started unearthing his silent story. Roizen discerned that there are a lot of unsaid and muted issues in his personal Hungarian past. Our paper, based on the authors' research in Hungarian archives and other sources reveals that not just Jellinek's personal but his transgenerational narrative has been not-yet-said. This silenced and silencing history appears an unfinished business of acculturation of the family, which started prior to four generations. Authors have been concluding that the issue of religious conversion is a critical point in the process of acculturation. They examine the counter move of loyalty to family values and driving force of assimilation making their story unspeakable. PMID:24443572

  5. Silence Is Consent, or Curse Ye Meroz!

    ERIC Educational Resources Information Center

    Levin, Richard

    1997-01-01

    Examines assumptions of "oppositional" literary criticism, namely the assumption that older-style "objective" literary criticism must, in its political silence, be supportive of dominant ideologies. (TB)

  6. "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…

  7. Quantification of Crack Interaction in Loading-Induced Rock Deformation Tests

    NASA Astrophysics Data System (ADS)

    Reyes-Montes, J. M.; Goodfellow, S. D.; Nasseri, M. H.; Young, R.

    2013-12-01

    The coalescence of microcracks into major fractures marks the onset of major damage in rock and induces significant changes in its geotechnical and sealing properties. The interaction between neighbouring fractures affects the deformation process leading to this major failure. This study presents the analysis of load-induced fracture using spatial and temporal patterns of recorded acoustic emission (AE) events to investigate the role of crack interaction during fracturing processes Two methods based on event separation are used to estimate correlation and interaction distances between events. The first method uses the spacing of sequential and non-sequential event pairs, thus, the distance bin where both distributions converge can be interpreted as an upper bound for the interaction distance of correlated events in each of the experiments. The second method compares the distribution of inter-event separation of recorded events with the distribution obtained for a random population of events, quantifying the divergence between both distributions and its variation with time. A positive deviation for a distance range including up to the interaction distance observed in the previous approach indicates spatial clustering and process dominated by interactive events. This deviation is used to quantify the degree of event interaction or degree of non-randomness (NR) during the process. Two load-stress induced fracturing tests, carried out at two different scales, are analysed in this study to investigate the role of crack interaction in the deformation and fracturing process. The first test registered a total of 15,198 AE events from a pillar created between two deposition holes at SKB's Aspö Pillar Stability Experiment. The results show a good correlation between maxima of NR and minima in b-value in the time period immediately preceding and during the development of the observed major damage in form of spalling. The result can be interpreted as an indication of spalling

  8. Systemic silencing signal(s).

    PubMed

    Fagard, M; Vaucheret, H

    2000-06-01

    Grafting experiments have revealed that transgenic plants that undergo co-suppression of homologous transgenes and endogenous genes or PTGS of exogenous transgenes produce a sequence-specific systemic silencing signal that is able to propagate from cell to cell and at long distance. Similarly, infection of transgenic plants by viruses that carry (part of) a transgene sequence results in global silencing (VIGS) of the integrated transgenes although viral infection is localized. Systemic PTGS and VIGS strongly resemble recovery from virus infection in non-transgenic plants, leading to protection against secondary infection in newly emerging leaves and PTGS of transiently expressed homologous transgenes. The sequence-specific PTGS signal is probably a transgene product (for example, aberrant RNA) or a secondary product (for example, RNA molecules produced by an RNA-dependent RNA polymerase with transgene RNA as a matrix) that mimics the type of viral RNA that is targeted for degradation by cellular defence. Whether some particular cases of transgene TGS could also rely on the production of such a mobile molecule is discussed. PMID:10999411

  9. 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…

  10. Venturis as silencers in a BOF stack

    SciTech Connect

    Kelsall, T.; Gerritsen, T.; Landon, T.

    1995-06-01

    Installation of a venture in a BOF stack reduced the sound level in a local community by 10 to 15 db. This application resulted in the development of a new type of fan silencer, called the modal silencer, having the inherent advantages of low pressure drop and less maintenance compared with conventional types.

  11. SILENCING POLYGALACTURONASE EXPRESSION INHIBITS TOMATO PETIOLE ABSCISSION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We used Virus Induced Gene Silencing (VIGS) as a tool for functional analysis of cell-wall associated genes that have been suggested to be involved in leaf abscission. Tobacco rattle virus (TRV) is an effective vector for VIGS in tomato (Lycopersicon esculentum). Silencing was more efficient when ...

  12. Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation

    PubMed Central

    Szittya, György; Silhavy, Dániel; Molnár, Attila; Havelda, Zoltán; Lovas, Ágnes; Lakatos, Lóránt; Bánfalvi, Zsófia; Burgyán, József

    2003-01-01

    Temperature dramatically affects plant–virus interactions. Outbreaks of virus diseases are frequently associated with low temperature, while at high temperature viral symptoms are often attenuated (heat masking) and plants rapidly recover from virus diseases. However, the underlying mechanisms of these well-known observations are not yet understood. RNA silencing is a conserved defence system of eukaryotic cells, which operates against molecular parasites including viruses and transgenes. Here we show that at low temperature both virus and transgene triggered RNA silencing are inhibited. Therefore, in cold, plants become more susceptible to viruses, and RNA silencing-based phenotypes of transgenic plants are lost. Consistently, the levels of virus- and transgene-derived small (21–26 nucleotide) interfering (si) RNAs—the central molecules of RNA silencing-mediated defence pathways—are dramatically reduced at low temperature. In contrast, RNA silencing was activated and the amount of siRNAs gradually increased with rising temperature. However, temperature does not influence the accumulation of micro (mi) RNAs, which play a role in developmental regulation, suggesting that the two classes of small (si and mi) RNAs are generated by different nuclease complexes. PMID:12554663

  13. Inhibition of IκB phosphorylation prevents load-induced cardiac dysfunction in mice.

    PubMed

    Tanaka, Tetsu; Ogawa, Masahito; Suzuki, Jun-ichi; Sekinishi, Asuka; Itai, Akiko; Hirata, Yasunobu; Nagai, Ryozo; Isobe, Mitsuaki

    2012-12-15

    Pressure overload is known to be a cause of cardiac hypertrophy that often transits to heart failure. Although nuclear factor (NF)-κB is a key factor in the progression of cardiac hypertrophy, its pathophysiology is yet to be elucidated. Thus, we aimed to show that inhibition of NF-κB activation improves pressure overload-induced cardiac dysfunction. To assess the effect of inhibition on NF-κB activation in pressure overload cardiac hypertrophy, we used IMD-1041 in a murine thoracic aortic constriction (TAC) model. IMD-1041 inhibits the phosphorylation of IκB via inhibition of IκB kinase-β. IMD-1041 (100 mg·kg(-1)·day(-1)) or vehicle was administered orally into mice once a day, and mice were euthanized on day 42 after TAC. TAC resulted in left ventricular wall thickening, cardiac dysfunction, and increases of heart and lung weight, whereas IMD-1041 significantly suppressed the development of cardiac hypertropy 6 wk after TAC. Histologically, developed cardiac fibrosis and cardiomyocyte hypertrophy occurred in the vehicle-treated group, whereas IMD-1041 significantly attenuated these changes. IMD-1041 suppressed the expression of p65-positive cells and nuclear translocation of p65 induced by TAC compared with vehicle. Matrix metalloproteinase-2 activity increased in the vehicle + TAC-treated group; however, it was suppressed in the IMD-1041 + TAC-treated group. IMD-1041 treatment from day 28 to day 42 after TAC significantly attenuated the decrease in the percentage of fractional shortening and cardiac fibrosis without an antihypertrophic effect. In conclusion, IMD-1041 may be useful for preventing pressure overload-induced cardiac dysfunction and the transition of cardiac hypertrophy to contraction failure via suppression of NF-κB activation. PMID:23042949

  14. Mammalian meiotic silencing exhibits sexually dimorphic features.

    PubMed

    Cloutier, J M; Mahadevaiah, S K; ElInati, E; Tóth, A; Turner, James

    2016-06-01

    During mammalian meiotic prophase I, surveillance mechanisms exist to ensure that germ cells with defective synapsis or recombination are eliminated, thereby preventing the generation of aneuploid gametes and embryos. Meiosis in females is more error-prone than in males, and this is in part because the prophase I surveillance mechanisms are less efficient in females. A mechanistic understanding of this sexual dimorphism is currently lacking. In both sexes, asynapsed chromosomes are transcriptionally inactivated by ATR-dependent phosphorylation of histone H2AFX. This process, termed meiotic silencing, has been proposed to perform an important prophase I surveillance role. While the transcriptional effects of meiotic silencing at individual genes are well described in the male germ line, analogous studies in the female germ line have not been performed. Here we apply single- and multigene RNA fluorescence in situ hybridization (RNA FISH) to oocytes from chromosomally abnormal mouse models to uncover potential sex differences in the silencing response. Notably, we find that meiotic silencing in females is less efficient than in males. Within individual oocytes, genes located on the same asynapsed chromosome are silenced to differing extents, thereby generating mosaicism in gene expression profiles across oocyte populations. Analysis of sex-reversed XY female mice reveals that the sexual dimorphism in silencing is determined by gonadal sex rather than sex chromosome constitution. We propose that sex differences in meiotic silencing impact on the sexually dimorphic prophase I response to asynapsis. PMID:26712235

  15. The gifts of silence and solitude.

    PubMed

    Schmidt Bunkers, Sandra

    2008-01-01

    In this column the author describes the importance of finding silence and solitude amid the noise and technology present today in the teaching-learning academy. Three gifts of silence and solitude are identified: the gift of comforting aloneness, the gift of vision for new horizons, and the gift of a sense of freedom. A humanbecoming perspective is used to explore the implications of these gifts. This column introduces a column by Diana Vander Woude describing her teaching-learning experience in leadership focusing on silence and solitude. PMID:18096981

  16. Rotary antenna attenuator

    NASA Technical Reports Server (NTRS)

    Dickinson, R. M.; Hardy, J. C.

    1969-01-01

    Radio frequency attenuator, having negligible insertion loss at minimum attenuation, can be used for making precise antenna gain measurements. It is small in size compared to a rotary-vane attenuator.

  17. Tomato mosaic virus replication protein suppresses virus-targeted posttranscriptional gene silencing.

    PubMed

    Kubota, Kenji; Tsuda, Shinya; Tamai, Atsushi; Meshi, Tetsuo

    2003-10-01

    Posttranscriptional gene silencing (PTGS), a homology-dependent RNA degradation system, has a role in defending against virus infection in plants, but plant viruses encode a suppressor to combat PTGS. Using transgenic tobacco in which the expression of green fluorescent protein (GFP) is posttranscriptionally silenced, we investigated a tomato mosaic virus (ToMV)-encoded PTGS suppressor. Infection with wild-type ToMV (L strain) interrupted GFP silencing in tobacco, coincident with visible symptoms, whereas some attenuated strains of ToMV (L(11) and L(11)A strains) failed to suppress GFP silencing. Analyses of recombinant viruses containing the L and L(11)A strains revealed that a single base change in the replicase gene, which causes an amino acid substitution, is responsible for the symptomless and suppressor-defective phenotypes of the attenuated strains. An agroinfiltration assay indicated that the 130K replication protein acts as a PTGS suppressor. Small interfering RNAs (siRNAs) of 21 to 25 nucleotides accumulated during ToMV infection, suggesting that the major target of the ToMV-encoded suppressor is downstream from the production of siRNAs in the PTGS pathway. Analysis with GFP-tagged recombinant viruses revealed that the suppressor inhibits the establishment of the ToMV-targeted PTGS system in the inoculated leaves but does not detectably suppress the activity of the preexisting, sequence-specific PTGS machinery there. Taken together, these results indicate that it is likely that the ToMV-encoded suppressor, the 130K replication protein, blocks the utilization of silencing-associated small RNAs, so that a homology-dependent RNA degradation machinery is not newly formed. PMID:14512550

  18. Gene silencing of TACE enhances plaque stability and improves vascular remodeling in a rabbit model of atherosclerosis

    PubMed Central

    Zhao, Xueqiang; Kong, Jing; Zhao, Yuxia; Wang, Xuping; Bu, Peili; Zhang, Cheng; Zhang, Yun

    2015-01-01

    We aimed to test the hypothesis that gene silencing of tumor necrosis factor alpha converting enzyme (TACE) may attenuate lesion inflammation and positive vascular remodeling and enhance plaque stability in a rabbit model of atherosclerosis. Lentivirus-mediated TACE shRNA was injected into the abdominal aortic plaques of rabbits which effectively down-regulated TACE expression and activities from week 8 to week 16. TACE gene silencing reduced remodeling index and plaque burden, and diminished the content of macrophages and lipids while increased that of smooth muscle cells and collagen in the aortic plaques. In addition, TACE gene silencing attenuated the local expression of P65, iNOS, ICAM-1, VEGF and Flt-1 and activities of MMP9 and MMP2 while increased the local expression of TGF-β1 together with reduced number of neovessels in the aorta. TACE shRNA treatment resulted in down-regulated expression of TACE in macrophages and blunted ERK-P38 phosphorylation and tube formation of co-cultured mouse vascular smooth muscle cells or human umbilical vein endothelial cells. In conclusion, gene silencing of TACE enhanced plaque stability and improved vascular positive remodeling. The mechanisms may involve attenuated local inflammation, neovascularization and MMP activation, as well as enhanced collagen production probably via down-regulated ERK-NF-κB and up-regulated TGF-β1 signaling pathways. PMID:26655882

  19. Replication and transcription. Silence of the ORCs.

    PubMed

    Kelly, T J; Jallepalli, P V; Clyne, R K

    1994-03-01

    The origin recognition complex, a multi-protein complex known to bind to replication origins, has now been implicated in transcriptional silencing, providing another link between DNA replication and transcription. PMID:7857395

  20. Design of steam silencers for geothermal applications

    SciTech Connect

    Lazalde-Crabtree, H.

    1985-01-01

    Steam silencers are a means of reducing the loud noise caused by venting steam into the atmosphere as a consequence of load-reductions in a geothermal power plant. For new plants, or for those in which noise measurements cannot be made, an analytical method is given to determine the unsilenced noise levels. Designs fo two types of steam silencers, based on experimental work and theoretical considerations, are presented.

  1. Common themes in mechanisms of gene silencing.

    PubMed

    Moazed, D

    2001-09-01

    The assembly of DNA into regions of inaccessible chromatin, called silent chromatin, is involved in the regulation of gene expression and maintenance of chromosome stability in eukaryotes. Recent studies on Sir2-containing silencing complexes in budding yeast and HP1- and Swi6-containing silencing complexes in metazoans and fission yeast suggest a common mechanism for the assembly of these domains, which involves the physical coupling of histone modifying enzymes to histone binding proteins. PMID:11583612

  2. 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.

  3. Satellite RNAs interfere with the function of viral RNA silencing suppressors

    PubMed Central

    Shen, Wan-Xia; Au, Phil Chi Khang; Shi, Bu-Jun; Smith, Neil A.; Dennis, Elizabeth S.; Guo, Hui-Shan; Zhou, Chang-Yong; Wang, Ming-Bo

    2015-01-01

    Viral satellite RNAs (satRNAs) are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs), Cucumber mosaic virus (CMV) 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA)-induced silencing of a β-glucuronidase (GUS) gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat) via the Y-Sat-derived small interfering RNAs (siRNAs), which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function. PMID:25964791

  4. Rescue of Mtp siRNA-induced hepatic steatosis by DGAT2 siRNA silencing.

    PubMed

    Tep, Samnang; Mihaila, Radu; Freeman, Alexander; Pickering, Victoria; Huynh, Felicia; Huyhn, Felicia; Tadin-Strapps, Marija; Stracks, Allison; Hubbard, Brian; Caldwell, Jeremy; Flanagan, W Michael; Kuklin, Nelly A; Ason, Brandon

    2012-05-01

    Microsomal triglyceride transfer protein (Mtp) inhibitors represent a novel therapeutic approach to lower circulating LDL cholesterol, although therapeutic development has been hindered by the observed increase in hepatic triglycerides and liver steatosis following treatment. Here, we used small interfering RNAs (siRNA) targeting Mtp to achieve target-specific silencing to study this phenomenon and to determine to what extent liver steatosis is induced by changes in Mtp expression. We observed that Mtp silencing led to a decrease in many genes involved in hepatic triglyceride synthesis. Given the role of diacylglycerol O-acyltransferase 2 (Dgat2) in regulating hepatic triglyceride synthesis, we then evaluated whether target-specific silencing of both Dgat2 and Mtp were sufficient to attenuate Mtp silencing-induced liver steatosis. We showed that the simultaneous inhibition of Dgat2 and Mtp led to a decrease in plasma cholesterol and a reduction in the accumulation of hepatic triglycerides caused by the inhibition of Mtp. Collectively, these findings provide a proof-of-principle for a triglyceride synthesis/Mtp inhibitor combination and represent a potentially novel approach for therapeutic development in which targeting multiple pathways can achieve the desired response. PMID:22355095

  5. Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA

    PubMed Central

    Rothé, Benjamin; Leal-Esteban, Lucia; Bernet, Florian; Urfer, Séverine; Doerr, Nicholas; Weimbs, Thomas; Iwaszkiewicz, Justyna

    2015-01-01

    Loss of the RNA-binding protein Bicaudal-C (Bicc1) provokes renal and pancreatic cysts as well as ectopic Wnt/β-catenin signaling during visceral left-right patterning. Renal cysts are linked to defective silencing of Bicc1 target mRNAs, including adenylate cyclase 6 (AC6). RNA binding of Bicc1 is mediated by N-terminal KH domains, whereas a C-terminal sterile alpha motif (SAM) self-polymerizes in vitro and localizes Bicc1 in cytoplasmic foci in vivo. To assess a role for multimerization in silencing, we conducted structure modeling and then mutated the SAM domain residues which in this model were predicted to polymerize Bicc1 in a left-handed helix. We show that a SAM-SAM interface concentrates Bicc1 in cytoplasmic clusters to specifically localize and silence bound mRNA. In addition, defective polymerization decreases Bicc1 stability and thus indirectly attenuates inhibition of Dishevelled 2 in the Wnt/β-catenin pathway. Importantly, aberrant C-terminal extension of the SAM domain in bpk mutant Bicc1 phenocopied these defects. We conclude that polymerization is a novel disease-relevant mechanism both to stabilize Bicc1 and to present associated mRNAs in specific silencing platforms. PMID:26217012

  6. DC attenuation meter

    DOEpatents

    Hargrove, Douglas L.

    2004-09-14

    A portable, hand-held meter used to measure direct current (DC) attenuation in low impedance electrical signal cables and signal attenuators. A DC voltage is applied to the signal input of the cable and feedback to the control circuit through the signal cable and attenuators. The control circuit adjusts the applied voltage to the cable until the feedback voltage equals the reference voltage. The "units" of applied voltage required at the cable input is the system attenuation value of the cable and attenuators, which makes this meter unique. The meter may be used to calibrate data signal cables, attenuators, and cable-attenuator assemblies.

  7. 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.

  8. "Stop the noise!" From voice to silence.

    PubMed

    Newton, Lorelei; Storch, Janet L; Makaroff, Kara Schick; Pauly, Bernadette

    2012-03-01

    Nurses are frequently portrayed in the literature as being silent about ethical concerns that arise in their practice. This silence is often represented as a lack of voice. However, in our study, we found that nurses who responded to questions about moral distress were not so much silent as silenced. These nurses were enacting their moral agency by engaging in diverse, multiple and time-consuming actions in response to situations identified as morally distressing with families, colleagues, physicians, educators or managers. In many situations, they took action by contacting other healthcare team members, making referrals and coordinating care with other departments such as home care and hospice, as well as initiating contact with groups such as professional regulatory bodies or unions. Examining the relationship between ethical climate, moral distress and voice offers insights into both the meaning and impact of being silenced in the workplace. PMID:22469764

  9. Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing.

    PubMed

    Spanò, Roberta; Mascia, Tiziana; Kormelink, Richard; Gallitelli, Donato

    2015-01-01

    RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper. PMID:26496695

  10. Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing

    PubMed Central

    Spanò, Roberta; Mascia, Tiziana; Kormelink, Richard; Gallitelli, Donato

    2015-01-01

    RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper. PMID:26496695

  11. Chemogenetic silencing of the midline and intralaminar thalamus blocks amygdala-kindled seizures.

    PubMed

    Wicker, Evan; Forcelli, Patrick A

    2016-09-01

    Temporal lobe epilepsy is the most common form of medically-intractable epilepsy. While seizures in TLE originate in structures such as hippocampus, amygdala, and temporal cortex, they propagate through a crucial relay: the midline/intralaminar thalamus. Prior studies have shown that pharmacological inhibition of midline thalamus attenuates limbic seizures. Here, we examined a recently developed technology, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), as a means of chemogenetic silencing to attenuate limbic seizures. Adult, male rats were electrically kindled from the amygdala, and injected with virus coding for inhibitory (hM4Di) DREADDs into the midline/intralaminar thalamus. When treated with the otherwise inert ligand Clozapine-N-Oxide (CNO) at doses of 2.5, 5, and 10mg/kg, electrographic and behavioral seizure manifestations were suppressed in comparison to vehicle. At higher doses, we found complete blockade of seizure activity in a subset of subjects. CNO displayed a sharp time-response profile, with significant seizure attenuation seen 20-30min post injection, in comparison to 10 and 40min post injection. Seizures in animals injected with a control vector (i.e., no DREADD) were unaffected by CNO administration. These data underscore the crucial role of the midline/intralaminar thalamus in the propagation of seizures, specifically in the amygdala kindling model, and provide validation of chemogenetic silencing of limbic seizures. PMID:27404844

  12. Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair

    PubMed Central

    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. PMID:25429216

  13. 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.

  14. Pressure surge attenuator

    DOEpatents

    Christie, Alan M.; Snyder, Kurt I.

    1985-01-01

    A pressure surge attenuation system for pipes having a fluted region opposite crushable metal foam. As adapted for nuclear reactor vessels and heads, crushable metal foam is disposed to attenuate pressure surges.

  15. Behold: Silence and Attention in Education

    ERIC Educational Resources Information Center

    Lewin, David

    2014-01-01

    Educators continually ask about the best means to engage students and how best to capture attention. These concerns often make the problematic assumption that students can directly govern their own attention. In order to address the role and limits of attention in education, some theorists have sought to recover the significance of silence or…

  16. Mainstream Television, Adolescent Homosexuality, and Significant Silence.

    ERIC Educational Resources Information Center

    Kielwasser, Alfred P.; Wolf, Michelle A.

    1992-01-01

    Argues that the symbolic annihilation of gay and lesbian youth exhibited by network television contributes to a dysfunctional isolation supported by the mutually reinforcing invisibility of homosexual adolescents on television and in the real world. Suggests that the spiral of silence also partially accounts for the inefficacy of oppositional…

  17. Effect of flow on the drumlike silencer

    NASA Astrophysics Data System (ADS)

    Choy, Y. S.; Huang, Lixi

    2005-11-01

    This study examines the effects of a mean flow and turbulent flow excitation on the performance of the recently conceived device which was tested under the no-flow condition [J. Acoust. Soc. Am. 112, 2014-2035 (2002)]. The silencer consists of two cavity-backed membranes lining part of the duct walls. When a certain optimal tension is applied, the silencer gives a broad stopband in the low-frequency regime. Similar performance is predicted for the condition with a mean flow, and tests conducted for flow speeds from 5 to 15 m/s validated these predictions. The spectrum of transmission loss without flow features three resonance peaks, and the mean flow is found to smooth out all peaks and shift two of them through cross-modal coupling. The silencer was tested in a wind tunnel, and no flow induced flexural instability was found on the membrane in the range of flow speeds tested. Insertion loss measurement was also conducted in a natural ventilation condition where a turbulence intensity of 3% was recorded, and the results were close to those without flow. It is concluded that no noticeable extra sound is produced by the turbulent excitation of the membrane under the optimal tension required by the silencer.

  18. Applying gene silencing technology to contraception.

    PubMed

    Dissen, G A; Lomniczi, A; Boudreau, R L; Chen, Y H; Davidson, B L; Ojeda, S R

    2012-12-01

    Population control of feral animals is often difficult, as it can be dangerous for the animals, labour intensive and expensive. Therefore, a useful tool for control of animal populations would be a non-surgical method to induce sterility. Our laboratories utilize methods aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A useful framework for design of a new approach will be the combination of these methods with the intended goal to produce a technique that can be used to non-invasively sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: the target gene must be essential for fertility; the method must include a mechanism to effectively and specifically silence the gene of interest; the method of delivering the silencing agent must be minimally invasive, and finally, the silencing effect must be sustained for the lifespan of the target species, so that expansion of the population can be effectively prevented. In this article, we discuss our work to develop gene silencing technology to induce sterility; we will use examples of our previous studies demonstrating that this approach is viable. These studies include (i) the use of viral vectors able to disrupt reproductive cyclicity when delivered to the regions of the brain involved in the control of reproduction and (ii) experiments with viral vectors that are able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy. PMID:23279544

  19. Silence in the Context of "Child Voice"

    ERIC Educational Resources Information Center

    Lewis, Ann

    2010-01-01

    Recent decades have seen growing enthusiasm internationally for the concept and practice of "child voice". This was encapsulated in, and stimulated, by Article 12 of the 1989 United Nations Convention on the Rights of the Child. This article presents the case for incorporating the equally important concept of "child silence" in both research and…

  20. Parenting a Precocious Preschooler: Breaking the Silence

    ERIC Educational Resources Information Center

    Fish, Leigh Ann

    2016-01-01

    Precocity in the very young should be a valid topic of discussion in parental and educational circles, yet too frequently those conversations are slow to occur or are absent altogether. Many parents and educators remain silent about raising and nurturing precocious preschoolers, and author Leigh Ann Fish believe that the silence is due to a lack…

  1. Muted Colors: Gender and Classroom Silence.

    ERIC Educational Resources Information Center

    Fredericksen, Elaine

    2000-01-01

    Highlights some causes for silence in schoolgirls and other marginalized students. Suggests ways teachers can help these students participate more fully as speakers and writers in language arts classes. Shows how language arts instruction can change students' attitudes about themselves as gendered subjects, agents, and communicators. (SR)

  2. 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…

  3. Complete Genome Sequence of Bacillus megaterium Siphophage Silence

    PubMed Central

    Solis, Jonathan A.; Farmer, Nicholas G.; Cahill, Jesse L.; Rasche, Eric S.

    2015-01-01

    Silence is a newly isolated siphophage that infects Bacillus megaterium, a soil bacterium that is used readily in research and commercial applications. A study of B. megaterium phage Silence will enhance our knowledge of the diversity of Bacillus phages. Here, we describe the complete genome sequence and annotated features of Silence. PMID:26450722

  4. Complete Genome Sequence of Bacillus megaterium Siphophage Silence.

    PubMed

    Solis, Jonathan A; Farmer, Nicholas G; Cahill, Jesse L; Rasche, Eric S; Kuty Everett, Gabriel F

    2015-01-01

    Silence is a newly isolated siphophage that infects Bacillus megaterium, a soil bacterium that is used readily in research and commercial applications. A study of B. megaterium phage Silence will enhance our knowledge of the diversity of Bacillus phages. Here, we describe the complete genome sequence and annotated features of Silence. PMID:26450722

  5. 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…

  6. 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…

  7. Silenced Voices and Extraordinary Conversations... Re-Imagining Schools.

    ERIC Educational Resources Information Center

    Fine, Michelle; Weis, Lois

    This collection of papers examines the crisis in public education, focusing on poor and minority children. There are seven chapters in two parts. After "Introduction: Silenced Voices and Extraordinary Conversations" (Michelle Fine and Lois Weis), Part 1, "Scenes of Silencing," includes: (1) "Silencing and Nurturing Voice in an Improbable Context:…

  8. The Coanda effect in gas-dynamic noise control. [pressure reduction by silencers

    NASA Technical Reports Server (NTRS)

    Vasilescu, G.

    1974-01-01

    The principle types of silencers are discussed for gas dynamic noise of free steam and gas expansions, as well as the results of research in gas dynamics of jets and applied acoustics. Gas dynamic noise attenuation by means of the Coanda effect is due to fluid decompression in a Coanda ejector of the external type, where a structural change takes place in the acoustic frequency spectrum and in its direction, as well as a substantial decrease in the fluid's velocity, temperature and concentration. This process is continued in the second phase with absorption of the acoustic waves by means of an active structure.

  9. Design and performance of resonant-cavity parallel baffles for duct silencing

    NASA Technical Reports Server (NTRS)

    Soderman, P. T.

    1981-01-01

    Resonant-cavity parallel baffles, either empty or with a thin absorbent lining, have been investigated as an alternative to fiberglass-filled baffles commonly used to control noise emission from large ducts. A method for predicting silencer attenuation is described, and it is shown that the new type of baffle is characterized by an acoustic performance similar to that of fibrous baffles, while being virtually immune to such problems as clogging, erosion, or settling. The emphasis of the study is on insertion loss measurements in a 7 by 10 ft wind tunnel.

  10. Vision-based estimation of vertical dynamic loading induced by jumping and bobbing crowds on civil structures

    NASA Astrophysics Data System (ADS)

    Mazzoleni, P.; Zappa, E.

    2012-11-01

    People's motion on civil structures induces dynamic loading that may lead to excessive vibrations. The complete characterization of this force distribution over a wide area due to a large number of people is still an unsolved issue. This work presents a measuring technique for the vertical load estimation in case of jumping and bobbing crowd, based on the evaluation of the vertical inertia of the human body. Laboratory experiments verify the proposed model on a single volunteer through standard inertial sensors and then extend it introducing the non-contact measuring technique. The method validation is carried out in a real environment: a stand of the G. Meazza stadium in Milan, dynamically characterized in terms of frequency response function. The load induced by groups of jumping people is estimated with the proposed method and the resulting structure accelerations are computed: the comparison between measured and estimated vibrations shows a very high correspondence in both time domain and main spectral components and, above all, the performances do not get worse as the number of volunteer increases.

  11. Tracer attenuation in groundwater

    NASA Astrophysics Data System (ADS)

    Cvetkovic, Vladimir

    2011-12-01

    The self-purifying capacity of aquifers strongly depends on the attenuation of waterborne contaminants, i.e., irreversible loss of contaminant mass on a given scale as a result of coupled transport and transformation processes. A general formulation of tracer attenuation in groundwater is presented. Basic sensitivities of attenuation to macrodispersion and retention are illustrated for a few typical retention mechanisms. Tracer recovery is suggested as an experimental proxy for attenuation. Unique experimental data of tracer recovery in crystalline rock compare favorably with the theoretical model that is based on diffusion-controlled retention. Non-Fickian hydrodynamic transport has potentially a large impact on field-scale attenuation of dissolved contaminants.

  12. Mild hypothermia combined with a scaffold of NgR-silenced neural stem cells/Schwann cells to treat spinal cord injury

    PubMed Central

    Wang, Dong; Liang, Jinhua; Zhang, Jianjun; Liu, Shuhong; Sun, Wenwen

    2014-01-01

    Because the inhibition of Nogo proteins can promote neurite growth and nerve cell differentiation, a cell-scaffold complex seeded with Nogo receptor (NgR)-silenced neural stem cells and Schwann cells may be able to improve the microenvironment for spinal cord injury repair. Previous studies have found that mild hypothermia helps to attenuate secondary damage in the spinal cord and exerts a neuroprotective effect. Here, we constructed a cell-scaffold complex consisting of a poly(D,L-lactide-co-glycolic acid) (PLGA) scaffold seeded with NgR-silenced neural stem cells and Schwann cells, and determined the effects of mild hypothermia combined with the cell-scaffold complexes on the spinal cord hemi-transection injury in the T9 segment in rats. Compared with the PLGA group and the NgR-silencing cells + PLGA group, hindlimb motor function and nerve electrophysiological function were clearly improved, pathological changes in the injured spinal cord were attenuated, and the number of surviving cells and nerve fibers were increased in the group treated with the NgR-silenced cell scaffold + mild hypothermia at 34°C for 6 hours. Furthermore, fewer pathological changes to the injured spinal cord and more surviving cells and nerve fibers were found after mild hypothermia therapy than in injuries not treated with mild hypothermia. These experimental results indicate that mild hypothermia combined with NgR gene-silenced cells in a PLGA scaffold may be an effective therapy for treating spinal cord injury. PMID:25657741

  13. Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis.

    PubMed

    Brosnan, C A; Mitter, N; Christie, M; Smith, N A; Waterhouse, P M; Carroll, B J

    2007-09-11

    In plants, silencing of mRNA can be transmitted from cell to cell and also over longer distances from roots to shoots. To investigate the long-distance mechanism, WT and mutant shoots were grafted onto roots silenced for an mRNA. We show that three genes involved in a chromatin silencing pathway, NRPD1a encoding RNA polymerase IVa, RNA-dependent RNA polymerase 2 (RDR2), and DICER-like 3 (DCL3), are required for reception of long-distance mRNA silencing in the shoot. A mutant representing a fourth gene in the pathway, argonaute4 (ago4), was also partially compromised in the reception of silencing. This pathway produces 24-nt siRNAs and resulted in decapped RNA, a known substrate for amplification of dsRNA by RDR6. Activation of silencing in grafted shoots depended on RDR6, but no 24-nt siRNAs were detected in mutant rdr6 shoots, indicating that RDR6 also plays a role in initial signal perception. After amplification of decapped transcripts, DCL4 and DCL2 act hierarchically as they do in antiviral resistance to produce 21- and 22-nt siRNAs, respectively, and these guide mRNA degradation. Several dcl genotypes were also tested for their capacity to transmit the mobile silencing signal from the rootstock. dcl1-8 and a dcl2 dcl3 dcl4 triple mutant are compromised in micro-RNA and siRNA biogenesis, respectively, but were unaffected in signal transmission. PMID:17785412

  14. Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing

    PubMed Central

    Cooper, Bret; Campbell, Kimberly B; McMahon, Michael B; Luster, Douglas G

    2013-01-01

    Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars. PMID:24401541

  15. 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. PMID:27386153

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

    PubMed

    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

  17. Cutting Edge: EZH2 Promotes Osteoclastogenesis by Epigenetic Silencing of the Negative Regulator IRF8.

    PubMed

    Fang, Celestia; Qiao, Yu; Mun, Se Hwan; Lee, Min Joon; Murata, Koichi; Bae, Seyeon; Zhao, Baohong; Park-Min, Kyung-Hyun; Ivashkiv, Lionel B

    2016-06-01

    Osteoclasts are resorptive cells that are important for homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role for epigenetic mechanisms in osteoclastogenesis. A recent study showed that epigenetic silencing of the negative regulator of osteoclastogenesis Irf8 by DNA methylation is required for osteoclast differentiation. In this study, we investigated the role of EZH2, which epigenetically silences gene expression by histone methylation, in osteoclastogenesis. Inhibition of EZH2 by the small molecule GSK126, or decreasing its expression using antisense oligonucleotides, impeded osteoclast differentiation. Mechanistically, EZH2 was recruited to the IRF8 promoter after RANKL stimulation to deposit the negative histone mark H3K27me3 and downregulate IRF8 expression. GSK126 attenuated bone loss in the ovariectomy mouse model of postmenopausal osteoporosis. Our findings provide evidence for an additional mechanism of epigenetic IRF8 silencing during osteoclastogenesis that likely works cooperatively with DNA methylation, further emphasizing the importance of IRF8 as a negative regulator of osteoclastogenesis. PMID:27183582

  18. Homology-dependent Gene Silencing in Paramecium

    PubMed Central

    Ruiz, Françoise; Vayssié, Laurence; Klotz, Catherine; Sperling, Linda; Madeddu, Luisa

    1998-01-01

    Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for all Paramecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant. PMID:9529389

  19. Silencing Nociceptor Neurons Reduces Allergic Airway Inflammation.

    PubMed

    Talbot, Sébastien; Abdulnour, Raja-Elie E; Burkett, Patrick R; Lee, Seungkyu; Cronin, Shane J F; Pascal, Maud A; Laedermann, Cedric; Foster, Simmie L; Tran, Johnathan V; Lai, Nicole; Chiu, Isaac M; Ghasemlou, Nader; DiBiase, Matthew; Roberson, David; Von Hehn, Christian; Agac, Busranour; Haworth, Oliver; Seki, Hiroyuki; Penninger, Josef M; Kuchroo, Vijay K; Bean, Bruce P; Levy, Bruce D; Woolf, Clifford J

    2015-07-15

    Lung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation, we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8(+) sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large-pore ion channels to specifically block nociceptors, substantially reduced ovalbumin- or house-dust-mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce the release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4(+) and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for asthma. PMID:26119026

  20. The Marijuana Phenomenon: Contradictions and Silence.

    PubMed

    Hall, Joanne M; Shattell, Mona M; McConnell, Elizabeth A

    2016-01-01

    The United States is trending toward more permissiveness regarding recreational and medicinal marijuana (MJ). Many conditions for which MJ is recommended, prescribed, or self-prescribed are symptoms that advanced practice nurses address daily. Yet, the silence of nursing scientists on ethics, practices, and policies regarding such clinical decisions is deafening. This is but one of many contradictions about MJ use that we discuss in this article. We do not propose to resolve these contradictions; that is left to the community of nurse scientists in interprofessional discourse. Collectively, we must explore these contradictions and, through evidence-based policy recommendations, overcome the silence about how providers view MJ, how it might be helpful, its risks, and cultural shifts that have accompanied a changed political/legal environment. Long term, we must close the gaps in the nursing knowledge base regarding MJ as it affects users and how it is used interventionally. PMID:26950836

  1. Silencing FAT10 inhibits metastasis of osteosarcoma.

    PubMed

    Ma, Chengbin; Zhang, Zhiyu; Cui, Yan; Yuan, Hongmou; Wang, Feng

    2016-08-01

    Metastasis is the main challenge of osteosarcoma treatment. Herein, we first reveal the oncogenic role of FAT10 in metastasis of osteosarcoma. FAT10 was upregulated in osteosarcoma, especially in metastatic osteosarcoma. High level of FAT10 was associated with poorer prognosis of osteosarcoma patients. Moreover, Transwell and Matrigel assays revealed that silencing FAT10 significantly inhibited the invasive and migratory abilities of osteosarcoma cells. Metastasis assay in vivo showed that silencing FAT10 decreased the number of mice with distant metastasis. We also found that FAT10 may act its oncogenic functions through regulating HOXB9. Collectively, the results suggested that FAT10 may be a novel therapeutic target for osteosarcoma patients. PMID:27279480

  2. Silencing the Singer. Antibioethics in Germany.

    PubMed

    Schöne-Seifert, B; Rippe, K P

    1991-01-01

    "Die Gedanken sind frei," in the words of the old song. But in Germany, thoughts are no longer free. Peter Singer, the "death ethicist," has become a special target for activists attempting to silence bioethical debate in Germany. In the context of the trauma inflicted by National Socialism, a profound unease over issues at the end of life is accompanied by an insistence that these issues are not to be discussed. PMID:1837289

  3. Targeted gene silencing to induce permanent sterility.

    PubMed

    Dissen, G A; Lomniczi, A; Boudreau, R L; Chen, Y H; Davidson, B L; Ojeda, S R

    2012-08-01

    A non-surgical method to induce sterility would be a useful tool to control feral populations of animals. Our laboratories have experience with approaches aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A combination/modification of these methods may provide a useful framework for the design of approaches that can be used to sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: it needs to target a gene essential for fertility. It must involve a method that can selectively silence the gene of interest. It also needs to deliver the silencing agent via a minimally invasive method. Finally, the silencing effect needs to be sustained for many years, so that expansion of the targeted population can be effectively prevented. In this article, we discuss this subject and provide a succinct account of our previous experience with: (i) molecular reagents able to disrupt reproductive cyclicity when delivered to regions of the brain involved in the control of reproduction and (ii) molecular reagents able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy. PMID:22827375

  4. Variable laser attenuator

    DOEpatents

    Foltyn, Stephen R.

    1988-01-01

    The disclosure relates to low loss, high power variable attenuators comprng one or more transmissive and/or reflective multilayer dielectric filters. The attenuator is particularly suitable to use with unpolarized lasers such as excimer lasers. Beam attenuation is a function of beam polarization and the angle of incidence between the beam and the filter and is controlled by adjusting the angle of incidence the beam makes to the filter or filters. Filters are selected in accordance with beam wavelength.

  5. Variable laser attenuator

    DOEpatents

    Foltyn, S.R.

    1987-05-29

    The disclosure relates to low loss, high power variable attenuators comprising one or more transmissive and/or reflective multilayer dielectric filters. The attenuator is particularly suitable to use with unpolarized lasers such as excimer lasers. Beam attenuation is a function of beam polarization and the angle of incidence between the beam and the filter and is controlled by adjusting the angle of incidence the beam makes to the filter or filters. Filters are selected in accordance with beam wavelength. 9 figs.

  6. Silencing of protein kinase D2 induces glioma cell senescence via p53-dependent and -independent pathways

    PubMed Central

    Bernhart, Eva; Damm, Sabine; Heffeter, Petra; Wintersperger, Andrea; Asslaber, Martin; Frank, Saša; Hammer, Astrid; Strohmaier, Heimo; DeVaney, Trevor; Mrfka, Manuel; Eder, Hans; Windpassinger, Christian; Ireson, Christopher R.; Mischel, Paul S.; Berger, Walter; Sattler, Wolfgang

    2014-01-01

    Background Glioblastoma multiforme (GBM) is a highly aggressive tumor of the central nervous system with a dismal prognosis for affected patients. Aberrant protein kinase C (PKC) signaling has been implicated in gliomagenesis, and a member of the PKC-activated protein kinase D (PRKD) family, PRKD2, was identified as mediator of GBM growth in vitro and in vivo. Methods The outcome of PRKD2 silencing and pharmacological inhibition on glioma cell proliferation was established with different glioma cell lines. Western blotting, senescence assays, co-immunoprecipitation, fluorescence activated cell sorting, quantitative PCR, and immunofluorescence microscopy were utilized to analyze downstream signaling. Results RNA-interference (21-mer siRNA) and pharmacological inhibition (CRT0066101) of PRKD2 profoundly inhibited proliferation of p53wt (U87MG, A172, and primary GBM2), and p53mut (GM133, T98G, U251, and primary Gli25) glioma cells. In a xenograft experiment, PRKD2 silencing significantly delayed tumor growth of U87MG cells. PRKD2 silencing in p53wt and p53mut cells was associated with typical hallmarks of senescence and cell cycle arrest in G1. Attenuated AKT/PKB phosphorylation in response to PRKD2 silencing was a common observation made in p53wt and p53mut GBM cells. PRKD2 knockdown in p53wt cells induced upregulation of p53, p21, and p27 expression, decreased phosphorylation of CDK2 and/or CDK4, hypophosphorylation of retinoblastoma protein (pRb), and reduced transcription of E2F1. In p53mut GM133 and primary Gli25 cells, PRKD2 silencing increased p27 and p15 and reduced E2F1 transcription but did not affect pRb phosphorylation. Conclusions PRKD2 silencing induces glioma cell senescence via p53-dependent and -independent pathways. PMID:24463355

  7. 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

  8. 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. PMID:25993336

  9. Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment.

    PubMed

    Le, Huy Quang; Ghatak, Sushmita; Yeung, Ching-Yan Chloé; Tellkamp, Frederik; Günschmann, Christian; Dieterich, Christoph; Yeroslaviz, Assa; Habermann, Bianca; Pombo, Ana; Niessen, Carien M; Wickström, Sara A

    2016-08-01

    Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. We show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. Our results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis. PMID:27398909

  10. Cohabitation of insulators and silencing elements in yeast subtelomeric regions.

    PubMed Central

    Fourel, G; Revardel, E; Koering, C E; Gilson, E

    1999-01-01

    In budding yeast, the telomeric DNA is flanked by a combination of two subtelomeric repetitive sequences, the X and Y' elements. We have investigated the influence of these sequences on telomeric silencing. The telomere-proximal portion of either X or Y' dampened silencing when located between the telomere and the reporter gene. These elements were named STARs, for subtelomeric anti-silencing regions. STARs can also counteract silencer-driven repression at the mating-type HML locus. When two STARs bracket a reporter gene, its expression is no longer influenced by surrounding silencing elements, although these are still active on a second reporter gene. In addition, an intervening STAR uncouples the silencing of neighboring genes. STARs thus display the hallmarks of insulators. Protection from silencing is recapitulated by multimerized oligonucleotides representing Tbf1p- and Reb1p-binding sites, as found in STARs. In contrast, sequences located more centromere proximal in X and Y' elements reinforce silencing. They can promote silencing downstream of an insulated expressed domain. Overall, our results suggest that the silencing emanating from telomeres can be propagated in a discontinuous manner via a series of subtelomeric relay elements. PMID:10228166

  11. RNA silencing and antiviral defense in plants.

    PubMed

    Agius, Claire; Eamens, Andrew L; Millar, Anthony A; Watson, John M; Wang, Ming-Bo

    2012-01-01

    Given the widespread impact of RNA silencing on the Arabidopsis thaliana genome, it is indeed remarkable that this means of gene regulation went undiscovered for so long. Since the publication of landmark papers in 1998 (Fire et al., Nature 391:806-811, 1998; Waterhouse et al., Proc Natl Acad Sci U S A 95:13959-13964, 1998), intense research efforts have resulted in much progress from the speculation of Mello and colleagues that "the mechanisms underlying RNA interference probably exist for a biological purpose" (Fire et al., Nature 391:806-811, 1998). Across the eukaryotic kingdom, with the notable exception of Saccharomyces cerevisiae (Moazed, Science 326:544-550, 2009), the importance of small RNA-driven gene regulation has been recognized and implicated in central developmental processes as well as in aberrant and diseased states. Plants have by far the most complex RNA-based control of gene expression (Wang et al., Floriculture, ornamental and plant biotechnology, vol. III, 2006). Four distinct RNA silencing pathways have been recognized in plants, albeit with considerable conservation of the molecular components. These pathways are directed by various small RNA species, including microRNAs (miRNAs), trans-acting small interfering RNAs (siRNA) (ta-siRNAs), repeat-associated siRNAs (ra-siRNAs), and natural antisense transcript siRNAs (nat-siRNAs). The effective functionality of each of these pathways appear to be fundamental to the integrity of A. thaliana. Furthermore, in response to viral invasion, plants synthesize viral sRNAs as a means of defense. This process may in fact reflect the ancient origins of RNA silencing: plants may have evolved RNA silencing pathways as a defense mechanism against foreign nucleic acid species in the absence of an immune system (Wang and Metzlaff, Curr Opin Plant Biol 8:216-222, 2005). The generation of viral siRNAs is a particularly interesting illustration of RNA silencing as it provides a context to explore the potential to

  12. Gender Differences in Self-Silencing and Psychological Distress in Informal Cancer Carers

    ERIC Educational Resources Information Center

    Ussher, Jane M.; Perz, Janette

    2010-01-01

    This study examined gender differences in self-silencing, the relationship between self-silencing and psychological distress, and reasons for self-silencing in informal cancer carers (329 women, 155 men), using a mixed-method design. Men reported greater self-silencing than women on the Silencing the Self Scale; however, women reported higher…

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

    PubMed

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

    2012-05-10

    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(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(PE) has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0(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. PMID:22361475

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

    SciTech Connect

    Fusaro, Adriana F.; Correa, Regis L.; Nakasugi, Kenlee; Jackson, Craig; Kawchuk, Lawrence; Vaslin, Maite F.S.; Waterhouse, Peter M.

    2012-05-10

    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 systemic 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.

  15. Suppressing RNA silencing with small molecules and the viral suppressor of RNA silencing protein p19.

    PubMed

    Danielson, Dana C; Filip, Roxana; Powdrill, Megan H; O'Hara, Shifawn; Pezacki, John P

    2015-08-01

    RNA silencing is a gene regulatory and host defense mechanism whereby small RNA molecules are engaged by Argonaute (AGO) proteins, which facilitate gene knockdown of complementary mRNA targets. Small molecule inhibitors of AGO represent a convenient method for reversing this effect and have applications in human therapy and biotechnology. Viral suppressors of RNA silencing, such as p19, can also be used to suppress the pathway. Here we assess the compatibility of these two approaches, by examining whether synthetic inhibitors of AGO would inhibit p19-siRNA interactions. We observe that aurintricarboxylic acid (ATA) is a potent inhibitor of p19's ability to bind siRNA (IC50 = 0.43 μM), oxidopamine does not inhibit p19:siRNA interactions, and suramin is a mild inhibitor of p19:siRNA interactions (IC50 = 430 μM). We observe that p19 and suramin are compatible inhibitors of RNA silencing in human hepatoma cells. Our data suggests that at least some inhibitors of AGO may be used in combination with p19 to inhibit RNA silencing at different points in the pathway. PMID:26079891

  16. 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

  17. Co-silencing the mirabilis antiviral protein permits virus-induced gene silencing in Mirabilis jalapa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is an attractive and rapid technique for loss of function assay that can reveal the phenotype of embryo-lethal sequences and avoids the need for time consuming transformation and regeneration processes. Among various VIGS vectors that have been explored, the tobac...

  18. Landing gear noise attenuation

    NASA Technical Reports Server (NTRS)

    Moe, Jeffrey W. (Inventor); Whitmire, Julia (Inventor); Kwan, Hwa-Wan (Inventor); Abeysinghe, Amal (Inventor)

    2011-01-01

    A landing gear noise attenuator mitigates noise generated by airframe deployable landing gear. The noise attenuator can have a first position when the landing gear is in its deployed or down position, and a second position when the landing gear is in its up or stowed position. The noise attenuator may be an inflatable fairing that does not compromise limited space constraints associated with landing gear retraction and stowage. A truck fairing mounted under a truck beam can have a compliant edge to allow for non-destructive impingement of a deflected fire during certain conditions.

  19. RADIO FREQUENCY ATTENUATOR

    DOEpatents

    Giordano, S.

    1963-11-12

    A high peak power level r-f attenuator that is readily and easily insertable along a coaxial cable having an inner conductor and an outer annular conductor without breaking the ends thereof is presented. Spaced first and second flares in the outer conductor face each other with a slidable cylindrical outer conductor portion therebetween. Dielectric means, such as water, contact the cable between the flares to attenuate the radio-frequency energy received thereby. The cylindrical outer conductor portion is slidable to adjust the voltage standing wave ratio to a low level, and one of the flares is slidable to adjust the attenuation level. An integral dielectric container is also provided. (AFC)

  20. 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

  1. Attenuator And Conditioner

    DOEpatents

    Anderson, Gene R.; Armendariz, Marcelino G.; Carson, Richard F.; Bryan, Robert P.; Duckett, III, Edwin B.; Kemme, Shanalyn Adair; McCormick, Frederick B.; Peterson, David W.

    2006-04-04

    An apparatus and method of attenuating and/or conditioning optical energy for an optical transmitter, receiver or transceiver module is disclosed. An apparatus for attenuating the optical output of an optoelectronic connector including: a mounting surface; an array of optoelectronic devices having at least a first end; an array of optical elements having at least a first end; the first end of the array of optical elements optically aligned with the first end of the array of optoelectronic devices; an optical path extending from the first end of the array of optoelectronic devices and ending at a second end of the array of optical elements; and an attenuator in the optical path for attenuating the optical energy emitted from the array of optoelectronic devices. Alternatively, a conditioner may be adapted in the optical path for conditioning the optical energy emitted from the array of optoelectronic devices.

  2. Fiber Optic Attenuators

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Mike Buzzetti designed a fiber optic attenuator while working at Jet Propulsion Laboratory, intended for use in NASA's Deep Space Network. Buzzetti subsequently patented and received an exclusive license to commercialize the device, and founded Nanometer Technologies to produce it. The attenuator functions without introducing measurable back-reflection or insertion loss, and is relatively insensitive to vibration and changes in temperature. Applications include cable television, telephone networks, other signal distribution networks, and laboratory instrumentation.

  3. Echoes of Silence: Empathy and Making Connections through Writing Process

    ERIC Educational Resources Information Center

    Freedman, Joel M.

    2009-01-01

    On April 25, 2008, students on college and public school campuses collectively committed to a vow of silence commemorating an event known as the National Day of Silence. This student-generated, nationwide action theatrically "speaks out" in solidarity with lesbian, gay, bisexual, and transgender (LGBT) people who for one reason or another fear…

  4. A Time for Silence: Booker T. Washington in Atlanta.

    ERIC Educational Resources Information Center

    Heath, Robert L.

    1978-01-01

    Discusses the rhetoric of silence as an alternative to participation in a discussion of social issues, particularly when participation confirms the efforts of those who seek social control. Concludes that Booker T. Washington should have employed the strategy of silence when invited to address an exposition in Atlanta in 1895. (JMF)

  5. 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…

  6. 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…

  7. 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…

  8. 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…

  9. 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…

  10. The Question of Silence: Techniques to Ensure Full Class Participation.

    ERIC Educational Resources Information Center

    Wildman, Stephanie M.

    1988-01-01

    Law school professors have a responsibility to monitor student participation and silence in the classroom, to consider why some students are silent, and to examine their role in contributing to that silence. A variety of techniques are available to encourage participation. (MSE)

  11. Silenced by Sex: Hard Truths & Taboos in Teaching Literature.

    ERIC Educational Resources Information Center

    Wolf, Shelby A.; Maniotes, Leslie K.

    2002-01-01

    Focuses on the silencing of young voices, both on and off the page, because of sexual trauma and even sexual curiosity. Considers the novel, "Speak," in which the protagonist's silence stems from the fact that she was raped. Explores two other literary texts that center on sexual issues for young teens. (SG)

  12. Virus-Induced Gene Silencing in Ornametal Plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-Induced Gene Silencing (VIGS) provides an attractive tool for high throughput analysis of the functional effects of gene knock-down. Virus genomes are engineered to include fragments of target host genes, and the infected plant recognizes and silences the target genes as part of its viral defe...

  13. Reflections on the Silencing the Self Scale and Its Origins

    ERIC Educational Resources Information Center

    Jack, Dana Crowley

    2011-01-01

    In this article, the author reflects on the Silencing the Self Scale (STSS) and blends her personal and professional thoughts about self-silencing, gender, and depression. For her, the despair of depression deeply involves questions of value and meaning, culture and freedom. The STSS grew from listening to depressed women's voices. From them, the…

  14. 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

  15. Virus-Induced gene silencing in ornamental plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-Induced Gene Silencing (VIGS) provides an attractive tool for high throughput analysis of the functional effects of gene knock-down. Virus genomes are engineered to include fragments of target host genes, and the infected plant recognizes and silences the target genes as part of its viral defe...

  16. Postnatal β-catenin deletion from Dmp1-expressing osteocytes/osteoblasts reduces structural adaptation to loading, but not periosteal load-induced bone formation.

    PubMed

    Kang, Kyung Shin; Hong, Jung Min; Robling, Alexander G

    2016-07-01

    Mechanical signal transduction in bone tissue begins with load-induced activation of several cellular pathways in the osteocyte population. A key pathway that participates in mechanotransduction is Wnt/Lrp5 signaling. A putative downstream mediator of activated Lrp5 is the nucleocytoplasmic shuttling protein β-catenin (βcat), which migrates to the nucleus where it functions as a transcriptional co-activator. We investigated whether osteocytic βcat participates in Wnt/Lrp5-mediated mechanotransduction by conducting ulnar loading experiments in mice with or without chemically induced βcat deletion in osteocytes. Mice harboring βcat floxed loss-of-function alleles (βcat(f/f)) were bred to the inducible osteocyte Cre transgenic (10)(kb)Dmp1-CreERt2. Adult male mice were induced to recombine the βcat alleles using tamoxifen, and intermittent ulnar loading sessions were applied over the following week. Although adult-onset deletion of βcat from Dmp1-expressing cells reduced skeletal mass, the bone tissue was responsive to mechanical stimulation as indicated by increased relative periosteal bone formation rates in recombined mice. However, load-induced improvements in cross sectional geometric properties were compromised in recombined mice. The collective results indicate that the osteoanabolic response to loading can occur on the periosteal surface when β-cat levels are significantly reduced in Dmp1-expressing cells, suggesting that either (i) only low levels of β-cat are required for mechanically induced bone formation on the periosteal surface, or (ii) other additional downstream mediators of Lrp5 might participate in transducing load-induced Wnt signaling. PMID:27143110

  17. An electronically tunable duct silencer using dielectric elastomer actuators.

    PubMed

    Lu, Zhenbo; Godaba, Hareesh; Cui, Yongdong; Foo, Choon Chiang; Debiasi, Marco; Zhu, Jian

    2015-09-01

    A duct silencer with tunable acoustic characteristics is presented in this paper. Dielectric elastomer, a smart material with lightweight, high elastic energy density and large deformation under high direct current/alternating current voltages, was used to fabricate this duct silencer. The acoustic performances and tunable mechanisms of this duct silencer were experimentally investigated. It was found that all the resonance peaks of this duct silencer could be adjusted using external control signals without any additional mechanical part. The physics of the tunable mechanism is further discussed based on the electro-mechanical interactions using finite element analysis. The present promising results also provide insight into the appropriateness of the duct silencer for possible use as next generation acoustic treatment device to replace the traditional acoustic treatment. PMID:26428819

  18. Silencing suppressors: viral weapons for countering host cell defenses.

    PubMed

    Song, Liping; Gao, Shijuan; Jiang, Wei; Chen, Shuai; Liu, Yanjun; Zhou, Ling; Huang, Wenlin

    2011-04-01

    RNA silencing is a conserved eukaryotic pathway involved in the suppression of gene expression via sequence-specific interactions that are mediated by 21-23 nt RNA molecules. During infection, RNAi can act as an innate immune system to defend against viruses. As a counter-defensive strategy, silencing suppressors are encoded by viruses to inhibit various stages of the silencing process. These suppressors are diverse in sequence and structure and act via different mechanisms. In this review, we discuss whether RNAi is a defensive strategy in mammalian host cells and whether silencing suppressors can be encoded by mammalian viruses. We also review the modes of action proposed for some silencing suppressors. PMID:21528352

  19. Transcriptional Silencing by Hairpin RNAs Complementary to a Gene Promoter

    PubMed Central

    Chu, Yongjun; Kalantari, Roya; Dodd, David W.

    2012-01-01

    Double-stranded RNAs can target gene promoters and inhibit transcription. To date, most research has focused on synthetic RNA duplexes. Transcriptional silencing by hairpin RNAs would facilitate a better understanding of endogenous RNA-mediated regulation of transcription within cells. Here we examine transcriptional silencing of progesterone receptor (PR) expression by hairpin RNAs. We identify the guide strand as the strand complementary to an antisense transcript at the PR promoter and that hairpin RNAs are active transcriptional silencing agents. The sequence of the hairpin loop affects activity, with the highest activity achieved when the loop has the potential for full complementarity to the antisense transcript target. Introduction of centrally mismatched bases relative to the target transcript does not prevent transcriptional silencing unless the mismatches are present on both the guide and passenger strands. These data demonstrate that hairpin RNAs can cause transcriptional silencing and offer insights into the mechanism of gene modulation by RNAs that target gene promoters. PMID:22703280

  20. Evaluating the ability of the barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley Stripe Mosaic Virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes afte...

  1. Evaluating the Ability of the Barley Stripe Mosaic Virus-Induced Gene Silencing System to Simultaneously Silence Two Wheat Genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley stripe mosaic virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes afte...

  2. GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells.

    PubMed

    Tchasovnikarova, Iva A; Timms, Richard T; Matheson, Nicholas J; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A; Lehner, Paul J

    2015-06-26

    Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing. PMID:26022416

  3. Antisense Gene Silencing: Therapy for Neurodegenerative Disorders?

    PubMed Central

    Nielsen, Troels T.; Nielsen, Jørgen E.

    2013-01-01

    Since the first reports that double-stranded RNAs can efficiently silence gene expression in C. elegans, the technology of RNA interference (RNAi) has been intensively exploited as an experimental tool to study gene function. With the subsequent discovery that RNAi could also be applied to mammalian cells, the technology of RNAi expanded from being a valuable experimental tool to being an applicable method for gene-specific therapeutic regulation, and much effort has been put into further refinement of the technique. This review will focus on how RNAi has developed over the years and how the technique is exploited in a pre-clinical and clinical perspective in relation to neurodegenerative disorders. PMID:24705213

  4. Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

    PubMed Central

    Méndez, Catalina; Ahlenstiel, Chantelle L; Kelleher, Anthony D

    2015-01-01

    While human immunodeficiency virus 1 (HIV-1) infection is controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from the body. Despite decades of research there is still no effective vaccine to prevent HIV-1 infection. Therefore, the possibility of an RNA interference (RNAi)-based cure has become an increasingly explored approach. Endogenous gene expression is controlled at both, transcriptional and post-transcriptional levels by non-coding RNAs, which act through diverse molecular mechanisms including RNAi. RNAi has the potential to control the turning on/off of specific genes through transcriptional gene silencing (TGS), as well as fine-tuning their expression through post-transcriptional gene silencing (PTGS). In this review we will describe in detail the canonical RNAi pathways for PTGS and TGS, the relationship of TGS with other silencing mechanisms and will discuss a variety of approaches developed to suppress HIV-1 via manipulation of RNAi. We will briefly compare RNAi strategies against other approaches developed to target the virus, highlighting their potential to overcome the major obstacle to finding a cure, which is the specific targeting of the HIV-1 reservoir within latently infected cells. PMID:26279984

  5. Bioinformatics tools for achieving better gene silencing in plants.

    PubMed

    Ahmed, Firoz; Dai, Xinbin; Zhao, Patrick Xuechun

    2015-01-01

    RNA interference (RNAi) is one of the most popular and effective molecular technologies for knocking down the expression of an individual gene of interest in living organisms. Yet the technology still faces the major issue of nonspecific gene silencing, which can compromise gene functional characterization and the interpretation of phenotypes associated with individual gene knockdown. Designing an effective and target-specific small interfering RNA (siRNA) for induction of RNAi is therefore the major challenge in RNAi-based gene silencing. A 'good' siRNA molecule must possess three key features: (a) the ability to specifically silence an individual gene of interest, (b) little or no effect on the expressions of unintended siRNA gene targets (off-target genes), and (c) no cell toxicity. Although several siRNA design and analysis algorithms have been developed, only a few of them are specifically focused on gene silencing in plants. Furthermore, current algorithms lack a comprehensive consideration of siRNA specificity, efficacy, and nontoxicity in siRNA design, mainly due to lack of integration of all known rules that govern different steps in the RNAi pathway. In this review, we first describe popular RNAi methods that have been used for gene silencing in plants and their serious limitations regarding gene-silencing potency and specificity. We then present novel, rationale-based strategies in combination with computational and experimental approaches to induce potent, specific, and nontoxic gene silencing in plants. PMID:25740355

  6. The effect of eccentricity and spatiotemporal energy on motion silencing.

    PubMed

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

    2016-03-01

    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. PMID:27019052

  7. Targeted silencing of CXCR4 inhibits epithelial-mesenchymal transition in oral squamous cell carcinoma

    PubMed Central

    Duan, Yuansheng; Zhang, Shu; Wang, Longlong; Zhou, Xuan; He, Qinghua; Liu, Su; Yue, Kai; Wang, Xudong

    2016-01-01

    Aberrant overexpression of C-X-C chemokine receptor type 4 (CXCR4) is a critical event during tumor metastasis. It has been previously reported that the expression of CXCR4 is linked with epithelial-mesenchymal transition (EMT) in oral squamous cell carcinoma (OSCC) tissues derived from patients. The present study addresses the role of CXCR4 in EMT in tongue squamous cell carcinoma (TSCCA) cells in vitro and in xenograft models. Small interfering (si) RNA sequences targeting the CXCR4 gene were transfected into TSCCA cells. Cell migration, invasion, apoptosis and EMT markers were determined in TSCCA cells using wound healing and Transwell assays, Annexin V/propdidum iodide double staining and western blot analysis, respectively. In vivo, tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice. Phenotypic EMT markers and regulatory factors were detected in the tumor tissues derived from the mice. In vitro, silencing of CXCR4 expression suppressed cell migration and invasion, and induced apoptosis. The protein expression of the EMT-associated markers N-cadherin and matrix metalloproteinases 2/9 were attenuated, while E-cadherin was increased. In vivo, CXCR4 siRNA inhibited tumor growth, and EMT-associated proteins had similar expression patterns to the experimental results observed in vitro. In conclusion, the present study demonstrated that CXCR4 silencing suppressed EMT in OSCC, thus affecting tumor metastasis. PMID:27602138

  8. Silencing of Eag1 Gene Inhibits Osteosarcoma Proliferation and Migration by Targeting STAT3-VEGF Pathway

    PubMed Central

    Wu, Xinyu; Chen, Zhida; Zeng, Wengrong; Zhong, Yuanfu; Liu, Qingjun; Wu, Jin

    2015-01-01

    So far, the role of Ether à go-go 1 (Eag1) potassium channels in migration and invasion progression of cancers remains elusive. In the present study, the effects of Eag1 knockdown on osteosarcoma cell proliferation, growth, and apoptosis were examined. Then, we evaluated the effects of Eag1 silencing on osteosarcoma cell migration and invasion. In addition, we detected the expression of vascular endothelial growth factor (VEGF) and signal transducer and activator of transcription 3 (STAT3) in osteosarcoma cell treated with Eag1 small interfering RNAs (siRNAs). Finally, STAT3 siRNA was employed to determine the influence of downregulation of STAT3 on cell proliferation and migration. The results showed that knockdown of Eag1 significantly suppressed osteosarcoma cell proliferation and osteosarcoma xenografts growth. However, Eag1 silencing had little effect on cell apoptosis. Additionally, osteosarcoma cell adhesion, migration, and invasion were also potently attenuated. Notably, the expression levels of VEGF decreased evidently upon Eag1 siRNAs treatment, paralleled with reductions in the expression levels of STAT3. Moreover, a similar pattern was observed in osteosarcoma cell proliferation and migration suppression between STAT3 siRNA and Eag1 siRNAs groups. Our data indicated that Eag1 promotes osteosarcoma proliferation and migration, at least in part, by targeting STAT3-VEGF pathway. PMID:26783521

  9. Radiofrequency attenuator and method

    DOEpatents

    Warner, Benjamin P.; McCleskey, T. Mark; Burrell, Anthony K.; Agrawal, Anoop; Hall, Simon B.

    2009-01-20

    Radiofrequency attenuator and method. The attenuator includes a pair of transparent windows. A chamber between the windows is filled with molten salt. Preferred molten salts include quarternary ammonium cations and fluorine-containing anions such as tetrafluoroborate (BF.sub.4.sup.-), hexafluorophosphate (PF.sub.6.sup.-), hexafluoroarsenate (AsF.sub.6.sup.-), trifluoromethylsulfonate (CF.sub.3SO.sub.3.sup.-), bis(trifluoromethylsulfonyl)imide ((CF.sub.3SO.sub.2).sub.2N.sup.-), bis(perfluoroethylsulfonyl)imide ((CF.sub.3CF.sub.2SO.sub.2).sub.2N.sup.-) and tris(trifluoromethylsulfonyl)methide ((CF.sub.3SO.sub.2).sub.3C.sup.-). Radicals or radical cations may be added to or electrochemically generated in the molten salt to enhance the RF attenuation.

  10. Radiofrequency attenuator and method

    DOEpatents

    Warner, Benjamin P.; McCleskey, T. Mark; Burrell, Anthony K.; Agrawal, Anoop; Hall, Simon B.

    2009-11-10

    Radiofrequency attenuator and method. The attenuator includes a pair of transparent windows. A chamber between the windows is filled with molten salt. Preferred molten salts include quarternary ammonium cations and fluorine-containing anions such as tetrafluoroborate (BF.sub.4.sup.-), hexafluorophosphate (PF.sub.6.sup.-), hexafluoroarsenate (AsF.sub.6.sup.-), trifluoromethylsulfonate (CF.sub.3SO.sub.3.sup.-), bis(trifluoromethylsulfonyl)imide ((CF.sub.3SO.sub.2).sub.2N.sup.-), bis(perfluoroethylsulfonyl)imide ((CF.sub.3CF.sub.2SO.sub.2).sub.2N.sup.-) and tris(trifluoromethylsulfonyl)methide ((CF.sub.3SO.sub.2).sub.3 C.sup.-). Radicals or radical cations may be added to or electrochemically generated in the molten salt to enhance the RF attenuation.

  11. Seismic attenuation in Florida

    SciTech Connect

    Bellini, J.J.; Bartolini, T.J.; Lord, K.M.; Smith, D.L. . Dept. of Geology)

    1993-03-01

    Seismic signals recorded by the expanded distribution of earthquake seismograph stations throughout Florida and data from a comprehensive review of record archives from stations GAI contribute to an initial seismic attenuation model for the Florida Plateau. Based on calculations of surface particle velocity, a pattern of attenuation exists that appears to deviate from that established for the remainder of the southeastern US. Most values suggest greater seismic attenuation within the Florida Plateau. However, a separate pattern may exist for those signals arising from the Gulf of Mexico. These results have important implications for seismic hazard assessments in Florida and may be indicative of the unique lithospheric identity of the Florida basement as an exotic terrane.

  12. RNA binding proteins implicated in Xist-mediated chromosome silencing.

    PubMed

    Moindrot, Benoit; Brockdorff, Neil

    2016-08-01

    Chromosome silencing by Xist RNA occurs in two steps; localisation in cis within the nuclear matrix to form a domain that corresponds to the territory of the inactive X chromosome elect, and transduction of silencing signals from Xist RNA to the underlying chromatin. Key factors that mediate these processes have been identified in a series of recent studies that harnessed comprehensive proteomic or genetic screening strategies. In this review we discuss these findings in light of prior knowledge both of Xist-mediated silencing and known functions/properties of the novel factors. PMID:26816113

  13. Analysis of trans-silencing interactions using transcriptional silencers of varying strength and targets with and without flanking nuclear matrix attachment regions.

    PubMed

    Ascenzi, Robert; Ulker, Bekir; Todd, Joselyn J; Sowinski, Dolores A; Schimeneck, Carolyn R; Allen, George C; Weissinger, Arthur K; Thompson, William F

    2003-06-01

    We investigated the effect of the Rb7 matrix attachment region (MAR) on trans-silencing in tobacco plants, comparing the effects of three transgene silencer loci on ten target loci. Two of the silencer loci, C40 and C190, contain complex and rearranged transgene arrays consisting of 35S:GUS or NOS:NPTII containing plasmids. The third silencer locus, V271, was previously characterized as a complex locus containing rearranged 35S:RiN sequences. Each of these silencers can reduce 35S promoter-driven expression at other loci, albeit with varying efficiencies. The presence of MARs at a target locus does not prevent trans-silencing by the V271 silencer. However, four of seven MAR-containing loci were at least partially resistant to silencing by the C40 and C190 loci. One MAR locus was unaffected by C40, our weakest silencer, and three were silenced only when the silencer locus was maternally inherited. Silencing is progressive in the F1 and F2 generations; two days after germination there is little or no difference between seedlings derived from crosses to silencing or control lines, but seedlings containing silencer loci slowly lose expression during subsequent development. These observations are compatible with the hypothesis that a product of the silencer locus must accumulate before unlinked loci can be affected. However, our silencer loci are themselves silenced for GUS transcription, and coding region homology is not required for their effects on target loci. Our results are consistent with a model in which transcriptional silencing is triggered by transcription of sequences during the early stages of embryo or seedling development. PMID:12779119

  14. Obesity-Induced Colorectal Cancer Is Driven by Caloric Silencing of the Guanylin-GUCY2C Paracrine Signaling Axis.

    PubMed

    Lin, Jieru E; Colon-Gonzalez, Francheska; Blomain, Erik; Kim, Gilbert W; Aing, Amanda; Stoecker, Brian; Rock, Justin; Snook, Adam E; Zhan, Tingting; Hyslop, Terry M; Tomczak, Michal; Blumberg, Richard S; Waldman, Scott A

    2016-01-15

    Obesity is a well-known risk factor for colorectal cancer but precisely how it influences risks of malignancy remains unclear. During colon cancer development in humans or animals, attenuation of the colonic cell surface receptor guanylyl cyclase C (GUCY2C) that occurs due to loss of its paracrine hormone ligand guanylin contributes universally to malignant progression. In this study, we explored a link between obesity and GUCY2C silencing in colorectal cancer. Using genetically engineered mice on different diets, we found that diet-induced obesity caused a loss of guanylin expression in the colon with subsequent GUCY2C silencing, epithelial dysfunction, and tumorigenesis. Mechanistic investigations revealed that obesity reversibly silenced guanylin expression through calorie-dependent induction of endoplasmic reticulum stress and the unfolded protein response in intestinal epithelial cells. In transgenic mice, enforcing specific expression of guanylin in intestinal epithelial cells restored GUCY2C signaling, eliminating intestinal tumors associated with a high calorie diet. Our findings show how caloric suppression of the guanylin-GUCY2C signaling axis links obesity to negation of a universal tumor suppressor pathway in colorectal cancer, suggesting an opportunity to prevent colorectal cancer in obese patients through hormone replacement with the FDA-approved oral GUCY2C ligand linaclotide. PMID:26773096

  15. In Vivo Silencing of A20 via TLR9-Mediated Targeted SiRNA Delivery Potentiates Antitumor Immune Response.

    PubMed

    Braun, Floriane C M; van den Brandt, Jens; Thomas, Sören; Lange, Sandra; Schrank, Juliane; Gand, Claudia; Przybylski, Grzegorz K; Schmoeckel, Katrin; Bröker, Barbara M; Schmidt, Christian A; Grabarczyk, Piotr

    2015-01-01

    A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling pathways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of dendritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of pro-inflammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infiltrating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucleotide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9+ mouse dendritic cells in vitro and in vivo. In the B16 mouse melanoma tumor model, silencing of A20 enhances the CpG-triggered induction of NFκB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimulatory potency of DCs and provide a strategy for simultaneous A20 silencing and TLR activation in vivo. PMID:26327508

  16. In Vivo Silencing of A20 via TLR9-Mediated Targeted SiRNA Delivery Potentiates Antitumor Immune Response

    PubMed Central

    Braun, Floriane C. M.; van den Brandt, Jens; Thomas, Sören; Lange, Sandra; Schrank, Juliane; Gand, Claudia; Przybylski, Grzegorz K.; Schmoeckel, Katrin; Bröker, Barbara M.; Schmidt, Christian A.; Grabarczyk, Piotr

    2015-01-01

    A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling pathways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of dendritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of pro-inflammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infiltrating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucleotide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9+ mouse dendritic cells in vitro and in vivo. In the B16 mouse melanoma tumor model, silencing of A20 enhances the CpG-triggered induction of NFκB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimulatory potency of DCs and provide a strategy for simultaneous A20 silencing and TLR activation in vivo. PMID:26327508

  17. Tritium Attenuation by Distillation

    SciTech Connect

    Wittman, N.E.

    2001-07-31

    The objective of this study was to determine how a 100 Area distillation system could be used to reduce to a satisfactory low value the tritium content of the dilute moderator produced in the 100 Area stills, and whether such a tritium attenuator would have sufficient capacity to process all this material before it is sent to the 400 Area for reprocessing.

  18. 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,"…

  19. New Construct Approaches for Efficient Gene Silencing in Plants

    PubMed Central

    Yan, Hua; Chretien, Robert; Ye, Jingsong; Rommens, Caius M.

    2006-01-01

    An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts. PMID:16766670

  20. Temperature-Responsive Gene Silencing by a Smart Polymer.

    PubMed

    Wang, Mingming; Cheng, Yiyun

    2016-03-16

    Intracellular siRNA release is a crucial step in efficient gene silencing mediated by cationic polymers. Here, we show an example of temperature change-induced intracellular siRNA release and silencing using a temperature-responsive polymer consisting of dendrimer, poly(N-isopropylacrylamide) and phenylboronic acid. The smart polymer can trigger the release of loaded siRNA in a controlled manner upon cooling the surrounding solution below its lower critical solution temperature. Gene silencing efficacy of the polymer was significantly increased by cool treatment after its cellular uptake. The polymer and the cool treatment cause minimal toxicity to the transfected cells. The results provide a facile and promising strategy to design stimuli-responsive polymers for efficient gene silencing. PMID:26783999

  1. Mutations in Ran system affected telomere silencing in Saccharomyces cerevisiae

    SciTech Connect

    Hayashi, Naoyuki Kobayashi, Masahiko; Shimizu, Hiroko; Yamamoto, Ken-ichi; Murakami, Seishi; Nishimoto, Takeharu

    2007-11-23

    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 high 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.

  2. Post-transcriptional gene silencing activity of human GIGYF2.

    PubMed

    Kryszke, Marie-Hélène; Adjeriou, Badia; Liang, Feifei; Chen, Hong; Dautry, François

    2016-07-01

    In mammalian post-transcriptional gene silencing, the Argonaute protein AGO2 indirectly recruits translation inhibitors, deadenylase complexes, and decapping factors to microRNA-targeted mRNAs, thereby repressing mRNA translation and accelerating mRNA decay. However, the exact composition and assembly pathway of the microRNA-induced silencing complex are not completely elucidated. As the GYF domain of human GIGYF2 was shown to bind AGO2 in pulldown experiments, we wondered whether GIGYF2 could be a novel protein component of the microRNA-induced silencing complex. Here we show that full-length GIGYF2 coimmunoprecipitates with AGO2 in human cells, and demonstrate that, upon tethering to a reporter mRNA, GIGYF2 exhibits strong, dose-dependent silencing activity, involving both mRNA destabilization and translational repression. PMID:27157137

  3. Variations in hypovirus interactions with the fungal-host RNA-silencing antiviral-defense response.

    PubMed

    Zhang, Xuemin; Shi, Diane; Nuss, Donald L

    2012-12-01

    Hypoviruses Cryphonectria hypovirus 1 (CHV-1)/EP713, CHV-1/Euro7, and CHV-1/EP721, which infect the chestnut blight fungus Cryphonectria parasitica, differ in their degrees of virulence attenuation (hypovirulence), symptom expression, and viral RNA accumulation, even though they share between 90% and 99% amino acid sequence identity. In this report we examine whether this variability is influenced by interactions with the C. parasitica Dicer gene dcl2-dependent RNA-silencing antiviral defense response. The mild symptoms exhibited by strains infected with CHV-1/Euro7 and CHV-1/EP721 relative to those with severe hypovirus CHV-1/EP713 did not correlate with a higher induction of the RNA-silencing pathway. Rather, dcl2 transcripts accumulated to a higher level (∼8-fold) following infection by CHV-1/EP713 than following infection by CHV-1/Euro7 (1.2-fold) or CHV-1/EP721 (1.4-fold). The differences in dcl2 transcript accumulation in response to CHV-1/EP713 and CHV-1/EP721 were unrelated to the suppressor of RNA silencing, p29, encoded by the two viruses. Moreover, the coding strand viral RNA levels increased by 33-, 32-, and 16-fold for CHV-1/EP713, CHV-1/Euro7, and CHV-1/EP721, respectively, in Δdcl2 mutant strains. This indicates that a very robust antiviral RNA-silencing response was induced against all three viruses, even though significant differences in the levels of dcl2 transcript accumulation were observed. Unexpectedly, the severe debilitation previously reported for CHV-1/EP713-infected Δdcl2 mutant strains, and observed here for the CHV-1/Euro7-infected Δdcl2 mutant strains, was not observed with infection by CHV-1/EP721. By constructing chimeric viruses containing portions of CHV-1/EP713 and CHV-1/EP721, it was possible to map the region that is associated with the severe debilitation of the Δdcl2 mutant hosts to a 4.1-kb coding domain located in the central part of the CHV-1/EP713 genome. PMID:22993160

  4. Improved design of PPRHs for gene silencing.

    PubMed

    Rodríguez, Laura; Villalobos, Xenia; Solé, Anna; Lliberós, Carolina; Ciudad, Carlos J; Noé, Véronique

    2015-03-01

    Nowadays, the modulation of gene expression by nucleic acids has become a routine tool in biomedical research for target validation and it is also used to develop new therapeutic approaches. Recently, we developed the so-called polypurine reverse Hoogsteen hairpins (PPRHs) that show high stability and a low immunogenic profile and we demonstrated their efficacy both in vitro and in vivo. In this work, we explored different characteristics of PPRHs to improve their usage as a tool for gene silencing. We studied the role of PPRH length in the range from 20 to 30 nucleotides. We also proved their higher affinity of binding and efficacy on cell viability compared to nonmodified TFOs. To overcome possible off-target effects, we tested wild-type PPRHs, which proved to be capable of binding to their target sequence with more affinity, displaying a higher stability of binding and a higher effect in terms of cell viability. Moreover, we developed a brand new molecule called Wedge-PPRH with the ability to lock the ds-DNA into the displaced structure and proved its efficacy in prostate and breast cancer cell lines. PMID:25615267

  5. A is for aphorism - the power of silence.

    PubMed

    Kreijkamp-Kaspers, Sanne; Glasziou, Paul

    2012-11-01

    'All you have to do is listen' is the title of Rob Kapilow's delightful book on classical music; but he could equally have been talking about general practice consultations. Listening requires several skills including attention, echoing and body language, but begins with silence. Well timed silences, used judiciously, can allow the patient adequate space to express symptoms and concerns, while allowing the general practitioner more time for attention, comprehension and synthesis. PMID:23145428

  6. RNA Silencing Is Resistant to Low-Temperature in Grapevine

    PubMed Central

    Romon, Marjorie; Soustre-Gacougnolle, Isabelle; Schmitt, Carine; Perrin, Mireille; Burdloff, Yannick; Chevalier, Elodie; Mutterer, Jérome; Himber, Christophe; Zervudacki, Jérôme; Montavon, Thomas; Zimmermann, Aude; Elmayan, Taline; Vaucheret, Hervé; Dunoyer, Patrice; Masson, Jean E.

    2013-01-01

    RNA silencing is a natural defence mechanism against viruses in plants, and transgenes expressing viral RNA-derived sequences were previously shown to confer silencing-based enhanced resistance against the cognate virus in several species. However, RNA silencing was shown to dysfunction at low temperatures in several species, questioning the relevance of this strategy in perennial plants such as grapevines, which are often exposed to low temperatures during the winter season. Here, we show that inverted-repeat (IR) constructs trigger a highly efficient silencing reaction in all somatic tissues in grapevines. Similarly to other plant species, IR-derived siRNAs trigger production of secondary transitive siRNAs. However, and in sharp contrast to other species tested to date where RNA silencing is hindered at low temperature, this process remained active in grapevine cultivated at 4°C. Consistently, siRNA levels remained steady in grapevines cultivated between 26°C and 4°C, whereas they are severely decreased in Arabidopsis grown at 15°C and almost undetectable at 4°C. Altogether, these results demonstrate that RNA silencing operates in grapevine in a conserved manner but is resistant to far lower temperatures than ever described in other species. PMID:24376561

  7. A compact rotary vane attenuator

    NASA Technical Reports Server (NTRS)

    Nixon, D. L.; Otosh, T. Y.; Stelzried, C. T.

    1969-01-01

    Rotary vane attenuator, when used as a front end attenuator, introduces an insertion loss that is proportional to the angle of rotation. New technique allows the construction of a shortened compact unit suitable for most installations.

  8. Scion on a Stock Producing siRNAs of Potato Spindle Tuber Viroid (PSTVd) Attenuates Accumulation of the Viroid

    PubMed Central

    Kasai, Atsushi; Sano, Teruo; Harada, Takeo

    2013-01-01

    Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock. PMID:23469061

  9. The Treatment of Fibrosis of Joint Synovium and Frozen Shoulder by Smad4 Gene Silencing in Rats

    PubMed Central

    Xue, MingFeng; Gong, SuiLiang; Dai, JiaPing; Chen, Gang; Hu, JunYu

    2016-01-01

    Soft tissue fibrosis at the joint induced by inflammation is the pathological basis of frozen shoulder. In the present study, we utilized a lentiviral approach to silence the Smad4 gene in an in vitro fibrosis model of fibroblasts and an in vivo frozen shoulder model. We observed the change in the fibrosis process and the biological indicators of frozen shoulder. The in vitro fibrosis models (Rat myoblasts L6, Rat synovial cell RSC-364 and Rat chondrocytes RCs) were established using TGF-β1 induction, and the effect of Smad4 gene silencing on fibrosis was analyzed. The method of Kanno A was employed to establish a rat model of frozen shoulder, and Smad4 in the relevant part was knocked down with the lentiviral approach. We then examined the abduction and rotation angles and the length of synovial intima and measured the inflammatory factors in effusion and the fibrotic markers of tissues. We found that Smad4 knockdown suppressed the proliferation and expression of fibrotic markers in L6, RSC-364 and RCs cells induced by TGF-β1. MMP activity measurements showed that Smad4 knockdown significantly reversed the decrease in MMP activity in these three cell lines that were induced by TGF-β1. Furthermore, using lentivirus in the rat frozen shoulder model, we found that Smad4 silencing attenuated the inflammatory response and fibrosis. It significantly inhibited the increase of the Vimentin, α-SMA, collagen I and III, Lama1 and Timp1 proteins in synovial tissue as well as the inflammatory factors of TNF-a, IL-1α/β, IL-6 and IL-10 in effusion. MMP acidity assays revealed that Smad4 silencing inhibited MMP activity in the synovial, cartilage and ligament tissues in the model animals. The assessment of the phosphorylated Smad2/3 in the nuclei isolated from the synovial tissues showed that Smad4 silencing significantly inhibited the phosphorylation and subsequent nuclear translocation of Smad2/3 proteins. Moreover, Smad4-shRNA lentivirus inhibited the decrease in both

  10. Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes

    PubMed Central

    Srinivasan, Padma P.; Parajuli, Ashutosh; Price, Christopher; Wang, Liyun; Duncan, Randall L.; Kirn-Safran, Catherine B.

    2015-01-01

    Voltage-sensitive calcium channels (VSCC) regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1) and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM) obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration. PMID:26011709

  11. Silencing PP2A inhibitor by lenti-shRNA interference ameliorates neuropathologies and memory deficits in tg2576 mice.

    PubMed

    Liu, Gong-Ping; Wei, Wei; Zhou, Xin; Shi, Hai-Rong; Liu, Xing-Hua; Chai, Gao-Shang; Yao, Xiu-Qing; Zhang, Jia-Yu; Peng, Cai-Xia; Hu, Juan; Li, Xia-Chun; Wang, Qun; Wang, Jian-Zhi

    2013-12-01

    Deficits of protein phosphatase-2A (PP2A) play a crucial role in tau hyperphosphorylation, amyloid overproduction, and synaptic suppression of Alzheimer's disease (AD), in which PP2A is inactivated by the endogenously increased inhibitory protein, namely inhibitor-2 of PP2A (I2(PP2A)). Therefore, in vivo silencing I2(PP2A) may rescue PP2A and mitigate AD neurodegeneration. By infusion of lentivirus-shRNA targeting I2(PP2A) (LV-siI2(PP2A)) into hippocampus and frontal cortex of 11-month-old tg2576 mice, we demonstrated that expression of LV-siI2(PP2A) decreased remarkably the elevated I2(PP2A) in both mRNA and protein levels. Simultaneously, the PP2A activity was restored with the mechanisms involving reduction of the inhibitory binding of I2(PP2A) to PP2A catalytic subunit (PP2AC), repression of the inhibitory Leu309-demethylation and elevation of PP2AC. Silencing I2(PP2A) induced a long-lasting attenuation of amyloidogenesis in tg2576 mice with inhibition of amyloid precursor protein hyperphosphorylation and β-secretase activity, whereas simultaneous inhibition of PP2A abolished the antiamyloidogenic effects of I2(PP2A) silencing. Finally, silencing I2(PP2A) could improve learning and memory of tg2576 mice with preservation of several memory-associated components. Our data reveal that targeting I2(PP2A) can efficiently rescue Aβ toxicities and improve the memory deficits in tg2576 mice, suggesting that I2(PP2A) could be a promising target for potential AD therapies. PMID:23922015

  12. Silencing of a Germin-Like Gene in Nicotiana attenuata Improves Performance of Native Herbivores1[W

    PubMed Central

    Lou, Yonggen; Baldwin, Ian T.

    2006-01-01

    Germins and germin-like proteins (GLPs) are known to function in pathogen resistance, but their involvement in defense against insect herbivores is poorly understood. In the native tobacco Nicotiana attenuata, attack from the specialist herbivore Manduca sexta or elicitation by adding larval oral secretions (OS) to wounds up-regulates transcripts of a GLP. To understand the function of this gene, which occurs as a single copy, we cloned the full-length NaGLP and silenced its expression in N. attenuata by expressing a 250-bp fragment in an antisense orientation with an Agrobacterium-based transformation system and by virus-induced gene silencing (VIGS). Homozygous lines harboring a single insert and VIGS plants had significantly reduced constitutive (measured in roots) and elicited NaGLP transcript levels (in leaves). Silencing NaGLP improved M. sexta larval performance and Tupiocoris notatus preference, two native herbivores of N. attenuata. Silencing NaGLP also attenuated the OS-induced hydrogen peroxide (H2O2), diterpene glycosides, and trypsin proteinase inhibitor responses, which may explain the observed susceptibility of antisense or VIGS plants to herbivore attack and increased nicotine contents, but did not influence the OS-elicited jasmonate and salicylate bursts, or the release of the volatile organic compounds (limonene, cis-α-bergamotene, and germacrene-A) that function as an indirect defense. This suggests that NaGLP is involved in H2O2 production and might also be related to ethylene production and/or perception, which in turn influences the defense responses of N. attenuata via H2O2 and ethylene-signaling pathways. PMID:16461381

  13. Functional characterization of neural-restrictive silencer element in mouse pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression.

    PubMed

    Sugawara, Hideki; Tominaga, Aiko; Inoue, Kazuhiko; Takeda, Yasuo; Yamada, Katsushi; Miyata, Atsuro

    2014-11-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is predominantly localized in the nervous system, but the underlying mechanism in its neuron-specific expression remains unclear. In addition to two neural-restrictive silencer-like element (NRSLE1 and 2), as reported previously, we have identified the third element in -1,601 to -1,581 bp from the translational initiation site of mouse PACAP gene and termed it as NRSLE3, of which, the sequence and location were highly conserved among mouse, rat, and human PACAP genes. In luciferase reporter assay, the deletion or site-directed mutagenesis of NRSLE3 in the reporter gene construct, driven by heterologous SV40 promoter, cancelled the repression of luciferase activity in non-neuronal Swiss-3T3 cells. Furthermore, its promoter activity was significantly repressed in Swiss-3T3 cells, but not in neuronal-differentiated PC12 cells. The electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells demonstrated a specific complex with NRSLE3 probe that exhibited the same migration with the neural-restrictive silencer element (NRSE) probe of rat type II sodium channel gene. During neuronal differentiation of PC12 cells, the increment of PACAP mRNA exhibited the correlation with that of REST4 mRNA, which is a neuron-specific variant form of neural-restrictive silencer factor (NRSF). In undifferentiated PC12 cells, trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which indirectly inhibits NRSF-mediated gene silencing, increased PACAP mRNA level and attenuated the repression of promoter activity of 5' flanking region of mouse PACAP gene containing NRSLEs. These suggest that the NRSE-NRSF system implicates in the regulatory mechanism of neuron-specific expression of PACAP gene. PMID:24939248

  14. A role for the Saccharomyces cerevisiae RENT complex protein Net1 in HMR silencing.

    PubMed Central

    Kasulke, Daniela; Seitz, Stefanie; Ehrenhofer-Murray, Ann E

    2002-01-01

    Silencing in the yeast Saccharomyces cerevisiae is known in three classes of loci: in the silent mating-type loci HML and HMR, in subtelomeric regions, and in the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD(+)-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1, as well as NET1 expression from a 2micro-plasmid, restored repression at silencing-defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins and recruit other silencing factors to the silencer. In agreement with this, Net1 provided ORC-dependent, Sir1-independent silencing when artificially tethered to the silencer. In contrast, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 from the silenced loci toward HMR. PMID:12196389

  15. Fluid dynamic bowtie attenuators

    NASA Astrophysics Data System (ADS)

    Szczykutowicz, Timothy P.; Hermus, James

    2015-03-01

    Fluence field modulated CT allows for improvements in image quality and dose reduction. To date, only 1-D modulators have been proposed, the extension to 2-D modulation is difficult with solid-metal attenuation-based modulators. This work proposes to use liquids and gas to attenuate the x-ray beam which can be arrayed allowing for 2-D fluence modulation. The thickness of liquid and the pressure for a given path length of gas were determined that provided the same attenuation as 30 cm of soft tissue at 80, 100, 120, and 140 kV. Gaseous Xenon and liquid Iodine, Zinc Chloride, and Cerium Chloride were studied. Additionally, we performed some proof-of-concept experiments in which (1) a single cell of liquid was connected to a reservoir which allowed the liquid thickness to be modulated and (2) a 96 cell array was constructed in which the liquid thickness in each cell was adjusted manually. Liquid thickness varied as a function of kV and chemical composition, with Zinc Chloride allowing for the smallest thickness; 1.8, 2.25, 3, and 3.6 cm compensated for 30 cm of soft tissue at 80, 100, 120, and 140 kV respectively. The 96 cell Iodine attenuator allowed for a reduction in both dynamic range to the detector and scatter to primary ratio. Successful modulation of a single cell was performed at 0, 90, and 130 degrees using a simple piston/actuator. The thickness of liquids and the Xenon gas pressure seem logistically implementable within the constraints of CBCT and diagnostic CT systems.

  16. Downhole pressure attenuation apparatus

    SciTech Connect

    Ricles, T.D.; Barton, J.A.

    1992-02-18

    This patent describes a process for preventing damage to tool strings and other downhole equipment in a well caused by pressures produced during detonation of one or more downhole explosive devices. It comprises adding to a tool string at least one pressure attenuating apparatus for attenuating the peak pressure wave and quasi-static pressure pulse produced by the explosive devices, the pressure attenuating apparatus including an initially closed relief vent including tubing means supporting a plurality of charge port assemblies each including an explosive filled shaped charge and a prestressed disc, the shaped charges interconnected by a detonating cord, the amount of explosive in each shaped charge being sufficient to rupture its associated disc without damaging surrounding tubular bodies in the well, and a vent chamber defined by the tubing means and providing a liquid free volume, and opening the relief vent substantially contemporaneously with downhole explosive device detonation by detonating the shaped charges to rupture the discs of the charge port assemblies.

  17. Flexible graphene based microwave attenuators.

    PubMed

    Byun, Kisik; Ju Park, Yong; Ahn, Jong-Hyun; Min, Byung-Wook

    2015-02-01

    We demonstrate flexible 3 dB and 6 dB microwave attenuators using multilayer graphene grown by the chemical vapor deposition method. On the basis of the characterized results of multilayer graphene and graphene-Au ohmic contacts, the graphene attenuators are designed and measured. The flexible graphene-based attenuators have 3 dB and 6 dB attenuation with a return loss of less than -15 dB at higher than 5 GHz. The devices have shown durability in a bending cycling test of 100 times. The circuit model of the attenuator based on the characterized results matches the experimental results well. PMID:25590144

  18. Control algorithms for dynamic attenuators

    SciTech Connect

    Hsieh, Scott S.; Pelc, Norbert J.

    2014-06-15

    Purpose: The authors describe algorithms to control dynamic attenuators in CT and compare their performance using simulated scans. Dynamic attenuators are prepatient beam shaping filters that modulate the distribution of x-ray fluence incident on the patient on a view-by-view basis. These attenuators can reduce dose while improving key image quality metrics such as peak or mean variance. In each view, the attenuator presents several degrees of freedom which may be individually adjusted. The total number of degrees of freedom across all views is very large, making many optimization techniques impractical. The authors develop a theory for optimally controlling these attenuators. Special attention is paid to a theoretically perfect attenuator which controls the fluence for each ray individually, but the authors also investigate and compare three other, practical attenuator designs which have been previously proposed: the piecewise-linear attenuator, the translating attenuator, and the double wedge attenuator. Methods: The authors pose and solve the optimization problems of minimizing the mean and peak variance subject to a fixed dose limit. For a perfect attenuator and mean variance minimization, this problem can be solved in simple, closed form. For other attenuator designs, the problem can be decomposed into separate problems for each view to greatly reduce the computational complexity. Peak variance minimization can be approximately solved using iterated, weighted mean variance (WMV) minimization. Also, the authors develop heuristics for the perfect and piecewise-linear attenuators which do not requirea priori knowledge of the patient anatomy. The authors compare these control algorithms on different types of dynamic attenuators using simulated raw data from forward projected DICOM files of a thorax and an abdomen. Results: The translating and double wedge attenuators reduce dose by an average of 30% relative to current techniques (bowtie filter with tube current

  19. Ultrasonic attenuation in pearlitic steel.

    PubMed

    Du, Hualong; Turner, Joseph A

    2014-03-01

    Expressions for the attenuation coefficients of longitudinal and transverse ultrasonic waves are developed for steel with pearlitic microstructure. This type of lamellar duplex microstructure influences attenuation because of the lamellar spacing. In addition, longitudinal attenuation measurements were conducted using an unfocused transducer with 10 MHz central frequency on the cross section of a quenched railroad wheel sample. The dependence of longitudinal attenuation on the pearlite microstructure is observed from the changes of longitudinal attenuation from the quenched tread surface to deeper locations. The results show that the attenuation value is lowest and relatively constant within the quench depth, then increases linearly. The experimental results demonstrate a reasonable agreement with results from the theoretical model. Ultrasonic attenuation provides an important non-destructive method to evaluate duplex microstructure within grains which can be implemented for quality control in conjunction with other manufacturing processes. PMID:24268679

  20. Reconstitution of Heterochromatin-Dependent Transcriptional Gene Silencing

    PubMed Central

    Johnson, Aaron; Li, Geng; Sikorski, Timothy W.; Buratowski, Stephen; Woodcock, Christopher L.; Moazed, Danesh

    2009-01-01

    Summary Heterochromatin assembly in budding yeast requires the SIR complex, which contains the NAD-dependent deacetylase Sir2 and the Sir3 and Sir4 proteins. Sir3 binds to nucleosomes containing deacetylated histone H4 lysine 16 (H4K16) and, with Sir4, promotes spreading of Sir2 and deacetylation along the chromatin fiber. Combined action of histone modifying and binding activities is a conserved hallmark of heterochromatin, but the relative contribution of each activity to silencing has remained unclear. Here we reconstitute SIR-chromatin complexes using purified components and show that the SIR complex efficiently deacetylates chromatin templates and promotes the assembly of altered structures that silence Gal4-VP16-activated transcription. Silencing requires all three Sir proteins, even with fully deacetylated chromatin, and involves the specific association of Sir3 with deacetylated H4K16. These results define a minimal set of components that mediate heterochromatic gene silencing and demonstrate distinct contributions for histone deacetylation and nucleosome binding in the silencing mechanism. PMID:19782027

  1. Is the Efficiency of RNA Silencing Evolutionarily Regulated?

    PubMed Central

    Ui-Tei, Kumiko

    2016-01-01

    Small interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate gene expression in a sequence-specific manner. Genes with partial complementarity to siRNA/miRNA sequences in their 3′-untranslated regions (UTRs) are suppressed by a mechanism referred to as the siRNA off-target effect or miRNA-mediated RNA silencing. However, the determinants of such RNA silencing efficiency are poorly understood. Previously, I and co-workers reported that the efficiency of RNA silencing is strongly correlated with the thermodynamic stability of base pairing in the duplex formed within an siRNA/miRNA and between the seed region and its target mRNA. In this review, I first summarize our previous studies that identified the thermodynamic parameter to estimate the silencing efficiency using the calculated base pairing stability: siRNAs downregulate the expression of off-target genes depending on the stability of binding between the siRNA seed region (nucleotides 2–8) and off-target mRNAs, and miRNAs downregulate target mRNA expression depending on the stability of the duplex formed between the 5′ terminus of the miRNA and its target mRNA. I further discuss the possibility that such thermodynamic features of silencing efficiency may have arisen during evolution with increasing body temperature in various organisms. PMID:27187367

  2. Study on plate silencer with general boundary conditions

    NASA Astrophysics Data System (ADS)

    Liu, Gongmin; Zhao, Xiaochen; Zhang, Wenping; Li, Shuaijun

    2014-09-01

    A plate silencer consists of an expansion chamber with two side-branch rigid cavities covered by plates. Previous studies showed that, in a duct, the introduction of simply supported or clamped plates into an air conveying system could achieve broadband quieting from low to medium frequencies. In this study, analytical formulation is extended to the plate silencer with general boundary conditions. A set of static beam functions, which are a combination of sine series and third-order polynomial, is employed as the trial functions of the plate vibration velocity. Greens function and Kirchhoff-Helmholtz integral are used to solve the sound radiation in the duct and the cavity, and then the vibration velocity of the plate is obtained. Having obtained the vibration velocity, the pressure perturbations induced by the plate oscillation and the transmission loss are found. Optimization is carried out in order to obtain the widest stopband. The transmission loss calculated by the analytical method agrees closely with the result of the finite element method simulation. Further studies with regard to the plate under several different classical boundary conditions based on the validated model show that a clamped-free plate silencer has the worst stopband. Attempts to release the boundary restriction of the plate are also made to study its effect on sound reflection. Results show that a softer end for a clamped-clamped plate silencer helps increase the optimal bandwidth, while the same treatment for simply supported plate silencer will result in performance degradation.

  3. 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. PMID:27225900

  4. Is the Efficiency of RNA Silencing Evolutionarily Regulated?

    PubMed

    Ui-Tei, Kumiko

    2016-01-01

    Small interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate gene expression in a sequence-specific manner. Genes with partial complementarity to siRNA/miRNA sequences in their 3'-untranslated regions (UTRs) are suppressed by a mechanism referred to as the siRNA off-target effect or miRNA-mediated RNA silencing. However, the determinants of such RNA silencing efficiency are poorly understood. Previously, I and co-workers reported that the efficiency of RNA silencing is strongly correlated with the thermodynamic stability of base pairing in the duplex formed within an siRNA/miRNA and between the seed region and its target mRNA. In this review, I first summarize our previous studies that identified the thermodynamic parameter to estimate the silencing efficiency using the calculated base pairing stability: siRNAs downregulate the expression of off-target genes depending on the stability of binding between the siRNA seed region (nucleotides 2-8) and off-target mRNAs, and miRNAs downregulate target mRNA expression depending on the stability of the duplex formed between the 5' terminus of the miRNA and its target mRNA. I further discuss the possibility that such thermodynamic features of silencing efficiency may have arisen during evolution with increasing body temperature in various organisms. PMID:27187367

  5. 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.

  6. Breaking the Silence: Protein Stabilization Uncovers Silenced Biosynthetic Gene Clusters in the Fungus Aspergillus nidulans

    PubMed Central

    Gerke, Jennifer; Bayram, Özgür; Feussner, Kirstin; Landesfeind, Manuel; Shelest, Ekaterina; Feussner, Ivo

    2012-01-01

    The genomes of filamentous fungi comprise numerous putative gene clusters coding for the biosynthesis of chemically and structurally diverse secondary metabolites (SMs), which are rarely expressed under laboratory conditions. Previous approaches to activate these genes were based primarily on artificially targeting the cellular protein synthesis apparatus. Here, we applied an alternative approach of genetically impairing the protein degradation apparatus of the model fungus Aspergillus nidulans by deleting the conserved eukaryotic csnE/CSN5 deneddylase subunit of the COP9 signalosome. This defect in protein degradation results in the activation of a previously silenced gene cluster comprising a polyketide synthase gene producing the antibiotic 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA). The csnE/CSN5 gene is highly conserved in fungi, and therefore, the deletion is a feasible approach for the identification of new SMs. PMID:23001671

  7. Digitally Controlled Beam Attenuator

    NASA Astrophysics Data System (ADS)

    Peppler, W. W.; Kudva, B.; Dobbins, J. T.; Lee, C. S.; Van Lysel, M. S.; Hasegawa, B. H.; Mistretta, C. A.

    1982-12-01

    In digital fluorographic techniques the video camera must accommodate a wide dynamic range due to the large variation in the subject thickness within the field of view. Typically exposure factors and the optical aperture are selected such that the maximum video signal is obtained in the most transmissive region of the subject. Consequently, it has been shown that the signal-to-noise ratio is severely reduced in the dark regions. We have developed a prototype digital beam attenuator (DBA) which will alleviate this and some related problems in digital fluorography. The prototype DBA consists of a 6x6 array of pistons which are individually controlled. A membrane containing an attenuating solu-tion of (CeC13) in water and the piston matrix are placed between the x-ray tube and the subject. Under digital control the pistons are moved into the attenuating material in order to adjust the beam intensity over each of the 36 cells. The DBA control unit which digitizes the image during patient positioning will direct the pistons under hydraulic control to produce a uniform x-ray field exiting the subject. The pistons were designed to produce very little structural background in the image. In subtraction studies any structure would be cancelled. For non-subtraction studies such as cine-cardiology we are considering higher cell densities (eg. 64x64). Due to the narrow range of transmission provided by the DBA, in such studies ultra-high contrast films could be used to produce a high resolution quasi-subtraction display. Additional benefits of the DBA are: 1) reduced dose to the bright image areas when the dark areas are properly exposed. 2) improved scatter and glare to primary ratios, leading to improved contrast in the dark areas.

  8. RNA interference for CFTR attenuates lung fluid absorption at birth in rats

    PubMed Central

    Li, Tianbo; Koshy, Shyny; Folkesson, Hans G

    2008-01-01

    Background Small interfering RNA (siRNA) against αENaC (α-subunit of the epithelial Na channel) and CFTR (cystic fibrosis transmembrane conductance regulator) was used to explore ENaC and CTFR function in newborn rat lungs. Methods Twenty-four hours after trans-thoracic intrapulmonary (ttip) injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C2), we measured CFTR and ENaC expression, extravascular lung water, and mortality. Results αENaC and CFTR mRNA and protein decreased by ~80% and ~85%, respectively, following αENaC and CFTR silencing. Extravascular lung water and mortality increased after αENaC and CFTR-silencing. In pSi-C2-transfected isolated DLE cells there were attenuated CFTR mRNA and protein. In pSi-4-transfected DLE cells αENaC mRNA and protein were both reduced. Interestingly, CFTR-silencing also reduced αENaC mRNA and protein. αENaC silencing, on the other hand, only slightly reduced CFTR mRNA and protein. Conclusion Thus, ENaC and CFTR are both involved in the fluid secretion to absorption conversion around at birth. PMID:18652671

  9. Radiation Imaging and Attenuation

    NASA Astrophysics Data System (ADS)

    Davison, Candace; Yocum, Douglas

    2008-03-01

    X-ray and neutron images are used to demonstrate materials' different radiation attenuation properties. This leads to discussion of applications in medicine, industry and research. The Penn State Radiation Science and Engineering Center (RSEC) uses neutron radioscopy to image the inside of a working hydrogen fuel cell. This is one of the many educational activities that are conducted when students visit the RSEC. To encourage pre-college students to apply these principles and learn more about nuclear technology, we are sponsoring a design competition. For more information visit www.rsec.psu.edu

  10. Temporal control of gene silencing by in ovo electroporation.

    PubMed

    Baeriswyl, Thomas; Mauti, Olivier; Stoeckli, Esther T

    2008-01-01

    The analysis of gene function during embryonic development asks for tight temporal control of gene expression. Classic genetic tools do not allow for this, as the absence of a gene during the early stages of development will preclude its functional analysis during later stages. In contrast, RNAi technology allows one to achieve temporal control of gene silencing especially when used with oviparous animal models. In contrast to mammals, reptiles and birds are easily accessible during embryonic development. We have developed approaches to use RNAi for the analysis of gene function during nervous system development in the chicken embryo. Although the protocol given here describes a method for gene silencing in the developing spinal cord, it can easily be adapted to other parts of the developing nervous system. The combination of the easy accessibility of the chicken embryo and RNAi provides a unique opportunity for temporal and spatial control of gene silencing during development. PMID:18369789

  11. 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

  12. Structure and Gene-Silencing Mechanisms of Small Noncoding RNAs

    NASA Astrophysics Data System (ADS)

    Chu, Chia-Ying; Rana, Tariq M.

    Small (19-31-nucleotides) noncoding RNAs were identified in the past 10 years for their distinct function in gene silencing. The best known gene-silencing phenomenon, RNA interference (RNAi), is triggered in a sequence-specific manner by endogenously produced or exogenously introduced small doubled-stranded RNAs. As knowledge of the structure and function of the RNAi machinery has expanded, this phenomenon has become a powerful tool for biochemical research; it has enormous potential for therapeutics. This chapter summarizes significant aspects of three major classes of small noncoding, regulatory RNAs: small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Here, we focus on the biogenesis of these small RNAs, their structural features and coupled effectors as well as the mechanisms of each small regulatory RNA pathway which reveal fascinating ways by which gene silencing is controlled and fine-tuned at an epigenetic level.

  13. Delivery of gene silencing agents for breast cancer therapy

    PubMed Central

    2013-01-01

    The discovery of RNA interference has opened the door for the development of a new class of cancer therapeutics. Small inhibitory RNA oligos are being designed to specifically suppress expression of proteins that are traditionally considered nondruggable, and microRNAs are being evaluated to exert broad control of gene expression for inhibition of tumor growth. Since most naked molecules are not optimized for in vivo applications, the gene silencing agents need to be packaged into delivery vehicles in order to reach the target tissues as their destinations. Thus, the selection of the right delivery vehicles serves as a crucial step in the development of cancer therapeutics. The current review summarizes the status of gene silencing agents in breast cancer and recent development of candidate cancer drugs in clinical trials. Nanotechnology-based delivery vectors for the formulation and packaging of gene silencing agents are also described. PMID:23659575

  14. Biomimetic RNA Silencing Nanocomplexes Overcome Multidrug Resistance in Cancer Cells**

    PubMed Central

    Wang, Zhongliang; Wang, Zhe; Liu, Dingbin; Yan, Xuefeng; Wang, Fu; Niu, Gang

    2015-01-01

    RNA interference (RNAi) is an RNA-dependent gene silencing approach controlled by RNA-induced silencing complex (RISC). Here we represent 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. The nanocomplexes targeting to multidrug resistance are able to not only bypass P-glycoprotein (Pgp) transporter due to their nano-size effect, but also effectively suppress the 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. PMID:24446433

  15. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

    PubMed

    Veit, Guido; Oliver, Kathryn; Apaja, Pirjo M; Perdomo, Doranda; Bidaud-Meynard, Aurélien; Lin, Sheng-Ting; Guo, Jingyu; Icyuz, Mert; Sorscher, Eric J; Hartman Iv, John L; Lukacs, Gergely L

    2016-05-01

    The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect. PMID:27168400

  16. Silencing miR-146a influences B cells and ameliorates experimental autoimmune myasthenia gravis

    PubMed Central

    Zhang, JunMei; Jia, Ge; Liu, Qun; Hu, Jue; Yan, Mei; Yang, BaiFeng; Yang, Huan; Zhou, WenBin; Li, Jing

    2015-01-01

    MicroRNAs have been shown to be important regulators of immune homeostasis as patients with aberrant microRNA expression appeared to be more susceptible to autoimmune diseases. We recently found that miR-146a was up-regulated in activated B cells in response to rat acetylcholine receptor (AChR) α-subunit 97-116 peptide, and this up-regulation was significantly attenuated by AntagomiR-146a. Our data also demonstrated that silencing miR-146a with its inhibitor AntagomiR-146a effectively ameliorated clinical myasthenic symptoms in mice with ongoing experimental autoimmune myasthenia gravis. Furthermore, multiple defects were observed after miR-146a was knocked down in B cells, including decreased anti-R97-116 antibody production and class switching, reduced numbers of plasma cells, memory B cells and B-1 cells, and weakened activation of B cells. Previously, miR-146a has been identified as a nuclear factor-κB-dependent gene and predicted to base pair with the tumour necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1) genes to regulate the immune response. However, our study proved that miR-146a inhibition had no effect on the expression of TRAF6 and IRAK1 in B cells. This result suggests that the function of miR-146a in B cells does not involve these two target molecules. We conclude that silencing miR-146a exerts its therapeutic effects by influencing the B-cell functions that contribute to the autoimmune pathogenesis of myasthenia gravis. PMID:24962817

  17. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect

    PubMed Central

    Veit, Guido; Oliver, Kathryn; Apaja, Pirjo M.; Perdomo, Doranda; Bidaud-Meynard, Aurélien; Guo, Jingyu; Icyuz, Mert; Sorscher, Eric J.; Hartman IV, John L.; Lukacs, Gergely L.

    2016-01-01

    The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect. PMID:27168400

  18. Chopping-Wheel Optical Attenuator

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B.

    1988-01-01

    Star-shaped rotating chopping wheel provides adjustable time-averaged attenuation of narrow beam of light without changing length of optical path or spectral distribution of light. Duty cycle or attenuation factor of chopped beam controlled by adjusting radius at which beam intersects wheel. Attenuation factor independent of wavelength. Useful in systems in which chopping frequency above frequency-response limits of photodetectors receiving chopped light. Used in systems using synchronous detection with lock-in amplifiers.

  19. Ultrasonic Attenuation in Zircaloy-4

    SciTech Connect

    Gomez, M.P.; Banchik, A.D.; Lopez Pumarega, M.I.; Ruzzante, J.E.

    2005-04-09

    In this work the relationship between Zircaloy-4 grain size and ultrasonic attenuation behavior was studied for longitudinal waves in the frequency range of 10-90 MHz. The attenuation was analyzed as a function of frequency for samples with different mechanical and heat treatments having recrystallized and Widmanstatten structures with different grain size. The attenuation behavior was analyzed by different scattering models, depending on grain size, wavelength and frequency.

  20. LINE-ABOVE-GROUND ATTENUATOR

    DOEpatents

    Wilds, R.B.; Ames, J.R.

    1957-09-24

    The line-above-ground attenuator provides a continuously variable microwave attenuator for a coaxial line that is capable of high attenuation and low insertion loss. The device consists of a short section of the line-above- ground plane type transmission lime, a pair of identical rectangular slabs of lossy material like polytron, whose longitudinal axes are parallel to and indentically spaced away from either side of the line, and a geared mechanism to adjust amd maintain this spaced relationship. This device permits optimum fineness and accuracy of attenuator control which heretofore has been difficult to achieve.

  1. 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

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

    PubMed

    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

    2006-06-21

    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

  3. An attenuated philosophical gentleman.

    PubMed

    Christie, John R R

    2014-06-20

    Dr. Joseph Black had at one time, a house near us to the west. He was a striking and beautiful person; tall, very thin, and cadaverously pale; his hair carefully powdered, though there was little of it except what was collected in a long thin queue; his eyes dark, clear and large, like deep pools of pure water. He wore black speckless clothes, silk stockings, silver buckles, and either a slim green umbrella, or a genteel brown cane. The general frame and air were feeble and slender. The wildest boy respected Black. No lad could be irreverent toward a man so pale, so gentle, so elegant and so illustrious. So he glided, like a spirit, through our rather mischievous sportiveness, unharmed. He died seated, with a bowl of milk upon his knee, of which his ceasing to be did not spill a drop; a departure which it seemed, after the event, might have been foretold of this attenuated philosophical gentleman. PMID:24921110

  4. Fiber optic attenuator

    NASA Technical Reports Server (NTRS)

    Buzzetti, Mike F. (Inventor)

    1994-01-01

    A fiber optic attenuator of the invention is a mandrel structure through which a bundle of optical fibers is wrapped around in a complete circle. The mandrel structure includes a flexible cylindrical sheath through which the bundle passes. A set screw on the mandrel structure impacts one side of the sheath against two posts on the opposite side of the sheath. By rotating the screw, the sheath is deformed to extend partially between the two posts, bending the fiber optic bundle to a small radius controlled by rotating the set screw. Bending the fiber optic bundle to a small radius causes light in each optical fiber to be lost in the cladding, the amount depending upon the radius about which the bundle is bent.

  5. Genetic diversity and silencing suppression effects of Rice yellow mottle virus and the P1 protein

    PubMed Central

    Siré, Christelle; Bangratz-Reyser, Martine; Fargette, Denis; Brugidou, Christophe

    2008-01-01

    Background PTGS (post-transcriptional gene silencing) is used to counter pathogenic invasions, particularly viruses. In return, many plant viruses produce proteins which suppress silencing directed against their RNA. The diversity of silencing suppression at the species level in natural hosts is unknown. Results We investigated the functional diversity of silencing suppression among isolates of the African RYMV (Rice yellow mottle virus) in rice. The RYMV-P1 protein is responsible for cell-to-cell movement and is a silencing suppressor. Transgenic gus-silencing rice lines were used to investigate intra-specific and serogroup silencing suppression diversity at two different levels: that of the virion and the P1 silencing suppressor protein. Our data provide evidence that silencing suppression is a universal phenomenon for RYMV species. However, we found considerable diversity in their ability to suppress silencing which was not linked to RYMV phylogeny, or pathogenicity. At the level of the silencing suppressor P1 alone, we found similar results to those previously found at the virion level. In addition, we showed that cell-to-cell movement of P1 was crucial for the efficiency of silencing suppression. Mutagenesis of P1 demonstrated a strong link between some amino acids and silencing suppression features with, one on the hand, the conserved amino acids C95 and C64 involved in cell-to-cell movement and the strength of suppression, respectively, and on the other hand, the non conserved F88 was involved in the strength of silencing suppression. Conclusion We demonstrated that intra-species diversity of silencing suppression is highly variable and by mutagenesis of P1 we established the first link between silencing suppression and genetic diversity. These results are potentially important for understanding virus-host interactions. PMID:18447922

  6. Strain Specific Factors Control Effector Gene Silencing in Phytophthora sojae.

    PubMed

    Shrestha, Sirjana Devi; Chapman, Patrick; Zhang, Yun; Gijzen, Mark

    2016-01-01

    The Phytophthora sojae avirulence gene Avr3a encodes an effector that is capable of triggering immunity on soybean plants carrying the resistance gene Rps3a. P. sojae strains that express Avr3a are avirulent to Rps3a plants, while strains that do not are virulent. To study the inheritance of Avr3a expression and virulence towards Rps3a, genetic crosses and self-fertilizations were performed. A cross between P. sojae strains ACR10 X P7076 causes transgenerational gene silencing of Avr3a allele, and this effect is meiotically stable up to the F5 generation. However, test-crosses of F1 progeny (ACR10 X P7076) with strain P6497 result in the release of silencing of Avr3a. Expression of Avr3a in the progeny is variable and correlates with the phenotypic penetrance of the avirulence trait. The F1 progeny from a direct cross of P6497 X ACR10 segregate for inheritance for Avr3a expression, a result that could not be explained by parental imprinting or heterozygosity. Analysis of small RNA arising from the Avr3a gene sequence in the parental strains and hybrid progeny suggests that the presence of small RNA is necessary but not sufficient for gene silencing. Overall, we conclude that inheritance of the Avr3a gene silenced phenotype relies on factors that are variable among P. sojae strains. PMID:26930612

  7. Tissue homogeneity requires inhibition of unequal gene silencing during development.

    PubMed

    Le, Hai H; Looney, Monika; Strauss, Benjamin; Bloodgood, Michael; Jose, Antony M

    2016-08-01

    Multicellular organisms can generate and maintain homogenous populations of cells that make up individual tissues. However, cellular processes that can disrupt homogeneity and how organisms overcome such disruption are unknown. We found that ∼100-fold differences in expression from a repetitive DNA transgene can occur between intestinal cells in Caenorhabditis elegans These differences are caused by gene silencing in some cells and are actively suppressed by parental and zygotic factors such as the conserved exonuclease ERI-1. If unsuppressed, silencing can spread between some cells in embryos but can be repeat specific and independent of other homologous loci within each cell. Silencing can persist through DNA replication and nuclear divisions, disrupting uniform gene expression in developed animals. Analysis at single-cell resolution suggests that differences between cells arise during early cell divisions upon unequal segregation of an initiator of silencing. Our results suggest that organisms with high repetitive DNA content, which include humans, could use similar developmental mechanisms to achieve and maintain tissue homogeneity. PMID:27458132

  8. Design curves for circular and annular duct silencers

    NASA Technical Reports Server (NTRS)

    Watson, Willie R.; Ramakrishnan, R.

    1989-01-01

    Conventional models of sound propagation between porous walls (Scott, 1946) are adapted in order to calculate design curves for the lined circular and annular-duct silencers used in HVAC systems. The derivation of the governing equations is outlined, and results for two typical cases are presented graphically. Good agreement with published experimental data is demonstrated.

  9. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  11. Virus-Induced Gene Silencing in Hexaploid Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Functional genomics analysis in hexaploid wheat is greatly impeded by the genetic redundancy of polyploidy and the difficulties in generating large numbers of transgenic plants required in insertional mutagenesis strategies. Virus-induced gene silencing (VIGS), however, is a strategy for creating g...

  12. A reciprocal identity method for large silencer analysis

    NASA Astrophysics Data System (ADS)

    Zhou, L.; Wu, T. W.; Ruan, K.; Herrin, D. W.

    2016-03-01

    Conventional techniques used in the boundary element method for evaluating muffler transmission loss have been limited by the cutoff frequency of the inlet and outlet ducts. Even though the boundary element method itself is a truly three-dimensional analysis tool, it has not been effectively used on large silencers due to the large inlet and outlet cross sections. In this paper, a numerical technique based on the reciprocal identity and the boundary element impedance matrix is proposed as a post-processing filter to extract the transmission loss of large silencers at all frequencies. Each reciprocal identity couples two different sound fields on the same silencer geometry. The first sound field has the analytical modal expansion in the inlet and outlet ducts, while the second sound field is the boundary element solution associated with a random boundary condition set. Depending on how many modes exist in the inlet and outlet ducts at a certain frequency, a minimum number of random boundary condition sets must be applied to the boundary element model. The boundary element impedance matrix provides more than enough such solution sets for the reciprocal identity coupling. The overdetermined system is then solved by a least-squares procedure. The proposed method is verified by comparing to the analytical solutions of a simple expansion chamber and a round bar silencer.

  13. Strain Specific Factors Control Effector Gene Silencing in Phytophthora sojae

    PubMed Central

    Shrestha, Sirjana Devi; Chapman, Patrick; Zhang, Yun; Gijzen, Mark

    2016-01-01

    The Phytophthora sojae avirulence gene Avr3a encodes an effector that is capable of triggering immunity on soybean plants carrying the resistance gene Rps3a. P. sojae strains that express Avr3a are avirulent to Rps3a plants, while strains that do not are virulent. To study the inheritance of Avr3a expression and virulence towards Rps3a, genetic crosses and self-fertilizations were performed. A cross between P. sojae strains ACR10 X P7076 causes transgenerational gene silencing of Avr3a allele, and this effect is meiotically stable up to the F5 generation. However, test-crosses of F1 progeny (ACR10 X P7076) with strain P6497 result in the release of silencing of Avr3a. Expression of Avr3a in the progeny is variable and correlates with the phenotypic penetrance of the avirulence trait. The F1 progeny from a direct cross of P6497 X ACR10 segregate for inheritance for Avr3a expression, a result that could not be explained by parental imprinting or heterozygosity. Analysis of small RNA arising from the Avr3a gene sequence in the parental strains and hybrid progeny suggests that the presence of small RNA is necessary but not sufficient for gene silencing. Overall, we conclude that inheritance of the Avr3a gene silenced phenotype relies on factors that are variable among P. sojae strains. PMID:26930612

  14. Bureaucratic Constructions of Sexual Diversity: "Sensitive", "Controversial" and Silencing

    ERIC Educational Resources Information Center

    Ullman, Jacqueline; Ferfolja, Tania

    2015-01-01

    National research illustrates the high degree of discrimination that prevails against lesbian, gay, bisexual, transgender and queer (LGBTQ) students resulting in diminished educational outcomes, both academic and social. This phenomenon is influenced by the prevalence of whole-school silences around LGBTQ topics in many Australian schools. This…

  15. Love Styles and Self-Silencing in Romantic Relationships

    ERIC Educational Resources Information Center

    Collins, Kerry A.; Cramer, Kenneth M.; Singleton-Jackson, Jill A.

    2005-01-01

    Six love styles have been theorized to be related to several personality constructs (e.g., self-esteem) (Lee, 1973). Despite the interpersonal nature of love, investigations have yet to evaluate related variables and their association to love styles in romantic relationships. As a stable cognitive schema, silencing the self is proposed to account…

  16. 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

  17. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

    PubMed

    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

  18. Multiple roles for Piwi in silencing Drosophila transposons

    PubMed Central

    Rozhkov, Nikolay V.; Hammell, Molly; Hannon, Gregory J.

    2013-01-01

    Silencing of transposons in the Drosophila ovary relies on three Piwi family proteins—Piwi, Aubergine (Aub), and Ago3—acting in concert with their small RNA guides, the Piwi-interacting RNAs (piRNAs). Aub and Ago3 are found in the germ cell cytoplasm, where they function in the ping-pong cycle to consume transposon mRNAs. The nuclear Piwi protein is required for transposon silencing in both germ and somatic follicle cells, yet the precise mechanisms by which Piwi acts remain largely unclear. We investigated the role of Piwi by combining cell type-specific knockdowns with measurements of steady-state transposon mRNA levels, nascent RNA synthesis, chromatin state, and small RNA abundance. In somatic cells, Piwi loss led to concerted effects on nascent transcripts and transposon mRNAs, indicating that Piwi acts through transcriptional gene silencing (TGS). In germ cells, Piwi loss showed disproportionate impacts on steady-state RNA levels, indicating that it also exerts an effect on post-transcriptional gene silencing (PTGS). Piwi knockdown affected levels of germ cell piRNAs presumably bound to Aub and Ago3, perhaps explaining its post-transcriptional impacts. Overall, our results indicate that Piwi plays multiple roles in the piRNA pathway, in part enforcing transposon repression through effects on local chromatin states and transcription but also participating in germ cell piRNA biogenesis. PMID:23392609

  19. Breaking Classroom Silences: A View from Linguistic Ethnography

    ERIC Educational Resources Information Center

    Rampton, Ben; Charalambous, Constadina

    2016-01-01

    This paper addresses potentially problematic classroom episodes in which someone foregrounds a social division that is normally taken for granted. It illustrates the way in which linguistic ethnography can unpack the layered processes that collide in the breaking of silence, showing how linguistic form and practice, individual positioning, local…

  20. Organizational Justice As a Predictor of Organizational Silence

    ERIC Educational Resources Information Center

    Tan, Çetin

    2014-01-01

    In this study, relation between teachers' perception for organizational justice and their organizational silence was examined. Sample of this study consists of 300 teachers who work at elementary schools in Siirt. Relational Scanning model was utilized in performance of this study. In this study, Organizational Justice Scale and…

  1. When to Shut Students Up: Civility, Silencing, and Free Speech

    ERIC Educational Resources Information Center

    Callan, Eamonn

    2011-01-01

    Teachers sometimes shut students up for the sake of civility. My question is whether silencing for the sake of civility can be morally justified when a student derogates fellow students as members of some widely stigmatized group, and the offending speech is not for any further reason to be deplored, for example, as a personally targeted insult.…

  2. Breaking the Silence: Helping Battered Moms and Their Children.

    ERIC Educational Resources Information Center

    Texas Child Care, 2000

    2000-01-01

    Discusses issues of domestic violence to help child caregivers care for children more effectively. Considers the causes of domestic violence and its impact on children and families, and offers resources for more information as well as suggestions for helping an abused person get help. Includes lists of "Do's and don'ts for breaking the silence"…

  3. Silencing of an arabidopsis Myb regulatory transgene in tobacco

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have been exploring the use of genetically engineered anthocyanin over-production as a visual indicator of gene activation and silencing in plants. Previous work demonstrated that constitutive over-expression of genes encoding specific Arabidopsis transcription factors from the myb family can pr...

  4. The Silenced Dialogue and Pre-Service Teachers

    ERIC Educational Resources Information Center

    Curry, Kristal

    2013-01-01

    In this article, the author reflects on the 1988 article "The Silenced Dialogue," by Lisa Delpit, which described the lack of communication dividing Black and White educators when it comes to the issue of race, specifically due to the disparity between reliance on theory (White) and reliance on cultural understanding (Black). Nearly a…

  5. Breaking the Silence Surrounding Mental Health on Campus

    ERIC Educational Resources Information Center

    Roper, Larry D.

    2013-01-01

    Mentally ill students are able to participate in higher education at unprecedented rates. While colleges and universities have been responsive to the therapeutic needs, we have failed to successfully create supportive campus climates. Campus leaders are challenged to demonstrate ethical leadership that breaks the silence and confronts the stigma…

  6. Breaking the Silence: Facing Undocumented Issues in Teacher Practice

    ERIC Educational Resources Information Center

    Jefferies, Julián; Dabach, Dafney Blanca

    2014-01-01

    This conceptual article addresses the need for educators to interrupt status-quo silences surrounding the role of immigration status in schools--an issue that disproportionately impacts Latina/os. In this article we: (a) articulate the need for teacher education to address the impact of undocumented status in school settings; (b) present…

  7. Range Restriction and Attenuation Corrections.

    ERIC Educational Resources Information Center

    Mumford, Michael D.; Mendoza, Jorge L.

    The present paper reviews the techniques commonly used to correct an observed correlation coefficient for the simultaneous influence of attenuation and range restriction effects. It is noted that the procedure which is currently in use may be somewhat biased because it treats range restriction and attenuation as independent restrictive influences.…

  8. Suicide Risk: Amplifiers and Attenuators.

    ERIC Educational Resources Information Center

    Plutchik, Robert; Van Praag, Herman M.

    1994-01-01

    Attempts to integrate findings on correlates of suicide and violent risk in terms of a theory called a two-stage model of countervailing forces, which assumes that the strength of aggressive impulses is modified by amplifiers and attenuators. The vectorial interaction of amplifiers and attenuators creates an unstable equilibrium making prediction…

  9. Adjustable Optical-Fiber Attenuator

    NASA Technical Reports Server (NTRS)

    Buzzetti, Mike F.

    1994-01-01

    Adjustable fiber-optic attenuator utilizes bending loss to reduce strength of light transmitted along it. Attenuator functions without introducing measurable back-reflection or insertion loss. Relatively insensitive to vibration and changes in temperature. Potential applications include cable television, telephone networks, other signal-distribution networks, and laboratory instrumentation.

  10. Different patterns of gene silencing in position-effect variegation.

    PubMed

    Lloyd, Vett K; Dyment, David; Sinclair, Donald A R; Grigliatti, Thomas A

    2003-12-01

    Position-effect variegation (PEV) results when a fully functional gene is moved from its normal position to a position near to a broken heterochromatic-euchromatic boundary. In this new position, the gene, while remaining unaltered at the DNA level, is transcriptionally silenced in some cells but active in others, producing a diagnostic mosaic phenotype. Many variegating stocks show phenotypic instability, in that the level of variegation is dramatically different in different isolates or when out crossed. To test if this phenotypic instability was due to segregation of spontaneously accumulated mutations that suppress variegation, four different and well-characterized strains showing PEV for the white+ gene (wm4, wmMc, wm51b, and wmJ) and representing both large and small spot variegators were repeatedly out crossed to a strain free of modifiers, and the phenotypes of these variegators were monitored for 30 generations. Once free of modifiers, these variegating strains were then allowed to reaccumulate modifiers. The spontaneous suppressors of variegation were found to include both dominant and recessive, autosomal and X-linked alleles selected to reduce the detrimental effects of silencing white+ and adjacent genes. The time of peak sensitivity to temperature during development was also determined for these four variegators. Although large and small spot variegators have previously been attributed to early and late silencing events, respectively, the variegators we examined all shared a common early period of peak sensitivity to temperature. Once free of their variegation suppressors, the different variegating strains showed considerable differences in the frequency of inactivation at a cellular level (the number of cells showing silencing of a given gene) and the extent of variegation within the cell (the number of silenced genes). These results suggest that large and small spot variegation may be a superficial consequence of spontaneous variegation suppressors

  11. Importance of coat protein and RNA silencing in satellite RNA/virus interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA silencing is a major defense mechanism that plants use to fight an invading virus. The silencing suppressor of Turnip crinkle virus (TCV) is the viral coat protein (CP), which obstructs the DCL2/DCL4 silencing pathway. TCV is associated with a virulent satellite RNA (satC) that represses the a...

  12. Trans-specific gene silencing between host and parasitic plants.

    PubMed

    Tomilov, Alexey A; Tomilova, Natalia B; Wroblewski, Tadeusz; Michelmore, Richard; Yoder, John I

    2008-11-01

    Species of Orobanchaceae parasitize the roots of nearby host plants to rob them of water and other nutrients. Parasitism can be debilitating to the host plant, and some of the world's most pernicious agricultural pests are parasitic weeds. We demonstrate here that interfering hairpin constructs transformed into host plants can silence expression of the targeted genes in the parasite. Transgenic roots of the hemi-parasitic plant Triphysaria versicolor expressing the GUS reporter gene were allowed to parasitize transgenic lettuce roots expressing a hairpin RNA containing a fragment of the GUS gene (hpGUS). When stained for GUS activity, Triphysaria roots attached to non-transgenic lettuce showed full GUS activity, but those parasitizing transgenic hpGUS lettuce lacked activity in root tissues distal to the haustorium. Transcript quantification indicated a reduction in the steady-state level of GUS mRNA in Triphysaria when they were attached to hpGUS lettuce. These results demonstrate that the GUS silencing signal generated by the host roots was translocated across the haustorium interface and was functional in the parasite. Movement across the haustorium was bi-directional, as demonstrated in double-junction experiments in which non-transgenic Triphysaria concomitantly parasitized two hosts, one transgenic for hpGUS and the other transgenic for a functional GUS gene. Observation of GUS silencing in the second host demonstrated that the silencing trigger could be moved from one host to another using the parasite as a physiological bridge. Silencing of parasite genes by generating siRNAs in the host provides a novel strategy for controlling parasitic weeds. PMID:18643992

  13. Mycoreovirus genome rearrangements associated with RNA silencing deficiency

    PubMed Central

    Eusebio-Cope, Ana; Suzuki, Nobuhiro

    2015-01-01

    Mycoreovirus 1 (MyRV1) has 11 double-stranded RNA genome segments (S1 to S11) and confers hypovirulence to the chestnut blight fungus, Cryphonectria parasitica. MyRV1 genome rearrangements are frequently generated by a multifunctional protein, p29, encoded by a positive-strand RNA virus, Cryphonectria hypovirus 1. One of its functional roles is RNA silencing suppression. Here, we explored a possible link between MyRV1 genome rearrangements and the host RNA silencing pathway using wild-type (WT) and mutant strains of both MyRV1 and the host fungus. Host strains included deletion mutants of RNA silencing components such as dicer-like (dcl) and argonaute-like (agl) genes, while virus strains included an S4 internal deletion mutant MyRV1/S4ss. Consequently, intragenic rearrangements with nearly complete duplication of the three largest segments, i.e. S1, S2 and S3, were observed even more frequently in the RNA silencing-deficient strains Δdcl2 and Δagl2 infected with MyRV1/S4ss, but not with any other viral/host strain combinations. An interesting difference was noted between genome rearrangement events in the two host strains, i.e. generation of the rearrangement required prolonged culture for Δagl2 in comparison with Δdcl2. These results suggest a role for RNA silencing that suppresses genome rearrangements of a dsRNA virus. PMID:25800742

  14. SOX30, a novel epigenetic silenced tumor suppressor, promotes tumor cell apoptosis by transcriptional activating p53 in lung cancer

    PubMed Central

    Han, F; Liu, W; Jiang, X; Shi, X; Yin, L; Ao, L; Cui, Z; Li, Y; Huang, C; Cao, J; Liu, J

    2015-01-01

    Although members of SOX family have been well documented for their essential roles in embryonic development, cell proliferation and disease, the functional role and molecular mechanism of SOX30 in cancer are largely unexplored. Here, we first identified SRY-box containing gene 30 (SOX30) as a novel preferentially methylated gene using genome-wide methylation screening. SOX30 hypermethylation was detected in 100% of lung cancer cell lines (9/9) and 70.83% (85/120) of primary lung tumor tissues compared with none (0/20) of normal and 8.0% (2/25) of peri-tumoral lung tissues (P<0.01). SOX30 was expressed in normal and peri-tumoral lung tissues in which SOX30 was unmethylated, but was silenced or downregulated in lung cancer cell lines and primary lung tumor tissues harboring a hypermethylated SOX30. De-methylation experiments further confirmed that silence of SOX30 was regulated by its hypermethylation. Ectopic expression of SOX30 induces cancer cell apoptosis with inhibiting proliferation in vitro and represses tumor formation in vivo, whereas knockdown of SOX30 demonstrates a reversed effect both in vitro and in vivo. At the molecular level, the antitumorigenic effect of SOX30 is mediated by directly binding to CACTTTG (+115 to +121) of p53 promoter region and activating p53 transcription, suggesting that SOX30 is a novel transcriptional activating factor of p53. Indeed, blockade of p53 attenuates the tumor inhibition of SOX30. Overall, these findings demonstrate that SOX30 is a novel epigenetic silenced tumor suppressor acting through direct regulation of p53 transcription and expression. This study provides novel insights on the mechanism of tumorigenesis in lung cancer. PMID:25435374

  15. Tobacco rattle virus 16K silencing suppressor binds ARGONAUTE 4 and inhibits formation of RNA silencing complexes.

    PubMed

    Fernández-Calvino, Lourdes; Martínez-Priego, Llúcia; Szabo, Edit Z; Guzmán-Benito, Irene; González, Inmaculada; Canto, Tomás; Lakatos, Lóránt; Llave, César

    2016-01-01

    The cysteine-rich 16K protein of tobacco rattle virus (TRV), the type member of the genus Tobravirus, is known to suppress RNA silencing. However, the mechanism of action of the 16K suppressor is not well understood. In this study, we used a GFP-based sensor strategy and an Agrobacterium-mediated transient assay in Nicotiana benthamiana to show that 16K was unable to inhibit the activity of existing small interfering RNA (siRNA)- and microRNA (miRNA)-programmed RNA-induced silencing effector complexes (RISCs). In contrast, 16K efficiently interfered with de novo formation of miRNA- and siRNA-guided RISCs, thus preventing cleavage of target RNA. Interestingly, we found that transiently expressed endogenous miR399 and miR172 directed sequence-specific silencing of complementary sequences of viral origin. 16K failed to bind small RNAs, although it interacted with ARGONAUTE 4, as revealed by bimolecular fluorescence complementation and immunoprecipitation assays. Site-directed mutagenesis demonstrated that highly conserved cysteine residues within the N-terminal and central regions of the 16K protein are required for protein stability and/or RNA silencing suppression. PMID:26498945

  16. A study of resonant-cavity and fiberglass-filled parallel baffles as duct silencers. [for wind tunnels

    NASA Technical Reports Server (NTRS)

    Soderman, P. T.

    1982-01-01

    Acoustical performance and pressure drop were measured for two types of splitters designed to attenuate sound propagating in ducts - resonant-cavity baffles and fiberglass-filled baffles. Arrays of four baffles were evaluated in the 7- by 10-foot wind tunnel number 1 at Ames Research Center at flow speeds from 0 to 41 m/sec. The baffles were 2.1 m high, 305 to 406 mm thick, and 3.1 to 4.4 m long. Emphasis was on measurements of silencer insertion loss as affected by variations of such parameters as baffle length, baffle thickness, perforated skin geometry, cavity size and shape, cavity damping, wind speed, and acoustic field directivity. An analytical method for predicting silencer performance is described and compared with measurements. With the addition of cavity damping in the form of 25-mm foam linings, the insertion loss above 250 Hz of the resonant-cavity baffles was improved 2 to 7 db compared with the undamped baffles; the loss became equal to or greater than the insertion loss of comparable size fiberglass baffles at frequencies above 250 Hz. Variations of cavity size and shape showed that a series of cavities with triangular cross-sections (i.e., variable depth) were superior to cavities with rectangular cross sections (i.e., constant depth). In wind, the undamped, resonant-cavity baffles generated loud cavity-resonance tones; the tones could be eliminated by cavity damping.

  17. Differential effects of two-pore channel protein 1 and 2 silencing in MDA-MB-468 breast cancer cells.

    PubMed

    Jahidin, Aisyah H; Stewart, Teneale A; Thompson, Erik W; Roberts-Thomson, Sarah J; Monteith, Gregory R

    2016-09-01

    Two-pore channel proteins, TPC1 and TPC2, are calcium permeable ion channels found localized to the membranes of endolysosomal calcium stores. There is increasing interest in the role of TPC-mediated intracellular signaling in various pathologies; however their role in breast cancer has not been extensively evaluated. TPC1 and TPC2 mRNA was present in all non-tumorigenic and tumorigenic breast cell lines assessed. Silencing of TPC2 but not TPC1 attenuated epidermal growth factor-induced vimentin expression in MDA-MB-468 breast cancer cells. This effect was not due to a general inhibition of epithelial to mesenchymal transition (EMT) as TPC2 silencing had no effect on epidermal growth factor (EGF)-induced changes on E-cadherin expression. TPC1 and TPC2 were also shown to differentially regulate cyclopiazonic acid (CPA)-mediated changes in cytosolic free Ca(2+). These findings indicate potential differential regulation of signaling processes by TPC1 and TPC2 in breast cancer cells. PMID:27353380

  18. The molecular origin of a loading-induced black layer in the deep region of articular cartilage at the magic angle

    PubMed Central

    Wang, Nian; Kahn, David; Badar, Farid; Xia, Yang

    2014-01-01

    Purpose To investigate the molecular origin of an unusual low-intensity layer in the deep region of articular cartilage as seen in MRI when the tissue is imaged under compression and oriented at the magic angle. Materials and Methods Microscopic MRI (μMRI) T2 and T1ρ experiments were carried out for both native and degraded (treated with trypsin) 18 specimens. The glycosaminoglycan (GAG) concentrations in the specimens were quantified by both sodium ICP-OES and μMRI Gd(DTPA)2--contrast methods. The mechanical modulus of the specimens was also measured. Results Native tissue shows no load-induced layer, while the trypsin-degraded tissue shows clearly the low-intensity line at the deep part of tissue. The GAG reductions are confirmed by the sodium ICP-OES (from 81.7 ± 5.4 mg/ml to 9.2 ± 3.4 mg/ml), MRI GAG quantification (from 72.4 ± 6.7 mg/ml to 11.2 ± 2.9 mg/ml). The modulus reduction is confirmed by biomechanics (from 4.3 ± 0.7 MPa to 0.3 ± 0.1 MPa). Conclusion Both T2 and T1ρ profiles in native and degraded cartilage show strongly strain-, depth-, and angle-dependent using high resolution MRI. The GAG reduction is responsible for the visualization of a low-intensity layer in deep cartilage when it is loaded and orientated at 55°. PMID:24833266

  19. Nanoparticle based galectin-1 gene silencing, implications in methamphetamine regulation of HIV-1 infection in monocyte derived macrophages.

    PubMed

    Reynolds, Jessica L; Law, Wing Cheung; Mahajan, Supriya D; Aalinkeel, Ravikumar; Nair, Bindukumar; Sykes, Donald E; Yong, Ken-Tye; Hui, Rui; Prasad, Paras N; Schwartz, Stanley A

    2012-09-01

    Galectin-1, an adhesion molecule, is expressed in macrophages and implicated in human immunodeficiency virus (HIV-1) viral adsorption. In this study, we investigated the effects of methamphetamine on galectin-1 production in human monocyte derived macrophages (MDM) and the role of galectin-1 in methamphetamine potentiation of HIV-1 infection. Herein we show that levels of galectin-1 gene and protein expression are significantly increased by methamphetamine. Furthermore, concomitant incubation of MDM with galectin-1 and methamphetamine facilitates HIV-1 infection compared to galectin-1 alone or methamphetamine alone. We utilized a nanotechnology approach that uses gold nanorod (GNR)-galectin-1 siRNA complexes (nanoplexes) to inhibit gene expression for galectin-1. Nanoplexes significantly silenced gene expression for galectin-1 and reversed the effects of methamphetamine on galectin-1 gene expression. Moreover, the effects of methamphetamine on HIV-1 infection were attenuated in the presence of the nanoplex in MDM. PMID:22689223

  20. Attenuation of Vaccinia Virus.

    PubMed

    Yakubitskiy, S N; Kolosova, I V; Maksyutov, R A; Shchelkunov, S N

    2015-01-01

    Since 1980, in the post-smallpox vaccination era the human population has become increasingly susceptible compared to a generation ago to not only the variola (smallpox) virus, but also other zoonotic orthopoxviruses. The need for safer vaccines against orthopoxviruses is even greater now. The Lister vaccine strain (LIVP) of vaccinia virus was used as a parental virus for generating a recombinant 1421ABJCN clone defective in five virulence genes encoding hemagglutinin (A56R), the IFN-γ-binding protein (B8R), thymidine kinase (J2R), the complement-binding protein (C3L), and the Bcl-2-like inhibitor of apoptosis (N1L). We found that disruption of these loci does not affect replication in mammalian cell cultures. The isogenic recombinant strain 1421ABJCN exhibits a reduced inflammatory response and attenuated neurovirulence relative to LIVP. Virus titers of 1421ABJCN were 3 lg lower versus the parent VACV LIVP when administered by the intracerebral route in new-born mice. In a subcutaneous mouse model, 1421ABJCN displayed levels of VACV-neutralizing antibodies comparable to those of LIVP and conferred protective immunity against lethal challenge by the ectromelia virus. The VACV mutant holds promise as a safe live vaccine strain for preventing smallpox and other orthopoxvirus infections. PMID:26798498

  1. SEISMIC ATTENUATION FOR RESERVOIR CHARACTERIZATION

    SciTech Connect

    Joel Walls; M.T. Taner; Gary Mavko; Jack Dvorkin

    2002-04-01

    Wave-induced variations of pore pressure in a partially-saturated reservoir result in oscillatory liquid flow. The viscous losses during this flow are responsible for wave attenuation. The same viscous effects determine the changes in the dynamic bulk modulus of the system versus frequency. These changes are necessarily linked to attenuation via the causality condition. We analytically quantify the frequency dependence of the bulk modulus of a partially saturated rock by assuming that saturation is patchy and then link these changes to the inverse quality factor. As a result, the P-wave attenuation is quantitatively linked to saturation and thus can serve as a saturation indicator.

  2. ssiRNA Induced Gene Silencing is Transmitted Between Cells From the Mammalian Central Nervous System

    PubMed Central

    Zhao, Tian-Yong; Zou, Shi-Ping; Alimova, Yelena V.; Wang, Guoying; Hauser, Kurt F.; Ghandour, M. Said; Knapp, Pamela E.

    2014-01-01

    Although siRNA induced gene silencing can be transmitted between cells in plants and in C. elegans, this phenomenon has been barely studied in mammalian cells. Both immortalized oligodendrocytes and SNB-19 glioblastoma cells were transfected with siRNA constructs for PTEN (phosphatase and tensin homolog deleted on chromosome 10) or Akt (Akt/protein kinase B). Co-cultures were established between silenced cells and non-silenced cells which were hygromycin resistant and/or expressed green fluorescent protein (GFP). After fluorescence sorting or hygromycin selection to remove the silenced cells, the expression of PTEN or Akt genes in the originally unsilenced cells was in all cases significantly decreased. Importantly, silencing did not occur in transwell culture studies, suggesting that transmission of the silencing signal requires a close association between cells. These results provide the first direct demonstration that an siRNA induced silencing signal can be transmitted between mammalian central nervous system (CNS) cells. PMID:16923165

  3. Ku-deficient yeast strains exhibit alternative states of silencing competence.

    PubMed

    Maillet, L; Gaden, F; Brevet, V; Fourel, G; Martin, S G; Dubrana, K; Gasser, S M; Gilson, E

    2001-03-01

    In Saccharomyces cerevisiae, efficient silencer function requires telomere proximity, i.e. compartments of the nucleoplasm enriched in silencing factors. Accordingly, silencers located far from telomeres function inefficiently. We show here that cells lacking yKu balance between two mitotically stable states of silencing competence. In one, a partial delocalization of telomeres and silencing factors throughout the nucleoplasm correlates with enhanced silencing at a non-telomeric locus, while in the other, telomeres retain their focal pattern of distribution and there is no repression at the non-telomeric locus, as observed in wild-type cells. The two states also differ in their level of residual telomeric silencing. These findings indicate the existence of a yKu-independent pathway of telomere clustering and Sir localization. Interestingly, this pathway appears to be under epigenetic control. PMID:11266361

  4. Tf-lipoplex-mediated c-Jun silencing improves neuronal survival following excitotoxic damage in vivo.

    PubMed

    Cardoso, A L C; Costa, P; de Almeida, L P; Simões, S; Plesnila, N; Culmsee, C; Wagner, E; de Lima, M C Pedroso

    2010-03-19

    Excitotoxicity is one of the main features responsible for neuronal cell death after acute brain injury and in several neurodegenerative disorders, for which only few therapeutic options are currently available. In this work, RNA interference was employed to identify and validate a potential target for successful treatment of excitotoxic brain injury, the transcription factor c-Jun. The nuclear translocation of c-Jun and its upregulation are early events following glutamate-induced excitotoxic damage in primary neuronal cultures. We present evidence for the efficient knockdown of this transcription factor using a non-viral vector consisting of cationic liposomes associated to transferrin (Tf-lipoplexes). Tf-lipoplexes were able to deliver anti-c-Jun siRNAs to neuronal cells in culture, resulting in efficient silencing of c-Jun mRNA and protein and in a significant decrease of cell death following glutamate-induced damage or oxygen-glucose deprivation. This formulation also leads to a significant c-Jun knockdown in the mouse hippocampus in vivo, resulting in the attenuation of both neuronal death and inflammation following kainic acid-mediated lesion of this region. Furthermore, a strong reduction of seizure activity and cytokine production was observed in animals treated with anti-c-Jun siRNAs. These findings demonstrate the efficient delivery of therapeutic siRNAs to the brain by Tf-lipoplexes and validate c-Jun as a promising therapeutic target in neurodegenerative disorders involving excitotoxic lesions. PMID:19913061

  5. Heterologous expression of viral suppressors of RNA silencing complements virulence of the HC-Pro mutant of clover yellow vein virus in pea.

    PubMed

    Atsumi, Go; Nakahara, Kenji S; Wada, Tomoko Sugikawa; Choi, Sun Hee; Masuta, Chikara; Uyeda, Ichiro

    2012-06-01

    Many plant viruses encode suppressors of RNA silencing, including the helper component-proteinase (HC-Pro) of potyviruses. Our previous studies showed that a D-to-Y mutation at amino acid position 193 in HC-Pro (HC-Pro-D193Y) drastically attenuated the virulence of clover yellow vein virus (ClYVV) in legume plants. Furthermore, RNA-silencing suppression (RSS) activity of HC-Pro-D193Y was significantly reduced in Nicotiana benthamiana. Here, we examine the effect of expression of heterologous suppressors of RNA silencing, i.e., tomato bushy stunt virus p19, cucumber mosaic virus 2b, and their mutants, on the virulence of the ClYVV point mutant with D193Y (Cl-D193Y) in pea. P19 and 2b fully and partially complemented Cl-D193Y multiplication and virulence, including lethal systemic HR in pea, respectively, but the P19 and 2b mutants with defects in their RSS activity did not. Our findings strongly suggest that the D193Y mutation exclusively affects RSS activity of HC-Pro and that RSS activity is necessary for ClYVV multiplication and virulence in pea. PMID:22398917

  6. Silencing of WIPK and SIPK mitogen-activated protein kinases reduces tobacco mosaic virus accumulation but permits systemic viral movement in tobacco possessing the N resistance gene.

    PubMed

    Kobayashi, Michie; Seo, Shigemi; Hirai, Katsuyuki; Yamamoto-Katou, Ayako; Katou, Shinpei; Seto, Hideharu; Meshi, Tetsuo; Mitsuhara, Ichiro; Ohashi, Yuko

    2010-08-01

    Infection of tobacco cultivars possessing the N resistance gene with Tobacco mosaic virus (TMV) results in confinement of the virus by necrotic lesions at the infection site. Although the mitogen-activated protein kinases WIPK and SIPK have been implicated in TMV resistance, evidence linking them directly to disease resistance is, as yet, insufficient. Viral multiplication was reduced slightly in WIPK- or SIPK-silenced plants but substantially in WIPK/SIPK-silenced plants, and was correlated with an increase in salicylic acid (SA) and a decrease in jasmonic acid (JA). Silencing of WIPK and SIPK in a tobacco cultivar lacking the N gene did not inhibit viral accumulation. The reduction in viral accumulation was attenuated by expressing a gene for an SA-degrading enzyme or by exogenously applying JA. Inoculation of lower leaves resulted in the systemic spread of TMV and formation of necrotic lesions in uninoculated upper leaves. These results suggested that WIPK and SIPK function to negatively regulate local resistance to TMV accumulation, partially through modulating accumulation of SA and JA in an N-dependent manner, but positively regulate systemic resistance. PMID:20615114

  7. 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

  8. Attachment issues: kinetochore transformations and spindle checkpoint silencing.

    PubMed

    Etemad, Banafsheh; Kops, Geert J P L

    2016-04-01

    Cell division culminates in the segregation of duplicated chromosomes in opposite directions prior to cellular fission. This process is guarded by the spindle assembly checkpoint (SAC), which prevents the anaphase of cell division until stable connections between spindle microtubules and the kinetochores of all chromosomes are established. The anaphase inhibitor is generated at unattached kinetochores and inhibitor production is prevented when microtubules are captured. Understanding the molecular changes in the kinetochore that are evoked by microtubule attachments is crucial for understanding the mechanisms of SAC signaling and silencing. Here, we highlight the most recent findings on these events, pinpoint some remaining mysteries, and argue for incorporating holistic views of kinetochore dynamics in order to understand SAC silencing. PMID:26947988

  9. Bacterial Cellular Engineering by Genome Editing and Gene Silencing

    PubMed Central

    Nakashima, Nobutaka; Miyazaki, Kentaro

    2014-01-01

    Genome editing is an important technology for bacterial cellular engineering, which is commonly conducted by homologous recombination-based procedures, including gene knockout (disruption), knock-in (insertion), and allelic exchange. In addition, some new recombination-independent approaches have emerged that utilize catalytic RNAs, artificial nucleases, nucleic acid analogs, and peptide nucleic acids. Apart from these methods, which directly modify the genomic structure, an alternative approach is to conditionally modify the gene expression profile at the posttranscriptional level without altering the genomes. This is performed by expressing antisense RNAs to knock down (silence) target mRNAs in vivo. This review describes the features and recent advances on methods used in genomic engineering and silencing technologies that are advantageously used for bacterial cellular engineering. PMID:24552876

  10. The weight of the word: knowing silences in obesity research.

    PubMed

    Warin, Megan J; Gunson, Jessica S

    2013-12-01

    In this article, we examine the ethical and methodological tensions entailed in doing qualitative research in obesity studies. Framing our own embodied engagements through critical social theory, we consider how cultural meanings associated with obesity are silenced and negotiated in the research process. This negotiation is fraught with linguistic and corporeal challenges, beginning with the decision to use (or not use) the word obesity in research materials. Obesity is a visible stigma, and we argue that silencing language does not erase the tacit judgments that accompany discursive categorization. It is in a broader context of power relations that we examine the relationship between researcher and participant bodies and the ways in which collective knowingness about fat bodies underpins methodological engagement. The simultaneous presence and absence of obesity have a significant impact on the research process, in shaping both participants' experiences and the researcher's actions and interpretations. PMID:24159004

  11. Silences and moral narratives: infanticide as reproductive disruption.

    PubMed

    Aengst, Jennifer

    2014-01-01

    Infanticide is a widespread practice, yet few ethnographic and theoretical works examine this. Drawing on ethnographic research conducted in the Indian Himalayas, I argue that infanticide is a form of reproductive disruption that elicits both public moral judgments and private silences. In this Himalayan context, the stigmas of abortion and premarital sex prevent community acknowledgement of infanticide and baby abandonment. Unmarried women hide their pregnancies, deliver and abandon their babies, and later are rushed to the hospital with postdelivery complications. While biomedical doctors deal with the debris of infanticide (postpartum hemorrhage), there is no formal accounting of the practice. I argue that by regarding infanticide as a form of reproductive disruption, we can open up women's narratives of pain and suffering that are silenced because of moral repugnance. PMID:24321033

  12. ZIP4 silencing improves bone loss in pancreatic cancer

    PubMed Central

    Yang, Jingxuan; Ding, Hao; LeBrun, Drake; Ding, Kai; Houchen, Courtney W.; Postier, Russell G.; Ambrose, Catherine G.; Li, Zhaoshen; Bi, Xiaohong; Li, Min

    2015-01-01

    Metabolic bone disorders are associated with several types of human cancers. Pancreatic cancer patients usually suffer from severe nutrition deficiency, muscle wasting, and loss of bone mass. We have previously found that silencing of a zinc transporter ZIP4 prolongs the survival and reduces the severity of the cachexia in vivo. However, the role of ZIP4 in the pancreatic cancer related bone loss remains unknown. In this study we investigated the effect of ZIP4 knockdown on the bone structure, composition and mechanical properties of femurs in an orthotopic xenograft mouse model. Our data showed that silencing of ZIP4 resulted in increased bone tissue mineral density, decreased bone crystallinity and restoration of bone strength through the RANK/RANKL pathway. The results further support the impact of ZIP4 on the progression of pancreatic cancer, and suggest its potential significance as a therapeutic target for treating patients with such devastating disease and cancer related disorders. PMID:26305676

  13. Drum silencer with shallow cavity filled with helium

    NASA Astrophysics Data System (ADS)

    Choy, Y. S.; Huang, Lixi

    2003-09-01

    The motivation of this study is twofold: (a) to produce a flow-through silencer with zero pressure loss for pressure-critical applications, and (b) to tackle low frequency noise with limited sideway space using cavities filled with helium. The work represents a further development of our recently conceived device of a drum-like silencer with conventional air cavity [Huang, J. Acoust. Soc. Am. 112, 2014-2025 (2002); Choy and Huang, ibid. 112, 2026-2035 (2002)]. Theoretical predictions are validated by experimental data. The new silencer consists of two highly tensioned membranes lining part of a duct, and each membrane is backed by a cavity filled with helium. For a typical configuration of a duct with height h, membrane length L=7h, cavity depth hc=0.2h, and tension T=0.52ρ0c02h2, where ρ0 and c0 are the ambient density and speed of sound in air, respectively, the transmission loss has a continuous stop band of TL>6.35 dB for frequency 0.03c0/h to 0.064c0/h, which is much better than traditional duct lining. In addition to the mechanisms at work for drum silencers with air cavity, the low density of helium reduces the masslike reactance of the cavity on the second in vacuo mode of membrane vibration. The reduction greatly enhances the membrane response at this mode, which is found to be critical for achieving a broadband performance in the low-frequency regime.

  14. Sumoylation of Sir2 differentially regulates transcriptional silencing in yeast

    PubMed Central

    Hannan, Abdul; Abraham, Neethu Maria; Goyal, Siddharth; Jamir, Imlitoshi; Priyakumar, U. Deva; Mishra, Krishnaveni

    2015-01-01

    Silent information regulator 2 (Sir2), the founding member of the conserved sirtuin family of NAD+-dependent histone deacetylase, regulates several physiological processes including genome stability, gene silencing, metabolism and life span in yeast. Within the nucleus, Sir2 is associated with telomere clusters in the nuclear periphery and rDNA in the nucleolus and regulates gene silencing at these genomic sites. How distribution of Sir2 between telomere and rDNA is regulated is not known. Here we show that Sir2 is sumoylated and this modification modulates the intra-nuclear distribution of Sir2. We identify Siz2 as the key SUMO ligase and show that multiple lysines in Sir2 are subject to this sumoylation activity. Mutating K215 alone counteracts the inhibitory effect of Siz2 on telomeric silencing. SUMO modification of Sir2 impairs interaction with Sir4 but not Net1 and, furthermore, SUMO modified Sir2 shows predominant nucleolar localization. Our findings demonstrate that sumoylation of Sir2 modulates distribution between telomeres and rDNA and this is likely to have implications for Sir2 function in other loci as well. PMID:26319015

  15. Identification of RNA silencing components in soybean and sorghum

    PubMed Central

    2014-01-01

    Background RNA silencing is a process triggered by 21–24 small RNAs to repress gene expression. Many organisms including plants use RNA silencing to regulate development and physiology, and to maintain genome stability. Plants possess two classes of small RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). The frameworks of miRNA and siRNA pathways have been established in the model plant, Arabidopsis thaliana (Arabidopsis). Results Here we report the identification of putative genes that are required for the generation and function of miRNAs and siRNAs in soybean and sorghum, based on knowledge obtained from Arabidopsis. The gene families, including DCL, HEN1, SE, HYL1, HST, RDR, NRPD1, NRPD2/NRPE2, NRPE1, and AGO, were analyzed for gene structures, phylogenetic relationships, and protein motifs. The gene expression was validated using RNA-seq, expressed sequence tags (EST), and reverse transcription PCR (RT-PCR). Conclusions The identification of these components could provide not only insight into RNA silencing mechanism in soybean and sorghum but also basis for further investigation. All data are available at http://sysbio.unl.edu/. PMID:24387046

  16. Epigenetic silencers are enriched in dormant desert frog muscle.

    PubMed

    Hudson, Nicholas J; Lonhienne, T G A; Franklin, Craig E; Harper, Gregory S; Lehnert, S A

    2008-08-01

    Green-striped burrowing frogs, Cyclorana alboguttata, survive droughts by entering a metabolic depression called aestivation, characterised by a reduction in resting oxygen consumption by 80%. Aestivation in C. alboguttata is manifest by transcriptional silencing of skeletal muscle bioenergetic genes, such as NADH ubiquinone oxidoreductase 1, ATP synthase and superoxide dismutase 2. In this study, we hypothesised that aestivation is associated with epigenetic change in frog muscle. We assessed mRNA transcript abundance of seven genes that code for proteins with established roles in epigenetically-mediated gene silencing [transcriptional co-repressor SIN3A, DNA (cytosine-5-) methyltransferase 1, methyl CpG binding protein 2, chromodomain helicase DNA binding protein 4, histone binding protein rbbp4, histone deacetylase 1 and nuclear receptor co-repressor 2] using qRT-PCR. These seven genes showed a modest (1.1-3.5-fold) but coordinated upregulation in 6-month aestivating muscle. This reached significance for SIN3A and DNA cytosine-5-methyltransferase 1 in standard pair-wise comparisons (p < 0.05), and the candidates as a whole when analysed by Fisher's combined probability test (p < 0.01). These data are consistent with the hypothesis that the transcriptional silencing and metabolic depression that occurs during seasonal dormancy are associated with chromatin remodelling, and present a novel example of an environmentally induced epigenetic modification in an adult vertebrate. PMID:18369641

  17. Epigenetic silencing of tumor suppressor genes: Paradigms, puzzles, and potential.

    PubMed

    Kazanets, Anna; Shorstova, Tatiana; Hilmi, Khalid; Marques, Maud; Witcher, Michael

    2016-04-01

    Cancer constitutes a set of diseases with heterogeneous molecular pathologies. However, there are a number of universal aberrations common to all cancers, one of these being the epigenetic silencing of tumor suppressor genes (TSGs). The silencing of TSGs is thought to be an early, driving event in the oncogenic process. With this in consideration, great efforts have been made to develop small molecules aimed at the restoration of TSGs in order to limit tumor cell proliferation and survival. However, the molecular forces that drive the broad epigenetic reprogramming and transcriptional repression of these genes remain ill-defined. Undoubtedly, understanding the molecular underpinnings of transcriptionally silenced TSGs will aid us in our ability to reactivate these key anti-cancer targets. Here, we describe what we consider to be the five most logical molecular mechanisms that may account for this widely observed phenomenon: 1) ablation of transcription factor binding, 2) overexpression of DNA methyltransferases, 3) disruption of CTCF binding, 4) elevation of EZH2 activity, 5) aberrant expression of long non-coding RNAs. The strengths and weaknesses of each proposed mechanism is highlighted, followed by an overview of clinical efforts to target these processes. PMID:27085853

  18. Epigenetic silencing of CYP24 in the tumor microenvironment

    PubMed Central

    Johnson, Candace S.; Chung, Ivy; Trump, Donald L.

    2010-01-01

    Calcitriol (1,25 dihydroxycholecalciferol) has significant antitumor activity in vitro and in vivo in a number of tumor model systems. We developed a system for isolation of fresh endothelial cells from tumors and Matrigel environments which demonstrate that CYP24, the catabolic enzyme involved in vitamin D signaling, is epigenetically silenced selectively in tumor-derived endothelial cells (TDEC). TDEC maintain phenotypic characteristics which are distinct from endothelial cells isolated from normal tissues and from Matrigel plugs (MDEC). In TDEC, calcitriol induces G0/G1 arrest, modulates p27 and p21, and induces apoptotic cell death and decreases P-Erk and P-Akt. In contrast, endothelial cells isolated from normal tissues and MDEC are unresponsive to calcitriol-mediated anti-proliferative effects despite intact signaling through the vitamin D receptor (VDR). In TDEC, which is sensitive to calcitriol, the CYP24 promoter is hypermethylated in two CpG island regions located at the 5′end; this hypermethylation may contribute to gene silencing of CYP24. The extent of methylation in these two regions is significantly less in MDEC. Lastly, treatment of TDEC with a DNA methyltransferase inhibitor restores calcitriol-mediated induction of CYP24 and resistance to calcitriol. These data suggest that epigenetic silencing of CYP24 modulates cellular responses to calcitriol. PMID:20304059

  19. Rhodopsin targeted transcriptional silencing by DNA-binding

    PubMed Central

    Botta, Salvatore; Marrocco, Elena; de Prisco, Nicola; Curion, Fabiola; Renda, Mario; Sofia, Martina; Lupo, Mariangela; Carissimo, Annamaria; Bacci, Maria Laura; Gesualdo, Carlo; Rossi, Settimio; Simonelli, Francesca; Surace, Enrico Maria

    2016-01-01

    Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations. DOI: http://dx.doi.org/10.7554/eLife.12242.001 PMID:26974343

  20. Virus-induced gene silencing in eggplant (Solanum melongena).

    PubMed

    Liu, Haiping; Fu, Daqi; Zhu, Benzhong; Yan, Huaxue; Shen, Xiaoying; Zuo, Jinhua; Zhu, Yi; Luo, Yunbo

    2012-06-01

    Eggplant (Solanum melongena) is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing (VIGS) has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS (PHYTOENE DESATURASE) in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su (Sulfur), CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function. PMID:22268843

  1. Exploring the spiral of silence in adjustable social networks

    NASA Astrophysics Data System (ADS)

    Wu, Yue; Du, Ya-Jun; Li, Xian-Yong; Chen, Xiao-Liang

    2015-03-01

    This study extends the understanding of the spiral of silence theory by taking into account four factors, including the topology of networks, the time factor of information transmission, the node degree of individuals and the freedom of expression. Simulation experiments analyze the silencers, public opinion in steady state and relaxation time in small-world networks, scale-free networks and community-structured networks by adjusting the initial conditions. Results highlight that individuals are easier to keep silent in scale-free network, especially when the individual with big degree and minority opinion starts the discussion. Conversely, there are only a few individuals keep silent in the community-structured network when the two communities hold opposite opinions. Moreover, the number of silencers grows as the degree of coupling increases, and it decreases as the freedom of expression goes up. By analyzing the public opinion evolution, we also find some important conditions, such as the network topology, the potential public opinion distribution, and the status and sides of the first speaker, can drive the minority reversal.

  2. Inhibitors of Histone Deacetylases Attenuate Noise-Induced Hearing Loss.

    PubMed

    Chen, Jun; Hill, Kayla; Sha, Su-Hua

    2016-08-01

    Loss of auditory sensory hair cells is the major pathological feature of noise-induced hearing loss (NIHL). Currently, no established clinical therapies for prevention or amelioration of NIHL are available. The absence of treatments is due to our lack of a comprehensive understanding of the molecular mechanisms underlying noise-induced damage. Our previous study indicates that epigenetic modification of histones alters hair cell survival. In this study, we investigated the effect of noise exposure on histone H3 lysine 9 acetylation (H3K9ac) in the inner ear of adult CBA/J mice and determined if inhibition of histone deacetylases by systemic administration of suberoylanilide hydroxamic acid (SAHA) could attenuate NIHL. Our results showed that H3K9ac was decreased in the nuclei of outer hair cells (OHCs) and marginal cells of the stria vascularis in the basal region after exposure to a traumatic noise paradigm known to induce permanent threshold shifts (PTS). Consistent with these results, levels of histone deacetylases 1, 2, and 3 (HDAC1, HDAC2 and HDAC3) were increased predominately in the nuclei of cochlear cells. Silencing of HDAC1, HDAC2, or HDAC3 with siRNA reduced the expression of the target HDAC in OHCs, but did not attenuate noise-induced PTS, whereas treatment with the pan-HDAC inhibitor SAHA, also named vorinostat, reduced OHC loss, and attenuated PTS. These findings suggest that histone acetylation is involved in the pathogenesis of noise-induced OHC death and hearing loss. Pharmacological targeting of histone deacetylases may afford a strategy for protection against NIHL. PMID:27095478

  3. 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

  4. A dual gene-silencing vector system for monocot and dicot plants.

    PubMed

    Liou, Ming-Ru; Huang, Ying-Wen; Hu, Chung-Chi; Lin, Na-Sheng; Hsu, Yau-Heiu

    2014-04-01

    Plant virus-based gene-silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus-induced gene-silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene-silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co-agro-inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat-shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV- but not BaMV-based vector could enhance gene-silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA-dependant RNA polymerase 6. The dual gene-silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants. PMID:24283212

  5. Memory formation and retrieval of neuronal silencing in the auditory cortex

    PubMed Central

    Nomura, Hiroshi; Hara, Kojiro; Abe, Reimi; Hitora-Imamura, Natsuko; Nakayama, Ryota; Sasaki, Takuya; Matsuki, Norio; Ikegaya, Yuji

    2015-01-01

    Sensory stimuli not only activate specific populations of cortical neurons but can also silence other populations. However, it remains unclear whether neuronal silencing per se leads to memory formation and behavioral expression. Here we show that mice can report optogenetic inactivation of auditory neuron ensembles by exhibiting fear responses or seeking a reward. Mice receiving pairings of footshock and silencing of a neuronal ensemble exhibited a fear response selectively to the subsequent silencing of the same ensemble. The valence of the neuronal silencing was preserved for at least 30 d and was susceptible to extinction training. When we silenced an ensemble in one side of auditory cortex for conditioning, silencing of an ensemble in another side induced no fear response. We also found that mice can find a reward based on the presence or absence of the silencing. Neuronal silencing was stored as working memory. Taken together, we propose that neuronal silencing without explicit activation in the cerebral cortex is enough to elicit a cognitive behavior. PMID:26199415

  6. Mariner Transposons Contain a Silencer: Possible Role of the Polycomb Repressive Complex 2

    PubMed Central

    Beauclair, Linda; Moiré, Nathalie; Arensbuger, Peter; Bigot, Yves

    2016-01-01

    Transposable elements are driving forces for establishing genetic innovations such as transcriptional regulatory networks in eukaryotic genomes. Here, we describe a silencer situated in the last 300 bp of the Mos1 transposase open reading frame (ORF) which functions in vertebrate and arthropod cells. Functional silencers are also found at similar locations within three other animal mariner elements, i.e. IS630-Tc1-mariner (ITm) DD34D elements, Himar1, Hsmar1 and Mcmar1. These silencers are able to impact eukaryotic promoters monitoring strong, moderate or low expression as well as those of mariner elements located upstream of the transposase ORF. We report that the silencing involves at least two transcription factors (TFs) that are conserved within animal species, NFAT-5 and Alx1. These cooperatively act with YY1 to trigger the silencing activity. Four other housekeeping transcription factors (TFs), neuron restrictive silencer factor (NRSF), GAGA factor (GAF) and GTGT factor (GTF), were also found to have binding sites within mariner silencers but their impact in modulating the silencer activity remains to be further specified. Interestingly, an NRSF binding site was found to overlap a 30 bp motif coding a highly conserved PHxxYSPDLAPxD peptide in mariner transposases. We also present experimental evidence that silencing is mainly achieved by co-opting the host Polycomb Repressive Complex 2 pathway. However, we observe that when PRC2 is impaired another host silencing pathway potentially takes over to maintain weak silencer activity. Mariner silencers harbour features of Polycomb Response Elements, which are probably a way for mariner elements to self-repress their transcription and mobility in somatic and germinal cells when the required TFs are expressed. At the evolutionary scale, mariner elements, through their exaptation, might have been a source of silencers playing a role in the chromatin configuration in eukaryotic genomes. PMID:26939020

  7. DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis.

    PubMed

    Mlotshwa, Sizolwenkosi; Pruss, Gail J; Peragine, Angela; Endres, Matthew W; Li, Junjie; Chen, Xuemei; Poethig, R Scott; Bowman, Lewis H; Vance, Vicki

    2008-01-01

    Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2-but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought. PMID:18335032

  8. High Temperature, High Ambient CO2 Affect the Interactions between Three Positive-Sense RNA Viruses and a Compatible Host Differentially, but not Their Silencing Suppression Efficiencies

    PubMed Central

    Del Toro, Francisco J.; Aguilar, Emmanuel; Hernández-Walias, Francisco J.; Tenllado, Francisco; Chung, Bong-Nam; Canto, Tomas

    2015-01-01

    We compared infection of Nicotiana benthamiana plants by the positive-sense RNA viruses Cucumber mosaic virus (CMV), Potato virus Y (PVY), and by a Potato virus X (PVX) vector, the latter either unaltered or expressing the CMV 2b protein or the PVY HCPro suppressors of silencing, at 25°C vs. 30°C, or at standard (~401 parts per million, ppm) vs. elevated (970 ppm) CO2 levels. We also assessed the activities of their suppressors of silencing under those conditions. We found that at 30°C, accumulation of the CMV isolate and infection symptoms remained comparable to those at 25°C, whereas accumulation of the PVY isolate and those of the three PVX constructs decreased markedly, even when expressing the heterologous suppressors 2b or HCPro, and plants had either very attenuated or no symptoms. Under elevated CO2 plants grew larger, but contained less total protein/unit of leaf area. In contrast to temperature, infection symptoms remained unaltered for the five viruses at elevated CO2 levels, but viral titers in leaf disks as a proportion of the total protein content increased in all cases, markedly for CMV, and less so for PVY and the PVX constructs. Despite these differences, we found that neither high temperature nor elevated CO2 prevented efficient suppression of silencing by their viral suppressors in agropatch assays. Our results suggest that the strength of antiviral silencing at high temperature or CO2 levels, or those of the viral suppressors that counteract it, may not be the main determinants of the observed infection outcomes. PMID:26313753

  9. 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 PMID:26727209

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

    PubMed

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

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. PMID:26747561

  11. 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

    2016-01-01

    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. PMID:27323202

  12. RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila.

    PubMed

    Pal-Bhadra, Manika; Bhadra, Utpal; Birchler, James A

    2002-02-01

    Two types of transgene silencing were found for the Alcohol dehydrogenase (Adh) transcription unit. Transcriptional gene silencing (TGS) is Polycomb dependent and occurs when Adh is driven by the white eye color gene promoter. Full-length Adh transgenes are silenced posttranscriptionally at high copy number or by a pulsed increase over a threshold. The posttranscriptional gene silencing (PTGS) exhibits molecular hallmarks typical of RNA interference (RNAi), including the production of 21--25 bp length sense and antisense RNAs homologous to the silenced RNA. Mutations in piwi, which belongs to a gene family with members required for RNAi, block PTGS and one aspect of TGS, indicating a connection between the two types of silencing. PMID:11864605

  13. Shots fired with silencers--a report on four cases and experimental testing.

    PubMed

    Missliwetz, J; Denk, W; Wieser, I

    1991-09-01

    Four cases of homicide, in which silenced firearms were used, are reported and supplemented by data from experimental investigations regarding wound features, marks, traces, and ballistic behavior. Wound features are largely determined by the construction of the silencer. In one case, even a muzzle imprint was produced by a silenced weapon fired at contact range. In general, silencers are likely to result in a decrease in bullet energy and accelerated energy release in the target (tissue). In terms of wound morphology, silencers produced a reduction in or even a lack of the contact ring (ring of dirt). In close-range and contact wounds, any features indicative of shots fired at close range were missing (such as soot deposit and powder tattooing). It is also worth mentioning that biological matter may get into the silencer in shots fired at contact range. PMID:1955830

  14. miR-34a Silences c-SRC to Attenuate Tumor Growth in Triple-Negative Breast Cancer.

    PubMed

    Adams, Brian D; Wali, Vikram B; Cheng, Christopher J; Inukai, Sachi; Booth, Carmen J; Agarwal, Seema; Rimm, David L; Győrffy, Balázs; Santarpia, Libero; Pusztai, Lajos; Saltzman, W Mark; Slack, Frank J

    2016-02-15

    Triple-negative breast cancer (TNBC) is an aggressive subtype with no clinically proven biologically targeted treatment options. The molecular heterogeneity of TNBC and lack of high frequency driver mutations other than TP53 have hindered the development of new and effective therapies that significantly improve patient outcomes. miRNAs, global regulators of survival and proliferation pathways important in tumor development and maintenance, are becoming promising therapeutic agents. We performed miRNA-profiling studies in different TNBC subtypes to identify miRNAs that significantly contribute to disease progression. We found that miR-34a was lost in TNBC, specifically within mesenchymal and mesenchymal stem cell-like subtypes, whereas expression of miR-34a targets was significantly enriched. Furthermore, restoration of miR-34a in cell lines representing these subtypes inhibited proliferation and invasion, activated senescence, and promoted sensitivity to dasatinib by targeting the proto-oncogene c-SRC. Notably, SRC depletion in TNBC cell lines phenocopied the effects of miR-34a reintroduction, whereas SRC overexpression rescued the antitumorigenic properties mediated by miR-34a. miR-34a levels also increased when cells were treated with c-SRC inhibitors, suggesting a negative feedback exists between miR-34a and c-SRC. Moreover, miR-34a administration significantly delayed tumor growth of subcutaneously and orthotopically implanted tumors in nude mice, and was accompanied by c-SRC downregulation. Finally, we found that miR-34a and SRC levels were inversely correlated in human tumor specimens. Together, our results demonstrate that miR-34a exerts potent antitumorigenic effects in vitro and in vivo and suggests that miR-34a replacement therapy, which is currently being tested in human clinical trials, represents a promising therapeutic strategy for TNBC. PMID:26676753

  15. Sound attenuation in magnetorheological fluids

    NASA Astrophysics Data System (ADS)

    Rodríguez-López, J.; Elvira, L.; Resa, P.; Montero de Espinosa, F.

    2013-02-01

    In this work, the attenuation of ultrasonic elastic waves propagating through magnetorheological (MR) fluids is analysed as a function of the particle volume fraction and the magnetic field intensity. Non-commercial MR fluids made with iron ferromagnetic particles and two different solvents (an olive oil based solution and an Araldite-epoxy) were used. Particle volume fractions of up to 0.25 were analysed. It is shown that the attenuation of sound depends strongly on the solvent used and the volume fraction. The influence of a magnetic field up to 212 mT was studied and it was found that the sound attenuation increases with the magnetic intensity until saturation is reached. A hysteretic effect is evident once the magnetic field is removed.

  16. A 22-nt artificial microRNA mediates widespread RNA silencing in Arabidopsis

    PubMed Central

    McHale, Marcus; Eamens, Andrew L; Finnegan, E Jean; Waterhouse, Peter M

    2013-01-01

    It is known that 22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger phased small-interfering RNA (phasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA)-mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA-mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis thaliana due to the obvious and non-lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over-expression of CHS in the pap1-D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA-DEPENDENT RNA POLYMERASE6-mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA-directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families. PMID:23937661

  17. SEISMIC ATTENUATION FOR RESERVOIR CHARACTERIZATION

    SciTech Connect

    Joel Walls; M.T. Taner; Naum Derzhi; Gary Mavko; Jack Dvorkin

    2003-04-01

    In this report we will show results of seismic and well log derived attenuation attributes from a deep water Gulf of Mexico data set. This data was contributed by Burlington Resources and Seitel Inc. The data consists of ten square kilometers of 3D seismic data and three well penetrations. We have computed anomalous seismic absorption attributes on the seismic data and have computed Q from the well log curves. The results show a good correlation between the anomalous absorption (attenuation) attributes and the presence of gas as indicated by well logs.

  18. A three dimensional investigation into the acoustic performance of dissipative splitter silencers.

    PubMed

    Kirby, Ray; Williams, Paul T; Hill, James

    2014-05-01

    Splitter silencers are found in ventilation and gas turbine systems and consist of parallel baffles of porous material placed within a duct so that they split the mean gas flow. Theoretical investigations into dissipative splitter silencers have generally been limited to two dimensions and this limits the analysis to finding the silencer eigenmodes or, for a finite length silencer, to rectangular baffles only. In this article a numerical point collocation approach is used to extend theoretical predictions to three dimensions. This facilitates the analysis of more complex silencer designs such as "bar" silencers and theoretical predictions are validated by comparison with experimental measurements. The insertion loss of different silencer designs is evaluated and the performance of a bar silencer is compared to traditional designs for rectangular and circular ducts. It is shown that a bar silencer with a volume of material identical to an equivalent parallel baffle design delivers a significant improvement in insertion loss at higher frequencies, although this is at the expense of a small reduction in performance at low frequencies. It is also shown that under most circumstances it is possible to get good agreement between prediction and experiment even for relatively large Helmholtz numbers. PMID:24815256

  19. Transcriptional silencing of geminiviral promoter-driven transgenes following homologous virus infection.

    PubMed

    Seemanpillai, Mark; Dry, Ian; Randles, John; Rezaian, Ali

    2003-05-01

    Promoters isolated from the Tomato leaf curl virus (TLCV) drive both constitutive and tissue-specific expression in transgenic tobacco. Following systemic TLCV infection of plants stably expressing TLCV promoter:GUS transgenes, transgene expression driven by all six TLCV promoters was silenced. Silencing in the TLCV coat protein promoter:GUS plants (V2:GUSdeltaC) was characterized in more detail. Transgene silencing observed in leaf, stem, and pre-anthesis floral tissue occurred with the continued replication of TLCV in host tissues. Infection of the V2:GUSdeltaC plants with heterologous geminiviruses did not result in transgene silencing, indicating that silencing was specifically associated with TLCV infection. Nuclear run-on assays indicated that silencing was due to the abolition of transcription from the V2:GUSdeltaC transgene. Bisulfite sequencing showed that silencing was associated with cytosine hypermethylation of the TLCV-derived promoter sequences of the V2:GUSdeltaC transgene. Progeny derived from V2:GUSdeltaC plants silenced by TLCV infection were analyzed. Transgene expression was silenced in progeny seedlings but was partially reactivated in the majority of plants by 75 days postgermination. Progeny seedlings treated with the nonmethylatable cytosine analog 5-azacytidine or the histone deacetylase inhibitor sodium butyrate exhibited partial reactivation of expression. This is the first report of the hypermethylation of a virus-derived transgene associated with a DNA virus infection. PMID:12744514

  20. Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing.

    PubMed

    Parent, Jean-Sébastien; Bouteiller, Nathalie; Elmayan, Taline; Vaucheret, Hervé

    2015-01-01

    Dicer proteins are central to the different mechanisms involving RNA interference. Plants have evolved multiple DICER-LIKE (DCL) copies, thus enabling functional diversification. In Arabidopsis, DCL2 and DCL4 process double-stranded RNA into 22 and 21 nucleotide small interfering (si)RNAs, respectively, and have overlapping functions with regards to virus and transgene silencing. Nonetheless, some studies have reported that dcl2 or dcl4 single mutations are sometimes sufficient to hinder silencing. To better dissect the role of DCL2 and DCL4, we analyzed silencing kinetics and efficiencies using different transgenic systems in single and double mutant backgrounds. The results indicate that DCL2 stimulates transitivity and secondary siRNA production through DCL4 while being sufficient for silencing on its own. Notably, silencing of 35S-driven transgenes functions more efficiently in dcl4 mutants, indicating that DCL4 mostly obscures DCL2 in wild-type plants. Nonetheless, in a dcl4 mutant compromised in phloem-originating silencing, ectopically expressed DCL2 allows restoration of silencing, suggesting that DCL2 is not, or poorly, expressed in phloem. Remarkably, this ectopic DCL2 contribution to phloem-originating silencing is dependent on the activity of RNA-DEPENDENT RNA POLYMERASE6. These results indicate that, despite differences in the silencing activity of their small RNA products, DCL2 and DCL4 mostly act redundantly yet hierarchically when present simultaneously. PMID:25376953

  1. Perturbation analysis of heterochromatin-mediated gene silencing and somatic inheritance.

    PubMed

    Schneiderman, Jonathan I; Goldstein, Sara; Ahmad, Kami

    2010-09-01

    Repetitive sequences in eukaryotic genomes induce chromatin-mediated gene-silencing of juxtaposed genes. Many components that promote or antagonize silencing have been identified, but how heterochromatin causes variegated and heritable changes in gene expression remains mysterious. We have used inducible mis-expression in the Drosophila eye to recover new factors that alter silencing caused by the bw(D) allele, an insertion of repetitive satellite DNA that silences a bw(+) allele on the homologous chromosome. Inducible modifiers allow perturbation of silencing at different times in development, and distinguish factors that affect establishment or maintenance of silencing. We find that diverse chromatin and RNA processing factors can de-repress silencing. Most factors are effective even in differentiated cells, implying that silent chromatin remains plastic. However, over-expression of the bantam microRNA or the crooked-legs (crol) zinc-finger protein only de-repress silencing when expressed in cycling cells. Over-expression of crol accelerates the cell cycle, and this is required for de-repression of silencing. Strikingly, continual over-expression of crol converts the speckled variegation pattern of bw(D) into sectored variegation, where de-repression is stably inherited through mitotic divisions. Over-expression of crol establishes an open chromatin state, but the factor is not needed to maintain this state. Our analysis reveals that active chromatin states can be efficiently inherited through cell divisions, with implications for the stable maintenance of gene expression patterns through development. PMID:20838586

  2. The Effect of Sortilin Silencing on Ovarian Carcinoma Cells

    PubMed Central

    Ghaemimanesh, Fatemeh; Ahmadian, Gholamreza; Talebi, Saeed; Zarnani, Amir-Hassan; Behmanesh, Mehrdad; Hemmati, Shayda; Hadavi, Reza; Jeddi-Tehrani, Mahmood; Farzi, Maryam; Akhondi, Mohammad Mehdi; Rabbani, Hodjattallah

    2014-01-01

    Background Our preliminary data on the protein expression of SORT1 in ovarian carcinoma tissues showed that sortilin was overexpressed in ovarian carcinoma patients and cell lines, while non-malignant ovaries expressed comparably lower amount of this protein. In spite of diverse ligands and also different putative functions of sortilin (NTR3), the function of overexpressed sortilin in ovarian carcinoma cells is an intriguing subject of inquiry. The aim of this study was, therefore, to investigate the functional role of sortilin in survival of ovarian carcinoma cell line. Methods Expression of sortilin was knocked down using RNAi technology in the ovarian carcinoma cell line, Caov-4. Silencing of SORT1 expression was assessed using real-time qPCR and Western blot analyses. Apoptosis induction was evaluated using flow cytometry by considering annexin-V FITC binding. [3H]-thymidine incorporation assay was also used to evaluate cell proliferation capacity. Results Real-time qPCR and Western blot analyses showed that expression of sortilin was reduced by nearly 70-80% in the siRNA transfected cells. Knocking down of sortilin expression resulted in increased apoptosis (27.5±0.48%) in siRNA-treated ovarian carcinoma cell line. Sortilin silencing led to significant inhibition of proliferation (40.1%) in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart (p < 0.05). Conclusion As it was suspected from overexpression of sortilin in ovarian tumor cells, a cell survival role for sortilin can be deduced from these results. In conclusion, the potency of apoptosis induction via silencing of sortilin expression in tumor cells may introduce sortilin as a potential candidate for developing a novel targeted therapy in patients with ovarian carcinoma. PMID:25215181

  3. Epigenetic silencing of AKAP12 in juvenile myelomonocytic leukemia.

    PubMed

    Wilhelm, Thomas; Lipka, Daniel B; Witte, Tania; Wierzbinska, Justyna A; Fluhr, Silvia; Helf, Monika; Mücke, Oliver; Claus, Rainer; Konermann, Carolin; Nöllke, Peter; Niemeyer, Charlotte M; Flotho, Christian; Plass, Christoph

    2016-02-01

    A-kinase anchor protein 12 (AKAP12) is a regulator of protein kinase A and protein kinase C signaling, acting downstream of RAS. Epigenetic silencing of AKAP12 has been demonstrated in different cancer entities and this has been linked to the process of tumorigenesis. Here, we used quantitative high-resolution DNA methylation measurement by MassARRAY to investigate epigenetic regulation of all three AKAP12 promoters (i.e., α, β, and γ) within a large cohort of juvenile myelomonocytic leukemia (JMML) patient samples. The AKAP12α promoter shows DNA hypermethylation in JMML samples, which is associated with decreased AKAP12α expression. Promoter methylation of AKAP12α correlates with older age at diagnosis, elevated levels of fetal hemoglobin and poor prognosis. In silico screening for transcription factor binding motifs around the sites of most pronounced methylation changes in the AKAP12α promoter revealed highly significant scores for GATA-2/-1 sequence motifs. Both transcription factors are known to be involved in the haematopoietic differentiation process. Methylation of a reporter construct containing this region resulted in strong suppression of AKAP12 promoter activity, suggesting that DNA methylation might be involved in the aberrant silencing of the AKAP12 promoter in JMML. Exposure to DNMT- and HDAC-inhibitors reactivates AKAP12α expression in vitro, which could potentially be a mechanism underlying clinical treatment responses upon demethylating therapy. Together, these data provide evidence for epigenetic silencing of AKAP12α in JMML and further emphasize the importance of dysregulated RAS signaling in JMML pathogenesis. PMID:26891149

  4. RNA based viral silencing suppression in plant pararetroviruses

    PubMed Central

    Hohn, Thomas

    2015-01-01

    The 35S promoter of cauliflower mosaic virus and that of other plant pararetroviruses gives rise to an RNA, which is both a pre-genome and a polycistronic mRNA. The 600 nucleotide long very structured leader of this RNA is also transcribed separately. The resulting 8S RNA is then converted to a double strand giving rise to a huge set of siRNAs, which suppress silencing. In this Mini-Review I discuss how this versatile stretch of 600 nts constitutes a masterpiece of evolution. PMID:26113850

  5. STEM policy and science education: scientistic curriculum and sociopolitical silences

    NASA Astrophysics Data System (ADS)

    Gough, Annette

    2015-06-01

    This essay responds to the contribution of Volny Fages and Virginia Albe, in this volume, to the field of research in science education, and places it in the context of the plethora of government and industry policy documents calling for more Science, Technology, Engineering and Mathematics (STEM) education in schools and universities and the tension between these and students' declining interest in studying STEM subjects. It also draws attention to the parallels between the silences around sociopolitical issues in government policies and curriculum related to STEM, including nanoscience, and those found with respect to environmental education two decades ago, and relates these to the resurgence of a scientific rationalist approach to curriculum.

  6. Sibling violence silenced: rivalry, competition, wrestling, playing, roughhousing, benign.

    PubMed

    Phillips, Debby A; Phillips, Kate H; Grupp, Kitty; Trigg, Lisa J

    2009-01-01

    In this article, sibling violence and the silence surrounding it is explicated through professional literature and research findings, exemplars from clinical practice, and statistics. Theoretical positions and discourse analysis have been used to help explain how regular broken bones, bruises, lacerations, and verbal humiliation can be minimized as normal sibling rivalry or roughhousing, which does not cause serious consequences. Nursing should be on the front lines of ending practices of violence. Recognizing sibling violence as such is part of this work and is a social justice issue. PMID:19461218

  7. NATURAL ATTENUATION OF CHLORINATED SOLVENTS

    EPA Science Inventory

    The protocol will simply describe in detail, with references and illustrations, the approach currently used by staff of the SPRD to evaluate natural attenuation of chlorinated solvents in ground water. Staff of SPRD, and staff of the Air Force Center for environmental excellence...

  8. SEISMIC ATTENUATION FOR RESERVOIR CHARACTERIZATION

    SciTech Connect

    Joel Walls; M.T. Taner; Gary Mavko; Jack Dvorkin

    2002-07-01

    In fully-saturated rock and at ultrasonic frequencies, the microscopic squirt flow induced between the stiff and soft parts of the pore space by an elastic wave is responsible for velocity-frequency dispersion and attenuation. In the seismic frequency range, it is the macroscopic cross-flow between the stiffer and softer parts of the rock. We use the latter hypothesis to introduce simple approximate equations for velocity-frequency dispersion and attenuation in a fully water saturated reservoir. The equations are based on the assumption that in heterogeneous rock and at a very low frequency, the effective elastic modulus of the fully-saturated rock can be estimated by applying a fluid substitution procedure to the averaged (upscaled) dry frame whose effective porosity is the mean porosity and the effective elastic modulus is the Backus-average (geometric mean) of the individual dry-frame elastic moduli of parts of the rock. At a higher frequency, the effective elastic modulus of the saturated rock is the Backus-average of the individual fully-saturated-rock elastic moduli of parts of the rock. The difference between the effective elastic modulus calculated separately by these two methods determines the velocity-frequency dispersion. The corresponding attenuation is calculated from this dispersion by using (e.g.) the standard linear solid attenuation model.

  9. Stormwater Attenuation by Green Roofs

    NASA Astrophysics Data System (ADS)

    Sims, A.; O'Carroll, D. M.; Robinson, C. E.; Smart, C. C.

    2014-12-01

    Innovative municipal stormwater management technologies are urgently required in urban centers. Inadequate stormwater management can lead to excessive flooding, channel erosion, decreased stream baseflows, and degraded water quality. A major source of urban stormwater is unused roof space. Green roofs can be used as a stormwater management tool to reduce roof generated stormwater and generally improve the quality of runoff. With recent legislation in some North American cities, including Toronto, requiring the installation of green roofs on large buildings, research on the effectiveness of green roofs for stormwater management is important. This study aims to assess the hydrologic response of an extensive sedum green roof in London, Ontario, with emphasis on the response to large precipitation events that stress municipal stormwater infrastructure. A green roof rapidly reaches field capacity during large storm events and can show significantly different behavior before and after field capacity. At field capacity a green roof has no capillary storage left for retention of stormwater, but may still be an effective tool to attenuate peak runoff rates by transport through the green roof substrate. The attenuation of green roofs after field capacity is linked to gravity storage, where gravity storage is the water that is temporarily stored and can drain freely over time after field capacity has been established. Stormwater attenuation of a modular experimental green roof is determined from water balance calculations at 1-minute intervals. Data is used to evaluate green roof attenuation and the impact of field capacity on peak flow rates and gravity storage. In addition, a numerical model is used to simulate event based stormwater attenuation. This model is based off of the Richards equation and supporting theory of multiphase flow through porous media.

  10. Modification of Small RNAs Associated with Suppression of RNA Silencing by Tobamovirus Replicase Protein▿

    PubMed Central

    Vogler, Hannes; Akbergenov, Rashid; Shivaprasad, Padubidri V.; Dang, Vy; Fasler, Monika; Kwon, Myoung-Ok; Zhanybekova, Saule; Hohn, Thomas; Heinlein, Manfred

    2007-01-01

    Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA. PMID:17634237

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

    SciTech Connect

    Xiong Ruyi; Wu Jianxiang; Zhou Yijun; Zhou Xueping

    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 long 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.

  12. SHREC Silences Heterochromatin via Distinct Remodeling and Deacetylation Modules.

    PubMed

    Job, Godwin; Brugger, Christiane; Xu, Tao; Lowe, Brandon R; Pfister, Yvan; Qu, Chunxu; Shanker, Sreenath; Baños Sanz, José I; Partridge, Janet F; Schalch, Thomas

    2016-04-21

    Nucleosome remodeling and deacetylation (NuRD) complexes are co-transcriptional regulators implicated in differentiation, development, and diseases. Methyl-CpG binding domain (MBD) proteins play an essential role in recruitment of NuRD complexes to their target sites in chromatin. The related SHREC complex in fission yeast drives transcriptional gene silencing in heterochromatin through cooperation with HP1 proteins. How remodeler and histone deacetylase (HDAC) cooperate within NuRD complexes remains unresolved. We determined that in SHREC the two modules occupy distant sites on the scaffold protein Clr1 and that repressive activity of SHREC can be modulated by the expression level of the HDAC-associated Clr1 domain alone. Moreover, the crystal structure of Clr2 reveals an MBD-like domain mediating recruitment of the HDAC module to heterochromatin. Thus, SHREC bi-functionality is organized in two separate modules with separate recruitment mechanisms, which work together to elicit transcriptional silencing at heterochromatic loci. PMID:27105116

  13. Efficient Virus-Induced Gene Silencing in Solanum rostratum

    PubMed Central

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a “super weed” that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  14. Sexuality and 'silence' among Khasi youth of Meghalaya, Northeast India.

    PubMed

    War, Ryntihlin Jennifer; Albert, Sandra

    2013-01-01

    The importance of sex education has been well documented in the literature, but there exists a lack of research involving indigenous youth in India. This paper describes perceptions, knowledge and attitudes towards sex education, sexuality, pre-marital sex, rape and homosexuality among indigenous students from the matrilineal Khasi tribe attending a university in Meghalaya in northeast India. Qualitative and quantitative data were collected during and after reproductive health, sexuality and life skills courses. Despite the impression of sexual permissiveness of indigenous peoples that exists in India, students reported a societal silence on issues related to sexuality. Lack of appropriate words in the indigenous language potentially contributes to this silence. Although co-habitation is common and culturally acceptable, students disapproved of pre-marital sex. The influence of Christianisation was also perceived in the frequent reference to sin and guilt associated with masturbation, homosexuality, pre-marital sex and abortion. Students reported that the sex education received in school was 'childish' and inadequate for their adult needs. Many had unrealistic images of what constituted 'normal' sex and also blamed women for rape. The majority of indigenous students expressed the need for non-judgmental fora for discussions on sexual health and for sexuality education. PMID:23758496

  15. Epigeneitc silencing of ribosomal RNA genes by Mybbp1a

    PubMed Central

    2012-01-01

    Background Transcription of the ribosomal RNA gene repeats by Pol I occurs in the nucleolus and is a fundamental step in ribosome biogenesis and protein translation. Due to tight coordination between ribosome biogenesis and cell proliferation, transcription of rRNA and stable maintenance of rDNA clusters are thought to be under intricate control by intercalated mechanisms, particularly at the epigenetic level. Methods and Results Here we identify the nucleolar protein Myb-binding protein 1a (Mybbp1a) as a novel negative regulator of rRNA expression. Suppression of rDNA transcription by Mybbp1a was linked to promoter regulation as illustrated by its binding to the chromatin around the hypermethylated, inactive rDNA gene promoters. Our data further showed that downregulation of Mybbp1a abrogated the local DNA methylation levels and histone marks associated with gene silencing, and altered the promoter occupancy of various factors such UBF and HDACs, consequently leading to elevated rRNA expression. Mechanistically, we propose that Mybbp1a maintains rDNA repeats in a silenced state while in association with the negative epigenetic modifiers HDAC1/2. Conclusions Results from our present work reveal a previously unrecognized co-repressor role of Mybbp1a in rRNA expression. They are further consistent with the scenario that Mybbp1a is an integral constituent of the rDNA epigenetic regulation that underlies the balanced state of rDNA clusters. PMID:22686419

  16. Epigenetic gene silencing in cancer: the DNA hypermethylome.

    PubMed

    Esteller, Manel

    2007-04-15

    Epigenetic gene inactivation in transformed cells involves many 'belts of silencing'. One of the best-known lesions of the malignant cell is the transcriptional repression of tumor-suppressor genes by promoter CpG island hypermethylation. We are in the process of completing the molecular dissection of the entire epigenetic machinery involved in methylation-associated silencing, such as DNA methyltransferases, methyl-CpG binding domain proteins, histone deacetylases, histone methyltransferases, histone demethylases and Polycomb proteins. The first indications are also starting to emerge about how the combination of cellular selection and targeted pathways leads to abnormal DNA methylation. One thing is certain already, promoter CpG island hypermethylation of tumor-suppressor genes is a common hallmark of all human cancers. It affects all cellular pathways with a tumor-type specific profile, and in addition to classical tumor-suppressor and DNA repair genes, it includes genes involved in premature aging and microRNAs with growth inhibitory functions. The importance of hypermethylation events is already in evidence at the bedside of cancer patients in the form of cancer detection markers and chemotherapy predictors, and in the approval of epigenetic drugs for the treatment of hematological malignancies. In the very near future, the synergy of candidate gene approaches and large-scale epigenomic technologies, such as methyl-DIP, will yield the complete DNA hypermethylome of cancer cells. PMID:17613547

  17. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    PubMed

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  18. The culture of silence: disruptive and impaired physicians.

    PubMed

    Mustard, Lewis W

    2009-01-01

    An experienced hospital CEO examines the emergence of disruptive and impaired physicians as an overwhelming problem for hospital medical staff nurses, and administrators. The poor behavior ranges from aggressive acts of yelling, swearing, or pushing to passive ones of being chronically late or providing inadequate chart notes. The Joint Commission on the Accreditation of Healthcare Organizations and the American Medical Association have standards and guidelines to minimize unprofessional behavior that negatively impacts hospital patient care. The hospital staff and employees fear retaliation and the stigma associated with "tattle telling," and demonstrate a reluctance to confront the physician or peer, resulting in a culture of silence. The healthcare industry has a "history of tolerance and indifference to intimidating and destructive behaviors." To combat this 'silent response," hospitals have created wellness committees composed of caregivers such as physicians, nurses, and therapists who are specifically trained by an outside entity skilled in hospital wellness committee response functioning. The culture of silence must be replaced with the culture of safety. PMID:20073169

  19. A Silenced vanA Gene Cluster on a Transferable Plasmid Caused an Outbreak of Vancomycin-Variable Enterococci

    PubMed Central

    Sivertsen, Audun; Pedersen, Torunn; Larssen, Kjersti Wik; Bergh, Kåre; Rønning, Torunn Gresdal; Radtke, Andreas

    2016-01-01

    We report an outbreak of vancomycin-variable vanA+ enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms for in vivo switching into vancomycin resistance and horizontal spread of the vanA cluster. Forty-eight vanA+ Enterococcus faecium isolates and one Enterococcus faecalis isolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and their vanA gene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 μg/ml for in vitro development of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer of vanA was assessed by filter mating. The only genetic difference between the vanA clusters of susceptible and resistant VVE was an ISL3-family element upstream of vanHAX, which silenced vanHAX gene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542 between orf2 and vanR that attenuated the expression of vanHAX. Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. The vanA gene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including one E. faecalis isolate. Horizontally transferable silenced vanA able to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci by vanA-PCR is advised. PMID:27139479

  20. Gli2 silencing enhances TRAIL-induced apoptosis and reduces tumor growth in human hepatoma cells in vivo

    PubMed Central

    Zhang, Da-wei; Li, Hai-yan; Lau, Wan-yee; Cao, Liang-qi; Li, Yue; Jiang, Xiao-feng; Yang, Xue-wei; Xue, Ping

    2014-01-01

    Our previous studies have showed that Gli2 played a predominant role in proliferation and apoptosis resistance to TRAIL in hepatoma cells. The purpose of this study was to explore whether Gli2 silencing enhances efficiency of TRAIL for hepatoma in vivo. SMMC-7721-shRNA cells were implanted subcutaneously into nude mices and TRAIL was injected into the peritoneal space. TUNEL assay was used to detect apoptosis of tumor cells. The expression of Gli2, c-FLIPL, c-FLIPS, and Bcl-2 protein was determined by immunohistochemistry, respectively. Apoptosis and the level of caspases proteins in SMMC-7721 and HepG2 cells were detected by Flow cytometry and Western blot. Transcriptional activity of c-FLIP induced by Gli2 was measured by luciferase reporter gene assay. The results showed that lower volumes and weights of tumor were found in mice xenografted with SMMC-7721-shRNA cells as compared with control cells in the presence of TRAIL (P < 0.05). TUNEL assay showed significantly higher apoptosis index (AI) in the SMMC-7721-shRNA group than in the control groups (P < 0.05). There were remarkable positive correlations between Gli2 and c-FLIPL, c-FLIPS, Bcl-2 protein expression. Over-expression of c-FLIP or Bcl-2 in HepG2 cells attenuated TRAIL-induced apoptosis via suppression of caspase-8 or caspase-9 activity, respectively. Luciferase reporter gene assay found a regulatory sequence by which Gli2 activated transcription between -1007 to -244 in the c-FLIP promoter region. This study demonstrates that Gli2 showed regulatory activity on transcription of c-FLIP gene, and Gli2 silencing enhances TRAIL-induced apoptosis via down-regulation of c-FLIP and Bcl-2 in human hepatoma cells in vivo. PMID:25535898

  1. Ferrite attenuator modulation improves antenna performance

    NASA Technical Reports Server (NTRS)

    Hooks, J. C.; Larson, S. G.; Shorkley, F. H.; Williams, B. T.

    1970-01-01

    Ferrite attenuator inserted into appropriate waveguide reduces the gain of the antenna element which is causing interference. Modulating the ferrite attenuator to change the antenna gain at the receive frequency permits ground tracking until the antenna is no longer needed.

  2. Virus-induced gene silencing of fiber-related genes in cotton.

    PubMed

    Tuttle, John R; Haigler, Candace H; Robertson, Dominique Niki

    2015-01-01

    Virus-Induced Gene Silencing (VIGS) is a useful method for transient downregulation of gene expression in crop plants. The geminivirus Cotton leaf crumple virus (CLCrV) has been modified to serve as a VIGS vector for persistent gene silencing in cotton. Here the use of Green Fluorescent Protein (GFP) is described as a marker for identifying silenced tissues in reproductive tissues, a procedure that requires the use of transgenic plants. Suggestions are given for isolating and cloning combinations of target and marker sequences so that the total length of inserted foreign DNA is between 500 and 750 bp. Using this strategy, extensive silencing is achieved with only 200-400 bp of sequence homologous to an endogenous gene, reducing the possibility of off-target silencing. Cotyledons can be inoculated using either the gene gun or Agrobacterium and will continue to show silencing throughout fruit and fiber development. CLCrV is not transmitted through seed, and VIGS is limited to genes expressed in the maternally derived seed coat and fiber in the developing seed. This complicates the use of GFP as a marker for VIGS because cotton fibers must be separated from unsilenced tissue in the seed to determine if they are silenced. Nevertheless, fibers from a large number of seeds can be rapidly screened following placement into 96-well plates. Methods for quantifying the extent of silencing using semiquantitative RT-PCR are given. PMID:25740368

  3. 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…

  4. 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.)

  5. RNA silencing as a tool to uncover gene function and engineer novel traits in soybean

    PubMed Central

    Kasai, Megumi; Kanazawa, Akira

    2012-01-01

    RNA silencing refers collectively to diverse RNA-mediated pathways of nucleotide-sequence-specific inhibition of gene expression. It has been used to analyze gene function and engineer novel traits in various organisms. Here, we review the application of RNA silencing in soybean. To produce soybean lines, in which a particular gene is stably silenced, researchers have frequently used a transgene that transcribes inverted repeats of a target gene segment. Suppression of gene expression in developing soybean embryos has been one of the main focuses of metabolic engineering using transgene-induced silencing. Plants that have enhanced resistance against diseases caused by viruses or cyst nematode have also been produced. Meanwhile, Agrobacterium rhizogenes-mediated transformation has been used to induce RNA silencing in roots, which enabled analysis of the roles of gene products in nodulation or disease resistance. RNA silencing has also been induced using viral vectors, which is particularly useful for gene function analysis. So far, three viral vectors for virus-induced gene silencing have been developed for soybean. One of the features of the soybean genome is the presence of a large number of duplicated genes. Potential use of RNA silencing technology in combination with forward genetic approaches for analyzing duplicated genes is discussed. PMID:23136487

  6. 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…

  7. Between Voice and Voicelessness: Transacting Silence in Laurie Halse Anderson's "Speak."

    ERIC Educational Resources Information Center

    O'Quinn, Elaine J.

    2001-01-01

    Describes how Laurie Halse Anderson's insightful novel, "Speak" (1999), reminds readers of the distance women have come in identifying the "oppressive and unhealthy behavior of the silences that so often betray them." Concludes that the oppression of silence which generations of women have stoically accepted is now being challenged publicly and…

  8. 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.

  9. The Effect of Imposed Silence on Food Consumption at a Nursery School.

    ERIC Educational Resources Information Center

    Knight, Kathy B.; Bomba, Anne K.

    1993-01-01

    Observed 19 preschool children at lunch for 20 days to determine the effects of imposed silence at lunch on the amount of food consumed during meals in a nursery school setting. Found that imposing silence during mealtime did not increase food consumption but did foster a stressful atmosphere. (MM)

  10. 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…

  11. Viral suppressors of RNA silencing in Wheat mosaic virus (WMoV)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA silencing is the most effective antiviral adaptive defense mechanism mounted in higher plants to combat viral infection and proliferation. However, viruses have developed a variety of efficient counter-defense mechanisms by suppression of RNA silencing (VSR) in order to successfully impede the h...

  12. 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…

  13. ENHANCEMENTS TO NATURAL ATTENUATION: SELECTED CASE STUDIES

    SciTech Connect

    Vangelas, K; W. H. Albright, W; E. S. Becvar, E; C. H. Benson, C; T. O. Early, T; E. Hood, E; P. M. Jardine, P; M. Lorah, M; E. Majche, E; D. Major, D; W. J. Waugh, W; G. Wein, G; O. R. West, O

    2007-05-15

    In 2003 the US Department of Energy (DOE) embarked on a project to explore an innovative approach to remediation of subsurface contaminant plumes that focused on introducing mechanisms for augmenting natural attenuation to achieve site closure. Termed enhanced attenuation (EA), this approach has drawn its inspiration from the concept of monitored natural attenuation (MNA).

  14. Identification and RNA binding characterization of plant virus RNA silencing suppressor proteins.

    PubMed

    Vargason, Jeffrey M; Burch, Carissa J; Wilson, Jesse W

    2013-11-01

    Suppression is a common mechanism employed by viruses to evade the antiviral effects of the host's RNA silencing pathway. The activity of suppression has commonly been localized to gene products in the virus, but the variety of mechanisms used in suppression by these viral proteins spans nearly the complete biochemical pathway of RNA silencing in the host. This review describes the agrofiltration assay and a slightly modified version of the agro-infiltration assay called co-infiltration, which are common methods used to observe RNA silencing and identify viral silencing suppressor proteins in plants, respectively. In addition, this review will provide an overview of two methods, electrophoretic mobility shift assay and fluorescence polarization, used to assess the binding of a suppressor protein to siRNA which has been shown to be a general mechanism to suppress RNA silencing by plant viruses. PMID:23981361

  15. Arabidopsis transcriptional repressor VAL1 triggers Polycomb silencing at FLC during vernalization.

    PubMed

    Qüesta, Julia I; Song, Jie; Geraldo, Nuno; An, Hailong; Dean, Caroline

    2016-07-29

    The determinants that specify the genomic targets of Polycomb silencing complexes are still unclear. Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC) accelerates flowering and involves a cold-dependent epigenetic switch. Here we identify a single point mutation at an intragenic nucleation site within FLC that prevents this epigenetic switch from taking place. The mutation blocks nucleation of plant homeodomain-Polycomb repressive complex 2 (PHD-PRC2) and indicates a role for the transcriptional repressor VAL1 in the silencing mechanism. VAL1 localizes to the nucleation region in vivo, promoting histone deacetylation and FLC transcriptional silencing, and interacts with components of the conserved apoptosis- and splicing-associated protein (ASAP) complex. Sequence-specific targeting of transcriptional repressors thus recruits the machinery for PHD-PRC2 nucleation and epigenetic silencing. PMID:27471304

  16. Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome.

    PubMed

    Colak, Dilek; Zaninovic, Nikica; Cohen, Michael S; Rosenwaks, Zev; Yang, Wang-Yong; Gerhardt, Jeannine; Disney, Matthew D; Jaffrey, Samie R

    2014-02-28

    Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide-repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5' untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA·DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA. PMID:24578575

  17. Promoter-Bound Trinucleotide Repeat mRNA Drives Epigenetic Silencing in Fragile X Syndrome

    PubMed Central

    Colak, Dilek; Zaninovic, Nikica; Cohen, Michael S.; Rosenwaks, Zev; Yang, Wang-Yong; Gerhardt, Jeannine; Disney, Matthew D.; Jaffrey, Samie R.

    2015-01-01

    Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide–repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5′ untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA·DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA. PMID:24578575

  18. RNA-mediated RNA degradation in transgene- and virus-induced gene silencing.

    PubMed

    Metzlaff, Michael

    2002-10-01

    In the 'RNA world' hypothesis it is postulated that RNA was the first genetic molecule. Recent discoveries in gene silencing research on plants, fungi and animals show that RNA indeed plays a key role not only in controlling invading nucleic acids, like viruses and transposable elements, but also in regulating the expression of transgenes and endogenous genes. Double-stranded RNAs were identified to be the triggering structures for the induction of a specific and highly efficient RNA silencing system, in which enzyme complexes, like Dicer and RISC, facilitate as 'molecular machines' the processing of dsRNA into characteristic small RNA species. RNA silencing can be transmitted rapidly from silenced to non-silenced cells by short and long distance signaling. There is evidence that at least one component of the signal is a specific, degradation-resistant RNA. PMID:12452426

  19. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    SciTech Connect

    Mann, David George James; McKnight, Timothy E; Mcpherson, Jackson; Hoyt, Peter R; Melechko, Anatoli Vasilievich; Simpson, Michael L; Sayler, Gary Steven

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and introduced alongside the yfp marker gene into Chinese hamster ovary cells using spatially indexed vertically aligned carbon nanofiber arrays (VACNFs) in a gene delivery process termed impalefection. The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. 24 hours after nanofiber-mediated delivery, 53.1% 10.4% of the cells that expressed the yfp marker gene were also fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  20. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    SciTech Connect

    Mann, David George James; McKnight, Timothy E; Mcpherson, Jackson; Hoyt, Peter R; Melechko, Anatoli Vasilievich; Simpson, Michael L; Sayler, Gary Steven

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  1. RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae.

    PubMed

    Moriwaki, Akihiro; Ueno, Makoto; Arase, Sakae; Kihara, Junichi

    2007-04-01

    The Ascomycetous fungus Bipolaris oryzae is the causal agent of brown leaf spot disease in rice and is a model for studying photomorphogenetic responses by near-UV radiation. Targeted gene disruption (knockout) for functional analysis of photomorphogenesis-related genes in B. oryzae can be achieved by homologous recombination with low efficiency. Here, the applicability of RNA silencing (knockdown) as a tool for targeting endogenous genes in B. oryzae is reported. A polyketide synthase gene (PKS1), involved in fungal DHN melanin biosynthesis pathways, was targeted by gene silencing as a marker. The silencing vector encoding hairpin RNA of the PKS1 fragment was constructed in a two-step PCR-based cloning, and introduced into the B. oryzae genomic DNA. Silencing of the PKS1 gene resulted in albino phenotypes and reduction of PKS1 mRNA expression. These results demonstrate the applicability of targeted gene silencing as a useful reverse-genetics approach in B. oryzae. PMID:17227462

  2. SEISMIC ATTENUATION FOR RESERVOIR CHARACTERIZATION

    SciTech Connect

    Joel Walls; M.T. Taner; Gary Mavko; Jack Dvorkin

    2002-01-01

    In Section 1 of this first report we will describe the work we are doing to collect and analyze rock physics data for the purpose of modeling seismic attenuation from other measurable quantities such as porosity, water saturation, clay content and net stress. This work and other empirical methods to be presented later, will form the basis for ''Q pseudo-well modeling'' that is a key part of this project. In Section 2 of this report, we will show the fundamentals of a new method to extract Q, dispersion, and attenuation from field seismic data. The method is called Gabor-Morlet time-frequency decomposition. This technique has a number of advantages including greater stability and better time resolution than spectral ratio methods.

  3. Chlorine signal attenuation in concrete.

    PubMed

    Naqvi, A A; Maslehuddin, M; ur-Rehman, Khateeb; Al-Amoudi, O S B

    2015-11-01

    The intensity of prompt gamma-ray was measured at various depths from chlorine-contaminated silica fume (SF) concrete slab concrete specimens using portable neutron generator-based prompt gamma-ray setup. The intensity of 6.11MeV chloride gamma-rays was measured from the chloride contaminated slab at distance of 15.25, 20.25, 25.25, 30.25 and 35.25cm from neutron target in a SF cement concrete slab specimens. Due to attenuation of thermal neutron flux and emitted gamma-ray intensity in SF cement concrete at various depths, the measured intensity of chlorine gamma-rays decreases non-linearly with increasing depth in concrete. A good agreement was noted between the experimental results and the results of Monte Carlo simulation. This study has provided useful experimental data for evaluating the chloride contamination in the SF concrete utilizing gamma-ray attenuation method. PMID:26218450

  4. Inhibition of Intracellular Clusterin Attenuates Cell Death in Nephropathic Cystinosis

    PubMed Central

    Li, Li

    2015-01-01

    Nephropathic cystinosis, characterized by accumulation of cystine in the lysosomes, is caused by mutations in CTNS. The molecular and cellular mechanisms underlying proximal tubular dysfunction and progressive renal failure in nephropathic cystinosis are largely unclear, and increasing evidence supports the notion that cystine accumulation alone is not responsible for the end organ injury in cystinosis. We previously identified clusterin as potentially involved in nephropathic cystinosis. Here, we studied the expression of clusterin in renal proximal tubular epithelial cells obtained from patients with nephropathic cystinosis. The cytoprotective secretory form of clusterin, as evaluated by Western blot analysis, was low or absent in cystinosis cells compared with normal primary cells. Confocal microscopy revealed elevated levels of intracellular clusterin in cystinosis cells. Clusterin in cystinosis cells localized to the nucleus and cytoplasm and showed a filamentous and punctate aggresome-like pattern compared with diffuse cytoplasmic staining in normal cells. In kidney biopsy samples from patients with nephropathic cystinosis, clusterin protein expression was mainly limited to the proximal tubular cells. Furthermore, expression of clusterin overlapped with the expression of apoptotic proteins (apoptosis-inducing factor and cleaved caspase-3) and autophagy proteins (LC3 II and p62). Silencing of the clusterin gene resulted in a significant increase in cell viability and attenuation of apoptosis in cystinosis cells. Results of this study identify clusterin as a pivotal factor in the cell injury mechanism of nephropathic cystinosis and provide evidence linking cellular stress and injury to Fanconi syndrome and progressive renal injury in nephropathic cystinosis. PMID:25071085

  5. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

    PubMed

    Ma, Yun-Yun; Sun, Lin; Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

    2016-01-01

    Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification. PMID:27589055

  6. Simple gene silencing using the trans-acting siRNA pathway.

    PubMed

    Jacobs, Thomas B; Lawler, Noah J; LaFayette, Peter R; Vodkin, Lila O; Parrott, Wayne A

    2016-01-01

    In plants, particular micro-RNAs (miRNAs) induce the production of a class of small interfering RNAs (siRNA) called trans-acting siRNA (ta-siRNA) that lead to gene silencing. A single miRNA target is sufficient for the production of ta-siRNAs, which target can be incorporated into a vector to induce the production of siRNAs, and ultimately gene silencing. The term miRNA-induced gene silencing (MIGS) has been used to describe such vector systems in Arabidopsis. Several ta-siRNA loci have been identified in soybean, but, prior to this work, few of the inducing miRNAs have been experimentally validated, much less used to silence genes. Nine ta-siRNA loci and their respective miRNA targets were identified, and the abundance of the inducing miRNAs varies dramatically in different tissues. The miRNA targets were experimentally verified by silencing a transgenic GFP gene and two endogenous genes in hairy roots and transgenic plants. Small RNAs were produced in patterns consistent with the utilization of the ta-siRNA pathway. A side-by-side experiment demonstrated that MIGS is as effective at inducing gene silencing as traditional hairpin vectors in soybean hairy roots. Soybean plants transformed with MIGS vectors produced siRNAs and silencing was observed in the T1 generation. These results complement previous reports in Arabidopsis by demonstrating that MIGS is an efficient way to produce siRNAs and induce gene silencing in other species, as shown with soybean. The miRNA targets identified here are simple to incorporate into silencing vectors and offer an effective and efficient alternative to other gene silencing strategies. PMID:25816689

  7. Construct design for efficient, effective and high-throughput gene silencing in plants.

    PubMed

    Wesley, S V; Helliwell, C A; Smith, N A; Wang, M B; Rouse, D T; Liu, Q; Gooding, P S; Singh, S P; Abbott, D; Stoutjesdijk, P A; Robinson, S P; Gleave, A P; Green, A G; Waterhouse, P M

    2001-09-01

    Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans. PMID:11576441

  8. A campaign against violence: USAID / Kenya funds "Breaking the Silence".

    PubMed

    1997-01-01

    From November 25 to December 10, 1996, USAID/Kenya supported 16 days of activism against gender violence. Called "Breaking the Silence," this annual national campaign was aimed at raising awareness of the rising level of violence against women in Kenya. At meeting places around the country, the campaign, organized by the Coalition on Violence Against Women/Kenya and partially funded by USAID/Kenya, brought together hundreds of participants for a dialogue about gender-based violence. Speakers sought to debunk the myths surrounding violence, including the tendency to blame the victims of gender violence. Victims of violence described their treatment at the hands of abusers. Information brochures discussing domestic battery, wife killing, incest, and other forms of violence against women were distributed. USAID/Kenya has provided funding for this coalition since 1995. PMID:12321054

  9. Lesbian body image perceptions: the context of body silence.

    PubMed

    Kelly, Laura

    2007-09-01

    As are all women, lesbians are influenced by Western society's expectations of what a woman should look like. However, they are also influenced by the lesbian subculture. One of the author's aims was to explore how the sociocultural contexts in which lesbians belong influence their perceptions of body image. She purposively selected 20 lesbians and used constant comparative analysis to guide data collection and analyze the resulting transcribed interviews. The internalization of the dominant culture's beauty expectations coupled with the adoption of a lesbian identity caused the participants to contend with a multitude of oppressive mandates from both sociocultural contexts. The author interpreted the outcomes of these oppressions through the lens of stigma theory. The results of the stigma assisted in the development of the context of Body Silence. The study results might serve to provide a better understanding of lesbian body image as an important component of lesbians' mental health. PMID:17724099

  10. Myoelectric silence following unopposed passive stretch in normal man.

    PubMed Central

    Angel, R W; Waxman, S G; Kocsis, J D

    1980-01-01

    The response to unopposed passive muscle stretch applied during sustained contraction was studied in normal man. When the subject did not resist the stretching force, the initial response was a brief cessation of EMG activity in the elongated muscle. The myoelectric silence was observed repeatedly in muscles of the upper and lower limbs. The response to passive stretch is discussed in relation to the lengthening reaction and the inverse myotatic reflex. The silent period observed under these experimental conditions is unlikely to be caused by Renshaw inhibition, a pause in spindle afferent discharge, or activity of the group II afferent reflex pathway. Possible mechanisms include autogenetic inhibition and a stretch-evoked decrease of fusimotor activity. PMID:7431031

  11. A peptidomimetic siRNA transfection reagent forhighly effectivegene silencing

    SciTech Connect

    Utku, Yeliz; Dehan, Elinor; Ouerfelli, Ouathek; Piano, Fabio; Zuckermann, Ronald N.; Pagano, Michele; Kirshenbaum, Kent

    2006-05-17

    RNA interference (RNAi) techniques hold forth great promisefor therapeutic silencing of deleterious genes. However, clinicalapplications of RNAi require the development of safe and efficientmethods for intracellular delivery of small interfering RNA (siRNA)oligonucleotides specific to targeted genes. We describe the use of alipitoid, a cationic oligopeptoid phospholipid conjugate, for non-viraltransfection of synthetic siRNA oligos in cell culture. Thispeptidomimetic delivery vehicle allows for efficient siRNA transfectionin a variety of human cell lines with negligible toxicity and promotesextensive downregulation of the targeted genes at both the protein andthe mRNA level. We compare the lipitoid reagent to a standard commercialtransfection reagent. The lipitoid is highly efficient even in primaryIMR-90 human lung fibroblasts in which other commercial reagents aretypically ineffective.

  12. Written formative assessment and silence in the classroom

    NASA Astrophysics Data System (ADS)

    Lee Hang, Desmond Mene; Bell, Beverley

    2015-09-01

    In this commentary, we build on Xinying Yin and Gayle Buck's discussion by exploring the cultural practices which are integral to formative assessment, when it is viewed as a sociocultural practice. First we discuss the role of assessment and in particular oral and written formative assessments in both western and Samoan cultures, building on the account of assessment practices in the Chinese culture given by Yin and Buck. Secondly, we document the cultural practice of silence in Samoan classroom's which has lead to the use of written formative assessment as in the Yin and Buck article. We also discuss the use of written formative assessment as a scaffold for teacher development for formative assessment. Finally, we briefly discuss both studies on formative assessment as a sociocultural practice.

  13. Silencing of CDC42 inhibits neuroblastoma cell proliferation and transformation

    PubMed Central

    Lee, Sora; Craig, Brian T.; Romain, Carmelle V.; Qiao, Jingbo; Chung, Dai H.

    2014-01-01

    Cell division cycle 42 (CDC42), a small GTPase of the Rho-subfamily, regulates diverse cellular functions including proliferation, cytoskeletal rearrangement and even promotes malignant transformation. Here, we found that increased expression of CDC42 correlated with undifferentiated neuroblastoma as compared to a more benign phenotype. CDC42 inhibition decreased cell growth and soft agar colony formation, and increased cell death in BE(2)-C and BE(2)-M17 cell lines, but not in SK-N-AS. In addition, silencing of CDC42 decreased expression of N-myc in BE(2)-C and BE(2)-M17 cells. Our findings suggest that CDC42 may play a role in the regulation of aggressive neuroblastoma behavior. PMID:25264923

  14. Editor meets silencer: crosstalk between RNA editing and RNA interference

    PubMed Central

    Nishikura, Kazuko

    2010-01-01

    The most prevalent type of RNA editing is mediated by ADAR (adenosine deaminase acting on RNA) enzymes, which convert adenosines to inosines (a process known as A→I RNA editing) in double-stranded (ds)RNA substrates. A→I RNA editing was long thought to affect only selected transcripts by altering the proteins they encode. However, genome-wide screening has revealed numerous editing sites within inverted Alu repeats in introns and untranslated regions. Also, recent evidence indicates that A→I RNA editing crosstalks with RNA-interference pathways, which, like A→I RNA editing, involve dsRNAs. A→I RNA editing therefore seems to have additional functions, including the regulation of retrotransposons and gene silencing, which adds a new urgency to the challenges of fully understanding ADAR functions. PMID:17139332

  15. Maintaining memory of silencing at imprinted differentially methylated regions.

    PubMed

    Voon, Hsiao P J; Gibbons, Richard J

    2016-05-01

    Imprinted genes are an exceptional cluster of genes which are expressed in a parent-of-origin dependent fashion. This allele-specific expression is dependent on differential DNA methylation which is established in the parental germlines in a sex-specific manner. The DNA methylation imprint is accompanied by heterochromatin modifications which must be continuously maintained through development. This review summarises the factors which are important for protecting the epigenetic modifications at imprinted differentially methylated regions (DMRs), including PGC7, ZFP57 and the ATRX/Daxx/H3.3 complex. We discuss how these factors maintain heterochromatin silencing, not only at imprinted DMRs, but also other heterochromatic regions in the genome. PMID:26883803

  16. Suppression of Transgene Silencing by Matrix Attachment Regions in Maize

    PubMed Central

    Brouwer, Cory; Bruce, Wesley; Maddock, Sheila; Avramova, Zoya; Bowen, Ben

    2002-01-01

    Matrix attachment regions (MARs) are DNA sequences that bind an internal nuclear network of nonhistone proteins called the nuclear matrix. Thus, they may define discrete gene-containing chromatin loops in vivo. We have studied the effects of flanking transgenes with MARs on transgene expression levels in maize callus and in transformed maize plants. Three MAR elements, two from maize (Adh1 5′ MAR and Mha1 5′ MAR) and one from yeast (ARS1), had very different effects on transgene expression that bore no relation to their affinity for the nuclear matrix in vitro. In callus, two of the MAR elements (Adh1 5′ MAR and ARS1) reduced transgene silencing but had no effect on the variability of expression. In transgenic plants, Adh1 5′ MAR had the effect of localizing β-glucuronidase expression to lateral root initiation sites. A possible model accounting for the function of Adh1 5′ MAR is discussed. PMID:12215518

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. Hypoxia Silences Retrotrapezoid Nucleus Respiratory Chemoreceptors via Alkalosis

    PubMed Central

    Basting, Tyler M.; Burke, Peter G.R.; Kanbar, Roy; Viar, Kenneth E.; Stornetta, Daniel S.; Stornetta, Ruth L.

    2015-01-01

    In conscious mammals, hypoxia or hypercapnia stimulates breathing while theoretically exerting opposite effects on central respiratory chemoreceptors (CRCs). We tested this theory by examining how hypoxia and hypercapnia change the activity of the retrotrapezoid nucleus (RTN), a putative CRC and chemoreflex integrator. Archaerhodopsin-(Arch)-transduced RTN neurons were reversibly silenced by light in anesthetized rats. We bilaterally transduced RTN and nearby C1 neurons with Arch (PRSx8-ArchT-EYFP-LVV) and measured the cardiorespiratory consequences of Arch activation (10 s) in conscious rats during normoxia, hypoxia, or hyperoxia. RTN photoinhibition reduced breathing equally during non-REM sleep and quiet wake. Compared with normoxia, the breathing frequency reduction (ΔfR) was larger in hyperoxia (65% FiO2), smaller in 15% FiO2, and absent in 12% FiO2. Tidal volume changes (ΔVT) followed the same trend. The effect of hypoxia on ΔfR was not arousal-dependent but was reversed by reacidifying the blood (acetazolamide; 3% FiCO2). ΔfR was highly correlated with arterial pH up to arterial pH (pHa) 7.5 with no frequency inhibition occurring above pHa 7.53. Blood pressure was minimally reduced suggesting that C1 neurons were very modestly inhibited. In conclusion, RTN neurons regulate eupneic breathing about equally during both sleep and wake. RTN neurons are the first putative CRCs demonstrably silenced by hypocapnic hypoxia in conscious mammals. RTN neurons are silent above pHa 7.5 and increasingly active below this value. During hyperoxia, RTN activation maintains breathing despite the inactivity of the carotid bodies. Finally, during hypocapnic hypoxia, carotid body stimulation increases breathing frequency via pathways that bypass RTN. PMID:25589748

  19. Flexible tools for gene expression and silencing in tomato.

    PubMed

    Fernandez, Ana I; Viron, Nicolas; Alhagdow, Moftah; Karimi, Mansour; Jones, Matthew; Amsellem, Ziva; Sicard, Adrien; Czerednik, Anna; Angenent, Gerco; Grierson, Donald; May, Sean; Seymour, Graham; Eshed, Yuval; Lemaire-Chamley, Martine; Rothan, Christophe; Hilson, Pierre

    2009-12-01

    As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources. PMID:19812183

  20. Polyphenoloxidase Silencing Affects Latex Coagulation in Taraxacum Species1[W

    PubMed Central

    Wahler, Daniela; Gronover, Christian Schulze; Richter, Carolin; Foucu, Florence; Twyman, Richard M.; Moerschbacher, Bruno M.; Fischer, Rainer; Muth, Jost; Prüfer, Dirk

    2009-01-01

    Latex is the milky sap that is found in many different plants. It is produced by specialized cells known as laticifers and can comprise a mixture of proteins, carbohydrates, oils, secondary metabolites, and rubber that may help to prevent herbivory and protect wound sites against infection. The wound-induced browning of latex suggests that it contains one or more phenol-oxidizing enzymes. Here, we present a comprehensive analysis of the major latex proteins from two dandelion species, Taraxacum officinale and Taraxacum kok-saghyz, and enzymatic studies showing that polyphenoloxidase (PPO) is responsible for latex browning. Electrophoretic analysis and amino-terminal sequencing of the most abundant proteins in the aqueous latex fraction revealed the presence of three PPO-related proteins generated by the proteolytic cleavage of a single precursor (pre-PPO). The laticifer-specific pre-PPO protein contains a transit peptide that can target reporter proteins into chloroplasts when constitutively expressed in dandelion protoplasts, perhaps indicating the presence of structures similar to plastids in laticifers, which lack genuine chloroplasts. Silencing the PPO gene by constitutive RNA interference in transgenic plants reduced PPO activity compared with wild-type controls, allowing T. kok-saghyz RNA interference lines to expel four to five times more latex than controls. Latex fluidity analysis in silenced plants showed a strong correlation between residual PPO activity and the coagulation rate, indicating that laticifer-specific PPO plays a major role in latex coagulation and wound sealing in dandelions. In contrast, very little PPO activity is found in the latex of the rubber tree Hevea brasiliensis, suggesting functional divergence of latex proteins during plant evolution. PMID:19605551

  1. Hypoxia silences retrotrapezoid nucleus respiratory chemoreceptors via alkalosis.

    PubMed

    Basting, Tyler M; Burke, Peter G R; Kanbar, Roy; Viar, Kenneth E; Stornetta, Daniel S; Stornetta, Ruth L; Guyenet, Patrice G

    2015-01-14

    In conscious mammals, hypoxia or hypercapnia stimulates breathing while theoretically exerting opposite effects on central respiratory chemoreceptors (CRCs). We tested this theory by examining how hypoxia and hypercapnia change the activity of the retrotrapezoid nucleus (RTN), a putative CRC and chemoreflex integrator. Archaerhodopsin-(Arch)-transduced RTN neurons were reversibly silenced by light in anesthetized rats. We bilaterally transduced RTN and nearby C1 neurons with Arch (PRSx8-ArchT-EYFP-LVV) and measured the cardiorespiratory consequences of Arch activation (10 s) in conscious rats during normoxia, hypoxia, or hyperoxia. RTN photoinhibition reduced breathing equally during non-REM sleep and quiet wake. Compared with normoxia, the breathing frequency reduction (Δf(R)) was larger in hyperoxia (65% FiO2), smaller in 15% FiO2, and absent in 12% FiO2. Tidal volume changes (ΔV(T)) followed the same trend. The effect of hypoxia on Δf(R) was not arousal-dependent but was reversed by reacidifying the blood (acetazolamide; 3% FiCO2). Δf(R) was highly correlated with arterial pH up to arterial pH (pHa) 7.5 with no frequency inhibition occurring above pHa 7.53. Blood pressure was minimally reduced suggesting that C1 neurons were very modestly inhibited. In conclusion, RTN neurons regulate eupneic breathing about equally during both sleep and wake. RTN neurons are the first putative CRCs demonstrably silenced by hypocapnic hypoxia in conscious mammals. RTN neurons are silent above pHa 7.5 and increasingly active below this value. During hyperoxia, RTN activation maintains breathing despite the inactivity of the carotid bodies. Finally, during hypocapnic hypoxia, carotid body stimulation increases breathing frequency via pathways that bypass RTN. PMID:25589748

  2. Natural and enhanced attenuation of metals

    SciTech Connect

    Rouse, J.V.; Pyrih, R.Z.

    1996-12-31

    The ability of natural earthen materials to attenuate the movement of contamination can be quantified in relatively simple geochemical experiments. In addition, the ability of subsurface material to attenuate potential contaminants can be enhanced through modifications to geochemical parameters such as pH or redox conditions. Such enhanced geochemical attenuation has been demonstrated at a number of sites to be a cost-effective alternative to conventional pump and treat operations. This paper describes the natural attenuation reactions which occur in the subsurface, and the way to quantify such attenuation. It also introduces the concept of enhanced geochemical attenuation, wherein naturally-occurring geochemical reactions can be used to achieve in situ fixation. The paper presents examples where such natural and enhanced attenuation have been implemented as a part of an overall remedy.

  3. Protective effect of caffeine against high sugar-induced transcription of microRNAs and consequent gene silencing: A study using lenses of galactosemic mice

    PubMed Central

    Kovtun, Svitlana

    2013-01-01

    Purpose Previous studies have shown that caffeine prevents the formation of cataracts induced by a high-galactose diet and consequent oxidative stress. The objective of this study was to investigate if this protective effect is reflected in the attenuation of the transcription of microRNAs (miRNAs) known to induce apoptosis and cell death by gene silencing. Methods Young CD-1 mice were fed either a normal laboratory diet or a diet containing 25% galactose with or without 1% caffeine. One week later, the animals were euthanized, and the lenses isolated and promptly processed for RNA isolation and subsequent preparation of cDNAs by reverse transcriptase reaction. Mature miRNA (miR)-specific cDNAs were then quantified with PCR in a 96-well microRNA-specific cassette using an ABI7900HT PCR machine. Results As expected from previous studies, the lenses were positive for all 84 miRs corresponding to the miRNA probes present in the cassette wells. However, the levels of at least 19 miRs were significantly elevated in galactosemic lenses compared to those in the normal lenses. The majority are proapoptotic. Such elevation was inhibited by caffeine. This has been demonstrated for the first time. Conclusions Since aberrant elevation of miRNAs silences various genes and consequently deactivates protein translation, and since caffeine downregulates such aberration, the beneficial effect of caffeine could be attributed to its ability to suppress elevation of toxic miRs and consequent gene silencing. PMID:23441122

  4. Gene silencing of non-obese diabetic receptor family (NLRP3) protects against the sepsis-induced hyper-bile acidaemia in a rat model

    PubMed Central

    Wu, Y; Ren, J; Zhou, B; Ding, C; Chen, J; Wang, G; Gu, G; Wu, X; Liu, S; Hu, D; Li, J

    2015-01-01

    The role of NOD-like receptor family (NLRP3) has been confirmed in various inflammatory diseases. The association between NLRP3 and hyper-bileacidaemia during the sepsis remains unclear. We aimed to investigate whether NLRP3 silencing protects against the sepsis-induced hyper-bileacidaemia. Sepsis was induced by caecum ligation and puncture (CLP). Gene silencing of NLRP3 was performed by injecting rats with NLRP3 short hairpin RNA plasmids (NLRP3 shRNA) 48 h before surgery. Rats were divided into four groups: group 1: sham; group 2: sepsis; group 3: NLRP3 shRNA + sepsis (called the ‘NLRP3 shRNA’ group); and group 4: scrambled shRNA + sepsis (called the ‘scrambled shRNA’ group). The serum levels of bile acids, hepatic expression of hepatocyte membrane transporters, hepatic cytokine levels and behaviours of immune cells were compared among the groups. Hepatic NLRP3 expression was increased dramatically during the sepsis, but was suppressed by pretreatment with NLRP3 shRNA. Compared with rats in the sepsis and the scrambled shRNA groups, rats in the NLRP3 shRNA group exhibited significantly decreased serum levels of glycine and taurine conjugated-bile acids, with rehabilitated expression of hepatocyte transporters, suppressed hepatic cytokine levels, decreased hepatic neutrophils infiltration and attenuated macrophages pyroptosis. Gene silencing of NLRP3 ameliorates sepsis-induced hyper-bileacidaemia by rehabilitating hepatocyte transporter expression, reducing hepatic cytokine levels, neutrophil infiltration and macrophages pyroptosis. NLRP3 may be a pivotal target for sepsis management. PMID:25228381

  5. Sequence homology requirements for transcriptional silencing of 35S transgenes and post-transcriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus.

    PubMed

    Thierry, D; Vaucheret, H

    1996-12-01

    The transgene locus of the tobacco plant 271 (271 locus) is located on a telomere and consists of multiple copies of a plasmid carrying an NptII marker gene driven by the cauliflower mosaic virus (CaMV) 19S promoter and the leaf-specific nitrite reductase Nii1 cDNA cloned in the antisense orientation under the control of the CaMV 35S promoter. Previous analysis of gene expression in leaves has shown that this locus triggers both post-transcriptional silencing of the host leaf-specific Nii genes and transcriptional silencing of transgenes driven by the 19S or 35S promoter irrespective of their coding sequence and of their location in the genome. In this paper we show that silencing of transgenes carrying Nii1 sequences occurs irrespective of the promoter driving their expression and of their location within the genome. This phenomenon occurs in roots as well as in leaves although root Nii genes share only 84% identity with leaf-specific Nii1 sequences carried by the 271 locus. Conversely, transgenes carrying the bean Nii gene (which shares 76% identity with the tobacco Nii1 gene) escape silencing by the 271 locus. We also show that transgenes driven by the figwort mosaic virus 34S promoter (which shares 63% identity with the 35S promoter) also escape silencing by the 271 locus. Taken together, these results indicate that a high degree of sequence similarity is required between the sequences of the silencing locus and of the target (trans)genes for both transcriptional and post-transcriptional silencing. PMID:9002606

  6. Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice.

    PubMed Central

    Kumpatla, S P; Teng, W; Buchholz, W G; Hall, T C

    1997-01-01

    Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btt cryIIIA and bar transgenes in rice (Oryza sativa L.) that displayed aberrant segregation in R1 progeny. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R1 plants had a DNA hybridization profile identical to that of the resistant R0 parent and R1 siblings. Genomic DNA analysis revealed substantial methylation of the Ubi1/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R2 progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubi1 promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences. PMID:9342860

  7. Translation Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA Oncogenes1

    PubMed Central

    Lee, Hyewon; Humann, Jodi L.; Pitrak, Jennifer S.; Cuperus, Josh T.; Parks, T. Dawn; Whistler, Cheryl A.; Mok, Machteld C.; Ream, L. Walt

    2003-01-01

    Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis. Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt. Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences. Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt, suggesting that transgene sequences influenced the effectiveness of PTGS. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5′ end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM. Silencing A. tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease. PMID:12972655

  8. A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae.

    PubMed Central

    McNally, F J; Rine, J

    1991-01-01

    Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus. Images PMID:1922068

  9. The Tnt1 Retrotransposon Escapes Silencing in Tobacco, Its Natural Host

    PubMed Central

    Hernández-Pinzón, Inmaculada; Cifuentes, Marta; Hénaff, Elizabeth; Santiago, Néstor; Espinás, M. Lluïsa; Casacuberta, Josep M.

    2012-01-01

    Retrotransposons' high capacity for mutagenesis is a threat that genomes need to control tightly. Transcriptional gene silencing is a general and highly effective control of retrotransposon expression. Yet, some retrotransposons manage to transpose and proliferate in plant genomes, suggesting that, as shown for plant viruses, retrotransposons can escape silencing. However no evidence of retrotransposon silencing escape has been reported. Here we analyze the silencing control of the tobacco Tnt1 retrotransposon and report that even though constructs driven by the Tnt1 promoter become silenced when stably integrated in tobacco, the endogenous Tnt1 elements remain active. Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction. Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host. PMID:22479451

  10. 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

  11. Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

    PubMed Central

    Butardo, Vito M.; Fitzgerald, Melissa A.; Bird, Anthony R.; Gidley, Michael J.; Flanagan, Bernadine M.; Larroque, Oscar; Resurreccion, Adoracion P.; Laidlaw, Hunter K. C.; Jobling, Stephen A.; Morell, Matthew K.; Rahman, Sadequr

    2011-01-01

    The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed. PMID:21791436

  12. Adenosine kinase inhibition and suppression of RNA silencing by geminivirus AL2 and L2 proteins.

    PubMed

    Wang, Hui; Buckley, Kenneth J; Yang, Xiaojuan; Buchmann, R Cody; Bisaro, David M

    2005-06-01

    Most plant viruses are initiators and targets of RNA silencing and encode proteins that suppress this adaptive host defense. The DNA-containing geminiviruses are no exception, and the AL2 protein (also known as AC2, C2, and transcriptional activator protein) encoded by members of the genus Begomovirus has been shown to act as a silencing suppressor. Here, a three-component, Agrobacterium-mediated transient assay is used to further examine the silencing suppression activity of AL2 from Tomato golden mosaic virus (TGMV, a begomovirus) and to determine if the related L2 protein of Beet curly top virus (BCTV, genus Curtovirus) also has suppression activity. We show that TGMV AL2, AL2(1-100) (lacking the transcriptional activation domain), and BCTV L2 can all suppress RNA silencing directed against a green fluorescent protein (GFP) reporter gene when silencing is induced by a construct expressing an inverted repeat GFP RNA (dsGFP). We previously found that these viral proteins interact with and inactivate adenosine kinase (ADK), a cellular enzyme important for adenosine salvage and methyl cycle maintenance. Using the GFP-dsGFP system, we demonstrate here that codelivery of a construct expressing an inverted repeat ADK RNA (dsADK), or addition of an ADK inhibitor (the adenosine analogue A-134974), suppresses GFP-directed silencing in a manner similar to the geminivirus proteins. In addition, AL2/L2 suppression phenotypes and nucleic acid binding properties are shown to be different from those of the RNA virus suppressors HC-Pro and p19. These findings provide strong evidence that ADK activity is required to support RNA silencing, and indicate that the geminivirus proteins suppress silencing by a novel mechanism that involves ADK inhibition. Further, since AL2(1-100) is as effective a suppressor as the full-length AL2 protein, activation and silencing suppression appear to be independent activities. PMID:15919897

  13. Heterologous virus-induced gene silencing as a promising approach in plant functional genomics.

    PubMed

    Hosseini Tafreshi, Seied Ali; Shariati, Mansour; Mofid, Mohammad Reza; Khayam Nekui, Mojtaba; Esmaeili, Abolghasem

    2012-03-01

    VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death. PMID:21655951

  14. Inhibition of FSS-induced actin cytoskeleton reorganization by silencing LIMK2 gene increases the mechanosensitivity of primary osteoblasts.

    PubMed

    Yang, Zhi; Tan, Shuyi; Shen, Yun; Chen, Rui; Wu, Changjing; Xu, Yajuan; Song, Zijun; Fu, Qiang

    2015-05-01

    Mechanical stimulation plays an important role in bone cell metabolic activity. However, bone cells lose their mechanosensitivity upon continuous mechanical stimulation (desensitization) and they can recover the sensitivity with insertion of appropriate rest period into the mechanical loading profiles. The concrete molecular mechanism behind the regulation of cell mechanosensitivity still remains unclear. As one kind of mechanosensitive cell to react to the mechanical stimulation, osteoblasts respond to fluid shear stress (FSS) with actin cytoskeleton reorganization, and the remodeling of actin cytoskeleton is closely associated with the alteration of cell mechanosensitivity. In order to find out whether inhibiting the actin cytoskeleton reorganization by silencing LIM-kinase 2 (LIMK2) gene would increase the mechanosensitivity of primary osteoblasts, we attenuated the formation of actin stress fiber under FSS in a more specific way: inhibiting the LIMK2 expression by RNA interference. We found that inhibition of LIMK2 expression by RNA interference attenuated the formation of FSS-induced actin stress fiber, and simultaneously maintained the integrity of actin cytoskeleton in primary osteoblasts. We confirmed that the decreased actin cytoskeleton reorganization in response to LIMK2 inhibition during FSS increased the mechanosensitivity of the osteoblasts, based on the increased c-Fos and COX-2 expression as well as the enhanced proliferative activity in response to FSS. These data suggest that osteoblasts can increase their mechanosensitivity under continuous mechanical stimulation by reducing the actin stress fiber formation through inhibiting the LIMK2 expression. This study provides us with a new and more specific method to regulate the osteoblast mechanosensitivity, and also a new therapeutic target to cure bone related diseases, which is of importance in maintaining bone mass and promoting osteogenesis. PMID:25549868

  15. SEISMIC ATTENUATION FOR RESERVOIR CHARACTERIZATION

    SciTech Connect

    Joel Walls; M.T. Taner; Naum Derzhi; Gary Mavko; Jack Dvorkin

    2003-04-01

    In this report we will show some new Q related seismic attributes on the Burlington-Seitel data set. One example will be called Energy Absorption Attribute (EAA) and is based on a spectral analysis. The EAA algorithm is designed to detect a sudden increase in the rate of exponential decay in the relatively higher frequency portion of the spectrum. In addition we will show results from a hybrid attribute that combines attenuation with relative acoustic impedance to give a better indication of commercial gas saturation.

  16. Imaging Rayleigh wave attenuation with USArray

    NASA Astrophysics Data System (ADS)

    Bao, Xueyang; Dalton, Colleen A.; Jin, Ge; Gaherty, James B.; Shen, Yang

    2016-04-01

    The EarthScope USArray provides an opportunity to obtain detailed images of the continental upper mantle at an unprecedented scale. The majority of mantle models derived from USArray data to date contain spatial variations in seismic-wave speed; however, in many cases these data sets do not by themselves allow a non-unique interpretation. Joint interpretation of seismic attenuation and velocity models can improve upon the interpretations based only on velocity and provide important constraints on the temperature, composition, melt content, and volatile content of the mantle. The surface-wave amplitudes that constrain upper-mantle attenuation are sensitive to factors in addition to attenuation, including the earthquake source excitation, focusing and defocusing by elastic structure, and local site amplification. Because of the difficulty of isolating attenuation from these other factors, little is known about the attenuation structure of the North American upper mantle. In this study, Rayleigh wave travel time and amplitude in the period range 25-100 s are measured using an interstation cross-correlation technique, which takes advantage of waveform similarity at nearby stations. Several estimates of Rayleigh wave attenuation and site amplification are generated at each period, using different approaches to separate the effects of attenuation and local site amplification on amplitude. It is assumed that focusing and defocusing effects can be described by the Laplacian of the travel-time field. All approaches identify the same large-scale patterns in attenuation, including areas where the attenuation values are likely contaminated by unmodelled focusing and defocusing effects. Regionally averaged attenuation maps are constructed after removal of the contaminated attenuation values, and the variations in intrinsic shear attenuation that are suggested by these Rayleigh wave attenuation maps are explored.

  17. Imaging Rayleigh wave attenuation with USArray

    NASA Astrophysics Data System (ADS)

    Bao, Xueyang; Dalton, Colleen A.; Jin, Ge; Gaherty, James B.; Shen, Yang

    2016-07-01

    The EarthScope USArray provides an opportunity to obtain detailed images of the continental upper mantle at an unprecedented scale. The majority of mantle models derived from USArray data to date contain spatial variations in seismic-wave speed; however, in many cases these data sets do not by themselves allow a non-unique interpretation. Joint interpretation of seismic attenuation and velocity models can improve upon the interpretations based only on velocity and provide important constraints on the temperature, composition, melt content, and volatile content of the mantle. The surface wave amplitudes that constrain upper-mantle attenuation are sensitive to factors in addition to attenuation, including the earthquake source excitation, focusing and defocusing by elastic structure, and local site amplification. Because of the difficulty of isolating attenuation from these other factors, little is known about the attenuation structure of the North American upper mantle. In this study, Rayleigh wave traveltime and amplitude in the period range 25-100 s are measured using an interstation cross-correlation technique, which takes advantage of waveform similarity at nearby stations. Several estimates of Rayleigh wave attenuation and site amplification are generated at each period, using different approaches to separate the effects of attenuation and local site amplification on amplitude. It is assumed that focusing and defocusing effects can be described by the Laplacian of the traveltime field. All approaches identify the same large-scale patterns in attenuation, including areas where the attenuation values are likely contaminated by unmodelled focusing and defocusing effects. Regionally averaged attenuation maps are constructed after removal of the contaminated attenuation values, and the variations in intrinsic shear attenuation that are suggested by these Rayleigh wave attenuation maps are explored.

  18. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  19. Honoring silence and valuing community: living leadership in 21st century teaching-learning.

    PubMed

    Backer Condon, Barbara; Hegge, Margaret

    2014-04-01

    Leadership is a very personal concept. The methods implemented by leaders often reflect philosophical beliefs and theoretical underpinnings. This column, while recognizing that leadership styles are indeed personal, proffers living leadership in nursing education through two key leadership attitudes. These attitudes are honoring silence and valuing community. Honoring silence is discussed as recognizing the multiple dimensions of silence, and valuing community is presented in light of the humanbecoming community models change concepts: moving-initiating, anchoring-shifting, and pondering-shaping. PMID:24740944

  20. SEISMIC ATTENUATION FOR RESERVOIR CHARACTERIZATION

    SciTech Connect

    Joel Walls; M.T. Taner; Naum Derzhi; Gary Mavko; Jack Dvorkin

    2003-12-01

    We have developed and tested technology for a new type of direct hydrocarbon detection. The method uses inelastic rock properties to greatly enhance the sensitivity of surface seismic methods to the presence of oil and gas saturation. These methods include use of energy absorption, dispersion, and attenuation (Q) along with traditional seismic attributes like velocity, impedance, and AVO. Our approach is to combine three elements: (1) a synthesis of the latest rock physics understanding of how rock inelasticity is related to rock type, pore fluid types, and pore microstructure, (2) synthetic seismic modeling that will help identify the relative contributions of scattering and intrinsic inelasticity to apparent Q attributes, and (3) robust algorithms that extract relative wave attenuation attributes from seismic data. This project provides: (1) Additional petrophysical insight from acquired data; (2) Increased understanding of rock and fluid properties; (3) New techniques to measure reservoir properties that are not currently available; and (4) Provide tools to more accurately describe the reservoir and predict oil location and volumes. These methodologies will improve the industry's ability to predict and quantify oil and gas saturation distribution, and to apply this information through geologic models to enhance reservoir simulation. We have applied for two separate patents relating to work that was completed as part of this project.

  1. Increased Expression of Chitinase 3-Like 1 in Aorta of Patients with Atherosclerosis and Suppression of Atherosclerosis in Apolipoprotein E-Knockout Mice by Chitinase 3-Like 1 Gene Silencing

    PubMed Central

    Gong, Zushun; Xing, Shanshan; Zheng, Fei; Xing, Qichong

    2014-01-01

    Introduction. The purpose of this study was to investigate the changes of chitinase 3-like 1 (CHI3L1) in the aorta of patients with coronary atherosclerosis and to determine whether inhibition of CHI3L1 by lentivirus-mediated RNA interference could stabilize atherosclerotic plaques in apolipoprotein E-knockout (ApoE−/−) mice. Methods. We collected discarded aortic specimens from patients undergoing coronary artery bypass graft surgery and renal arterial tissues from kidney donors. A lentivirus carrying small interfering RNA targeting the expression of CHI3L1 was constructed. Fifty ApoE−/− mice were divided into control group and CHI3L1 gene silenced group. A constrictive collar was placed around carotid artery to induce plaques formation. Then lentivirus was transfected into carotid plaques. Results. We found that CHI3L1 was overexpressed in aorta of patients with atherosclerosis and its expression was correlated with the atherosclerotic risk factors. After lentivirus transduction, mRNA and protein expression of CHI3L1 were attenuated in carotid plaques, leading to reduced plaque content of lipids and macrophages, and increased plaque content of collagen and smooth muscle cells. Moreover, CHI3L1 gene silencing downregulated the expression of local proinflammatory mediators. Conclusions. CHI3L1 is overexpressed in aorta from patients with atherosclerosis and the lentivirus-mediated CHI3L1 gene silencing could represent a new strategy to inhibit plaques progression. PMID:24729664

  2. Global Attenuation Model of the Upper Mantle

    NASA Astrophysics Data System (ADS)

    Adenis, A.; Debayle, E.; Ricard, Y. R.

    2015-12-01

    We present a three-dimensional shear attenuation model based on a massive surface wave data-set (372,629 Rayleigh waveforms analysed in the period range 50-300s by Debayle and Ricard, 2012). For each seismogram, this approach yields depth-dependent path average models of shear velocity and quality factor, and a set of fundamental and higher-mode dispersion and attenuation curves. We combine these attenuation measurements in a tomographic inversion after a careful rejection of the noisy data. We first remove data likely to be biased by a poor knowledge of the source. Then we assume that waves corresponding to events having close epicenters and recorded at the same station sample the same elastic and anelastic structure, we cluster the corresponding rays and average the attenuation measurements. Logarithms of the attenuations are regionalized using the non-linear east square formalism of Tarantola and Valette (1982), resulting in attenuation tomographic maps between 50s and 300s. After a first inversion, outlyers are rejected and a second inversion yields a moderate variance reduction of about 20%. We correct the attenuation curves for focusing effect using the linearized ray theory of Woodhouse and Wong (1986). Accounting for focussing effects allows building tomographic maps with variance reductions reaching 40%. In the period range 120-200s, the root mean square of the model perturbations increases from about 5% to 20%. Our 3-D attenuation models present strong agreement with surface tectonics at period lower than 200s. Areas of low attenuation are located under continents and areas of high attenuation are associated with oceans. Surprisingly, although mid oceanic ridges are located in attenuating regions, their signature, even if enhanced by focusing corrections, remains weaker than in the shear velocity models. Synthetic tests suggests that regularisation contributes to damp the attenuation signature of ridges, which could therefore be underestimated.

  3. General relationships between ultrasonic attenuation and dispersion

    NASA Technical Reports Server (NTRS)

    Odonnell, M.; Jaynes, E. T.; Miller, J. G.

    1978-01-01

    General relationships between the ultrasonic attenuation and dispersion are presented. The validity of these nonlocal relationships hinges only on the properties of causality and linearity, and does not depend upon details of the mechanism responsible for the attenuation and dispersion. Approximate, nearly local relationships are presented and are demonstrated to predict accurately the ultrasonic dispersion in solutions of hemoglobin from the results of attenuation measurements.

  4. Calculation Of Pneumatic Attenuation In Pressure Sensors

    NASA Technical Reports Server (NTRS)

    Whitmore, Stephen A.

    1991-01-01

    Errors caused by attenuation of air-pressure waves in narrow tubes calculated by method based on fundamental equations of flow. Changes in ambient pressure transmitted along narrow tube to sensor. Attenuation of high-frequency components of pressure wave calculated from wave equation derived from Navier-Stokes equations of viscous flow in tube. Developed to understand and compensate for frictional attenuation in narrow tubes used to connect aircraft pressure sensors with pressure taps on affected surfaces.

  5. NOX2 Antisense Attenuates Hypoxia-Induced Oxidative Stress and Apoptosis in Cardiomyocyte

    PubMed Central

    Yu, Bo; Meng, Fanbo; Yang, Yushuang; Liu, Dongna; Shi, Kaiyao

    2016-01-01

    Heart ischemia is a hypoxia related disease. NOX2 and HIF-1α proteins were increased in cardiomyocytes after acute myocardial infarction. However, the relationship of the hypoxia-induced HIF-1α. NOX2-derived oxidative stress and apoptosis in cardiomyocyte remains unclear. In the current study, we use NOX2 antisense strategy to investigate the role of NOX2 in hypoxia-induced oxidative stress and apoptosis in rat cardiomyocytes. Here, we show that transduction of ADV-NOX2-AS induces potent silencing of NOX2 in cardiomyocytes, and resulting in attenuation of hypoxia-induced oxidative stress and apoptosis. This study indicates the potential of antisense-based therapies and validates NOX2 as a potent therapeutic candidate for heart ischemia. PMID:27499697

  6. Lentiviral Vector-Mediated RNA Silencing in the Central Nervous System

    PubMed Central

    Foster, Edmund; Moon, Lawrence D.F.

    2014-01-01

    Abstract RNA silencing is an established method for investigating gene function and has attracted particular interest because of the potential for generating RNA-based therapeutics. Using lentiviral vectors as an efficient delivery system that offers stable, long-term expression in postmitotic cells further enhances the applicability of an RNA-based gene therapy for the CNS. In this review we provide an overview of both lentiviral vectors and RNA silencing along with design considerations for generating lentiviral vectors capable of RNA silencing. We go on to describe the current preclinical data regarding lentiviral vector-mediated RNA silencing for CNS disorders and discuss the concerns of side effects associated with lentiviral vectors and small interfering RNAs and how these might be mitigated. PMID:24090197

  7. Cohesin and Polycomb Proteins Functionally Interact to Control Transcription at Silenced and Active Genes

    PubMed Central

    Schaaf, Cheri A.; Misulovin, Ziva; Gause, Maria; Koenig, Amanda; Gohara, David W.; Watson, Audrey; Dorsett, Dale

    2013-01-01

    Cohesin is crucial for proper chromosome segregation but also regulates gene transcription and organism development by poorly understood mechanisms. Using genome-wide assays in Drosophila developing wings and cultured cells, we find that cohesin functionally interacts with Polycomb group (PcG) silencing proteins at both silenced and active genes. Cohesin unexpectedly facilitates binding of Polycomb Repressive Complex 1 (PRC1) to many active genes, but their binding is mutually antagonistic at silenced genes. PRC1 depletion decreases phosphorylated RNA polymerase II and mRNA at many active genes but increases them at silenced genes. Depletion of cohesin reduces long-range interactions between Polycomb Response Elements in the invected-engrailed gene complex where it represses transcription. These studies reveal a previously unrecognized role for PRC1 in facilitating productive gene transcription and provide new insights into how cohesin and PRC1 control development. PMID:23818863

  8. viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence.

    PubMed

    Csorba, Tibor; Kontra, Levente; Burgyán, József

    2015-05-01

    RNA silencing is a homology-dependent gene inactivation mechanism that regulates a wide range of biological processes including antiviral defense. To deal with host antiviral responses viruses evolved mechanisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Besides working as silencing suppressors, these proteins may also fulfill other functions during infection. In many cases the interplay between the suppressor function and other "unrelated" functions remains elusive. We will present host factors implicated in antiviral pathways and summarize the current status of knowledge about the diverse viral suppressors' strategies acting at various steps of antiviral silencing in plants. Besides, we will consider the multi-functionality of these versatile proteins and related biochemical processes in which they may be involved in fine-tuning the plant-virus interaction. Finally, we will present the current applications and discuss perspectives of the use of these proteins in molecular biology and biotechnology. PMID:25766638

  9. Adult-specific electrical silencing of pacemaker neurons uncouples the molecular oscillator from circadian outputs

    PubMed Central

    Depetris-Chauvin, Ana; Berni, Jimena; Aranovich, Ezequiel J.; Muraro, Nara I.; Beckwith, Esteban J.; Ceriani, María Fernanda

    2011-01-01

    Summary Background Circadian rhythms regulate physiology and behavior through transcriptional feedback loops of clock genes running within specific pacemaker cells. In Drosophila, molecular oscillations in the small ventral Lateral Neurons (sLNvs) command rhythmic behavior under free-running conditions releasing the neuropeptide PIGMENT DISPERSING FACTOR (PDF) in a circadian fashion. Electrical activity in the sLNvs is also required for behavioral rhythmicity. Yet, how temporal information is transduced into behavior remains unclear. Results Here we developed a new tool for temporal control of gene expression to obtain adult-restricted electrical silencing of the PDF circuit, which led to reversible behavioral arrhythmicity. Remarkably, PER oscillations during the silenced phase remained unaltered, indicating that arrhythmicity is a direct consequence of the silenced activity. Accordingly, circadian axonal remodeling and PDF accumulation were severely affected during the silenced phase. Conclusions Although electrical activity of the sLNvs is not a clock component it coordinates circuit outputs leading to rhythmic behavior. PMID:22018542

  10. Trans-Reactivation: A New Epigenetic Phenomenon Underlying Transcriptional Reactivation of Silenced Genes

    PubMed Central

    Cavalieri, Vincenzo; Ingrassia, Antonia M. R.; Pavesi, Giulio; Corona, Davide F. V.

    2015-01-01

    In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin. We found that non-functional alleles of white, unable to produce a coding transcript, could reactivate in trans the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional trans-reactivation is heritable, relies on the presence of homologous RNA’s and is affected by mutations in genes involved in post-transcriptional gene silencing. Our data suggest a general new unexpected level of gene expression control mediated by homologous RNA molecules in the context of heterochromatic genes. PMID:26292210

  11. Trans-Reactivation: A New Epigenetic Phenomenon Underlying Transcriptional Reactivation of Silenced Genes.

    PubMed

    Onorati, Maria Cristina; Arancio, Walter; Cavalieri, Vincenzo; Ingrassia, Antonia M R; Pavesi, Giulio; Corona, Davide F V

    2015-08-01

    In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin. We found that non-functional alleles of white, unable to produce a coding transcript, could reactivate in trans the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional trans-reactivation is heritable, relies on the presence of homologous RNA's and is affected by mutations in genes involved in post-transcriptional gene silencing. Our data suggest a general new unexpected level of gene expression control mediated by homologous RNA molecules in the context of heterochromatic genes. PMID:26292210

  12. Atmospheric attenuation calibrations of surface weather observations

    NASA Technical Reports Server (NTRS)

    Sanii, Babak

    2001-01-01

    A correlation between near-IR atmospheric attenuation measurements made by the Atmospheric Visibility Monitor (AVM) at the Table Mountain Facility and airport surface weather observations at Edwards Air Force Base has been performed. High correlations (over 0.93) exist between the simultaneous Edwards observed sky cover and the average AVM measured attenuations over the course of the 10 months analyzed. The statistical relationship between the data-sets allows the determination of coarse attenuation statistics from the surface observations, suggesting that such statistics may be extrapolated from any surface weather observation site, Furthermore, a superior technique for converting AVM images to attenuation values by way of MODTRAN predictions has been demonstrated.

  13. Differential dust attenuation in CALIFA galaxies

    NASA Astrophysics Data System (ADS)

    Vale Asari, N.; Cid Fernandes, R.; Amorim, A. L.; Lacerda, E. A. D.; Schlickmann, M.; Wild, V.; Kennicutt, R. C.

    2016-06-01

    Dust attenuation has long been treated as a simple parameter in SED fitting. Real galaxies are, however, much more complicated: The measured dust attenuation is not a simple function of the dust optical depth, but depends strongly on galaxy inclination and the relative distribution of stars and dust. We study the nebular and stellar dust attenuation in CALIFA galaxies, and propose some empirical recipes to make the dust treatment more realistic in spectral synthesis codes. By adding optical recombination emission lines, we find better constraints for differential attenuation. Those recipes can be applied to unresolved galaxy spectra, and lead to better recovered star formation rates.

  14. Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

    SciTech Connect

    Lv, Peng; Xue, Peng; Dong, Jian; Peng, Hui; Clewell, Rebecca; Wang, Aiping; Wang, Yue; Peng, Shuangqing; Qu, Weidong; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

    2013-11-01

    Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation.

  15. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing.

    PubMed

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P; Low, Audrey; Yoshida, Masayuki; Bennett, C Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

    2015-01-01

    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  16. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling

    PubMed Central

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA–DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx−/− pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration. PMID:27462424

  17. Structure of the guide-strand-containing argonaute silencing complex

    SciTech Connect

    Wang, Yanli; Sheng, Gang; Juranek, Stefan; Tuschl, Thomas; Patel, Dinshaw J.

    2009-01-15

    The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5'-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3'-end-binding pocket made up of the PAZ domain show little effect.

  18. Tgfbi/Bigh3 silencing activates ERK in mouse retina.

    PubMed

    Allaman-Pillet, Nathalie; Oberson, Anne; Bustamante, Mauro; Tasinato, Andrea; Hummler, Edith; Schorderet, Daniel F

    2015-11-01

    BIGH3 is a secreted protein, part of the extracellular matrix where it interacts with collagen and integrins on the cell surface. BIGH3 can play opposing roles in cancer, acting as either tumor suppressor or promoter, and its mutations lead to different forms of corneal dystrophy. Although many studies have been carried out, little is known about the physiological role of BIGH3. Using the cre-loxP system, we generated a mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing did not result in any apparent phenotype modifications, the mice remained viable and fertile. We were able to determine the presence of BIGH3 in the retinal pigment epithelium (RPE). In the absence of BIGH3, a transient decrease in the apoptotic process involved in retina maturation was observed, leading to a transient increase in the INL thickness at P15. This phenomenon was accompanied by an increased activity of the pro-survival ERK pathway. PMID:26387839

  19. Optical imaging of RNAi-mediated silencing of cancer

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

  20. Silencing of DELLA induces facultative parthenocarpy in tomato fruits.

    PubMed

    Martí, Cristina; Orzáez, Diego; Ellul, Philippe; Moreno, Vicente; Carbonell, Juan; Granell, Antonio

    2007-12-01

    DELLA proteins are plant nuclear factors that restrain growth and proliferation in response to hormonal signals. The effects of the manipulation of the DELLA pathway in the making of a berry-like fruit were investigated. The expression of the Arabidopsis thaliana gain-of-function DELLA allele Atgai (del) in tomato (Solanum lycopersicum L.) produced partially sterile dwarf plants and compacted influorescences, as expected for a constitutively activated growth repressor. In contrast, antisense silencing of the single endogenous tomato DELLA gene homologue (SlDELLA) produced slender-like plants with elongated flower trusses. Interestingly, the depletion of SlDELLA in tomato was sufficient to overcome the growth arrest normally imposed on the ovary at anthesis, resulting in parthenocarpic fruits in the absence of pollination. Antisense SlDELLA-engineered fruits were smaller in size and elongated in shape compared with wild type. Cell number estimations showed that fruit set, resulting from reduced SlDELLA expression, arose from activated cell elongation at the longitudinal and lateral axes of the fruit pericarp, bypassing phase-II (post-pollination) cell divisions. Parthenocarpy caused by SlDELLA depletion is facultative, as hand pollination restored wild-type fruit phenotype. This indicates that fertilization-associated SlDELLA-independent signals are operational in ovary-fruit transitions. SlDELLA was also found to restrain growth in other reproductive structures, affecting style elongation, stylar hair primordial growth and stigma development. PMID:17883372

  1. Synergy from Silence in a Combinatorial Neural Code

    PubMed Central

    Schneidman, Elad; Puchalla, Jason L.; Segev, Ronen; Harris, Robert A.; Bialek, William; Berry, Michael J.

    2011-01-01

    The manner in which groups of neurons represent events in the external world is a central question in neuroscience. Estimation of the information encoded by small groups of neurons has shown that in many neural systems, cells carry mildly redundant information. These measures average over all the activity patterns of a neural population. Here, we analyze the population code of the salamander and guinea pig retinas by quantifying the information conveyed by specific multi-cell activity patterns. Synchronous spikes, even though they are relatively rare and highly informative, convey less information than the sum of either spike alone, making them redundant coding symbols. Instead, patterns of spiking in one cell and silence in others, which are relatively common and often overlooked as special coding symbols, were found to be mostly synergistic. Our results reflect that the mild average redundancy between ganglion cells that was previously reported is actually the result of redundant and synergistic multi-cell patterns, whose contributions partially cancel each other when taking the average over all patterns. We further show that similar coding properties emerge in a generic model of neural responses, suggesting that this form of combinatorial coding, in which specific compound patterns carry synergistic or redundant information, may exist in other neural circuits. PMID:22049416

  2. The capacity of target silencing by Drosophila PIWI and piRNAs

    PubMed Central

    Post, Christina; Clark, Josef P.; Sytnikova, Yuliya A.; Chirn, Gung-Wei

    2014-01-01

    Although Piwi proteins and Piwi-interacting RNAs (piRNAs) genetically repress transposable elements (TEs), it is unclear how the highly diverse piRNA populations direct Piwi proteins to silence TE targets without silencing the entire transcriptome. To determine the capacity of piRNA-mediated silencing, we introduced reporter genes into Drosophila OSS cells, which express microRNAs (miRNAs) and piRNAs, and compared the Piwi pathway to the Argonaute pathway in gene regulation. Reporter constructs containing several target sites that were robustly silenced by miRNAs were not silenced to the same degrees by piRNAs. However, another set of reporters we designed to enable a large number of both TE-directed and genic piRNAs to bind were robustly silenced by the PIWI/piRNA complex in OSS cells. These reporters show that a bulk of piRNAs are required to pair to the reporter's transcripts and not the reporter's DNA sequence to engage PIWI-mediated silencing. Following our genome-wide study of PIWI-regulated targets in OSS cells, we assessed candidate gene elements with our reporter platform. These results suggest TE sequences are the most direct of PIWI regulatory targets while coding genes are less directly affected by PIWI targeting. Finally, our study suggests that the PIWI transcriptional silencing mechanism triggers robust chromatin changes on targets with sufficient piRNA binding, and preferentially regulates TE transcripts because protein-coding transcripts lack a threshold of targeting by piRNA populations. This reporter platform will facilitate future dissections of the PIWI-targeting mechanism. PMID:25336588

  3. Graft Transmission of RNA Silencing to Non-Transgenic Scions for Conferring Virus Resistance in Tobacco

    PubMed Central

    Md. Ali, Emran; Kobayashi, Kappei; Yamaoka, Naoto; Ishikawa, Masayuki; Nishiguchi, Masamichi

    2013-01-01

    RNA silencing is a mechanism of gene regulation by sequence specific RNA degradation and is involved in controlling endogenous gene expression and defense against invasive nucleic acids such as viruses. RNA silencing has been proven to be transmitted between scions and rootstocks through grafting, mostly using transgenic plants. It has been reported that RNA silencing of tobacco endogenous genes, NtTOM1 and NtTOM3, that are required for tobamovirus multiplication, resulted in high resistance against several tobamoviruses. In the present study, we examined the graft transmission of RNA silencing for conferring virus resistance to non-transgenic scions of the same and different Nicotiana species grafted onto rootstocks in which both NtTOM1 and NtTOM3 were silenced. Non-transgenic Nicotiana tabacum (cvs. Samsun and Xanthi nc) and N. benthamiana were used as scions for grafting onto the rootstocks silenced with both genes. Short interfering RNA (siRNA) of NtTOM1 and NtTOM3 was detected in both the scions and the rootstocks eight weeks after grafting. The leaves were detached from the scions and inoculated with several tobamoviruses. The virus accumulation was tested by ELISA and northern blot analysis. The viruses were detected in grafted scions at extremely low levels, showing that virus resistance was conferred. These results suggest that RNA silencing was induced in and virus resistance was conferred to the non-transgenic scions by grafting onto silenced rootstocks. The effect of low temperature on siRNA accumulation and virus resistance was not significantly observed in the scions. PMID:23717405

  4. Characterization of Arabidopsis Genes Involved in Gene Silencing. Final Progress Report

    SciTech Connect

    Grant, S. R.

    1999-02-05

    Enhancer of gene silencing 1 (egs1) is an Arabidopsis mutant that enhances post-transcriptional gene silencing of the rolB gene introduced by genetic engineering (transgene). The goal of our proposal was cloning EGS1 based on its map position. Although we screened more than 2000 chromosomes for recombination, we were unable to get closer than 2 cM to the gene. We experienced an unexpected tendency of the post-transcriptionally silenced transgene to switch to a more stable silenced state. This made it impossible to select egs1 homozygotes for map based cloning. This forced us to reconsider our cloning strategy. One possibility would have been to use a different transgene as the target of gene silencing. We tested two other transgenes. Both encoded proteins unrelated to the first but they were all expressed from the same type of promoter and they all had a similar tendency to become post-transcriptionally silenced. After screening over 80 F2 segregants from each cross between our egs1 mutant and Arabidopsis of the same ecotype homozygous for the new transgene, we were disappointed to find that the egs1 mutation did not enhance post-transcription silencing of the two new genes. In 80 plants we expected to have between 4 and 6 plants that were homozygous for the transgene and for the mutant egs1 allele. If egs1 mutations could enhance gene silencing of the new transgene, these plants would not express it. However all the double homozygotes still expressed the transgene. Therefore, we could not change the target transgene for mapping. This was the state of the cloning at the time for renewal of the grant in 1999. Because the selection of new meaningful recombinant plants had become extremely inefficient using the original rolB transgene, we abandoned the attempt at map based cloning and did not apply for further funding.

  5. SERRATE is required for intron suppression of RNA silencing in Arabidopsis

    PubMed Central

    Christie, Michael; Carroll, Bernard J.

    2011-01-01

    Transposons and viruses are generally devoid of introns and are prime targets for small interfering RNAs (siRNAs) and RNA silencing. Conversely, endogenous genes often contain introns and are not usually subjected to post-transcriptional gene silencing by siRNAs. In a recent study, we reported that efficient intron splicing directly suppresses siRNA biogenesis and RNA silencing of a Green Fluorescence Protein (GFP) transgene. Splicing-mediated suppression of GFP silencing was dependent on ABH1, the Arabidopsis ortholog of human mRNA cap-binding protein 80. Furthermore, genome-wide analyses of Arabidopsis small RNA libraries showed that exons from intron-containing genes accumulate less small RNAs than exons from intronless genes. Our in silico analysis therefore suggested that intron splicing has a fundamental role in protecting endogenous genes from becoming templates for siRNA biogenesis and RNA silencing. Here, we show that SERRATE (SE) is also required for splicing-mediated suppression of RNA silencing in Arabidopsis. SE encodes a zinc finger protein that, like ABH1, functions in micro-RNA (miRNA) biogenesis and intron splicing. The implications of our findings are also discussed in a broader context. PMID:22112452

  6. Sim4: a novel fission yeast kinetochore protein required for centromeric silencing and chromosome segregation.

    PubMed

    Pidoux, Alison L; Richardson, William; Allshire, Robin C

    2003-04-28

    Fission yeast centromeres are composed of two domains: the central core and the outer repeats. Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins. Genes placed within either region are transcriptionally silenced, reflecting the formation of a functional kinetochore complex and flanking centromeric heterochromatin. Here, transcriptional silencing was exploited to identify components involved in central core silencing and kinetochore assembly or structure. The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region. Sim4 coimmunoprecipitates with the central core component Mis6 and, like Mis6, affects Cnp1CENP-A association with the central domain. Functional Mis6 is required for Sim4 localization at the kinetochore. Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore. PMID:12719471

  7. Sea urchin arylsulfatase insulator exerts its anti-silencing effect without interacting with the nuclear matrix.

    PubMed

    Hino, Shinjiro; Akasaka, Koji; Matsuoka, Masao

    2006-03-17

    Chromatin insulators have been shown to stabilize transgene expression. Although insulators have been suggested to regulate the subcellular localization of chromosomes, it is still unclear whether this property is important for their anti-silencing activity. To investigate the underlying mechanisms governing the anti-silencing function of insulators, we studied the association of sea urchin arylsulfatase insulator (ArsI) with the nuclear matrix, which is a key component of the subnuclear localization of the genome. ArsI did not potentiate the nuclear matrix association with the transgene, even though it showed strong anti-silencing activity. This observation was in clear contrast to the results of the experiment using a human interferon-beta scaffold attachment region, in which the anti-silencing effect coincided with the enhanced matrix association. Chromatin immunoprecipitation analyses suggested that the absence of the matrix binding by ArsI was due to a lack of its binding to CCCTC-binding factor (CTCF), a protein known to be associated with matrix binding by chicken beta-globin insulator. Furthermore, ArsI maintained the nucleosome occupancy within the transgene at a constant level during long-term culture, although ArsI itself was not a nucleosome-excluding sequence. Taken together, these results suggest that this insulator exerts its anti-silencing activity by counteracting silencing-associated factors to maintain local chromatin environment, rather than by remodeling the subnuclear localization of the transgene locus. PMID:16426632

  8. A Polycomb and Gaga Dependent Silencer Adjoins the Fab-7 Boundary in the Drosophila Bithorax Complex

    PubMed Central

    Hagstrom, K.; Muller, M.; Schedl, P.

    1997-01-01

    The homeotic genes of the Drosophila bithorax complex are controlled by a large cis-regulatory region that ensures their segmentally restricted pattern of expression. A deletion that removes the Frontabdominal-7 cis-regulatory region (Fab-7(1)) dominantly transforms parasegment 11 into parasegment 12. Previous studies suggested that removal of a domain boundary element on the proximal side of Fab-7(1) is responsible for this gain-of-function phenotype. In this article we demonstrate that the Fab-7(1) deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment and plays a different role in regulating parasegment-specific expression patterns of the Abd-B gene. The iab-7 PRE mediates pairing-sensitive silencing of mini-white, and can maintain the segmentally restricted expression pattern of a BXD, Ubx/lacZ reporter transgene. Both silencing activities depend upon Polycomb Group proteins. Pairing-sensitive silencing is relieved by removing the transvection protein Zeste, but is enhanced in a novel pairing-independent manner by the zeste(1) allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans a nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor. PMID:9258680

  9. Control of heterochromatin localization and silencing by the nuclear membrane protein Lem2

    PubMed Central

    Barrales, Ramón Ramos; Forn, Marta; Georgescu, Paula Raluca; Sarkadi, Zsuzsa; Braun, Sigurd

    2016-01-01

    Transcriptionally silent chromatin localizes to the nuclear periphery, which provides a special microenvironment for gene repression. A variety of nuclear membrane proteins interact with repressed chromatin, yet the functional role of these interactions remains poorly understood. Here, we show that, in Schizosaccharomyces pombe, the nuclear membrane protein Lem2 associates with chromatin and mediates silencing and heterochromatin localization. Unexpectedly, we found that these functions can be separated and assigned to different structural domains within Lem2, excluding a simple tethering mechanism. Chromatin association and tethering of centromeres to the periphery are mediated by the N-terminal LEM (LAP2–Emerin–MAN1) domain of Lem2, whereas telomere anchoring and heterochromatin silencing require exclusively its conserved C-terminal MSC (MAN1–Src1 C-terminal) domain. Particularly, silencing by Lem2 is epistatic with the Snf2/HDAC (histone deacetylase) repressor complex SHREC at telomeres, while its necessity can be bypassed by deleting Epe1, a JmjC protein with anti-silencing activity. Furthermore, we found that loss of Lem2 reduces heterochromatin association of SHREC, which is accompanied by increased binding of Epe1. This reveals a critical function of Lem2 in coordinating these antagonistic factors at heterochromatin. The distinct silencing and localization functions mediated by Lem2 suggest that these conserved LEM-containing proteins go beyond simple tethering to play active roles in perinuclear silencing. PMID:26744419

  10. 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

  11. 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. PMID:27524609

  12. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

    PubMed Central

    Kirov, Julia V.; Adkisson, Michael; Nava, A. J.; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K.; Lloyd, K. C. Kent; de Jong, Pieter; West, David B.

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  13. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation.

    PubMed

    Kirov, Julia V; Adkisson, Michael; Nava, A J; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K; Lloyd, K C Kent; de Jong, Pieter; West, David B

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  14. Resilience among women with HIV: Impact of silencing the self and socioeconomic factors.

    PubMed

    Dale, Sannisha K; Cohen, Mardge H; Kelso, Gwendolyn A; Cruise, Ruth C; Weber, Kathleen M; Watson, Cheryl; Burke-Miller, Jane K; Brody, Leslie R

    2014-03-01

    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

  15. Importance of coat protein and RNA silencing in satellite RNA/virus interactions.

    PubMed

    Manfre, Alicia J; Simon, Anne E

    2008-09-15

    RNA silencing is a major defense mechanism plants use to fight an invading virus. The silencing suppressor of Turnip crinkle virus (TCV) is the viral coat protein (CP), which obstructs the DCL2/DCL4 silencing pathway. TCV is associated with a virulent satellite RNA (satC) that represses the accumulation of TCV genomic RNA and whose accumulation is repressed by the TCV CP. To investigate if reduced TCV accumulation due to satC involves RNA silencing and/or the suppressor activity of the CP, TCV was altered to contain a mutation reported to target CP silencing suppressor activity (Deleris et al., Science 313, 68, 2006). However, the mutation did not cause an exclusive defect in silencing suppression, but rather produced a generally non-functional protein. We demonstrate that a functional CP, but not DCL2/DCL4, is required for satC-mediated repression of TCV. In addition, enhancement of satC accumulation in the absence of a functional CP requires DCL2/DCL4. PMID:18639914

  16. The Citrus leaf blotch virus movement protein acts as silencing suppressor.

    PubMed

    Renovell, Águeda; Vives, Mari Carmen; Ruiz-Ruiz, Susana; Navarro, Luis; Moreno, Pedro; Guerri, José

    2012-02-01

    To counteract plant antiviral defense based on RNA silencing, many viruses express proteins that inhibit this mechanism at different levels. The genome of Citrus leaf blotch virus (CLBV) encodes a 227-kDa protein involved in replication, a 40-kDa movement protein (MP), and a 41-kDa coat protein (CP). To determine if any of these proteins might have RNA silencing suppressor activities, we have used Agrobacterium-mediated transient assays in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c. Only CLBV MP was able to suppress intracellular GFP silencing induced by expression of either single- or double-stranded (ds) GFP RNA, but not cell-to-cell or long distance spread of the silencing signal. The MP suppressor activity was weak compared to other characterized viral suppressor proteins. Overall our data indicate that MP acts as a suppressor of local silencing probably by interfering in the silencing pathway downstream of the steps of dsRNA and small RNAs generation. PMID:21948005

  17. H19ICR mediated transcriptional silencing does not require target promoter methylation.

    PubMed

    Gebert, Claudia; Rong, Qi; Jeong, Sangkyun; Iben, James; Pfeifer, Karl

    2016-07-29

    Transcription of the reciprocally imprinted genes Insulin-like growth factor 2 (Igf2) and H19 is orchestrated by the 2.4-kb H19 Imprinting Control Region (H19ICR) located upstream of H19. Three known functions are associated with the H19ICR: (1) it is a germline differentially methylated region, (2) it is a transcriptional insulator, and (3) it is a transcriptional silencer. The molecular mechanisms of the DMR and insulator functions have been well characterized but the basis for the ICR's silencer function is less well understood. In order to study the role the H19ICR intrinsically plays in gene silencing, we transferred the 2.4-kb H19ICR to a heterologous non-imprinted location on chromosome 5, upstream of the alpha fetoprotein (Afp) promoter. Independent of its orientation, the 2.4-kb H19ICR silences transcription from the paternal Afp promoter. Thus silencing is a function intrinsic to this DNA element. Further, ICR mediated silencing is a developmental process that, unexpectedly, does not occur through DNA methylation at the target promoter. PMID:27178213

  18. A role for DNA polymerase alpha in epigenetic control of transcriptional silencing in fission yeast.

    PubMed

    Nakayama Ji; Allshire, R C; Klar, A J; Grewal, S I

    2001-06-01

    In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase alpha (pol(alpha)) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that pol(alpha) mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polalpha and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Pol(alpha) in vitro. Moreover, silencing-defective mutant Pol(alpha) displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Pol(alpha), in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  19. Underwing compression vortex attenuation device

    NASA Technical Reports Server (NTRS)

    Patterson, James C., Jr. (Inventor)

    1993-01-01

    A vortex attenuation device is presented which dissipates a lift-induced vortex generated by a lifting aircraft wing. The device consists of a positive pressure gradient producing means in the form of a compression panel attached to the lower surface of the wing and facing perpendicular to the airflow across the wing. The panel is located between the midpoint of the local wing cord and the trailing edge in the chord-wise direction and at a point which is approximately 55 percent of the wing span as measured from the fuselage center line in the spanwise direction. When deployed in flight, this panel produces a positive pressure gradient aligned with the final roll-up of the total vortex system which interrupts the axial flow in the vortex core and causes the vortex to collapse.

  20. SEISMIC ATTENUATION FOR RESERVOIR CHARACTERIZATION

    SciTech Connect

    Joel Walls; M.T. Taner; Naum Derzhi; Gary Mavko; Jack Dvorkin

    2002-10-01

    RSI has access to two synthetic seismic programs: Osiris seismic modeling system provided by Odegaard (Osiris) and synthetic seismic program, developed by SRB, implementing the Kennett method for normal incidence. Achieving virtually identical synthetic seismic traces from these different programs serves as cross-validation for both. The subsequent experiments have been performed with the Kennett normal incidence code because: We have access to the source code, which allowed us to easily control computational parameters and integrate the synthetics computations with our graphical and I/O systems. This code allows to perform computations and displays on a PC in MatLab or Octave environment, which is faster and more convenient. The normal incidence model allows us to exclude from the synthetic traces some of the physical effects that take place in 3-D models (like inhomogeneous waves) but have no relevance to the topic of our investigation, which is attenuation effects on seismic reflection and transmission.

  1. Attenuation Relationship of Arias Intensity for Taiwan

    NASA Astrophysics Data System (ADS)

    Sung, C.; Hsieh, P.; Lin, P.; Lee, C.

    2008-12-01

    Arias intensity (AI) reflects the complete acceleration time history duration of ground vibrations. It correlates well with several commonly used demand measure of structural performance, liquefaction, and seismic slope stability. A good attenuation equation can reflect the characteristics of the ground-motion attenuation for a region, and can be used to predict the ground-motion value of a specific site for seismic resistance design. This study analyzed two local empirical attenuation relationships, one for the crustal earthquakes and the other for the subduction zone earthquakes, based on the strong ground-motion data from TSMIP and SMART1 array in Taiwan. Maximum likelihood method and mixed-effect model were used with non-linear regression analyses to determine coefficients. The result shows that adding terms of Vs30 and focal mechanism can effectively reduce the standard deviation in the attenuation models. To compare with other AI attenuation equations, the AI value predicted by our crustal earthquake attenuation equation is higher in the near field and is lower in the far field than the researches in other regions. The subduction zone earthquake attenuation equation predicts higher AI value than the crustal earthquake attenuation equation does.

  2. LONG TERM MONITORING FOR NATURAL ATTENUATION

    EPA Science Inventory

    We have good statistical methods to: (1) determine whether concentrations of a contaminant are attenuating over time, (2) determine the rate of attenuation and confidence interval on the rate, and (3) determine whether concentrations have met a particular clean up goal. We do no...

  3. Underwing Compression Vortex-Attenuation Device

    NASA Technical Reports Server (NTRS)

    Patterson, James C., Jr.

    1994-01-01

    Underwing compression vortex-attenuation device designed to provide method for attenuating lift-induced vortex generated by wings of airplane. Includes compression panel attached to lower surface of wing, facing perpendicular to streamwise airflow. Concept effective on all types of aircraft. Causes increase in wing lift rather than reduction when deployed. Device of interest to aircraft designers and enhances air safety in general.

  4. Docking-mechanism attenuator with electromechanical damper

    NASA Technical Reports Server (NTRS)

    Syromyatnikov, V. S.

    1971-01-01

    Theoretical and practical problems involved in the application of electromechanical damping for spacecraft docking-mechanism attenuation are discussed. Some drawbacks of hydraulic dampers used for the purpose are pointed out. The basic scheme of the attenuator with the electromechanical damper is given.

  5. Attenuation coefficients for water quality trading.

    PubMed

    Keller, Arturo A; Chen, Xiaoli; Fox, Jessica; Fulda, Matt; Dorsey, Rebecca; Seapy, Briana; Glenday, Julia; Bray, Erin

    2014-06-17

    Water quality trading has been proposed as a cost-effective approach for reducing nutrient loads through credit generation from agricultural or point source reductions sold to buyers facing costly options. We present a systematic approach to determine attenuation coefficients and their uncertainty. Using a process-based model, we determine attenuation with safety margins at many watersheds for total nitrogen (TN) and total phosphorus (TP) loads as they transport from point of load reduction to the credit buyer. TN and TP in-stream attenuation generally increases with decreasing mean river flow; smaller rivers in the modeled region of the Ohio River Basin had TN attenuation factors per km, including safety margins, of 0.19-1.6%, medium rivers of 0.14-1.2%, large rivers of 0.13-1.1%, and very large rivers of 0.04-0.42%. Attenuation in ditches transporting nutrients from farms to receiving rivers is 0.4%/km for TN, while for TP attenuation in ditches can be up to 2%/km. A 95 percentile safety margin of 30-40% for TN and 6-10% for TP, applied to the attenuation per km factors, was determined from the in-stream sensitivity of load reductions to watershed model parameters. For perspective, over 50 km a 1% per km factor would result in 50% attenuation = 2:1 trading ratio. PMID:24866482

  6. Relating P-wave attenuation to permeability

    SciTech Connect

    Akbar, N.; Dvorkin, J.; Nur, A. . Dept. of Geophysics)

    1993-01-01

    To relate P-wave attenuation to permeability, the authors examine a three-dimensional (3-D) theoretical model of a cylindrical pore filled with viscous fluid and embedded in an infinite isotropic elastic medium. They calculate both attenuation and permeability as functions of the direction of wave propagation. Attenuation estimates are based on the squirt flow mechanism; permeability is calculated using the Kozeny-Carmen relation. They find that in the case when a plane P-wave propagates parallel to this orientation (Q[sup [minus]1][delta] = 90[degree]), attenuation is always higher than when a wave propagates parallel to this orientation (Q[sup [minus]1][delta] = 0[degree]). The ratio of these two attenuation values Q[sup [minus]1][delta] = 90[degree]/Q[sup [minus]1] = 0[degree] increases with an increasing pore radius and decreasing frequency and saturation. By changing permeability, varying the radius of the pore, they find that the permeability-attenuation relation is characterized by a peak that shifts toward lower permeabilities as frequency decreases. Therefore, the attenuation of a low-frequency wave decreases with increasing permeability. They observe a similar trend on relations between attenuation and permeability experimentally obtained on sandstone samples.

  7. Seismic attenuation anisotropy in reservoir sedimentary rocks

    SciTech Connect

    Best, A.I.

    1994-12-31

    Seismic attenuation is a fundamental property of reservoir sedimentary rocks; it is strongly related to reservoir permeability. Knowledge of its variation with lithology, with burial depth, and with wave propagation direction is vital for understanding the attenuation mechanism. Given this information, realistic theoretical models may be constructed for predicting attenuation, and hence permeability, over a wide frequency range. Accurate ultrasonic attenuation measurements were made in the laboratory over a range of effective pressures on sandstone samples with different amounts of humic organic matter. The organic matter formed fine laminations along the bedding planes of the sandstones. The results show that the sandstones are highly attenuating at 5 MPa mainly because of the presence of grain contact microcracks giving rise to squirt flow; at 40 MPa, when most of the microcracks are closed, the clean sandstones are poorly attenuating, but the organic-rich sandstones remain highly attenuating. It is postulated that the compliant organic matter is responsible for causing squirt flow at high and at low pressures. The results also show that the maximum attenuation occurs when the particle motion of the propagating wave is perpendicular to the planes of the organic matter laminations. These results are consistent with the squirt flow theory of Akbar et al (1993) for compressional waves.

  8. Secrecy and silence: why women hide contraceptive use.

    PubMed

    1998-09-01

    A recent Population Council survey of 1860 married women and 1056 of their husbands in urban Zambia found that many women who use contraception do so without their husbands' knowledge and that those women who hid their practice of contraception from their husbands did so because they found it very difficult to bring up the subject of family planning with them. These findings indicate that low levels of contraceptive use are not the result of a simple communication matter. Sex and sexuality are often the exclusive domain of African husbands. As such, if a wife initiates a discussion of family planning, she may threaten her husband's sense of control and create discord within the family. The culture of silence about sex and sexuality is very strong in Africa. 57% of women stated that were they to propose contraceptive use with their husbands and the husband opposed such practice, they would nonetheless use them without his knowledge. 7% of the women stated that if their husbands disapproved of contraceptive use, they would nonetheless openly use a method against his wishes. The majority of women correctly perceived their husbands' views on family planning use and fertility preferences. In focus groups, both men and women said that they did not believe that women have the right to independently act upon their reproductive preferences. A husband's inadequate financial support of his children could, however, justify clandestine contraceptive use. These findings point to the need to include easily hidden methods in the mix of contraceptives family planning programs offer. Moreover, service providers should not automatically encourage husbands' involvement. A client's right to privacy should always be respected. PMID:12321878

  9. Histone deacetylases (HDACs) in XPC gene silencing and bladder cancer

    PubMed Central

    2011-01-01

    Bladder cancer is one of the most common malignancies and causes hundreds of thousands of deaths worldwide each year. Bladder cancer is strongly associated with exposure to environmental carcinogens. It is believed that DNA damage generated by environmental carcinogens and their metabolites causes development of bladder cancer. Nucleotide excision repair (NER) is the major DNA repair pathway for repairing bulk DNA damage generated by most environmental carcinogens, and XPC is a DNA damage recognition protein required for initiation of the NER process. Recent studies demonstrate reduced levels of XPC protein in tumors for a majority of bladder cancer patients. In this work we investigated the role of histone deacetylases (HDACs) in XPC gene silencing and bladder cancer development. The results of our HDAC inhibition study revealed that the treatment of HTB4 and HTB9 bladder cancer cells with the HDAC inhibitor valproic acid (VPA) caused an increase in transcription of the XPC gene in these cells. The results of our chromatin immunoprecipitation (ChIP) studies indicated that the VPA treatment caused increased binding of both CREB1 and Sp1 transcription factors at the promoter region of the XPC gene for both HTB4 and HTB9 cells. The results of our immunohistochemistry (IHC) staining studies further revealed a strong correlation between the over-expression of HDAC4 and increased bladder cancer occurrence (p < 0.001) as well as a marginal significance of increasing incidence of HDAC4 positivity seen with an increase in severity of bladder cancer (p = 0.08). In addition, the results of our caspase 3 activation studies demonstrated that prior treatment with VPA increased the anticancer drug cisplatin-induced activation of caspase 3 in both HTB4 and HTB9 cells. All of these results suggest that the HDACs negatively regulate transcription of the XPC gene in bladder cancer cells and contribute to the severity of bladder tumors. PMID:21507255

  10. Tumor Selective Silencing Using an RNAi-Conjugated Polymeric Nanopharmaceutical.

    PubMed

    Svenson, Sonke; Case, Roy I; Cole, Roderick O; Hwang, Jungyeon; Kabir, Sujan R; Lazarus, Douglas; Lim Soo, Patrick; Ng, Pei-Sze; Peters, Christian; Shum, Pochi; Sweryda-Krawiec, Beata; Tripathi, Snehlata; van der Poll, Derek; Eliasof, Scott

    2016-03-01

    Small interfering RNA (siRNA) therapeutics have potential advantages over traditional small molecule drugs such as high specificity and the ability to inhibit otherwise "undruggable" targets. However, siRNAs have short plasma half-lives in vivo, can induce a cytokine response, and show poor cellular uptake. Formulating siRNA into nanoparticles offers two advantages: enhanced siRNA stability against nuclease degradation beyond what chemical modification alone can provide; and improved site-specific delivery that takes advantage of the enhanced permeability and retention (EPR) effect. Existing delivery systems generally suffer from poor delivery to tumors. Here we describe the formation and biological activity of polymeric nanopharmaceuticals (PNPs) based on biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) conjugated to siRNA via an intracellular cleavable disulfide linker (PLGA-siRNA). Additionally, these PNPs contain (1) PLGA conjugated to polyethylene glycol (PEG) for enhanced pharmacokinetics of the nanocarrier; (2) a cation for complexation of siRNA and charge compensation to avoid high negative zeta potential; and (3) neutral poly(vinyl alcohol) (PVA) to stabilize the PNPs and support the PEG shell to prevent particle aggregation and protein adsorption. The biological data demonstrate that these PNPs achieve prolonged circulation, tumor accumulation that is uniform throughout the tumor, and prolonged tumor-specific knockdown. PNPs employed in this study had no effect on body weight, blood cell count, serum chemistry, or cytokine response at doses >10 times the effective dose. PNPs, therefore, constitute a promising solution for achieving durable siRNA delivery and gene silencing in tumors. PMID:26835715

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

    PubMed

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

    2016-04-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system. PMID:26787723

  12. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.

    PubMed

    McHugh, Colleen A; Chen, Chun-Kan; Chow, Amy; Surka, Christine F; Tran, Christina; McDonel, Patrick; Pandya-Jones, Amy; Blanco, Mario; Burghard, Christina; Moradian, Annie; Sweredoski, Michael J; Shishkin, Alexander A; Su, Julia; Lander, Eric S; Hess, Sonja; Plath, Kathrin; Guttman, Mitchell

    2015-05-14

    Many long non-coding RNAs (lncRNAs) affect gene expression, but the mechanisms by which they act are still largely unknown. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell. Here we develop a method to purify a lncRNA from cells and identify proteins interacting with it directly using quantitative mass spectrometry. We identify ten proteins that specifically associate with Xist, three of these proteins--SHARP, SAF-A and LBR--are required for Xist-mediated transcriptional silencing. We show that SHARP, which interacts with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of RNA polymerase II (Pol II) from the inactive X. Both SMRT and HDAC3 are also required for silencing and Pol II exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for Xist-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X chromosome. Our results suggest that Xist silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude Pol II across the X chromosome. PMID:25915022

  13. Lipidoid nanoparticle mediated silencing of Mcl-1 induces apoptosis in mantle cell lymphoma.

    PubMed

    Knapp, Christopher M; He, Jia; Lister, John; Whitehead, Kathryn A

    2016-05-01

    Conventional chemo-immunotherapy fails to cure the majority of mantle cell lymphoma patients and causes substantial toxicity. Resistant mantle cell lymphoma cells commonly overexpress and are dependent on the anti-apoptotic protein, Mcl-1, for survival. In this study, we use potent lipidoid nanoparticles to deliver siRNA to silence Mcl-1 expression. Studies were conducted using two different mantle cell lymphoma cell lines, a normal (JeKo-1) and an aggressive (MAVER-1) line, to assess the ability of lipidoid nanoparticles to be used broadly in the treatment of mantle cell lymphoma. Mcl-1 mRNA silencing and protein knockdown was observed as early as one day after treatment and the lipidoid nanoparticles achieved sustained silencing of Mcl-1 mRNA for at least four days in both JeKo-1 and MAVER-1 cells. Eighty percent silencing was achieved at three days post-transfection in JeKo-1 cells while 50% silencing was achieved in MAVER-1 cells, which are more resistant to transfection. Interestingly, silencing of Mcl-1 induced apoptosis in nearly 30% of both JeKo-1 and MAVER-1 cells three days post-transfection. Additionally, Mcl-1 silencing and the resultant apoptosis in mantle cell lymphoma cells were dose dependent. These data suggest that lipidoid nanoparticles siRNA therapy targeting Mcl-1 has potential as a new treatment modality for mantle cell lymphoma and many other cancers that overexpress Mcl-1. The combination of anti-Mcl-1 lipidoid nanoparticles with other forms of targeted therapy offers hope for reducing or replacing cytotoxic chemotherapy as standard treatment for mantle cell lymphoma. PMID:27022142

  14. The Xist lncRNA directly interacts with SHARP to silence transcription through HDAC3

    PubMed Central

    McHugh, Colleen A.; Chen, Chun-Kan; Chow, Amy; Surka, Christine F.; Tran, Christina; McDonel, Patrick; Pandya-Jones, Amy; Blanco, Mario; Burghard, Christina; Moradian, Annie; Sweredoski, Michael J.; Shishkin, Alexander A.; Su, Julia; Lander, Eric S.; Hess, Sonja; Plath, Kathrin; Guttman, Mitchell

    2015-01-01

    Many long non-coding RNAs (lncRNAs) affect gene expression1, but the mechanisms by which they act are still largely unknown2. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X-chromosome during development in female mammals3,4. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role3. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell5. Here we develop a method to purify a lncRNA and identify its direct interacting proteins using quantitative mass spectrometry. We identify 10 proteins that specifically associate with Xist, three of these proteins – SHARP, SAF-A, and LBR – are required for Xist-mediated transcriptional silencing. We show that SHARP, which interacts with the SMRT co-repressor6 that activates HDAC37, is not only essential for silencing, but is also required for the exclusion of RNA Polymerase II (PolII) from the inactive X. Both SMRT and HDAC3 are also required for silencing and PolII exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for Xist-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X-chromosome. Our results suggest that Xist silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude PolII across the X-chromosome. PMID:25915022

  15. Development of a gene silencing DNA vector derived from a broad host range geminivirus

    PubMed Central

    Golenberg, Edward M; Sather, D Noah; Hancock, Leandria C; Buckley, Kenneth J; Villafranco, Natalie M; Bisaro, David M

    2009-01-01

    Background Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems. Results The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV), named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS), transketolase, the sulfur allele of magnesium chelatase (ChlI), and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant. Conclusion The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility. PMID:19573239

  16. 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

  17. High-Performance Genetically Targetable Optical Neural Silencing via Light-Driven Proton Pumps

    PubMed Central

    Chow, Brian Y.; Han, Xue; Dobry, Allison S.; Qian, Xiaofeng; Chuong, Amy S.; Li, Mingjie; Henninger, Michael A.; Belfort, Gabriel M.; Lin, Yingxi; Monahan, Patrick E.; Boyden, Edward S.

    2009-01-01

    The ability to silence the activity of genetically specified neurons in a temporally precise fashion would open up the ability to investigate the causal role of specific cell classes in neural computations, behaviors, and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate very powerful, safe, multiple-color silencing of neural activity. The gene archaerhodopsin-31 (Arch) from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. In addition, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourally-relevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins2,3 or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue-green light-drivable proton pump from the fungus Leptosphaeria maculans4 (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents the potential for independent silencing of two neural populations by blue vs. red light. Light-driven proton pumps thus represent a high-performance and extremely versatile class of “optogenetic” voltage and ion modulator, which will broadly empower new neuroscientific, biological, neurological, and psychiatric investigations. PMID:20054397

  18. Plasmodium falciparum: attenuation by irradiation

    SciTech Connect

    Waki, S.; Yonome, I.; Suzuki, M.

    1983-12-01

    The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10(6) parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.

  19. Attenuation Tomography of the Upper Mantle

    NASA Astrophysics Data System (ADS)

    Adenis, A.; Debayle, E.; Ricard, Y. R.

    2014-12-01

    We present a 3-D model of surface wave attenuation in the upper mantle. The model is constrained by a large data set of fundamental and higher Rayleigh mode observations. This data set consists of about 1,800,000 attenuation curves measured in the period range 50-300s by Debayle and Ricard (2012). A careful selection allows us to reject data for which measurements are likely biased by the poor knowledge of the scalar seismic moment or by a ray propagation too close to a node of the source radiation pattern. For each epicenter-station path, elastic focusing effects due to seismic heterogeneities are corrected using DR2012 and the data are turned into log(1/Q). The selected data are then combined in a tomographic inversion using the non-linear least square formalism of Tarantola and Valette (1982). The obtained attenuation maps are in agreement with the surface tectonic for periods and modes sensitive to the top 200km of the upper mantle. Low attenuation regions correlate with continental shields while high attenuation regions are located beneath young oceanic regions. The attenuation pattern becomes more homogeneous at depths greater than 200 km and the maps are dominated by a high quality factor signature beneath slabs. We will discuss the similarities and differences between the tomographies of seismic velocities and of attenuations.

  20. Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis

    PubMed Central

    Chandrasekhara, Chinmayi; Mohannath, Gireesha; Blevins, Todd; Pontvianne, Frederic; Pikaard, Craig S.

    2016-01-01

    In eukaryotes, scores of excess ribosomal RNA (rRNA) genes are silenced by repressive chromatin modifications. Given the near sequence identity of rRNA genes within a species, it is unclear how specific rRNA genes are reproducibly chosen for silencing. Using Arabidopsis thaliana ecotype (strain) Col-0, a systematic search identified sequence polymorphisms that differ between active and developmentally silenced rRNA gene subtypes. Recombinant inbred mapping populations derived from three different ecotype crosses were then used to map the chromosomal locations of silenced and active RNA gene subtypes. Importantly, silenced and active rRNA gene subtypes are not intermingled. All silenced rRNA gene subtypes mapped to the nucleolus organizer region (NOR) on chromosome 2 (NOR2). All active rRNA gene subtypes mapped to NOR4. Using an engineered A. thaliana line in which a portion of Col-0 chromosome 4 was replaced by sequences of another ecotype, we show that a major rRNA gene subtype silenced at NOR2 is active when introgressed into the genome at NOR4. Collectively, these results reveal that selective rRNA gene silencing is not regulated gene by gene based on mechanisms dependent on subtle gene sequence variation. Instead, we propose that a subchromosomal silencing mechanism operates on a multimegabase scale to inactivate NOR2. PMID:26744421

  1. Insight into the relationship between the cell culture model, cell trafficking and siRNA silencing efficiency.

    PubMed

    Capel, Victoria; Vllasaliu, Driton; Watts, Peter; Stolnik, Snow

    2016-08-19

    Despite research efforts, cell uptake processes determining siRNA silencing efficiency remain unclear. Here, we examine the relationship between in vitro cell culture models, cellular trafficking and siRNA silencing efficiency to provide a mechanistic insight on siRNA delivery system design. Model siRNA-polyplexes, based on chitosan as a 'classical' condensing agent, were applied to a panel of lung epithelial cell lines, H1299, A549 and Calu-3 and cell internalization levels, trafficking pathways and gene silencing assessed on exposure to pharmacological inhibitors. The data reveal striking differences in the internalization behaviour and gene silencing efficiency in the tested cell lines, despite their common lung epithelial origins. The model system's silencing was lower where clathrin internalization pathway predominated in Calu-3, relative to silencing in H1299 cells where a non-clathrin internalization appears dominant. Increased silencing on endosomal disruption was apparent in Calu-3 cells, but absent when cellular internalization was not predominantly clathrin-mediated in A549 cells. This highlights that identifying cell trafficking pathways before incorporation of functional components to siRNA delivery systems (e.g. endosomolytic compounds) is crucial. The study hence stresses the importance of selection of appropriate cell culture model, relevant to in vivo target, to assess the gene silencing efficiency and decide which functionalities the 'stratified siRNA silencing vector' requires. PMID:27349867

  2. Natural attenuation general data guide. Final report

    SciTech Connect

    Kram, M.L.; Goetz, F.

    1999-02-01

    This guide is a decision-making tool to help remedial project managers (RPMs) determine whether natural attenuation can be used as a remedial option at contaminant release sites. Data requirements and methodology to evaluate the potential for using natural attenuation are presented. For sites where the natural attenuation remedial option is implemented, tables of commonly measured parameters, general data collection rationale, and interpretation guidance are included. This format allows the RPM to recognize data gaps, interpret data, construct a conceptual site model, and develop a sampling and analysis plan for evaluation and monitoring purposes.

  3. Spectral attenuation length of scintillating fibers

    NASA Astrophysics Data System (ADS)

    Drexlin, Guido; Eberhard, Veit; Hunkel, Dirk; Zeitnitz, B.

    1995-02-01

    A double spectrometer allows the precise measurement of the spectral attenuation length of scintillating fibers. Exciting the fibers with a N 2-laser at different points and measuring the wavelength dependent light intensity on both ends of the fiber simultaneously, enables a measurement of the attenuation length which is practically independent of systematic uncertainties. The experimental setup can additionally be used for the measurement of the relative light output. Six types of scintillating fibers from four manufactures (Bicron, Kuraray, Pol.Hi.Tech, and Plastifo) were tested. For different fibers the wavelength dependent attenuation lengths were measured from 0.3 m up to 20 m with an accuracy as good as 1%.

  4. Identification of high-copy disruptors of telomeric silencing in Saccharomyces cerevisiae.

    PubMed Central

    Singer, M S; Kahana, A; Wolf, A J; Meisinger, L L; Peterson, S E; Goggin, C; Mahowald, M; Gottschling, D E

    1998-01-01

    The ends of chromosomes in Saccharomyces cerevisiae initiate a repressive chromatin structure that spreads internally and inhibits the transcription of nearby genes, a phenomenon termed telomeric silencing. To investigate the molecular basis of this process, we carried out a genetic screen to identify genes whose overexpression disrupts telomeric silencing. We thus isolated 10 DOT genes (disruptor of telomeric silencing). Among these were genes encoding chromatin component Sir4p, DNA helicase Dna2p, ribosomal protein L32, and two proteins of unknown function, Asf1p and Ifh1p. The collection also included genes that had not previously been identified: DOT1, DOT4, DOT5, DOT6, and TLC1, which encodes the RNA template component of telomerase. With the exception of TLC1, all these genes, particularly DOT1 and DOT4, also reduced silencing at other repressed loci (HM loci and rDNA) when overexpressed. Moreover, deletion of the latter two genes weakened silencing as well, suggesting that DOT1 and DOT4 normally play important roles in gene repression. DOT1 deletion also affected telomere tract length. The function of Dot1p is not known. The sequence of Dot4p suggests that it is a ubiquitin-processing protease. Taken together, the DOT genes include both components and regulators of silent chromatin. PMID:9755194

  5. Epigenetic silencing of EYA2 in pancreatic adenocarcinomas promotes tumor growth

    PubMed Central

    Vincent, Audrey; Hong, Seung-Mo; Hu, Chaoxin; Omura, Noriyuki; Young, Angela; Kim, Haeryoung; Yu, Jun; Knight, Spencer; Ayars, Michael; Griffith, Margaret; Van Seuningen, Isabelle; Maitra, Anirban; Goggins, Michael

    2014-01-01

    To identify potentially important genes dysregulated in pancreatic cancer, we analyzed genome-wide transcriptional analysis of pancreatic cancers and normal pancreatic duct samples and identified the transcriptional coactivator, EYA2 (Drosophila Eyes Absent Homologue-2) as silenced in the majority of pancreatic cancers. We investigated the role of epigenetic mechanisms of EYA2 gene silencing in pancreatic cancers, performed in vitro and in vivo proliferation and migration assays to assess the effect of EYA2 silencing on tumor cell growth and metastasis formation, and expression analysis to identify genes transcriptionally regulated by EYA2. We found loss of tumoral Eya2 expression in 63% of pancreatic cancers (120/189 cases). Silencing of EYA2 expression in pancreatic cancer cell lines correlated with promoter methylation and histone deacetylation and was reversible with DNA methyltransferase and HDAC inhibitors. EYA2 knockdown in pancreatic cancer cell lines increased cell proliferation. Compared to parental pancreatic cancer cells, pancreatic cancers stably-expressing EYA2 grew more slowly and had fewer metastases in orthotopic models. The transcriptional changes after stable expression of EYA2 in pancreatic cancer cells included induction of genes in the TGFbeta pathway. Epigenetic silencing of EYA2 is a common event in pancreatic cancers and stable expression EYA2 limits the growth and metastases of pancreatic adenocarcinoma. PMID:24810906

  6. Removal of Spindly from microtubule-attached kinetochores controls spindle checkpoint silencing in human cells

    PubMed Central

    Gassmann, Reto; Holland, Andrew J.; Varma, Dileep; Wan, Xiaohu; Çivril, Filiz; Cleveland, Don W.; Oegema, Karen; Salmon, Edward D.; Desai, Arshad

    2010-01-01

    The spindle checkpoint generates a “wait anaphase” signal at unattached kinetochores to prevent premature anaphase onset. Kinetochore-localized dynein is thought to silence the checkpoint by transporting checkpoint proteins from microtubule-attached kinetochores to spindle poles. Throughout metazoans, dynein recruitment to kinetochores requires the protein Spindly. Here, we identify a conserved motif in Spindly that is essential for kinetochore targeting of dynein. Spindly motif mutants, expressed following depletion of endogenous Spindly, target normally to kinetochores but prevent dynein recruitment. Spindly depletion and Spindly motif mutants, despite their similar effects on kinetochore dynein, have opposite consequences on chromosome alignment and checkpoint silencing. Spindly depletion delays chromosome alignment, but Spindly motif mutants ameliorate this defect, indicating that Spindly has a dynein recruitment-independent role in alignment. In Spindly depletions, the checkpoint is silenced following delayed alignment by a kinetochore dynein-independent mechanism. In contrast, Spindly motif mutants are retained on microtubule-attached kinetochores along with checkpoint proteins, resulting in persistent checkpoint signaling. Thus, dynein-mediated removal of Spindly from microtubule-attached kinetochores, rather than poleward transport per se, is the critical reaction in checkpoint silencing. In the absence of Spindly, a second mechanism silences the checkpoint; this mechanism is likely evolutionarily ancient, as fungi and higher plants lack kinetochore dynein. PMID:20439434

  7. Gene silencing activity of siRNA polyplexes based on biodegradable polymers.

    PubMed

    Varkouhi, Amir K; Lammers, Twan; Schiffelers, Raymond M; van Steenbergen, Mies J; Hennink, Wim E; Storm, Gert

    2011-04-01

    Cationic polymers are used as non-viral vectors for nucleic acid delivery. In this study, two biodegradable cationic polymers were evaluated for the purpose of siRNA delivery: pHPMA-MPPM (poly((2-hydroxypropyl) methacrylamide 1-methyl-2-piperidine methanol)) and TMC (O-methyl-free N,N,N-trimethylated chitosan). The silencing activity and the cellular cytotoxicity of polyplexes based on these biodegradable polymers were compared with those based on non-biodegradable pDMAEMA (poly(2-dimethylamino)ethyl methacrylate) and PEI (polyethylenimine) and with the regularly used lipidic transfection agent Lipofectamine. To promote endosomal escape, either the endosomolytic peptide diINF-7 was added to the formulations or photochemical internalization (PCI) was applied. Incubation of H1299 human lung cancer cells expressing firefly luciferase with polyplexes based on pHPMA-MPPM and TMC showed 30-40% silencing efficiency. This silencing activity was equal to or better than that obtained with the standard transfectants. Under all experimental conditions tested, the cytotoxicity of the biodegradable polymers was low. The application of PCI, as well as the addition of the diINF-7 peptide to the formulations increased their silencing activity up to 70-80%. This demonstrates that pHPMA-MPPM- and TMC-based polyplexes benefit substantially from endosomal escape enhancement. Importantly, the polyplexes retained their silencing activity in the presence of serum, and they showed low cytotoxicity. These biodegradable vectors are therefore attractive systems for further in vivo evaluations. PMID:21118719

  8. Epigenetic Inheritance of Transcriptional Silencing and Switching Competence in Fission Yeast

    PubMed Central

    Thon, G.; Friis, T.

    1997-01-01

    Epigenetic events allow the inheritance of phenotypic changes that are not caused by an alteration in DNA sequence. Here we characterize an epigenetic phenomenon occuring in the mating-type region of fission yeast. Cells of fission yeast switch between the P and M mating-type by interconverting their expressed mating-type cassette between two allelic forms, mat1-P and mat1-M. The switch results from gene conversions of mat1 by two silent cassettes, mat2-P and mat3-M, which are linked to each other and to mat1. GREWAL and KLAR observed that the ability to both switch mat1 and repress transcription near mat2-P and mat3-M was maintained epigenetically in a strain with an 8-kb deletion between mat2 and mat3. Using a strain very similar to theirs, we determined that interconversions between the switching-and silencing-proficient state and the switching and silencing-deficient state occurred less frequently than once per 1000 cell divisions. Although transcriptional silencing was alleviated by the 8-kb deletion, it was not abolished. We performed a mutant search and obtained a class of trans-acting mutations that displayed a strong cumulative effect with the 8-kb deletion. These mutations allow to assess the extent to which silencing is affected by the deletion and provide new insights on the redundancy of the silencing mechanism. PMID:9055078

  9. Polycomb PRC2 complex mediates epigenetic silencing of a critical osteogenic master regulator in the hippocampus.

    PubMed

    Aguilar, Rodrigo; Bustos, Fernando J; Saez, Mauricio; Rojas, Adriana; Allende, Miguel L; van Wijnen, Andre J; van Zundert, Brigitte; Montecino, Martin

    2016-08-01

    During hippocampal neuron differentiation, the expression of critical inducers of non-neuronal cell lineages must be efficiently silenced. Runx2 transcription factor is the master regulator of mesenchymal cells responsible for intramembranous osteoblast differentiation and formation of the craniofacial bone tissue that surrounds and protects the central nervous system (CNS) in mammalian embryos. The molecular mechanisms that mediate silencing of the Runx2 gene and its downstream target osteogenic-related genes in neuronal cells have not been explored. Here, we assess the epigenetic mechanisms that mediate silencing of osteoblast-specific genes in CNS neurons. In particular, we address the contribution of histone epigenetic marks and histone modifiers on the silencing of the Runx2/p57 bone-related isoform in rat hippocampal tissues at embryonic to adult stages. Our results indicate enrichment of repressive chromatin histone marks and of the Polycomb PRC2 complex at the Runx2/p57 promoter region. Knockdown of PRC2 H3K27-methyltransferases Ezh2 and Ezh1, or forced expression of the Trithorax/COMPASS subunit Wdr5 activates Runx2/p57 mRNA expression in both immature and mature hippocampal cells. Together these results indicate that complementary epigenetic mechanisms progressively and efficiently silence critical osteoblastic genes during hippocampal neuron differentiation. PMID:27216774

  10. Functional analysis of gene-silencing suppressors from tomato yellow leaf curl disease viruses.

    PubMed

    Luna, Ana P; Morilla, Gabriel; Voinnet, Olivier; Bejarano, Eduardo R

    2012-10-01

    Tomato yellow leaf curl disease (TYLCD) is caused by a complex of phylogenetically related Begomovirus spp. that produce similar symptoms when they infect tomato plants but have different host ranges. In this work, we have evaluated the gene-silencing-suppression activity of C2, C4, and V2 viral proteins isolated from the four main TYLCD-causing strains in Spain in Nicotiana benthamiana. We observed varying degrees of local silencing suppression for each viral protein tested, with V2 proteins from all four viruses exhibiting the strongest suppression activity. None of the suppressors were able to avoid the spread of the systemic silencing, although most produced a delay. In order to test the silencing-suppression activity of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) proteins in a shared (tomato) and nonshared (bean) host, we established novel patch assays. Using these tools, we found that viral proteins from TYLCV were able to suppress silencing in both hosts, whereas TYLCSV proteins were only effective in tomato. This is the first time that viral suppressors from a complex of disease-causing geminiviruses have been subject to a comprehensive analysis using two economically important crop hosts, as well as the established N. benthamiana plant model. PMID:22712505

  11. 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

  12. RNA Quality Control as a Key to Suppressing RNA Silencing of Endogenous Genes in Plants.

    PubMed

    Liu, Lin; Chen, Xuemei

    2016-06-01

    RNA quality control of endogenous RNAs is an integral part of eukaryotic gene expression and often relies on exonucleolytic degradation to eliminate dysfunctional transcripts. In parallel, exogenous and selected endogenous RNAs are degraded through RNA silencing, which is a genome defense mechanism used by many eukaryotes. In plants, RNA silencing is triggered by the production of double-stranded RNAs (dsRNAs) by RNA-DEPENDENT RNA POLYMERASEs (RDRs) and proceeds through small interfering (si) RNA-directed, ARGONAUTE (AGO)-mediated cleavage of homologous transcripts. Many studies revealed that plants avert inappropriate posttranscriptional gene silencing of endogenous coding genes by using RNA surveillance mechanisms as a safeguard to protect their transcriptome profiles. The tug of war between RNA surveillance and RNA silencing ensures the appropriate partitioning of endogenous RNA substrates among these degradation pathways. Here we review recent advances on RNA quality control and its role in the suppression of RNA silencing at endogenous genes and discuss the mechanisms underlying the crosstalk among these pathways. PMID:27045817

  13. Applications and advantages of virus-induced gene silencing for gene function studies in plants.

    PubMed

    Burch-Smith, Tessa M; Anderson, Jeffrey C; Martin, Gregory B; Dinesh-Kumar, S P

    2004-09-01

    Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. The approach involves cloning a short sequence of a targeted plant gene into a viral delivery vector. The vector is used to infect a young plant, and in a few weeks natural defense mechanisms of the plant directed at suppressing virus replication also result in specific degradation of mRNAs from the endogenous plant gene that is targeted for silencing. VIGS is rapid (3-4 weeks from infection to silencing), does not require development of stable transformants, allows characterization of phenotypes that might be lethal in stable lines, and offers the potential to silence either individual or multiple members of a gene family. Here we briefly review the discoveries that led to the development of VIGS and what is known about the experimental requirements for effective silencing. We describe the methodology of VIGS and how it can be optimized and used for both forward and reverse genetics studies. Advantages and disadvantages of VIGS compared with other loss-of-function approaches available for plants are discussed, along with how the limitations of VIGS might be overcome. Examples are reviewed where VIGS has been used to provide important new insights into the roles of specific genes in plant development and plant defense responses. Finally, we examine the future prospects for VIGS as a powerful tool for assessing and characterizing the function of plant genes. PMID:15315635

  14. Cytorhabdovirus P protein suppresses RISC-mediated cleavage and RNA silencing amplification in planta.

    PubMed

    Mann, Krin S; Johnson, Karyn N; Carroll, Bernard J; Dietzgen, Ralf G

    2016-03-01

    Plant viruses have evolved to undermine the RNA silencing pathway by expressing suppressor protein(s) that interfere with one or more key components of this antiviral defense. Here we show that the recently identified RNA silencing suppressor (RSS) of lettuce necrotic yellows virus (LNYV), phosphoprotein P, binds to RNA silencing machinery proteins AGO1, AGO2, AGO4, RDR6 and SGS3 in protein-protein interaction assays when transiently expressed. In planta, we demonstrate that LNYV P inhibits miRNA-guided AGO1 cleavage and translational repression, and RDR6/SGS3-dependent amplification of silencing. Analysis of LNYV P deletion mutants identified a C-terminal protein domain essential for both local RNA silencing suppression and interaction with AGO1, AGO2, AGO4, RDR6 and SGS3. In contrast to other viral RSS known to disrupt AGO activity, LNYV P sequence does not contain any recognizable GW/WG or F-box motifs. This suggests that LNYV P may represent a new class of AGO binding proteins. PMID:26808923

  15. 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

  16. Restriction of Retrotransposon Mobilization in Schizosaccharomyces pombe by Transcriptional Silencing and Higher-Order Chromatin Organization.

    PubMed

    Murton, Heather E; Grady, Patrick J R; Chan, Tsun Ho; Cam, Hugh P; Whitehall, Simon K

    2016-08-01

    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

  17. Breaking an Epigenetic Chromatin Switch: Curious Features of Hysteresis in Saccharomyces cerevisiae Telomeric Silencing

    PubMed Central

    Nagaraj, Vijayalakshmi H.; Mukhopadhyay, Swagatam; Dayarian, Adel; Sengupta, Anirvan M.

    2014-01-01

    In addition to gene network switches, local epigenetic modifications to DNA and histones play an important role in all-or-none cellular decision-making. Here, we study the dynamical design of a well-characterized epigenetic chromatin switch: the yeast SIR system, in order to understand the origin of the stability of epigenetic states. We study hysteresis in this system by perturbing it with a histone deacetylase inhibitor. We find that SIR silencing has many characteristics of a non-linear bistable system, as observed in conventional genetic switches, which are based on activities of a few promoters affecting each other through the abundance of their gene products. Quite remarkably, our experiments in yeast telomeric silencing show a very distinctive pattern when it comes to the transition from bistability to monostability. In particular, the loss of the stable silenced state, upon increasing the inhibitor concentration, does not seem to show the expected saddle node behavior, instead looking like a supercritical pitchfork bifurcation. In other words, the ‘off’ state merges with the ‘on’ state at a threshold concentration leading to a single state, as opposed to the two states remaining distinct up to the threshold and exhibiting a discontinuous jump from the ‘off’ to the ‘on’ state. We argue that this is an inevitable consequence of silenced and active regions coexisting with dynamic domain boundaries. The experimental observations in our study therefore have broad implications for the understanding of chromatin silencing in yeast and beyond. PMID:25536038

  18. Dual miRNA Targeting Restricts Host Range and Attenuates Neurovirulence of Flaviviruses

    PubMed Central

    Tsetsarkin, Konstantin A.; Liu, Guangping; Kenney, Heather; Bustos-Arriaga, Jose; Hanson, Christopher T.; Whitehead, Stephen S.; Pletnev, Alexander G.

    2015-01-01

    Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3’NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4) replication in mosquito cell lines and adult Aedes mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics. PMID:25906260

  19. Electrically tunable hot-silicon terahertz attenuator

    NASA Astrophysics Data System (ADS)

    Wang, Minjie; Vajtai, Robert; Ajayan, Pulickel M.; Kono, Junichiro

    2014-10-01

    We have developed a continuously tunable, broadband terahertz attenuator with a transmission tuning range greater than 103. Attenuation tuning is achieved electrically, by simply changing the DC voltage applied to a heating wire attached to a bulk silicon wafer, which controls its temperature between room temperature and ˜550 K, with the corresponding free-carrier density adjusted between ˜1011 cm-3 and ˜1017 cm-3. This "hot-silicon"-based terahertz attenuator works most effectively at 450-550 K (corresponding to a DC voltage variation of only ˜7 V) and completely shields terahertz radiation above 550 K in a frequency range of 0.1-2.5 THz. Both intrinsic and doped silicon wafers were tested and demonstrated to work well as a continuously tunable attenuator. All behaviors can be understood quantitatively via the free-carrier Drude model taking into account thermally activated intrinsic carriers.

  20. UHF Radio Wave Attenuation Factor Database

    NASA Astrophysics Data System (ADS)

    Khomenko, S. I.; Kostina, V. L.; Mytsenko, I. M.; Roenko, A. N.

    2007-07-01

    As is known each sea-going vessel is equipped with navigation, communication and other radio engineering facilities that serve to secure the safety of navigation and are chiefly operated at UHF-wave band. In developing these systems and calculating the energy potential for a necessary coverage range one should be well aware of the radio signal attenuation processes on a propagation path. The key parameter of this path is the (radio) wave attenuation factor V and its distance dependence V(R). A diversity of factors influencing the radio signal attenuation over the oceanic expanses, especially well pronounced and quite stable tropospheric ducts, and the lack of experimental data were the compelling reasons why the researchers of the Institute for Radiophysics and Electronics, NASU, had spent many years on comprehensive radiophysical investigations carried out in different regions of the Atlantic, Indian, Arctic and Pacific Oceans. The experimental data obtained allow creating the database of radio wave attenuation factor V.

  1. Evolution of Natural Attenuation Evaluation Protocols

    EPA Science Inventory

    Traditionally the evaluation of the efficacy of natural attenuation was based on changes in contaminant concentrations and mass reduction. Statistical tools and models such as Bioscreen provided evaluation protocols which now are being approached via other vehicles including m...

  2. Attenuation of human influenza a viruses

    PubMed Central

    Beare, A. S.; Bynoe, M. L.

    1969-01-01

    The attenuation of two human influenza A viruses has been carried out, using the selection of inhibitor-resistant strains and multiple passages at low temperatures. A virus related to A2/Tokyo/3/67 was obtained in an inhibitor-resistant form. When this was compared with the inhibitor-sensitive strain in a volunteer trial it was relatively non-pathogenic. The second virus, A2/Hongkong/1/68, was subjected to much longer treatment, but nevertheless remained slightly sensitive to serum inhibitor. When given to volunteers it was less pathogenic than before but attenuation was incomplete. A2/Hongkong/1/68 was also modified by passage at low temperatures. Many of these passages are apparently necessary for full attenuation. All attenuated viruses were infective and antigenic. PMID:4900146

  3. Attenuation of external Bremsstrahlung in metallic absorbers

    SciTech Connect

    Dhaliwal, A.S.; Powar, M.S.; Singh, M. )

    1990-12-01

    In this paper attenuation of bremsstrahlung from {sup 147}Pm and {sup 170}Tm beta emitters has been studied in aluminum, copper, tin, and lead metallic absorbers. Bremsstrahlung spectra and mass attenuation coefficients for monoenergetic gamma rays are used to calculate theoretical attenuation curves. Magnetic deflection and beta stopping techniques are used to measure the integral bremsstrahlung intensities above 30 keV in different target thicknesses. Comparison of measured and calculated attenuation curves shows a good agreement for various absorbers, thus providing a test of this technique, which may be useful in understanding bremsstrahlung intensity buildup and in the design of optimum shielding for bremsstrahlung sources. It is found that the absorption of bremsstrahlung in metallic absorbers does not obey an exponential law and that absorbers act as energy filters.

  4. Attenuation layer for magnetostatic wave (MSW) absorbers

    NASA Astrophysics Data System (ADS)

    Glass, H. L.; Adkins, L. R.; Stearns, F. S.

    1984-09-01

    A new technique has been developed for the suppression of MSW end reflections which give rise to passband ripple. The basic idea is to provide a thin film of highly attenuating epitaxial material at the ends of a MSW delay line while preserving high quality YIG in the active region of the device. The GGG wafer preparation is a three step process which involves: (1) the growth of the attenuation layer, (2) the removal of this layer from the central region of the wafer and (3) the growth of high quality YIG on the remaining structure. Delay lines using the attenuation layer for end terminations have been evaluated experimentally and compared to devices utilizing other termination methods. The results indicate that the attenuation layer method produces ripple suppression characteristics which are the equal of those obtained with other termination techniques. The advantage of this new method lies in its suitability for large quantity fabrication requirements.

  5. The role of GW182 proteins in miRNA-mediated gene silencing.

    PubMed

    Braun, Joerg E; Huntzinger, Eric; Izaurralde, Elisa

    2013-01-01

    GW182 family proteins are essential for microRNA-mediated gene silencing in animal cells. They are recruited to miRNA targets through direct interactions with Argonaute proteins and promote target silencing. They do so by repressing translation and enhancing mRNA turnover. Although the precise mechanism of action of GW182 proteins is not fully understood, these proteins have been shown to interact with the cytoplasmic poly(A)-binding protein (PABP) and with the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These findings suggest that GW182 proteins function as scaffold proteins for the assembly of the multiprotein complex that silences miRNA targets. PMID:23224969

  6. Use of guanidinopropyl-modified siRNAs to silence gene expression.

    PubMed

    Buff, Maximilian C R; Bernhardt, Stefan; Marimani, Musa D; Ely, Abdullah; Engels, Joachim W; Arbuthnot, Patrick

    2015-01-01

    Silencing gene expression by harnessing the RNA interference (RNAi) pathway with short interfering RNAs (siRNAs) has useful analytical and potentially therapeutic application. To augment silencing efficacy of siRNAs, chemical modification has been employed to improve stability, target specificity, and delivery to target tissues. siRNAs incorporating guanidinopropyl (GP) moieties have demonstrated enhanced target gene silencing in cell culture and in vivo models of hepatitis B virus replication. Here we describe the synthesis of GP-modified siRNAs and use of 5' rapid amplification of cDNA ends (5' RACE) to verify an RNAi-mediated mechanism of action of these novel chemically modified siRNAs. PMID:25319654

  7. Different Dicer-like protein components required for intracellular and systemic antiviral silencing in Arabidopsis thaliana.

    PubMed

    Andika, Ida Bagus; Maruyama, Kazuyuki; Sun, Liying; Kondo, Hideki; Tamada, Tetsuo; Suzuki, Nobuhiro

    2015-01-01

    Eukaryotes employ RNA silencing as an innate defense system against invading viruses. Dicer proteins play the most crucial role in initiating this antiviral pathway as they recognize and process incoming viral nucleic acids into small interfering RNAs. Generally, 2 successive infection stages constitute viral infection in plants. First, the virus multiplies in initially infected cells or organs after viral transmission and then the virus subsequently spreads systemically through the vasculature to distal plant tissues or organs. Thus, antiviral silencing in plants must cope with both local and systemic invasion of viruses. In a recent study using 2 sets of different experiments, we clearly demonstrated the differential requirement for Dicer-like 4 (DCL4) and DCL2 proteins in the inhibition of intracellular and systemic infection by potato virus X in Arabidopsis thaliana. Taken together with the results of other studies, here we further discuss the functional specificity of DCL proteins in the antiviral silencing pathway. PMID:26273728

  8. Being silenced: the impact of negative social reactions on the disclosure of rape.

    PubMed

    Ahrens, Courtney E

    2006-12-01

    Rape survivors who speak out about their assault experiences are often punished for doing so when they are subjected to negative reactions from support providers. These negative reactions may thereby serve a silencing function, leading some rape survivors to stop talking about their experiences to anyone at all. The current study sought to examine this worst case scenario. Focusing on the qualitative narratives of eight rape survivors who initially disclosed the assault but then stopped disclosing for a significant period of time, this study sought to provide an in-depth description of how negative reactions silenced these survivors. Three routes to silence were identified: 1) negative reactions from professionals led survivors to question whether future disclosures would be effective; 2) negative reactions from friends and family reinforced feelings of self-blame; and 3) negative reactions from either source reinforced uncertainty about whether their experiences qualified as rape. Implications for future research and practice are discussed. PMID:17111229

  9. Silencing of VAMP3 inhibits cell migration and integrin-mediated adhesion

    SciTech Connect

    Luftman, Kevin; Hasan, Nazarul; Day, Paul; Hardee, Deborah; Hu Chuan

    2009-02-27

    Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the present study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of {beta}1 integrin at the cell surface but had no effect on total cellular {beta}1 integrin, indicating that VAMP3 is required for trafficking of {beta}1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.

  10. How abusive supervisors influence employees' voice and silence: the effects of interactional justice and organizational attribution.

    PubMed

    Wang, Rong; Jiang, Jiang

    2015-01-01

    In this research we investigated the influence of abusive supervision on employees' prosocial voice and silence, as well as clarified the roles of interactional justice (as a mediator) and organizational attribution (as a moderator). Moreover, we examined a mediated moderating model stipulating that interactional justice mediated the moderating effect of organizational attribution on the focal relationship. A scenario experiment was employed in Study 1, and after analyzing data from 196 employees, we found that abusive supervision influenced employees' prosocial voice and silence via interactional justice. In Study 2, data were collected from 379 employees in two waves separated by 1 week. The results not only replicated the findings of Study 1 but also indicated that organizational attribution buffered the abusive supervision-voice and silence relationship, and that interactional justice mediated this moderating effect. PMID:25492100

  11. Silencing of R-Spondin1 increases radiosensitivity of glioma cells

    PubMed Central

    Ma, Guoda; Cui, Lili; Li, You; Zhou, Haihong; Liang, Wandong; Zhao, Bin; Li, Keshen

    2015-01-01

    Although radiation therapy is the most effective postoperative adjuvant treatment, it does not substantially improve the long-term outcomes of glioma patients because of the characteristic radioresistance of glioma. We found that R-Spondin1 (Rspo1) expression was elevated in high-grade gliomas and was associated with worse overall survival and disease-free survival. Rspo1 expression was also associated with reduced survival rates in glioma patients after treatment with radiotherapy and temozolomide (RT-TMZ). Importantly, Rspo1 was dramatically upregulated after radiation treatment in patients with glioma. Rspo1 silencing by shRNA potentiated glioma cell death upon radiation treatment. In a xenograft nude mouse model, combining radiation and silencing of Rspo1 potentiated tumor growth inhibition. Thus, combining radiotherapy with silencing of Rspo1 is a potential therapeutic approach. PMID:25865226

  12. Excavating silences and tensions of agency|passivity in science education reform

    NASA Astrophysics Data System (ADS)

    Rivera Maulucci, Maria S.

    2010-12-01

    I reflect on studies by Rodriguez and Carlone, Haun-Frank, and Kimmel to emphasize the ways in which they excavate silences in the science education literature related to linguistic and cultural diversity and situating the problem of reform in teachers rather than contextual factors, such as traditional schooling discourses and forces that serve to marginalize science. I propose that the current push for top-down reform and accountability diminishes opportunities for receptivity, learning with and from students in order to transform teachers' practices and promote equity in science education. I discuss tensions of agency and passivity in science education reform and argue that attention to authentic caring constitutes another silence in the science education literature. I conclude that the current policy context positions teachers and science education researchers as tempered radicals struggling against opp(reg)ressive reforms and that there is a need for more studies to excavate these and other silences.

  13. Something Blossoms in Between: Silence-Phenomena as a Bordering Notions in Psychology.

    PubMed

    Lehmann Oliveros, Olga V

    2016-03-01

    Mysterious yet unavoidable, silence-phenomena appear to us in inherent ambiguity. In its plurality of meanings, phenomena related to silence are often perceived as overwhelming because they transcend the communicative capacity of language making it a challenge for cultural psychology to understand its involvement in our processes of making sense of experience and existence. Human growth and development involve processes where presence, void and content, voice, sound and noise, motion, transition and stillness, have dialectic interactions. In this article I discuss silence-phenomena as a bordering notion in terms of its discursive quality, the silent quality of speech, and the awareness of the ineffable. In addition, I highlight the possible implications of such notion in the understanding of affect from the perspective of Semiotic Cultural Psychology. I also emphasize the importance of considering psychological borders as multi-dimensional, taking the phenomenological experience of temporality as an illustration, which is also related to high emotional involvement of attention. PMID:26232279

  14. Small-RNA loading licenses Argonaute for assembly into a transcriptional silencing complex

    PubMed Central

    Holoch, Daniel; Moazed, Danesh

    2015-01-01

    Argonautes and their small-RNA cofactors form the core effectors of ancient and diverse gene-silencing mechanisms whose roles include regulation of gene expression and defense against foreign genetic elements. Although Argonautes generally act within multisubunit complexes, what governs their assembly into these machineries is not well defined. Here, we show that loading of small RNAs onto Argonaute is a checkpoint for Argonaute’s association with conserved GW-protein components of silencing complexes. We demonstrate that the Argonaute small interfering RNA chaperone (ARC) complex mediates loading of small RNAs onto Ago1 in Schizosaccharomyces pombe and that deletion of its subunits, or mutations in Ago1 that prevent small-RNA loading, abolish the assembly of the GW protein–containing RNA-induced transcriptional silencing (RITS) complex. Our studies uncover a mechanism that ensures that Argonaute loading precedes RITS assembly and thereby averts the formation of inert and potentially deleterious complexes. PMID:25730778

  15. Persistent virus-induced gene silencing in asymptomatic accessions of Arabidopsis.

    PubMed

    Flores, Miguel A; Reyes, Maria I; Robertson, Dominique Niki; Kjemtrup, Susanne

    2015-01-01

    Coupled with the advantages afforded by the model plant Arabidopsis, virus-induced gene silencing (VIGS) offers a rapid means to assess gene function. The geminivirus vector based on Cabbage leaf curl virus described here has the benefits of small insert size and persistent silencing of the target gene through the life cycle of the plant. Here, we show that genetic variation in the vast collection of Arabidopsis accessions can be leveraged to ameliorate viral symptomology that accompanies the VIGS procedure. The plasticity of phenotypes under different day lengths or temperature conditions can be exploited to achieve maximum silencing efficacy in either vegetative or inflorescence tissue, according to the question being asked. Protocols and vectors for Agro-infiltration of primary leaves, subapical pricking in older plants, and microprojectile bombardment are described. PMID:25757779

  16. 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

  17. RNA interference silencing of CHS greatly alters the growth pattern of apple (Malus x domestica).

    PubMed

    Dare, Andrew P; Hellens, Roger P

    2013-08-01

    Plants produce a vast array of phenolic compounds which are essential for their survival on land. One major class of polyphenols are the flavonoids and their formation is dependent on the enzyme chalcone synthase (CHS). In a recent study we silenced the CHS genes of apple (Malus × domestica Borkh.) and observed a loss of pigmentation in the fruit skin, flowers and stems. More surprisingly, highly silenced lines were significantly reduced in size, with small leaves and shortened internode lengths. Chemical analysis also revealed that the transgenic shoots contained greatly reduced concentrations of flavonoids which are known to modulate auxin flow. An auxin transport study verified this, with an increased auxin transport in the CHS-silenced lines. Overall, these findings suggest that auxin transport in apple has adapted to take place in the presence of high endogenous concentrations of flavonoids. Removal of these compounds therefore results in abnormal auxin movement and a highly disrupted growth pattern. PMID:23733058

  18. Gene Silencing and Polycomb Group Proteins: An Overview of their Structure, Mechanisms and Phylogenetics

    PubMed Central

    Majid, Nazia Abdul; Hassandarvish, Pouya; Hajrezaie, Maryam; Abdulla, Mahmood Ameen; Hadi, A. Hamid A.

    2013-01-01

    Abstract DNA methylation, histone modifications, and chromatin configuration are crucially important in the regulation of gene expression. Among these epigenetic mechanisms, silencing the expression of certain genes depending on developmental stage and tissue specificity is a key repressive system in genome programming. Polycomb (Pc) proteins play roles in gene silencing through different mechanisms. These proteins act in complexes and govern the histone methylation profiles of a large number of genes that regulate various cellular pathways. This review focuses on two main Pc complexes, Pc repressive complexes 1 and 2, and their phylogenetic relationship, structures, and function. The dynamic roles of these complexes in silencing will be discussed herein, with a focus on the recruitment of Pc complexes to target genes and the key factors involved in their recruitment. PMID:23692361

  19. Gene silencing and Polycomb group proteins: an overview of their structure, mechanisms and phylogenetics.

    PubMed

    Golbabapour, Shahram; Majid, Nazia Abdul; Hassandarvish, Pouya; Hajrezaie, Maryam; Abdulla, Mahmood Ameen; Hadi, A Hamid A

    2013-06-01

    DNA methylation, histone modifications, and chromatin configuration are crucially important in the regulation of gene expression. Among these epigenetic mechanisms, silencing the expression of certain genes depending on developmental stage and tissue specificity is a key repressive system in genome programming. Polycomb (Pc) proteins play roles in gene silencing through different mechanisms. These proteins act in complexes and govern the histone methylation profiles of a large number of genes that regulate various cellular pathways. This review focuses on two main Pc complexes, Pc repressive complexes 1 and 2, and their phylogenetic relationship, structures, and function. The dynamic roles of these complexes in silencing will be discussed herein, with a focus on the recruitment of Pc complexes to target genes and the key factors involved in their recruitment. PMID:23692361

  20. Different Dicer-like protein components required for intracellular and systemic antiviral silencing in Arabidopsis thaliana

    PubMed Central

    Andika, Ida Bagus; Maruyama, Kazuyuki; Sun, Liying; Kondo, Hideki; Tamada, Tetsuo; Suzuki, Nobuhiro

    2015-01-01

    Eukaryotes employ RNA silencing as an innate defense system against invading viruses. Dicer proteins play the most crucial role in initiating this antiviral pathway as they recognize and process incoming viral nucleic acids into small interfering RNAs. Generally, 2 successive infection stages constitute viral infection in plants. First, the virus multiplies in initially infected cells or organs after viral transmission and then the virus subsequently spreads systemically through the vasculature to distal plant tissues or organs. Thus, antiviral silencing in plants must cope with both local and systemic invasion of viruses. In a recent study using 2 sets of different experiments, we clearly demonstrated the differential requirement for Dicer-like 4 (DCL4) and DCL2 proteins in the inhibition of intracellular and systemic infection by potato virus X in Arabidopsis thaliana. Taken together with the results of other studies, here we further discuss the functional specificity of DCL proteins in the antiviral silencing pathway. PMID:26273728

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

    PubMed

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

    2015-12-01

    This is the first report of gene silencing by RNA interference (RNAi) in the European house dust mite, Dermatophagoides pteronyssinus, Trouessart, 1897. Using a non-invasive immersion method first developed for the honey bee mite, Varroa destructor, a significant reduction in the expression of D. pteronyssinus glutathione-S-transferase mu-class 1 enzyme (DpGST-mu1) was achieved following overnight immersion in double stranded RNA encoding DpGST-mu1. Although no detrimental phenotypic changes were observed following silencing, this technique can now be used to address fundamental physiological questions and assess the potential therapeutic benefit in silencing D. pteronyssinus target genes in selected domestic situations of high human-mite interface. PMID:26212476

  2. Study of the dispersion of natural gas issuing from compressor stations through silencers with upper cover.

    PubMed

    García, J; Migoya, E; Lana, J A; Crespo, A

    2008-04-15

    The aim of the present study is the simulation of the dispersion of natural gas issuing from the silencer of compressor stations during vent operations. The objective is to analyze the dispersion of the gas emitted under different conditions of mass flow rate at the exit and ambient cross-flow velocity. We have considered a silencer with an upper cover to protect it from the rain and the fall of objects. The influence of the upper cover of the silencer on the dispersion of natural gas has also been studied, and non-dimensional approaches of the model have been proposed to simplify the problem. Seven different cases have been solved, using two models: a 3D model based on the commercial code FLUENT, and a simplified quasi-one-dimensional model. The results obtained in both cases have been compared, and the range of validity of the one-dimensional model in non-dimensional form has been discussed. PMID:17875364

  3. Electron Effective-Attenuation-Length Database

    National Institute of Standards and Technology Data Gateway

    SRD 82 NIST Electron Effective-Attenuation-Length Database (PC database, no charge)   This database provides values of electron effective attenuation lengths (EALs) in solid elements and compounds at selected electron energies between 50 eV and 2,000 eV. The database was designed mainly to provide EALs (to account for effects of elastic-eletron scattering) for applications in surface analysis by Auger-electron spectroscopy (AES) and X-ray photoelectron spectroscopy (XPS).

  4. Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence.

    PubMed

    Chen, Jen-Chih; Jiang, Cai-Zhong; Gookin, Timothy E; Hunter, Donald A; Clark, David G; Reid, Michael S

    2004-07-01

    Agrobacterium-mediated infection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petunia genes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes. Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues. Infection with TRV containing a chalcone synthase (CHS) fragment resulted in silencing of anthocyanin production in infected flowers. The silencing phenotype ranged from scattered white spots on the normal purple background to entirely white flowers. Symptoms in the V26 cultivar were a diffuse mosaic, but infection of some purple-flowered commercial cultivars resulted in large white sectors and even entirely white flowers. Abundance of CHS transcripts in the white flowers was less than 4% of that in purple flowers on the same plant. Infection with TRV containing a tandem construct of PDS and CHS resulted in leaf photobleaching and white patterns on the flowers. Transcripts of CHS and PDS were reduced both in leaves and in flowers confirming simultaneous silencing of both genes by the tandem construct. We tested the effects of infection with TRV containing CHS and a fragment of a petunia gene encoding for 1-aminocyclopropane-1-carboxylate oxidase (ACO4) Abundance of transcripts encoding ACO4 and ACO1 were reduced (by 5% and 20%, respectively) in infected flowers. Whether the flowers were treated with ACC or pollinated, the white (silenced) flowers or flower sectors produced less ethylene and senesced later than purple (non-silenced) tissues. These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes. PMID:15604697

  5. Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana

    PubMed Central

    Bond, Donna M.; Baulcombe, David C.

    2015-01-01

    In plants, RNA-directed DNA methylation (RdDM), a mechanism where epigenetic modifiers are guided to target loci by small RNAs, plays a major role in silencing of transposable elements (TEs) to maintain genome integrity. So far, two RdDM pathways have been identified: RNA Polymerase IV (PolIV)-RdDM and RNA-dependent RNA Polymerase 6 (RDR6)-RdDM. PolIV-RdDM involves a self-reinforcing feedback mechanism that maintains TE silencing, but cannot explain how epigenetic silencing is first initiated. A function of RDR6-RdDM is to reestablish epigenetic silencing of active TEs, but it is unknown if this pathway can induce DNA methylation at naïve, non-TE loci. To investigate de novo establishment of RdDM, we have used virus-induced gene silencing (VIGS) of an active FLOWERING WAGENINGEN epiallele. Using genetic mutants we show that unlike PolIV-RdDM, but like RDR6-RdDM, establishment of VIGS-mediated RdDM requires PolV and DRM2 but not Dicer like-3 and other PolIV pathway components. DNA methylation in VIGS is likely initiated by a process guided by virus-derived small (s) RNAs that are 21/22-nt in length and reinforced or maintained by 24-nt sRNAs. We demonstrate that VIGS-RdDM as a tool for gene silencing can be enhanced by use of mutant plants with increased production of 24-nt sRNAs to reinforce the level of RdDM. PMID:25561534

  6. Hypoxia Reduces Arylsulfatase B Activity and Silencing Arylsulfatase B Replicates and Mediates the Effects of Hypoxia

    PubMed Central

    Bhattacharyya, Sumit; Tobacman, Joanne K.

    2012-01-01

    This report presents evidence of 1) a role for arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) in mediating intracellular oxygen signaling; 2) replication between the effects of ARSB silencing and hypoxia on sulfated glycosaminoglycan content, cellular redox status, and expression of hypoxia-associated genes; and 3) a mechanism whereby changes in chondroitin-4-sulfation that follow either hypoxia or ARSB silencing can induce transcriptional changes through galectin-3. ARSB removes 4-sulfate groups from the non-reducing end of chondroitin-4-sulfate and dermatan sulfate and is required for their degradation. For activity, ARSB requires modification of a critical cysteine residue by the formylglycine generating enzyme and by molecular oxygen. When primary human bronchial and human colonic epithelial cells were exposed to 10% O2×1 h, ARSB activity declined by ∼41% and ∼30% from baseline, as nuclear hypoxia inducible factor (HIF)-1α increased by ∼53% and ∼37%. When ARSB was silenced, nuclear HIF-1α increased by ∼81% and ∼61% from baseline, and mRNA expression increased to 3.73 (±0.34) times baseline. Inversely, ARSB overexpression reduced nuclear HIF-1α by ∼37% and ∼54% from baseline in the epithelial cells. Hypoxia, like ARSB silencing, significantly increased the total cellular sulfated glycosaminoglycans and chondroitin-4-sulfate (C4S) content. Both hypoxia and ARSB silencing had similar effects on the cellular redox status and on mRNA expression of hypoxia-associated genes. Transcriptional effects of both ARSB silencing and hypoxia may be mediated by reduction in galectin-3 binding to more highly sulfated C4S, since the galectin-3 that co-immunoprecipitated with C4S declined and the nuclear galectin-3 increased following ARSB knockdown and hypoxia. PMID:22428001

  7. Satellite RNA pathogens of plants: impacts and origins-an RNA silencing perspective.

    PubMed

    Wang, Ming-Bo; Smith, Neil A

    2016-01-01

    Viral satellite RNAs (satRNAs) are among the smallest RNA pathogens in plants. They have little or no protein-coding capacity but can have a major impact on the host plants through trilateral interactions with helper viruses and host plants. Studies around the 1980s revealed much of what we know about satRNAs: they can affect helper virus accumulation, modulate helper virus-induced disease symptoms, and induce their own symptoms with the assistance of helper viruses which depend on specific nucleotide sequences of their genome and host species. The molecular basis of these satRNA-caused impacts and the origin of satRNAs have yet to be fully understood and revealed, but recent understanding of the antiviral RNA silencing pathways and advancement in RNA and DNA sequencing technologies have provided new avenues and opportunities to examine these unanswered questions. These RNA silencing-based studies have revealed the existence of cross silencing between some satRNAs and helper viruses, the downregulation of helper virus-encoded suppressor (VSR) of RNA silencing or inhibition/enhancement of VSR activity by satRNAs, the silencing of host-encoded genes by satRNA-derived small interfering RNA (siRNAs), and the presence of satRNA-like small RNAs in uninfected host plants. These findings have provided alternative RNA silencing-based models to explain the pathogenicity and origin of satRNAs. WIREs RNA 2016, 7:5-16. doi: 10.1002/wrna.1311 For further resources related to this article, please visit the WIREs website. PMID:26481458

  8. From silencing the self to action: experiences of women living with HIV/AIDS.

    PubMed

    DeMarco, R F; Miller, K H; Patsdaughter, C A; Chisholm, M; Grindel, C G

    1998-01-01

    Feminist literature has demonstrated that women often maintain behaviors that support silencing of their voices. The critical issue is whether the silencing experience is (a) a destructive process of burying feelings and needs, (b) a protective strategy to preserve personal and professional relationships which they value, (c) a coping mechanism to divorce themselves from an androcentric/ethnocentric health care culture, or all of these. The transition from silence to action may be a process of reacting to a threat to self (i.e., HIV/AIDS diagnosis) where gender normative behaviors become irrelevant and self-advocacy becomes paramount for survival. Alternatively, the transition may be a conscious process of gaining insight into past behaviors that have been learned and culturally supported and making purposeful changes. Data for this study were extracted for secondary analysis from data from a larger study on experiences and needs of persons living with HIV/AIDS. Data were obtained from transcripts from three focus groups (N = 14 women) and six individual interviews. Women ranged in age from 21 to 55; 9 were European American, 7 were African American, and 4 were Latina American. Data were content analyzed and organized using four categories proposed by Jack (1991): (a) externalized self-perception, (b) care as self-sacrifice, (c) silencing the self, and (d) the divided self. Data supported that women with HIV/AIDS reported all four categories of silencing behaviors, particularly early in the HIV trajectory. For some women, an HIV/AIDS diagnosis ignited them to speak for themselves and to shape their own lives based on feelings and needs. For others, peer or professional support or both was the catalyst for the transition from silence to action. Findings suggest interventions that would assist women in judging themselves by internal versus external standards, putting their own needs before the perceived needs of others, expressing themselves toward action rather than

  9. 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

  10. Attenuation of noise by motorcycle safety helmets.

    PubMed

    Młyński, Rafał; Kozłowski, Emil; Zera, Jan

    2009-01-01

    For workers such as police motorcyclists or couriers, traffic and engine noise reaching the ears is an important factor contributing to the overall condition of their work. This noise can be reduced with motorcycle helmets. In this study, insertion loss of motorcycle helmets was measured with the microphone-in-real-ear technique and sound attenuation with the real-ear-at-threshold method. Results for 3 Nolan helmets show essentially no protection against external noise in the frequency range <250 Hz. In the frequency range >500 Hz, attenuation increases linearly at a rate of 8-9 dB per octave, to ~30 dB at 8 kHz. Lack of attenuation in the low-frequency range may cause annoying effects. In addition, high attenuation in the high-frequency range may decrease intelligibility of speech signals for a rider in a helmet. Attenuation measured in this study does not take into account noise generated by turbulent wind around the helmet. Thus, the measured values of attenuation represent a motorcycle rider's best conditions of hearing. PMID:19744370

  11. Transcriptional silencing induced by Arabidopsis T-DNA mutants is associated with 35S promoter siRNAs and requires genes involved in siRNA-mediated chromatin silencing.

    PubMed

    Mlotshwa, Sizolwenkosi; Pruss, Gail J; Gao, Zhihuan; Mgutshini, Nomathamsanqa L; Li, Junjie; Chen, Xuemei; Bowman, Lewis H; Vance, Vicki

    2010-11-01

    The utility of many T-DNA insertion mutant lines of Arabidopsis is compromised by their propensity to trigger transcriptional silencing of transgenes expressed from the CaMV 35S promoter. To try to circumvent this problem, we characterized the genetic requirements for maintenance of 35S promoter homology-dependent transcriptional gene silencing induced by the dcl3-1 (SALK_005512) T-DNA insertion mutant line. Surprisingly, even though DCL3 and RDR2 are known components of the siRNA-dependent transcriptional gene silencing pathway, transcriptional gene silencing of a 35S promoter-driven GUS hairpin transgene did occur in plants homozygous for the dcl3-1 T-DNA insertion and was unaffected by loss of function of RDR2. However, the transcriptional gene silencing was alleviated in dcl2 dcl3 dcl4 triple mutant plants and also by mutations in AGO4, NRPD2, HEN1 and MOM1. Thus, some T-DNA insertion mutant lines induce 35S promoter homology-dependent transcriptional silencing that requires neither DCL3 nor RDR2, but involves other genes known to function in siRNA-dependent transcriptional silencing. Consistent with these results, we detected 35S promoter siRNAs in dcl3-1 SALK line plants, suggesting that the 35S promoter homology-dependent silencing induced by some T-DNA insertion mutant lines is siRNA-mediated. PMID:21070421

  12. Silencing microRNA-155 ameliorates experimental autoimmune encephalomyelitis.

    PubMed

    Murugaiyan, Gopal; Beynon, Vanessa; Mittal, Akanksha; Joller, Nicole; Weiner, Howard L

    2011-09-01

    IFN-γ-producing Th1 and IL-17-producing Th17 cells are the key participants in various autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Although both of these T cell subsets are known to be regulated by specific transcription factors and cytokines, the role of microRNAs that control these two inflammatory T cell subsets and whether targeting microRNAs can have therapeutic effects are not known. In this study, we show that microRNA-155 (Mir-155) expression is elevated in CD4(+) T cells during EAE, and Mir-155(-/-) mice had a delayed course and reduced severity of disease and less inflammation in the CNS. The attenuation of EAE in Mir-155(-/-) mice was associated with a decrease in Th1 and Th17 responses in the CNS and peripheral lymphoid organs. The T cell-intrinsic function of Mir-155(-/-) was demonstrated by the resistance of Mir-155(-/-) CD4(+) T cell-repleted Rag-1(-/-) mice to EAE. Finally, we found that anti-Mir-155 treatment reduced clinical severity of EAE when given before and after the appearance of clinical symptoms. These findings demonstrate that Mir-155 confers susceptibility to EAE by affecting inflammatory T cell responses and identify Mir-155 as a new target for therapeutic intervention in multiple sclerosis. PMID:21788439

  13. Selective repression of gene expression in neuropathic pain by the neuron-restrictive silencing factor/repressor element-1 silencing transcription (NRSF/REST).

    PubMed

    Willis, Dianna E; Wang, Meng; Brown, Elizabeth; Fones, Lilah; Cave, John W

    2016-06-20

    Neuropathic pain often develops following nerve injury as a result of maladaptive changes that occur in the injured nerve and along the nociceptive pathways of the peripheral and central nervous systems. Multiple cellular and molecular mechanisms likely account for these changes; however, the exact nature of these mechanisms remain largely unknown. A growing number of studies suggest that alteration in gene expression is an important step in the progression from acute to chronic pain states and epigenetic regulation has been proposed to drive this change in gene expression. In this review, we discuss recent evidence that the DNA-binding protein neuron-restrictive silencing factor/repressor element-1 silencing transcription factor (NRSF/REST) is an important component in the development and maintenance of neuropathic pain through its role as a transcriptional regulator for a select subset of genes that it normally represses during development. PMID:26679228

  14. PGC-1α Silencing Compounds the Perturbation of Mitochondrial Function Caused by Mutant SOD1 in Skeletal Muscle of ALS Mouse Model

    PubMed Central

    Qi, Yan; Yin, Xiang; Wang, Shuyu; Jiang, Hongquan; Wang, Xudong; Ren, Ming; Su, Xiang-ping; Lei, Shi; Feng, Honglin

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease causing death of motor neurons. This study investigated the roles of energy metabolism in the pathogenesis of ALS in the SOD1(G93A) transgenic mouse model. Control and SOD1(G93A) mice were administered with shcontrol or shPGC-1α in combination with PBS or thiazolidinedione (TZD) for 8 weeks. Gene expression was analyzed by quantitative real-time PCR and Western blot. ROS and fibrosis were assessed with a colorimetric kit and Sirius staining, respectively. Inflammatory cytokines were measured using ELISA kits. The levels of tissue ROS and serum inflammatory cytokines were significantly higher in SOD1(G93A) mice compared to control mice, and knocking down peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) drastically increased cytokine levels in both control and SOD1(G93A) mice. Muscle fibrosis was much severer in SOD1(G93A) mice, and worsened by silencing PGC-1α and attenuated by TZD. The expression levels of PGC-1α, SOD1, UCP2, and cytochrome C were substantially reduced by shPGC-1α and increased by TZD in muscle of both control and SOD1(G93A) mice, whereas the level of NF-κB was significantly elevated in SOD1(G93A) mice, which was further increased by PGC-1α silencing. These data indicated that disruption of energy homeostasis would exacerbate the pathological changes caused by SOD1 mutations to promote the pathogenesis of ALS. PMID:26539112

  15. Mass loading induced dephasing in nanomechanical resonators

    NASA Astrophysics Data System (ADS)

    Atalaya, Juan

    2012-11-01

    This paper presents a study of dephasing of an underdamped nanomechanical resonator subject to random mass loading of small particles. A frequency noise model is presented which describes dephasing due to the attachment and detachment of particles at random points and particle diffusion along the resonator. This situation is commonly encountered in current mass measurement experiments using nanoelectromechanical (NEM) resonators. The conditions which can lead to inhomogeneous broadening and fine structure in the modes’ absorption spectra are discussed. It is also shown that the spectra of the higher-order cumulants of the (complex) vibrational mode amplitude are sensitive to the parameters characterizing the frequency noise process. Hence, measurement of these cumulants can provide information not only about the mass but also about other parameters of the particles (diffusion coefficient and attachment-detachment rates).

  16. Cohesin and Polycomb: Cooperative Checks and Balances in Gene Silencing and Transcription

    PubMed Central

    Dorsett, Dale; Kassis, Judith A.

    2014-01-01

    The cohesin protein complex was discovered for its roles in sister chromatid cohesion and segregation, and the Polycomb group (PcG) proteins for their roles in epigenetic gene silencing during development. Cohesin also controls gene transcription via multiple mechanisms. Genetic and molecular evidence from Drosophila argue that cohesin and the PRC1 PcG complex interact to control transcription of many active genes that are critical for development, and that via these interactions cohesin also controls the availability of PRC1 for gene silencing. PMID:24892918

  17. Templated assembly of albumin-based nanoparticles for simultaneous gene silencing and magnetic resonance imaging.

    PubMed

    Mertz, Damien; Affolter-Zbaraszczuk, Christine; Barthès, Julien; Cui, Jiwei; Caruso, Frank; Baumert, Thomas F; Voegel, Jean-Claude; Ogier, Joelle; Meyer, Florent

    2014-10-21

    In this article, we address the design of innovative human serum albumin (HSA)-based nanoparticles loaded with silencing RNA and grafted with gadolinium complexes having average sizes ranging from ca. 50 to 150 nm according to the siRNA/HSA composition. The non-covalent siRNA/HSA assembly is formed on isobutyramide-modified mesoporous silica and the self-supported HSA-based nanoparticles are obtained following the silica template dissolution. These original protein particles provide simultaneous magnetic resonance imaging contrast enhancement and cellular in vitro gene silencing. PMID:25163585

  18. Induction of stable epigenetic gene silencing in plants using a virus vector.

    PubMed

    Kanazawa, Akira; Kasai, Megumi

    2015-01-01

    Gene silencing through transcriptional repression can be induced by double-stranded RNA targeted to a gene promoter, a process known as RNA-mediated transcriptional gene silencing (TGS). This phenomenon is associated with epigenetic changes involving cytosine methylation of the promoter. Plant virus vectors have been used to induce RNA-mediated TGS. Here, we describe methods relevant to the induction of epigenetic changes and RNA-mediated TGS in plants using a virus vector, which include inoculation of recombinant virus, detection of short interfering RNAs, bisulfite sequencing analysis, and nuclear run-on transcription assay. PMID:25740361

  19. 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

    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

  20. 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