Use of high-resolution mass spectrometry to investigate a metabolite interference during liquid chromatography/tandem mass spectrometric quantification of a small molecule in toxicokinetic study samples.

PubMed

Furlong, Michael; Bessire, Andrew; Song, Wei; Huntington, Christopher; Groeber, Elizabeth

2010-07-15

During routine liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalysis of a small molecule analyte in rat serum samples from a toxicokinetic study, an unexpected interfering peak was observed in the extracted ion chromatogram of the internal standard. No interfering peaks were observed in the extracted ion chromatogram of the analyte. The dose-dependent peak area response and peak area response versus time profiles of the interfering peak suggested that it might have been related to a metabolite of the dosed compound. Further investigation using high-resolution mass spectrometry led to unequivocal identification of the interfering peak as an N-desmethyl metabolite of the parent analyte. High-resolution mass spectrometry (HRMS) was also used to demonstrate that the interfering response of the metabolite in the multiple reaction monitoring (MRM) channel of the internal standard was due to an isobaric relationship between the (13)C-isotope of the metabolite and the internal standard (i.e., common precursor ion mass), coupled with a metabolite product ion with identical mass to the product ion used in the MRM transition of the internal standard. These results emphasize (1) the need to carefully evaluate internal standard candidates with regard to potential interferences from metabolites during LC/MS/MS method development, validation and bioanalysis of small molecule analytes in biological matrices; (2) the value of HRMS as a tool to investigate unexpected interferences encountered during LC/MS/MS analysis of small molecules in biological matrices; and (3) the potential for interference regardless of choice of IS and therefore the importance of conducting assay robustness on incurred in vitro or in vivo study samples. Copyright 2010 John Wiley & Sons, Ltd.

A positive circuit of VEGF increases Glut-1 expression by increasing HIF-1α gene expression in human retinal endothelial cells.

PubMed

Choi, Yoon Kyung

2017-12-01

Treatment of human retinal microvascular endothelial cells (HRMECs) with vascular endothelial growth factor 165 (VEGF 165 ) increased hypoxia-inducible factor 1α (HIF-1α), VEGF, and glucose transporter 1 (Glut-1) mRNA expression and Glut-1 protein localization to the membrane. In contrast, treatment of human retinal pigment epithelium cells with VEGF 165 did not induce HIF-1α, VEGF, and Glut-1 gene expression. Microvascular endothelial cells are surrounded by astrocytic end feet in the retina. Astrocyte-derived A-kinase anchor protein 12 overexpression during hypoxia downregulated VEGF secretion, and this conditioned medium reduced VEGF and Glut-1 expression in HRMECs, suggesting that communications between astrocytes and endothelial cells may be the determinants of the blood vessel network. In HRMECs, HIF-1α small interfering RNA transfection blocked the VEGF 165 -mediated increase in VEGF and Glut-1 gene expression. Inhibition of protein kinase C (PKC) with inhibitor GF109203X or with a small interfering RNA targeting PKCζ attenuated the VEGF 165 -induced Glut-1 protein expression and VEGF and Glut-1 mRNA expression. In addition, results of an immunoprecipitation assay imply an interaction between VEGF receptor 2 (VEGFR2) and PKCζ in HRMECs. Therefore, VEGF secretion by hypoxic astrocytes may upregulate HIF-1α gene expression, inducing VEGF and Glut-1 expression via the VEGFR2-PKCζ axis in HRMECs.

Combination of small RNAs for skeletal muscle regeneration.

PubMed

Kim, NaJung; Yoo, James J; Atala, Anthony; Lee, Sang Jin

2016-03-01

Selectively controlling the expression of the target genes through RNA interference (RNAi) has significant therapeutic potential for injuries or diseases of tissues. We used this strategy to accelerate and enhance skeletal muscle regeneration for the treatment of muscular atrophy. In this study, we used myostatin small interfering (si)RNA (siGDF-8), a major inhibitory factor in the development and postnatal regeneration of skeletal muscle and muscle-specific microRNAs (miR-1 and -206) to further accelerate muscle regeneration. This combination of 3 small RNAs significantly improved the gene expression of myogenic regulatory factors in vitro, suggesting myogenic activation. Moreover, cell proliferation and myotube formation improved without compromising each other, which indicates the myogenic potential of this combination of small RNAs. The recovery of chemically injured tibialis anterior muscles in rats was significantly accelerated, both functionally and structurally. This novel combination of siRNA and miRNAs has promising therapeutic potential to improve in situ skeletal muscle regeneration. © FASEB.

An endogenous small interfering RNA pathway in Drosophila

PubMed Central

Czech, Benjamin; Malone, Colin D.; Zhou, Rui; Stark, Alexander; Schlingeheyde, Catherine; Dus, Monica; Perrimon, Norbert; Kellis, Manolis; Wohlschlegel, James A.; Sachidanandam, Ravi; Hannon, Gregory J.; Brennecke, Julius

2009-01-01

Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of ~22 nucleotides in length, which arise from structured precursors through the action of Drosha–Pasha and Dicer-1–Loquacious complexes1–7. These join Argonaute-1 to regulate gene expression8,9. A second endogenous small RNA class, the Piwi-interacting RNAs, bind Piwi proteins and suppress transposons10,11. Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of single-stranded RNAs12,13. Here we show that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious1,4,5 rather than the canonical Dicer-2 partner, R2D2 (ref. 14). Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles. PMID:18463631

Novel small interfering RNA-containing solution protecting donor organs in heart transplantation.

PubMed

Zheng, Xiufen; Lian, Dameng; Wong, Arthur; Bygrave, Michael; Ichim, Thomas E; Khoshniat, Mahdieh; Zhang, Xusheng; Sun, Hongtao; De Zordo, Tobias; Lacefield, James C; Garcia, Bertha; Jevnikar, Anthony M; Min, Wei-Ping

2009-09-22

Ischemia/reperfusion injury is a major factor in graft quality and subsequent function in the transplantation setting. We hypothesize that the process of RNA interference may be used to "engineer" a graft to suppress expression of genes associated with inflammation, apoptosis, and complement, which are believed to cause ischemia/reperfusion injury. Such manipulation of pathological gene expression may be performed by treatment of the graft ex vivo with small interfering RNA (siRNA) as part of the preservation procedure. Heart grafts from BALB/c mice were preserved in UW solution (control) or UW solution containing siRNAs targeting tumor necrosis factor-alpha, C3, and Fas genes (siRNA solution) at 4 degrees C for 48 hours and subsequently transplanted into syngeneic recipients. Tumor necrosis factor-alpha, C3, and Fas genes were elevated by ischemia/reperfusion injury after 48 hours of preservation in UW solution. Preservation in siRNA solution knocked down gene expression at the level of messenger RNA and protein in the grafts after transplantation. All grafts preserved in siRNA solution showed strong contraction, whereas grafts preserved in control solution demonstrated no detectable contraction by high-frequency ultrasound scanning. siRNA solution-treated organs exhibited improved histology and diminished neutrophil and lymphocyte infiltration compared with control solution-treated organs. Furthermore, the treated heart grafts retained strong beating up to the end of the observation period (>100 days), whereas all control grafts lost function within 8 days. Incorporation of siRNA into organ storage solution is a feasible and effective method of attenuating ischemia/reperfusion injury, protecting cardiac function, and prolonging graft survival.

HIF1α regulated expression of XPA contributes to cisplatin resistance in lung cancer

PubMed Central

Liu, Yanbin; Bernauer, Amanda M.; Yingling, Christin M.; Belinsky, Steven A.

2012-01-01

Factors regulating nucleotide excision repair probably contribute to the heterogenous response of advanced stage lung cancer patients to drugs such as cisplatin. Studies to identify the genes in the nucleotide excision repair pathway most closely associated with resistance to cisplatin have not been conclusive. We hypothesized that Xeroderma pigmentosum complementation group A (XPA), because of its dual role in sensing and recruiting other DNA repair proteins to the damaged template, would be critical in defining sensitivity to cisplatin. Studies were conducted to identify factors regulating transcription of XPA, to assess its role in modulating sensitivity to cisplatin and its expression in primary lung tumors. Hypoxia-inducible factor 1 alpha (HIF1α) subunit was found to bind with strong affinity to a hypoxia response element sequence in the promoter of XPA. Modulating expression of HIF1α by small interfering RNA or cobalt chloride markedly reduced or increased transcription of XPA in lung cancer cell lines, respectively. Protein levels of XPA were strongly correlated with sensitivity to cisplatin (r = 0.88; P < 0.001) in cell lines and sensitivity could be increased by small interfering RNA depletion of XPA. Expression of XPA determined in 54 primary lung tumors was elevated on average 5.2-fold when compared with normal bronchial epithelial cells and correlated with levels of HIF1α (r = 0.58; P < 0.01). Together, these studies identify XPA as a novel target for regulation by HIF1α whose modulation could impact lung cancer therapy. PMID:22467238

Kelch-like ECH-associated Protein 1-dependent Nuclear Factor-E2-related Factor 2 Activation in Relation to Antioxidation Induced by Sevoflurane Preconditioning.

PubMed

Cai, Min; Tong, Li; Dong, Beibei; Hou, Wugang; Shi, Likai; Dong, Hailong

2017-03-01

The authors have reported that antioxidative effects play a crucial role in the volatile anesthetic-induced neuroprotection. Accumulated evidence shows that endogenous antioxidation could be up-regulated by nuclear factor-E2-related factor 2 through multiple pathways. However, whether nuclear factor-E2-related factor 2 activation is modulated by sevoflurane preconditioning and, if so, what is the signaling cascade underlying upstream of this activation are still unknown. Sevoflurane preconditioning in mice was performed with sevoflurane (2.5%) 1 h per day for five consecutive days. Focal cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion. Expression of nuclear factor-E2-related factor 2, kelch-like ECH-associated protein 1, manganese superoxide dismutase, thioredoxin-1, and nicotinamide adenine dinucleotide phosphate quinolone oxidoreductase-1 was detected (n = 6). The antioxidant activities and oxidative product expression were also examined. To determine the role of kelch-like ECH-associated protein 1 inhibition-dependent nuclear factor-E2-related factor 2 activation in sevoflurane preconditioning-induced neuroprotection, the kelch-like ECH-associated protein 1-nuclear factor-E2-related factor 2 signal was modulated by nuclear factor-E2-related factor 2 knockout, kelch-like ECH-associated protein 1 overexpression lentivirus, and kelch-like ECH-associated protein 1 deficiency small interfering RNA (n = 8). The infarct volume, neurologic scores, and cellular apoptosis were assessed. Sevoflurane preconditioning elicited neuroprotection and increased nuclear factor-E2-related factor 2 nuclear translocation, which in turn up-regulated endogenous antioxidation and reduced oxidative injury. Sevoflurane preconditioning reduced kelch-like ECH-associated protein 1 expression. Nuclear factor-E2-related factor 2 ablation abolished neuroprotection and reversed sevoflurane preconditioning by mediating the up-regulation of antioxidants. Kelch-like ECH-associated protein 1 overexpression reversed nuclear factor-E2-related factor 2 up-regulation and abolished the neuroprotection induced by sevoflurane preconditioning. Kelch-like ECH-associated protein 1 small interfering RNA administration improved nuclear factor-E2-related factor 2 expression and the outcome of mice subjected to ischemia/reperfusion injury. Kelch-like ECH-associated protein 1 down-regulation-dependent nuclear factor-E2-related factor 2 activation underlies the ability of sevoflurane preconditioning to activate the endogenous antioxidant response, which elicits its neuroprotection.

Northern Blot Detection of Virus-Derived Small Interfering RNAs in Caenorhabditis elegans Using Nonradioactive Oligo Probes.

PubMed

Long, Tianyun; Lu, Rui

2017-01-01

Northern blot analysis has been widely used as a tool for detection and characterization of specific RNA molecules. When coupled with radioactive probe northern blot allows for robust detection and characterization of small RNA molecules of trace amount. Here, we describe the detection and size characterization of virus-derived small interfering RNAs (vsiRNAs) in C. elegans using nonradioactive DNA oligo probes in northern blotting. Our protocol allows for the detection and characterization of not only primary vsiRNAs but also secondary vsiRNAs, a class of single-stranded vsiRNAs that has distinct migration pattern, and can be easily adapted to the detection of vsiRNAs in other organisms.

Graft-transmissible movement of inverted-repeat-induced siRNA signals into flowers.

PubMed

Zhang, Wenna; Kollwig, Gregor; Stecyk, Ewelina; Apelt, Federico; Dirks, Rob; Kragler, Friedrich

2014-10-01

In plants, small interfering RNAs (siRNA) and microRNAs move to distant tissues where they control numerous developmental and physiological processes such as morphogenesis and stress responses. Grafting techniques and transient expression systems have been employed to show that sequence-specific siRNAs with a size of 21-24 nucleotides traffic to distant organs. We used inverted-repeat constructs producing siRNA targeting the meiosis factor DISRUPTED MEIOTIC cDNA 1 (DMC1) and GFP to test whether silencing signals move into meiotically active tissues. In grafted Nicotiana tabacum, a transgenic DMC1 siRNA signal made in source tissues preferably entered the anthers formed in the first flowers. Here, the DMC1 siRNA interfered with meiotic progression and, consequently, the flowers were at least partially sterile. In agro-infiltrated N. benthamiana plants, a GFP siRNA signal produced in leaves was allocated and active in most flower tissues including anthers. In hypocotyl-grafted Arabidopsis thaliana plants, the DMC1 silencing signal consistently appeared in leaves, petioles, and stem, and only a small number of plants displayed DMC1 siRNA signals in flowers. In all three tested plant species the systemic silencing signal penetrated male sporogenic tissues suggesting that plants harbour an endogenous long-distance small RNA transport pathway facilitating siRNA signalling into meiotically active cells. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Targeting focal adhesion kinase with small interfering RNA prevents and reverses load-induced cardiac hypertrophy in mice.

PubMed

Clemente, Carolina F M Z; Tornatore, Thais F; Theizen, Thais H; Deckmann, Ana C; Pereira, Tiago C; Lopes-Cendes, Iscia; Souza, José Roberto M; Franchini, Kleber G

2007-12-07

Hypertrophy is a critical event in the onset of failure in chronically overloaded hearts. Focal adhesion kinase (FAK) has attracted particular attention as a mediator of hypertrophy induced by increased load. Here, we demonstrate increased expression and phosphorylation of FAK in the hypertrophic left ventricles (LVs) of aortic-banded mice. We used an RNA interference strategy to examine whether FAK signaling plays a role in the pathophysiology of load-induced LV hypertrophy and failure. Intrajugular delivery of specific small interfering RNA induced prolonged FAK silencing ( approximately 70%) in both normal and hypertrophic LVs. Myocardial FAK silencing was accompanied by prevention, as well as reversal, of load-induced left ventricular hypertrophy. The function of LVs was preserved and the survival rate was higher in banded mice treated with small interfering RNA targeted to FAK, despite the persistent pressure overload. Studies in cardiac myocytes and fibroblasts harvested from LVs confirmed the ability of the systemically administered specific small interfering RNA to silence FAK in both cell types. Further analysis indicated attenuation of cardiac myocyte hypertrophic growth and of the rise in the expression of beta-myosin heavy chain in overloaded LVs. Moreover, FAK silencing was demonstrated to attenuate the rise in the fibrosis, collagen content, and activity of matrix metalloproteinase-2 in overloaded LVs, as well as the rise of matrix metalloproteinase-2 protein expression in fibroblasts harvested from overloaded LVs. This study provides novel evidence that FAK may be involved in multiple aspects of the pathophysiology of cardiac hypertrophy and failure induced by pressure overload.

Improved silencing properties using small internally segmented interfering RNAs

PubMed Central

Bramsen, Jesper B.; Laursen, Maria B.; Damgaard, Christian K.; Lena, Suzy W.; Ravindra Babu, B.; Wengel, Jesper; Kjems, Jørgen

2007-01-01

RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19–27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10–12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo. PMID:17726057

Two pore channels control Ebolavirus host cell entry and are drug targets for disease treatment

PubMed Central

Sakurai, Yasuteru; Kolokoltsov, Andrey A.; Chen, Cheng-Chang; Tidwell, Michael W.; Bauta, William E.; Klugbauer, Norbert; Grimm, Christian; Wahl-Schott, Christian; Biel, Martin; Davey, Robert A.

2015-01-01

Ebolavirus causes sporadic outbreaks of lethal hemorrhagic fever in humans with no currently approved therapy. Cells take up Ebolavirus by macropinocytosis, followed by trafficking through endosomal vesicles. However, few factors controlling endosomal virus movement are known. Here we find that Ebolavirus entry into host cells requires the endosomal calcium channels called two pore channels (TPCs). Disrupting TPC function by gene knockout, small interfering RNAs or small molecule inhibitors halted virus trafficking and prevented infection. Tetrandrine, the most potent small molecule we tested, inhibited infection of human macrophages, the primary target of Ebolavirus in vivo, and also showed therapeutic efficacy in mice. Therefore, TPC proteins play a key role in Ebolavirus infection and may be effective targets for antiviral therapy. PMID:25722412

Exploring the trans-acting short interfering RNAs (ta-siRNAs) technology for virus control in plants

USDA-ARS?s Scientific Manuscript database

Small ribonucleic acid (RNAs) (~20-24nt) processed from double-stranded RNA in plants can trigger degradation of the target mRNAs in cytoplasm or de novo DNA methylation in nucleus leading to gene silencing. Trans-acting short-interfering RNAs (ta-siRNAs) have been shown to enhance the target mRNA d...

Lidocaine attenuates lipopolysaccharide-induced inflammatory responses in microglia.

PubMed

Yuan, Tong; Li, Zhiwen; Li, Xinbai; Yu, Gaoqi; Wang, Na; Yang, Xige

2014-11-01

Lidocaine has been used as a local anesthetic with anti-inflammatory properties, but its effects on neuroinflammation have not been well defined. In the present study, we investigated the prophylactic effects of lidocaine on lipopolysaccharide (LPS)-activated microglia and explored the underlying mechanisms. Microglial cells were incubated with or without 1 μg/mL LPS in the presence or absence of lidocaine, a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580), a nuclear factor-kappa B (NF-κB) inhibitor (pyrrolidine dithiocarbamate), or small interfering RNA. The protein and expression levels of inflammatory mediators, such as monocyte chemotactic protein 1, nitric oxide, prostaglandin E2, interleukin 1β, and tumor necrosis factor α were measured using enzyme-linked immunosorbent assays and real-time polymerase chain reaction. The effect of lidocaine on NF-κB and p38 MAPK activation was evaluated using enzyme-linked immunosorbent assays, Western blot analysis, and electrophoretic mobility shift assay. Lidocaine (≥2 μg/mL) significantly inhibited the release and expression of nitric oxide, monocyte chemotactic protein 1, prostaglandin E2, interleukin 1β, and tumor necrosis factor α in LPS-activated microglia. Treatment with lidocaine also significantly inhibited the phosphorylation of p38 MAPK and the nuclear translocation of NF-κB p50/p65, increased the protein levels of inhibitor kappa B-α. Furthermore, our study shows that the LPS-induced release of inflammatory mediators was suppressed by SB203580, pyrrolidine dithiocarbamate, and small interfering RNA. Prophylactic treatment with lidocaine inhibits LPS-induced release of inflammatory mediators from microglia, and these effects may be mediated by blockade of p38 MAPK and NF-κB signaling pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

Arterial and venous endothelia display differential functional fractalkine (CX3CL1) expression by angiotensin-II.

PubMed

Rius, Cristina; Piqueras, Laura; González-Navarro, Herminia; Albertos, Fernando; Company, Chantal; López-Ginés, Concha; Ludwig, Andreas; Blanes, Jose-Ignacio; Morcillo, Esteban J; Sanz, Maria-Jesus

2013-01-01

Angiotensin-II (Ang-II) promotes the interaction of mononuclear cells with arterioles and neutrophils with postcapillary venules. To investigate the mechanisms underlying this dissimilar response, the involvement of fractalkine (CX(3)CL1) was explored. Enhanced CX(3)CL1 expression was detected in both cremasteric arterioles and postcapillary venules 24 hours after Ang-II intrascrotal injection. Arteriolar leukocyte adhesion was the unique parameter significantly reduced (83%) in animals lacking CX(3)CL1 receptor (CX(3)CR1). Human umbilical arterial and venous endothelial cell stimulation with 1 μmol/L Ang-II increased CX(3)CL1 expression, yet neutralization of CX(3)CL1 activity only significantly inhibited Ang-II-induced mononuclear cell-human umbilical arterial endothelial cell interactions (73%) but not with human umbilical venous endothelial cells. The use of small interfering RNA revealed the involvement of tumor necrosis factor-α in Ang-II-induced CX(3)CL1 upregulation and mononuclear cell arrest. Nox5 knockdown with small interfering RNA or pharmacological inhibition of extracellular signal-regulated kinases1/2, p38 mitogen-activated protein kinase, and nuclear factor-κB also abolished these responses. Finally, when human umbilical arterial endothelial cells were costimulated with Ang-II, tumor necrosis factor-α, and interferon-γ, CX(3)CL1 expression and mononuclear cell adhesiveness were more pronounced than when each stimulus was provided alone. These results suggest that Ang-II induces functional CX(3)CL1 expression in arterial but not in venous endothelia. Thus, targeting endothelial CX(3)CL1-mononuclear leukocyte CX(3)CR1 interactions may constitute a new therapeutic strategy in the treatment of Ang-II-associated cardiovascular diseases.

Ocular Angiogenesis: Vascular Endothelial Growth Factor and Other Factors.

PubMed

Rubio, Roman G; Adamis, Anthony P

2016-01-01

Systematic study of the mechanisms underlying pathological ocular neovascularization has yielded a wealth of knowledge about pro- and anti-angiogenic factors that modulate diseases such as neovascular age-related macular degeneration. The evidence implicating vascular endothelial growth factor (VEGF) in particular has led to the development of a number of approved anti-VEGF therapies. Additional proangiogenic targets that have emerged as potential mediators of ocular neovascularization include hypoxia-inducible factor-1, angiopoietin-2, platelet-derived growth factor-B and components of the alternative complement pathway. As for VEGF, knowledge of these factors has led to a product pipeline of many more novel agents that are in various stages of clinical development in the setting of ocular neovascularization. These agents are represented by a range of drug classes and, in addition to novel small- and large-molecule VEGF inhibitors, include gene therapies, small interfering RNA agents and tyrosine kinase inhibitors. In addition, combination therapy is beginning to emerge as a strategy to improve the efficacy of individual therapies. Thus, a variety of agents, whether administered alone or as adjunctive therapy with agents targeting VEGF, offer the promise of expanding the range of treatments for ocular neovascular diseases. © 2016 S. Karger AG, Basel.

Improving Small Interfering RNA Delivery In Vivo Through Lipid Conjugation.

PubMed

Osborn, Maire F; Khvorova, Anastasia

2018-05-10

RNA interference (RNAi)-based therapeutics are approaching clinical approval for genetically defined diseases. Current clinical success is a result of significant innovations in the development of chemical architectures that support sustained, multi-month efficacy in vivo following a single administration. Conjugate-mediated delivery has established itself as the most promising platform for safe and targeted small interfering RNA (siRNA) delivery. Lipophilic conjugates represent a major class of modifications that improve siRNA pharmacokinetics and enable efficacy in a broad range of tissues. Here, we review current literature and define key features and limitations of this approach for in vivo modulation of gene expression.

Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells.

PubMed

Gou, Qing; He, ShuJiao; Zhou, ZeJian

2017-02-01

Hepatocellular carcinoma is the most common subtype of liver cancer. Protein arginine N-methyltransferase 1 was shown to be upregulated in various cancers. However, the role of protein arginine N-methyltransferase 1 in hepatocellular carcinoma progression remains incompletely understood. We investigated the clinical and functional significance of protein arginine N-methyltransferase 1 in a series of clinical hepatocellular carcinoma samples and a panel of hepatocellular carcinoma cell lines. We performed suppression analysis of protein arginine N-methyltransferase 1 using small interfering RNA to determine the biological roles of protein arginine N-methyltransferase 1 in hepatocellular carcinoma. In addition, the expression of epithelial-mesenchymal transition indicators was verified by western blotting in hepatocellular carcinoma cell lines after small interfering RNA treatment. Protein arginine N-methyltransferase 1 expression was found to be significantly upregulated in hepatocellular carcinoma cell lines and clinical tissues. Moreover, downregulation of protein arginine N-methyltransferase 1 in hepatocellular carcinoma cells by small interfering RNA could inhibit cell proliferation, migration, and invasion in vitro. These results indicate that protein arginine N-methyltransferase 1 may contribute to hepatocellular carcinoma progression and serves as a promising target for the treatment of hepatocellular carcinoma patients.

RISC assembly: Coordination between small RNAs and Argonaute proteins.

PubMed

Kobayashi, Hotaka; Tomari, Yukihide

2016-01-01

Non-coding RNAs generally form ribonucleoprotein (RNP) complexes with their partner proteins to exert their functions. Small RNAs, including microRNAs, small interfering RNAs, and PIWI-interacting RNAs, assemble with Argonaute (Ago) family proteins into the effector complex called RNA-induced silencing complex (RISC), which mediates sequence-specific target gene silencing. RISC assembly is not a simple binding between a small RNA and Ago; rather, it follows an ordered multi-step pathway that requires specific accessory factors. Some steps of RISC assembly and RISC-mediated gene silencing are dependent on or facilitated by particular intracellular platforms, suggesting their spatial regulation. In this review, we summarize the currently known mechanisms for RISC assembly of each small RNA class and propose a revised model for the role of the chaperone machinery in the duplex-initiated RISC assembly pathway. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Copyright © 2015 Elsevier B.V. All rights reserved.

Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer.

PubMed

Yang, Tianzhi; Fogarty, Brittany; LaForge, Bret; Aziz, Salma; Pham, Thuy; Lai, Leanne; Bai, Shuhua

2017-03-01

Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.

Inhibition effect of small interfering RNA of connective tissue growth factor on the expression of extracellular matrix molecules in cultured human renal proximal tubular cells.

PubMed

Liu, Yuyuan; Li, Weiwei; Liu, Hong; Peng, Youming; Yang, Qiu; Xiao, Li; Liu, Yinghong; Liu, Fuyou

2014-03-01

In this study, we investigated the effect of small interfering RNA (siRNA) of connective tissue growth factor (CTGF) by pRetro-Super (PRS) retrovirus vector on the expression of CTGF and related extracellular matrix molecules in human renal proximal tubular cells (HKCs) induced by high glucose, to provide help for renal tubulointerstitial fibrosis therapy. HKCs were exposed to d-glucose to observe their dose and time effect, while the mannitol as osmotic control. Retrovirus producing CTGF siRNA were constructed from the inverted oligonucleotides and transferred into packaging cell line PT67 with lipofectamine, and the virus supernatant was used to infect HKC. The expression of CTGF, fibronectin (FN) and collagen-type I (col1) were measured by semi-quantitative RT-PCR and Western blot. In response to high glucose, CTGF expression in HKCs was increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Introduction of PRS-CTGF-siRNA resulted in the significant reduction of CTGF, FN, col1 mRNA (p < 0.01, respectively) and CTGF, col1 protein (p < 0.05, respectively) expression, while PRS void vector group did not have these effects (p > 0.05). CTGF siRNA therapy can effectively reduce the levels of CTGF, FN and col1 induced by high glucose in cultured HKCs, which suggested that it may be a potential therapeutic strategy to prevent the renal interstitial fibrosis in the future.

Microarray pathway analysis indicated that mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin growth factor 1 signaling pathways were inhibited by small interfering RNA against AT-rich interactive domain 1A in endometrial cancer

PubMed Central

Yang, Ye; Bao, Wei; Sang, Zhengyu; Yang, Yongbing; Lu, Meng; Xi, Xiaowei

2018-01-01

Mutations in the gene encoding AT-rich interactive domain 1A (ARID1A) are frequently observed in endometrial cancer (EC) but the molecular mechanisms linking the genetic changes remain to be fully understood. The present study aimed to elucidate the influence of ARID1A mutations on signaling pathways. Missense, synonymous and nonsense heterozygous ARID1A mutations in the EC HEC-1-A cell line were verified by Sanger sequencing. Mutated ARID1A small interfering RNA was transfected into HEC-1-A cells. Biochemical microarray analysis revealed 13 upregulated pathways, 17 downregulated pathways, 14 significantly affected disease states and functions, 662 upstream and 512 downstream genes in mutated ARID1A-depleted HEC-1-A cells, among which the mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin-like growth factor-1 (IGF1) signaling pathways were the 2 most downregulated pathways. Furthermore, the forkhead box protein O1 pathway was upregulated, while the IGF1 receptor, insulin receptor substrate 1 and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit b pathways were downregulated. Carcinoma tumorigenesis, tumor cell mitosis and tumor cell death were significantly upregulated disease states and functions, while cell proliferation and tumor growth were significantly downregulated. The results of the present study suggested that ARID1A may be a potential prognostic and therapeutic molecular drug target for the prevention of EC progression. PMID:29399196

Galactosylated magnetic nanovectors for regulation of lipid metabolism based on biomarker-specific RNAi and MR imaging.

PubMed

Heo, Dan; Lee, Chanjoo; Ku, Minhee; Haam, Seungjoo; Suh, Jin-Suck; Huh, Yong-Min; Park, Sahng Wook; Yang, Jaemoon

2015-08-21

The specific delivery of ribonucleic acid (RNA) interfering molecules to disease-related cells is still a critical blockade for in vivo systemic treatment. Here, this study suggests a robust delivery carrier for targeted delivery of RNA-interfering molecules using galactosylated magnetic nanovectors (gMNVs). gMNVs are an organic-inorganic polymeric nanomaterial composed of polycationics and magnetic nanocrystal for delivery of RNA-interfering molecules and tracking via magnetic resonance (MR) imaging. In particular, the surface of gMNVs was modified by galactosylgluconic groups for targeted delivering to asialoglycoprotein receptor (ASGPR) of hepatocytes. Moreover, the small interfering RNAs were used to regulate target proteins related with low-density lipoprotein level and in vivo MR imaging was conducted for tracking of nanovectors. The obtained results show that the prepared gMNVs demonstrate potential as a systemic theragnostic nanoplatform for RNA interference and MR imaging.

Unidentified Factors in Jojoba Meal Prevent Oviduct Development in Broiler Breeder Females.

PubMed

Vermaut; Onagbesan; Bruggeman; Verhoeven; Berghman; Flo; Cokelaere; Decuypere

1998-01-19

Supplementation of feed with jojoba meal, as a means for autonomous feed restriction, was successful in depressing feed intake and controlling body weight of broiler breeder pullets to the extent recommended by the breeder company. However, these broiler breeders never produced eggs. At the level of ovary, normal follicle development and maturation did occur. A considerable number of ovulations occurred which were not followed by oviposition. After ovulation, the ova could not be captured by the oviduct, because of the small size of the oviduct, resulting in "internal laying". The virtual absence of oviduct development cannot be explained presently but it must be due to some yet unidentified factor(s) in jojoba meal which prevent(s) the normal development of the oviduct. These factors may be acting by abnormally increasing plasma progesterone or triiodothyronin levels and/or directly by themselves interfering with oviduct development. The nature of these factors requires further investigations.

Therapeutic opportunities of small interfering RNA.

PubMed

Goyal, Bhoomika R; Patel, Mayur M; Soni, Mithil K; Bhadada, Shraddha V

2009-08-01

Formation of small interfering RNA (siRNA) occurs in two steps involving binding of the RNA nucleases to a large double-stranded RNA (dsRNA) and its cleavage into fragments called siRNA. In the second step, these siRNAs join a multinuclease complex, which degrades the homologous single-stranded mRNAs. The delivery of siRNA involves viral- and non-viral-mediated delivery systems; the approaches for chemical modifications have also been developed. It has various therapeutic applications for disorders like cardiovascular diseases, central nervous system (CNS) disorders, cancer, human immunodeficiency virus (HIV), hepatic disorders, etc. The present review gives an overview of the applications of siRNA and their potential for treating many hitherto untreatable diseases.

Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment.

PubMed

Sakurai, Yasuteru; Kolokoltsov, Andrey A; Chen, Cheng-Chang; Tidwell, Michael W; Bauta, William E; Klugbauer, Norbert; Grimm, Christian; Wahl-Schott, Christian; Biel, Martin; Davey, Robert A

2015-02-27

Ebola virus causes sporadic outbreaks of lethal hemorrhagic fever in humans, but there is no currently approved therapy. Cells take up Ebola virus by macropinocytosis, followed by trafficking through endosomal vesicles. However, few factors controlling endosomal virus movement are known. Here we find that Ebola virus entry into host cells requires the endosomal calcium channels called two-pore channels (TPCs). Disrupting TPC function by gene knockout, small interfering RNAs, or small-molecule inhibitors halted virus trafficking and prevented infection. Tetrandrine, the most potent small molecule that we tested, inhibited infection of human macrophages, the primary target of Ebola virus in vivo, and also showed therapeutic efficacy in mice. Therefore, TPC proteins play a key role in Ebola virus infection and may be effective targets for antiviral therapy. Copyright © 2015, American Association for the Advancement of Science.

Manipulation of Cell Physiology Enables Gene Silencing in Well-differentiated Airway Epithelia

PubMed Central

Krishnamurthy, Sateesh; Behlke, Mark A; Ramachandran, Shyam; Salem, Aliasger K; McCray Jr, Paul B; Davidson, Beverly L

2012-01-01

The application of RNA interference-based gene silencing to the airway surface epithelium holds great promise to manipulate host and pathogen gene expression for therapeutic purposes. However, well-differentiated airway epithelia display significant barriers to double-stranded small-interfering RNA (siRNA) delivery despite testing varied classes of nonviral reagents. In well-differentiated primary pig airway epithelia (PAE) or human airway epithelia (HAE) grown at the air–liquid interface (ALI), the delivery of a Dicer-substrate small-interfering RNA (DsiRNA) duplex against hypoxanthine–guanine phosphoribosyltransferase (HPRT) with several nonviral reagents showed minimal uptake and no knockdown of the target. In contrast, poorly differentiated cells (2–5-day post-seeding) exhibited significant oligonucleotide internalization and target knockdown. This finding suggested that during differentiation, the barrier properties of the epithelium are modified to an extent that impedes oligonucleotide uptake. We used two methods to overcome this inefficiency. First, we tested the impact of epidermal growth factor (EGF), a known enhancer of macropinocytosis. Treatment of the cells with EGF improved oligonucleotide uptake resulting in significant but modest levels of target knockdown. Secondly, we used the connectivity map (Cmap) database to correlate gene expression changes during small molecule treatments on various cells types with genes that change upon mucociliary differentiation. Several different drug classes were identified from this correlative assessment. Well-differentiated epithelia treated with DsiRNAs and LY294002, a PI3K inhibitor, significantly improved gene silencing and concomitantly reduced target protein levels. These novel findings reveal that well-differentiated airway epithelia, normally resistant to siRNA delivery, can be pretreated with small molecules to improve uptake of synthetic oligonucleotide and RNA interference (RNAi) responses. PMID:23344182

Knockdown of the placental growth factor gene inhibits laser induced choroidal neovascularization in a murine model.

PubMed

Nourinia, Ramin; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Akrami, Hassan; Rezaei Kanavi, Mozhgan; Samiei, Shahram

2013-01-01

To evaluate the effect of placental growth factor (PlGF) gene knockdown in a murine model of laser-induced choroidal neovascularization. Choroidal neovascularization was induced in the left eyes of 11 mice by infrared laser. Small interfering RNA (siRNA, 20 picomoles/10 μl) corresponding to PlGF mRNA was administered intravitreally by Hamilton syringe in all subjects. One month later, fluorescein angiography and histolologic examination were performed. No leakage was apparent in the 11 eyes treated with siRNA cognate to PlGF. The results of histological evaluation were consistent with angiographic findings showing absence of choroidal neovascularization. Knockdown of the PlGF gene can inhibit the growth of laser-induced choroidal neovascularization in mice.

Manipulating the NF-κB pathway in macrophages using mannosylated, siRNA-delivering nanoparticles can induce immunostimulatory and tumor cytotoxic functions.

PubMed

Ortega, Ryan A; Barham, Whitney; Sharman, Kavya; Tikhomirov, Oleg; Giorgio, Todd D; Yull, Fiona E

2016-01-01

Tumor-associated macrophages (TAMs) are critically important in the context of solid tumor progression. Counterintuitively, these host immune cells can often support tumor cells along the path from primary tumor to metastatic colonization and growth. Thus, the ability to transform protumor TAMs into antitumor, immune-reactive macrophages would have significant therapeutic potential. However, in order to achieve these effects, two major hurdles would need to be overcome: development of a methodology to specifically target macrophages and increased knowledge of the optimal targets for cell-signaling modulation. This study addresses both of these obstacles and furthers the development of a therapeutic agent based on this strategy. Using ex vivo macrophages in culture, the efficacy of mannosylated nanoparticles to deliver small interfering RNA specifically to TAMs and modify signaling pathways is characterized. Then, selective small interfering RNA delivery is tested for the ability to inhibit gene targets within the canonical or alternative nuclear factor-kappaB pathways and result in antitumor phenotypes. Results confirm that the mannosylated nanoparticle approach can be used to modulate signaling within macrophages. We also identify appropriate gene targets in critical regulatory pathways. These findings represent an important advance toward the development of a novel cancer therapy that would minimize side effects because of the targeted nature of the intervention and that has rapid translational potential.

The influence of cyclooxygenase-1 expression on the efficacy of cyclooxygenase-2 inhibition in head and neck squamous cell carcinoma cell lines.

PubMed

Park, Seok-Woo; Kim, Hyo-Sun; Choi, Myung-Sun; Kim, Ji-Eun; Jeong, Woo-Jin; Heo, Dae-Seog; Sung, Myung-Whun

2011-06-01

We have previously observed that cyclooxygenase-2 (COX-2) inhibition blocked the production of vascular endothelial growth factor (VEGF) in some head and neck squamous cell carcinoma (HNSCC) cells. However, as some HNSCC cells showed little response to COX-2 inhibition, although they highly expressed COX-2 and prostaglandin E2, we set out to elucidate what made this difference between them and focused on the possibility of the differential expression of COX-1. In western blotting, we found that COX-1 was expressed in SNU-1041 and SNU-1066, but not in SNU-1076 and PCI-50. Only in those cell lines without expression of COX-1 was VEGF production blocked meaningfully by small interfering RNA of COX-2. However, by cotreating with small interfering RNAs of COX-2 and COX-1, VEGF synthesis and prostaglandin E2 were inhibited in SNU-1041 and SNU-1066, similarly in SNU-1076 and PCI-50 with high expression of only COX-2. We also found that there was no difference in the pattern of prostaglandin synthesis between COX-2 and COX-1 through enzyme-linked immunosorbent assay for various prostaglandins. Our study suggests that, as COX-1 and COX-2 express and affect VEGF synthesis in HNSCC cells, we should check COX-1 expression in investigations on cancer treatment by inhibiting COX-2-induced prostaglandins.

Novel liposomal combination treatments using dual genes knockdown in oral cancer treatment

NASA Astrophysics Data System (ADS)

Wu, Jyun-Sian; Yeh, Chia-Hsien; Huang, Leaf; Hsu, Yih-Chih

2018-02-01

Small interfering RNA (siRNA) can be used to treat tumor because it can effectively knockdown target oncoprotein expression and it leads to cancer cell death and apoptosis. Hypoxia-inducible factors-1 (HIF-1) is a transcription factor gene. Its high expression of tumor hypoxia cells, activation of transcription factor HIF-1α and angiogenesis found in most cancerous tissues. HIF-1α protein in cancer cells are critical to cell survival, tumor growth and proliferation. Epidermal growth factor receptor (EGFR) gene is another common head and neck oncogene. The dual self-designed siRNA sequences were encapsulated in the lipid-calcium-phosphate (LCP) and targeted to sigma receptors on the surface of cancer cells via binding to amino ethyl anisamide (AEAA). We used human oral cancer cells to establish the xenograft animal model to study the combination therapy for therapeutic results.

Work-family conflict and burnout among Chinese female nurses: the mediating effect of psychological capital.

PubMed

Wang, Yang; Chang, Ying; Fu, Jialiang; Wang, Lie

2012-10-29

Burnout among nurses not only threatens their own health, but also that of their patients. Exploring risk factors of nurse' burnout is important to improve nurses' health and to increase the quality of health care services. This study aims to explore the relationship between work-family conflict and burnout among Chinese female nurses and the mediating role of psychological capital in this relationship. This cross-sectional study was performed during the period of September and October 2010. A questionnaire that consisted of the Maslach Burnout Inventory-General Survey (MBI-GS), the work-family conflict scale and the psychological capital questionnaire (PCQ-24) scale, as well as demographic and working factors, was distributed to nurses in Liaoning province, China. A total of 1,332 individuals (effective response rate: 78.35%) became our subjects. Hierarchical linear regression analyses were performed to explore the mediating role of psychological capital. Both work interfering family conflict and family interfering work conflict were positively related with emotional exhaustion and cynicism. However, work interfering family conflict was positively related with professional efficacy whereas family interfering work conflict was negatively related with it. Psychological capital partially mediated the relationship of work interfering family conflict with emotional exhaustion and cynicism; and partially mediated the relationship of family interfering work conflict with emotional exhaustion, cynicism and professional efficacy. Work-family conflict had effects on burnout and psychological capital was a mediator in this relationship among Chinese nurses. Psychological capital was a positive resource for fighting against nurses' burnout.

Apple miRNAs and tasiRNAs with novel regulatory networks

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown. Results We performed deep small RNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that 10 of the 19 miR828-targeted MYBs undergo small interfering RNA (siRNA) biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis. Conclusions Our work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family. PMID:22704043

Colored petri net modeling of small interfering RNA-mediated messenger RNA degradation.

PubMed

Nickaeen, Niloofar; Moein, Shiva; Heidary, Zarifeh; Ghaisari, Jafar

2016-01-01

Mathematical modeling of biological systems is an attractive way for studying complex biological systems and their behaviors. Petri Nets, due to their ability to model systems with various levels of qualitative information, have been wildly used in modeling biological systems in which enough qualitative data may not be at disposal. These nets have been used to answer questions regarding the dynamics of different cell behaviors including the translation process. In one stage of the translation process, the RNA sequence may be degraded. In the process of degradation of RNA sequence, small-noncoding RNA molecules known as small interfering RNA (siRNA) match the target RNA sequence. As a result of this matching, the target RNA sequence is destroyed. In this context, the process of matching and destruction is modeled using Colored Petri Nets (CPNs). The model is constructed using CPNs which allow tokens to have a value or type on them. Thus, CPN is a suitable tool to model string structures in which each element of the string has a different type. Using CPNs, long RNA, and siRNA strings are modeled with a finite set of colors. The model is simulated via CPN Tools. A CPN model of the matching between RNA and siRNA strings is constructed in CPN Tools environment. In previous studies, a network of stoichiometric equations was modeled. However, in this particular study, we modeled the mechanism behind the silencing process. Modeling this kind of mechanisms provides us with a tool to examine the effects of different factors such as mutation or drugs on the process.

Modeling Interactions between Transposable Elements and the Plant Epigenetic Response: A Surprising Reliance on Element Retention.

PubMed

Roessler, Kyria; Bousios, Alexandros; Meca, Esteban; Gaut, Brandon S

2018-03-01

Transposable elements (TEs) compose the majority of angiosperm DNA. Plants counteract TE activity by silencing them epigenetically. One form of epigenetic silencing requires 21-22 nt small interfering RNAs that act to degrade TE mRNA and may also trigger DNA methylation. DNA methylation is reinforced by a second mechanism, the RNA-dependent DNA methylation (RdDM) pathway. RdDM relies on 24 nt small interfering RNAs and ultimately establishes TEs in a quiescent state. These host factors interact at a systems level, but there have been no system level analyses of their interactions. Here, we define a deterministic model that represents the propagation of active TEs, aspects of the host response and the accumulation of silenced TEs. We describe general properties of the model and also fit it to biological data in order to explore two questions. The first is why two overlapping pathways are maintained, given that both are likely energetically expensive. Under our model, RdDM silenced TEs effectively even when the initiation of silencing was weak. This relationship implies that only a small amount of RNAi is needed to initiate TE silencing, but reinforcement by RdDM is necessary to efficiently counter TE propagation. Second, we investigated the reliance of the host response on rates of TE deletion. The model predicted that low levels of deletion lead to few active TEs, suggesting that silencing is most efficient when methylated TEs are retained in the genome, thereby providing one explanation for the large size of plant genomes.

Small-Molecule Inhibitors of the SOX18 Transcription Factor.

PubMed

Fontaine, Frank; Overman, Jeroen; Moustaqil, Mehdi; Mamidyala, Sreeman; Salim, Angela; Narasimhan, Kamesh; Prokoph, Nina; Robertson, Avril A B; Lua, Linda; Alexandrov, Kirill; Koopman, Peter; Capon, Robert J; Sierecki, Emma; Gambin, Yann; Jauch, Ralf; Cooper, Matthew A; Zuegg, Johannes; Francois, Mathias

2017-03-16

Pharmacological modulation of transcription factors (TFs) has only met little success over the past four decades. This is mostly due to standard drug discovery approaches centered on blocking protein/DNA binding or interfering with post-translational modifications. Recent advances in the field of TF biology have revealed a central role of protein-protein interaction in their mode of action. In an attempt to modulate the activity of SOX18 TF, a known regulator of vascular growth in development and disease, we screened a marine extract library for potential small-molecule inhibitors. We identified two compounds, which inspired a series of synthetic SOX18 inhibitors, able to interfere with the SOX18 HMG DNA-binding domain, and to disrupt HMG-dependent protein-protein interaction with RBPJ. These compounds also perturbed SOX18 transcriptional activity in a cell-based reporter gene system. This approach may prove useful in developing a new class of anti-angiogenic compounds based on the inhibition of TF activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

The effects of small interfering RNA–targeting tissue factor on an in vitro model of neovascularization

PubMed Central

Peng, Wenyan; Yu, Ying; Li, Tiejun; Zhu, Yuanyuan

2013-01-01

Purpose Tissue factor (TF) plays an important role in neovascularization (NV). This study aimed to determine whether small interfering RNA–targeting TF (TF-siRNA) could knock down TF expression and inhibit cell proliferation, cell migration, and tube formation in an in vitro model of NV. Methods Lipopolysaccharide (LPS) was used to stimulate human umbilical vein endothelial cell (HUVEC) lines to express TF and mimic certain phenotypes of NV in vitro. HUVECs were transfected with TF-siRNAs and control siRNAs using LipofectamineTM 2000. The inhibitory effect of the siRNAs on the expression of TF mRNA and protein was evaluated by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and western blot analysis. The effects on the cell viability, migration, and tube formation of siRNA-treated cells were examined by MTT assay, wound-healing assay, and Matrigel-induced capillary tube formation. Results Lipopolysaccharide treatment increased the expression of TF. TF-siRNAs effectively knocked down TF expression, with the most efficient TF-siRNA reducing 78.9% of TF expression. TF protein was also notably curtailed by TF-siRNA. The MTT and wound-healing assays showed that the TF-siRNA substantially inhibited the proliferation and migration of HUVECs. Tube formation was decreased by 47.4% and 59.4% in cells treated with the TF-siRNA and vascular endothelial growth factor–siRNA, respectively, compared with the blank control. Conclusions TF-siRNA can knockdown TF expression and inhibit cell proliferation, migration, and tube formation in vitro. TF-siRNA may provide a novel therapeutic candidate for NV-related diseases. PMID:23805036

Mechanisms of estradiol-induced insulin secretion by the G protein-coupled estrogen receptor GPR30/GPER in pancreatic beta-cells.

PubMed

Sharma, Geetanjali; Prossnitz, Eric R

2011-08-01

Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes.

Haplodeficiency of Klotho Gene Causes Arterial Stiffening via Upregulation of Scleraxis Expression and Induction of Autophagy.

PubMed

Chen, Kai; Zhou, Xiaoli; Sun, Zhongjie

2015-11-01

The prevalence of arterial stiffness increases with age, whereas the level of the aging-suppressor protein klotho decreases with age. The objective of this study is to assess whether haplodeficiency of klotho gene causes arterial stiffness and to investigate the underlying mechanism. Pulse wave velocity, a direct measure of arterial stiffness, was increased significantly in klotho heterozygous (klotho(+/-)) mice versus their age-matched wild-type (WT) littermates, suggesting that haplodeficiency of klotho causes arterial stiffening. Notably, plasma aldosterone levels were elevated significantly in klotho(+/-) mice. Treatment with eplerenone (6 mg/kg per day IP), an aldosterone receptor blocker, abolished klotho deficiency-induced arterial stiffening in klotho(+/-) mice. Klotho deficiency was associated with increased collagen and decreased elastin contents in the media of aortas. In addition, arterial matrix metalloproteinase-2, matrix metalloproteinase-9, and transforming growth factor-β1 expression and myofibroblast differentiation were increased in klotho(+/-) mice. These klotho deficiency-related changes can be blocked by eplerenone. Protein expression of scleraxis, a transcription factor for collagen synthesis, and LC3-II/LC3-I, an index of autophagy, were upregulated in aortas of klotho(+/-) mice, which can be abolished by eplerenone. In cultured mouse aortic smooth muscle cells, aldosterone increased collagen-1 expression that can be completely eliminated by small interfering RNA knockdown of scleraxis. Interestingly, aldosterone decreased elastin levels in smooth muscle cells, which can be abolished by small interfering RNA knockdown of Beclin-1, an autophagy-related gene. In conclusion, this study demonstrated for the first time that klotho deficiency-induced arterial stiffening may involve aldosterone-mediated upregulation of scleraxis and induction of autophagy, which led to increased collagen-1 expression and decreased elastin levels, respectively. © 2015 American Heart Association, Inc.

Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway

PubMed Central

Wu, Shouhai; Zhang, Tianpeng; Du, Jingsheng

2016-01-01

Background Combinations of adjuvant sensitizers with anticancer drugs is a promising new strategy to reverse chemoresistance. Ursolic acid (UA) is one of the natural pentacyclic triterpene compounds known to have many pharmacological characteristics such as anti-inflammatory and anticancer properties. This study investigates whether UA can sensitize hepatocellular carcinoma cells to cisplatin. Materials and methods Cells were transfected with nuclear factor erythroid-2-related factor 2 (Nrf2) small interfering RNA and Nrf2 complementary DNA by using Lipofectin 2000. The cytotoxicity of cells was investigated by Cell Counting Kit 8 assay. Cell apoptosis, cell cycle, reactive oxygen species, and mitochondrial membrane potential were detected by flow cytometry fluorescence-activated cell sorting. The protein level of Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and heme oxygenase-1 (HO-1) was detected by Western blot analysis. Results The results showed that the reverse index was 2.9- and 9.69-fold by UA of 1.125 μg/mL and 2.25 μg/mL, respectively, for cisplatin to HepG2/DDP cells. UA–cisplatin combination induced cell apoptosis and reactive oxygen species, blocked the cell cycle in G0/G1 phase, and reduced the mitochondrial membrane potential. Mechanistically, UA–cisplatin dramatically decreased the expression of Nrf2 and its downstream genes. The sensibilization of UA–cisplatin combination was diminished in Nrf2 small interfering RNA-transfected HepG2/DDP cells, as well as in Nrf2 complementary DNA-transfected HepG2/DDP cells. Conclusion The results confirmed the sensibilization of UA on HepG2/DDP cells to cisplatin, which was possibly mediated via the Nrf2/antioxidant response element pathway. PMID:27822011

Targeting Micrornas With Small Molecules: A Novel Approach to Treating Breast Cancer

DTIC Science & Technology

2010-10-01

ribozymes and the DNAzymes, small interfering RNAs and short hairpin RNAs, and anti-miRNA agents such as antisense oligo- nucleotides, locked nucleic...of the antagomir Preclinical studies Ribozymes or DNAzymes A ribozyme , or RNA enzyme, is an RNA molecule that can catalyze a chemical reaction. A

Work-family conflict and burnout among Chinese female nurses: the mediating effect of psychological capital

PubMed Central

2012-01-01

Background Burnout among nurses not only threatens their own health, but also that of their patients. Exploring risk factors of nurse’ burnout is important to improve nurses’ health and to increase the quality of health care services. This study aims to explore the relationship between work-family conflict and burnout among Chinese female nurses and the mediating role of psychological capital in this relationship. Methods This cross-sectional study was performed during the period of September and October 2010. A questionnaire that consisted of the Maslach Burnout Inventory-General Survey (MBI-GS), the work-family conflict scale and the psychological capital questionnaire (PCQ-24) scale, as well as demographic and working factors, was distributed to nurses in Liaoning province, China. A total of 1,332 individuals (effective response rate: 78.35%) became our subjects. Hierarchical linear regression analyses were performed to explore the mediating role of psychological capital. Results Both work interfering family conflict and family interfering work conflict were positively related with emotional exhaustion and cynicism. However, work interfering family conflict was positively related with professional efficacy whereas family interfering work conflict was negatively related with it. Psychological capital partially mediated the relationship of work interfering family conflict with emotional exhaustion and cynicism; and partially mediated the relationship of family interfering work conflict with emotional exhaustion, cynicism and professional efficacy. Conclusion Work-family conflict had effects on burnout and psychological capital was a mediator in this relationship among Chinese nurses. Psychological capital was a positive resource for fighting against nurses’ burnout. PMID:23107113

Knockdown of the Placental Growth Factor Gene Inhibits Laser Induced Choroidal Neovascularization in a Murine Model

PubMed Central

Nourinia, Ramin; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Akrami, Hassan; Rezaei Kanavi, Mozhgan; Samiei, Shahram

2013-01-01

Purpose To evaluate the effect of placental growth factor (PlGF) gene knockdown in a murine model of laser-induced choroidal neovascularization. Methods Choroidal neovascularization was induced in the left eyes of 11 mice by infrared laser. Small interfering RNA (siRNA, 20 picomoles/10 μl) corresponding to PlGF mRNA was administered intravitreally by Hamilton syringe in all subjects. One month later, fluorescein angiography and histolologic examination were performed. Results No leakage was apparent in the 11 eyes treated with siRNA cognate to PlGF. The results of histological evaluation were consistent with angiographic findings showing absence of choroidal neovascularization. Conclusion Knockdown of the PlGF gene can inhibit the growth of laser-induced choroidal neovascularization in mice. PMID:23825706

Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice.

PubMed

Overman, Jeroen; Fontaine, Frank; Moustaqil, Mehdi; Mittal, Deepak; Sierecki, Emma; Sacilotto, Natalia; Zuegg, Johannes; Robertson, Avril Ab; Holmes, Kelly; Salim, Angela A; Mamidyala, Sreeman; Butler, Mark S; Robinson, Ashley S; Lesieur, Emmanuelle; Johnston, Wayne; Alexandrov, Kirill; Black, Brian L; Hogan, Benjamin M; De Val, Sarah; Capon, Robert J; Carroll, Jason S; Bailey, Timothy L; Koopman, Peter; Jauch, Ralf; Smyth, Mark J; Cooper, Matthew A; Gambin, Yann; Francois, Mathias

2017-01-31

Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics.

Gene Suppression of Mouse Testis In Vivo Using Small Interfering RNA Derived from Plasmid Vectors

PubMed Central

Takizawa, Takami; Ishikawa, Tomoko; Kosuge, Takuji; Mizuguchi, Yoshiaki; Sato, Yoko; Koji, Takehiko; Araki, Yoshihiko; Takizawa, Toshihiro

2012-01-01

We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules. PMID:22489107

Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA.

PubMed

Wang, Xushan; Mader, Mary M; Toth, John E; Yu, Xiaohong; Jin, Najia; Campbell, Robert M; Smallwood, Jeffrey K; Christe, Michael E; Chatterjee, Arindam; Goodson, Theodore; Vlahos, Chris J; Matter, William F; Bloem, Laura J

2005-05-13

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1beta or tumor necrosis factor-alpha-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

PubMed

Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

2015-12-01

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

MicroRNA superfamilies descended from miR390 and their roles in secondary small interfering RNA biogenesis in eudicots

USDA-ARS?s Scientific Manuscript database

MiRNAs have been demonstrated to regulate diverse biological processes through cleavage of gene transcripts. Some of miRNAs acquire additional function and their cleavage can incite production of secondary small RNAs which possibly provoke a novel regulatory cascade. In this study, we investigated...

SoMART, a web server for miRNA, tasiRNA and target gene analysis in Solanaceae plants

USDA-ARS?s Scientific Manuscript database

Plant micro(mi)RNAs and trans-acting small interfering (tasi)RNAs mediate posttranscriptional silencing of genes and play important roles in a variety of biological processes. Although bioinformatics prediction and small (s)RNA cloning are the key approaches used for identification of miRNAs, tasiRN...

Ligand-targeted delivery of small interfering RNAs to malignant cells and tissues.

PubMed

Thomas, Mini; Kularatne, Sumith A; Qi, Longwu; Kleindl, Paul; Leamon, Christopher P; Hansen, Michael J; Low, Philip S

2009-09-01

Potential clinical applications of small interfering RNA (siRNA) are hampered primarily by delivery issues. We have successfully addressed the delivery problems associated with off-site targeting of highly toxic chemotherapeutic agents by attaching the drugs to tumor-specific ligands that will carry the attached cargo into the desired cancer cell. Indeed, several such tumor-targeted drugs are currently undergoing human clinical trials. We now show that efficient targeting of siRNA to malignant cells and tissues can be achieved by covalent conjugation of small-molecular-weight, high-affinity ligands, such as folic acid and DUPA (2-[3-(1, 3-dicarboxy propyl)-ureido] pentanedioic acid), to siRNA. The former ligand binds a folate receptor that is overexpressed on a variety of cancers, whereas the latter ligand binds to prostate-specific membrane antigen that is overexpressed specifically on prostate cancers and the neovasculature of all solid tumors. Using these ligands, we show remarkable receptor-mediated targeting of siRNA to cancer tissues in vitro and in vivo.

Inhibition of West Nile Virus replication by retrovirus-delivered small interfering RNA in human neuroblastoma cells.

PubMed

Yang, Yongbo; Wu, Chengxiang; Wu, Jianguo; Nerurkar, Vivek R; Yanagihara, Richard; Lu, Yuanan

2008-05-01

West Nile virus (WNV) has been responsible for the largest outbreaks of arboviral encephalitis in U.S. history. No specific drug is currently available for the effective treatment of WNV infection. To exploit RNA interference as a potential therapeutic approach, a Moloney murine leukemia virus-based retrovirus vector was used to effectively deliver WNV-specific small interfering RNA (siRNA) into human neuroblastoma HTB-11 cells. Viral plaque assays demonstrated that transduced cells were significantly refractory to WNV replication, as compared to untransduced control cells (P < 0.05), which correlated with the reduced expression of target viral genes and respective viral proteins. Therefore, retrovirus-mediated delivery of siRNA for gene silencing can be used to study the specific functions of viral genes associated with replication and may have potential therapeutic applications.

Bcl-XL small interfering RNA suppresses the proliferation of 5-fluorouracil-resistant human colon cancer cells.

PubMed

Zhu, Hongbo; Guo, Wei; Zhang, Lidong; Davis, John J; Teraishi, Fuminori; Wu, Shuhong; Cao, Xiaobo; Daniel, Jonathan; Smythe, W Roy; Fang, Bingliang

2005-03-01

5-Fluorouracil (5-FU) is commonly used to treat human colon cancers but resistance to this compound is frequently observed in clinics. To characterize mechanisms of resistance to 5-FU and to develop new strategies for overcoming it, we established two cell lines that were resistant to 5-FU but not other chemotherapeutic agents from parental 5-FU-sensitive cell lines. Western blot analysis revealed that these resistant cells overexpressed the proteins Bcl-XL, Bcl-Xs, and Bik, and further data showed that the cells were resistant to 5-FU-induced DNA damage and cell cycle disorder. However, in parental cells, enforced expression of Bcl-XL protein provided only limited protection from 5-FU-induced apoptosis and overexpression of Bcl-XL protein did not affect 5-FU-induced DNA damage or cell cycle changes; these findings suggested that overexpression of Bcl-XL protein was not the major contributor to 5-FU resistance in any of our cells lines. Even so, knockdown of Bcl-XL protein expression by Bcl-XL-specific small interfering RNA could inhibit proliferation more effectively in 5-FU-resistant cells than in 5-FU-sensitive cells, and the combination of Bcl-XL-specific small interfering RNA and 5-FU had additive effect on the inhibition of 5-FU-resistant cells. These results suggest that down-regulation of Bcl-XL protein expression might provide a new treatment strategy for human 5-FU-resistant colon cancer therapy.

Interference of oxygen, carbon dioxide, and water vapor on the analysis for oxides of nitrogen by chemiluminescence

NASA Technical Reports Server (NTRS)

Maahs, H. G.

1975-01-01

The interference of small concentrations (less than 4 percent by volume) of oxygen, carbon dioxide, and water vapor on the analysis for oxides of nitrogen by chemiluminescence was measured. The sample gas consisted primarily of nitrogen, with less than 100 parts per million concentration of nitric oxide, and with small concentrations of oxygen, carbon dioxide, and water vapor added. Results obtained under these conditions indicate that although oxygen does not measurably affect the analysis for nitric oxide, the presence of carbon dioxide and water vapor causes the indicated nitric oxide concentration to be too low. An interference factor - defined as the percentage change in indicated nitric oxide concentration (relative to the true nitric oxide concentration) divided by the percent interfering gas present - was determined for carbon dioxide to be -0.60 + or - 0.04 and for water vapor to be -2.1 + or - 0.3.

Small silencing RNAs: an expanding universe.

PubMed

Ghildiyal, Megha; Zamore, Phillip D

2009-02-01

Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, their modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected, and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats.

Quality of glucose measurement with blood glucose meters at the point-of-care: relevance of interfering factors.

PubMed

Heinemann, Lutz

2010-11-01

A good understanding of the relevance of interfering factors having an impact on blood glucose (BG) measurement is needed to obtain the required quality. This depends on the application in which meters designed for self-monitoring of BG (SMBG) are used. By means of a literature search all publications (from January 1, 1980 to August 10, 2009) were identified that report about the influence of potentially interfering substances/factors on the measurement quality of BG meters. Certain substances (e.g., maltose) can have a profound and misleading impact on the BG measurement result when the enzymatic reaction embedded on the given test strips cross-reacts. Also, a number of other drugs (e.g., acetaminophen) and factors (like temperature and altitude) affect the reliability of BG measurement massively. However, the susceptibility of the BG meter (depending on the enzyme technology of the test strips) differs significantly. In daily practice the factors that have a relevant impact on the reliability of BG measurements with modern BG meters are rarely met. Clearly this also depends on the intended use (SMBG in patient hands vs. point-of-care testing in hospitals). To avoid misleading measurement results requires adequate training of all people involved.

Using small RNA deep sequencing data to detect siRNA duplexes induced by plant viruses

USDA-ARS?s Scientific Manuscript database

Small interfering RNA (siRNA) duplexes are produced in plants during virus infection, which are short (usually 21 to 24-base pair) double-stranded RNAs (dsRNAs) with several overhanging nucleotides on the 5' end and 3' end. The investigation of the siRNA duplexes is useful to better understand the R...

Divergent patterns of endogenous small RNA populations from seed and vegetative tissues of Glycine max

USDA-ARS?s Scientific Manuscript database

Background: Small non-coding RNAs (smRNAs) are known to have major roles in gene regulation in eukaryotes. In plants, knowledge of the biogenesis and mechanisms of action of smRNA classes including microRNAs (miRNAs), short interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs) has been gaine...

Small RNAs Targeting Transcription Start Site Induce Heparanase Silencing through Interference with Transcription Initiation in Human Cancer Cells

PubMed Central

Pu, Jiarui; Mei, Hong; Zhao, Jun; Huang, Kai; Zeng, Fuqing; Tong, Qiangsong

2012-01-01

Heparanase (HPA), an endo-h-D-glucuronidase that cleaves the heparan sulfate chain of heparan sulfate proteoglycans, is overexpressed in majority of human cancers. Recent evidence suggests that small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in human cells. In this study, transfection of siRNA against −9/+10 bp (siH3), but not −174/−155 bp (siH1) or −134/−115 bp (siH2) region relative to transcription start site (TSS) locating at 101 bp upstream of the translation start site, resulted in TGS of heparanase in human prostate cancer, bladder cancer, and gastric cancer cells in a sequence-specific manner. Methylation-specific PCR and bisulfite sequencing revealed no DNA methylation of CpG islands within heparanase promoter in siH3-transfected cells. The TGS of heparanase did not involve changes of epigenetic markers histone H3 lysine 9 dimethylation (H3K9me2), histone H3 lysine 27 trimethylation (H3K27me3) or active chromatin marker acetylated histone H3 (AcH3). The regulation of alternative splicing was not involved in siH3-mediated TGS. Instead, siH3 interfered with transcription initiation via decreasing the binding of both RNA polymerase II and transcription factor II B (TFIIB), but not the binding of transcription factors Sp1 or early growth response 1, on the heparanase promoter. Moreover, Argonaute 1 and Argonaute 2 facilitated the decreased binding of RNA polymerase II and TFIIB on heparanase promoter, and were necessary in siH3-induced TGS of heparanase. Stable transfection of the short hairpin RNA construct targeting heparanase TSS (−9/+10 bp) into cancer cells, resulted in decreased proliferation, invasion, metastasis and angiogenesis of cancer cells in vitro and in athymic mice models. These results suggest that small RNAs targeting TSS can induce TGS of heparanase via interference with transcription initiation, and significantly suppress the tumor growth, invasion, metastasis and angiogenesis of cancer cells. PMID:22363633

The role of PACT in the RNA silencing pathway

PubMed Central

Lee, Yoontae; Hur, Inha; Park, Seong-Yeon; Kim, Young-Kook; Suh, Mi Ra; Kim, V Narry

2006-01-01

Small RNA-mediated gene silencing (RNA silencing) has emerged as a major regulatory pathway in eukaryotes. Identification of the key factors involved in this pathway has been a subject of rigorous investigation in recent years. In humans, small RNAs are generated by Dicer and assembled into the effector complex known as RNA-induced silencing complex (RISC) by multiple factors including hAgo2, the mRNA-targeting endonuclease, and TRBP (HIV-1 TAR RNA-binding protein), a dsRNA-binding protein that interacts with both Dicer and hAgo2. Here we describe an additional dsRNA-binding protein known as PACT, which is significant in RNA silencing. PACT is associated with an ∼500 kDa complex that contains Dicer, hAgo2, and TRBP. The interaction with Dicer involves the third dsRNA-binding domain (dsRBD) of PACT and the N-terminal region of Dicer containing the helicase motif. Like TRBP, PACT is not required for the pre-microRNA (miRNA) cleavage reaction step. However, the depletion of PACT strongly affects the accumulation of mature miRNA in vivo and moderately reduces the efficiency of small interfering RNA-induced RNA interference. Our study indicates that, unlike other RNase III type proteins, human Dicer may employ two different dsRBD-containing proteins that facilitate RISC assembly. PMID:16424907

Antitheft container for instruments

NASA Technical Reports Server (NTRS)

Kerley, J. J., Jr.

1979-01-01

Antitheft container is used to prevent theft of calculators, portable computers, and other small instruments. Container design is simple and flexible enough to allow easy access to display or input systems of instruments, while not interfering with power input to device.

Therapeutic implications of small interfering RNA in cardiovascular diseases.

PubMed

Raghunathan, Suchi; Patel, Bhoomika M

2013-02-01

Cardiovascular diseases (CVDs) place a heavy burden on the economies of low- and middle-income countries. Comprehensive action requires combining approaches that seek to reduce the risks throughout the entire population with strategies that target individuals at high risk or with established disease. Small interfering RNA (siRNA) as a functional mediator for regulation of gene expression has been evaluated for potential therapeutic targets for the treatment of various cardiovascular diseases such as hypertension, atherosclerosis, heart failure etc. The present review attempts have been made to provide a brief outline of the current understanding of the mechanism of RNAi and the delivery system and describe the therapeutic application of siRNAs and their potential for treating CVDs which are taking a heavy toll on human life. © 2012 The Authors Fundamental and Clinical Pharmacology © 2012 Société Française de Pharmacologie et de Thérapeutique.

Systematic coarse-grained modeling of complexation between small interfering RNA and polycations

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

Wei, Zonghui; Luijten, Erik, E-mail: luijten@northwestern.edu; Department of Materials Science and Engineering, Northwestern University, Evanston, Illinois 60208

All-atom molecular dynamics simulations can provide insight into the properties of polymeric gene-delivery carriers by elucidating their interactions and detailed binding patterns with nucleic acids. However, to explore nanoparticle formation through complexation of these polymers and nucleic acids and study their behavior at experimentally relevant time and length scales, a reliable coarse-grained model is needed. Here, we systematically develop such a model for the complexation of small interfering RNA (siRNA) and grafted polyethyleneimine copolymers, a promising candidate for siRNA delivery. We compare the predictions of this model with all-atom simulations and demonstrate that it is capable of reproducing detailed bindingmore » patterns, charge characteristics, and water release kinetics. Since the coarse-grained model accelerates the simulations by one to two orders of magnitude, it will make it possible to quantitatively investigate nanoparticle formation involving multiple siRNA molecules and cationic copolymers.« less

Small interfering ribonucleic acid induces liquid-to-ripple phase transformation in a phospholipid membrane

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

Choubey, Amit; Nomura, Ken-ichi; Kalia, Rajiv K.

Small interfering ribonucleic acid (siRNA) molecules play a pivotal role in silencing gene expression via the RNA interference mechanism. A key limitation to the widespread implementation of siRNA therapeutics is the difficulty of delivering siRNA-based drugs to cells. Here, we examine changes in the structure and dynamics of a dipalmitoylphosphatidylcholine bilayer in the presence of a siRNA molecule and mechanical barriers to siRNA transfection in the bilayer. Our all-atom molecular dynamics simulation shows that siRNA induces a liquid crystalline-to-ripple phase transformation in the bilayer. The ripple phase consists of a major region of non-interdigitated and a minor region of interdigitatedmore » lipid molecules with an intervening kink. In the ripple phase, hydrocarbon chains of lipid molecules have large compressive stresses, which present a considerable barrier to siRNA transfection.« less

Silencing heat shock factor 1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia-reperfusion injury in mice.

PubMed

Yin, Chang; Xi, Lei; Wang, Xiaoyin; Eapen, Mareen; Kukreja, Rakesh C

2005-10-01

Induction of heat shock factor 1 (HSF1) is known to associate with cellular response to divergent pathophysiological stresses including whole body hyperthermia (WBH) and ischemia-reperfusion. However, a direct cause-effect relationship between HSF1 activation and cytoprotection induced by myocardial preconditioning has not been conclusively established, mainly due to the limitations of available experiment tools. In the present studies, we used a novel approach to block HSF1 with small interfering RNA (siRNA) technique in vivo. Male adult ICR mice were treated intraperitoneally with amine (vehicle) or siRNA specific to HSF1 (siRNA-HSF1). Three days later, WBH preconditioning protocol (rectal temperature 42 degrees C for 15 min) was applied to these mice under light anesthesia. WBH preconditioning resulted in 2.7-fold and 3.4-fold increase in cardiac HSF1 mRNA and protein expression respectively 2 hours after WBH, which was inhibited in the siRNA-treated mice. The silencing effect of siRNA on HSF1 was associated with complete loss of the infarct- limiting protection by WBH preconditioning after 48 hours. Pretreatment with siRNA-HSF1 had no effect on infarct size in the sham control animals as compared with the amine-treated group. DNA micro-array analysis revealed that siRNA-HSF1 caused a general inhibition on multiple members of HSP family, except Hsp32, Hsp47 and Hsp60. In addition, the silencing effect of siRNA on HSF1 and HSPs gene expression was transient and its inhibitory effect disappeared by 10 days after treatment. siRNA-HSF1 also impaired the thermotolerance of the heat shocked mice as indicated by higher mortality following WBH. For the first time, we have applied siRNA technique in the field of myocardial preconditioning to demonstrate HSF1 activation as an essential step in WBH preconditioning against cardiac ischemia-reperfusion injury.

The chance of small interfering RNAs as eligible candidates for a personalized treatment of prostate cancer.

PubMed

Pietschke, Katharina; Walker, Tobias; Krajewski, Stefanie; Kurz, Julia; Aufderklamm, Stefan; Schwentner, Christian; Schlensak, Christian; Stenzl, Arnulf; Wendel, Hans P; Nolte, Andrea

2014-01-01

Prostate cancer is one of the leading malignant tumors in men. Current therapies are associated with severe side effects making it problematic for many multi-morbid patients to receive treatment. Prostate specific antigen, serum response factor (SRF), signal transducer and activator of transcription-3 (STAT3), hypoxia-inducible factor-1α (HIF-1α), HIF-2α, E2F1 and Survivin are well known proteins being overexpressed in cancer cells, expediting cell growth and also demonstrated in prostate cancer cells. Targeting these genes using the RNA-Interference pathway could be a new approach for prostate cancer therapy with fewer side effects. Three prostate cancer cell lines were cultured under standard conditions and transfected with three different concentrations (25 nM, 50 nM, 100 nM) of specific small interfering RNAs (siRNAs) targeting SRF, STAT3, HIF1α, HIF2α, E2F1 and Survivin in a non-viral manner. Cells treated with non-specific siRNA (SCR-siRNA) served as control. Changes of messenger RNA (mRNA) levels were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The analysis of the effect of siRNA on the number of cells was detected using CASY cell counter system. Transfections of the PC-3 cell line with specific siRNA especially against Survivin, E2F1, HIF1α- and HIF2α-siRNA resulted in a significant reduction of intracellular mRNA concentration together with a significant decreased number of cells. In the LnCAP and DU-145 cell lines Survivin and E2F1 showed similar effects. The impact of silencing STAT3 or SRF showed little influence on the amount of cells in all three cell lines. This study shows that RNAi succeeds in silencing gene expression and reducing the number of cells in differing dimensions depending on the transfected cell line and used siRNA.

Mechanisms of Estradiol-Induced Insulin Secretion by the G Protein-Coupled Estrogen Receptor GPR30/GPER in Pancreatic β-Cells

PubMed Central

Sharma, Geetanjali

2011-01-01

Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes. PMID:21673097

Silencing tumor necrosis factor-alpha in vitro from small interfering RNA-decorated titanium nanotube array can facilitate osteogenic differentiation of mesenchymal stem cells.

PubMed

Wang, Zhenlin; Hu, Zhiqiang; Zhang, Dawei; Zhuo, Mengchuan; Cheng, Jiwei; Xu, Xingping; Xing, Yongming; Fan, Jie

2016-01-01

Titanium implants are known for their bone bonding ability. However, the osseointegration may be severely disturbed in the inflammation environment. In order to enhance osseointegration of the implant in an inflamed environment, the small interfering RNA (siRNA) targeting tumor necrosis factor alpha (TNF-α) was used to functionalize titanium surface for gene silencing. The chitosan-tripolyphosphate-hyaluronate complexes were used to formulate nanoparticles (NPs) with siRNA, which were adsorbed directly by the anodized titanium surface. The surface characterization was analyzed by scanning electron microscope, atomic force microscopy, as well as contact angle measurement. The fluorescence microscope was used to monitor the degradation of the layer. The coculture system was established with mesenchymal stem cells (MSCs) grown directly on functionalized titanium surface and RAW264.7 cells (preactivated by lipopolysaccharide) grown upside in a transwell chamber. The transfection and knockdown efficiency of TNF-α in RAW264.7 cells were determined by fluorescence microscope, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. The cytoskeleton and osteogenic differentiation of MSCs were also analyzed. Regular vertical aligned nanotubes (~100 nm diameter and ~300 nm length) were generated after anodization of polished titanium. After loading with NPs, the nanotubes were filled and covered by a layer of amorphous particles. The surface topography changed and wettability decreased after covering with NPs. As expected, a burst degradation of the film was observed, which could provide sufficient NPs in the released supernatant and result in transfection and knockdown effects in RAW264.7 cells. The cytoskeleton arrangement of MSCs was elongated and the osteogenic differentiation was also significantly improved on NPs loading surface. In conclusion, the siRNA decorated titanium implant could simultaneously suppress inflammation and improve osteogenesis, which may be suitable for peri-implant bone formation under inflammatory conditions.

Mammalian genome-wide loss-of-function screens using arrayed small interfering RNA expression libraries.

PubMed

Zheng, Lianxing; Ding, Sheng

2004-04-01

Extract: RNA interference (RNAi), first discovered in Caenorhabdtitis elegans and now widely found and applied in a variety of organisms such as Drosophila, zebrafish and mammalian systems, has emerged to revolutionize the field of functional genomics by inducing specific and effective post-transcriptional gene silencing for loss-of-function studies. Mechanistic investigations of RNAi suggest that long double-stranded RNAs (dsRNAs) are first cleaved by the RNase III-like enzyme, Dicer, to 21-23 base pair (bp) small interfering RNAs (siRNAs). These siRNAs are resolved by ATP-dependent RNA helicase, and the resulting single-stranded RNAs are then incorporated into the RNA-induced silencing complex (RISC). The antisense strand of the siRNA duplex guides the RISC to the homologous mRNA, where the RISC-associated endoribonuclease cleaves the target mRNA, resulting in silencing of the target gene. The approach of using long dsRNA (up to 1-2 kb) in C. elegans and Drosophila to induce gene silencing cannot be similarly used in mammalian cells, where introduction of long dsRNA activates the dsRNA-dependent protein kinase PKR. PKR phosphorylates and inactivates the translation initiation factor eIF2, resulting in a non-specific gene-silencing effect. Development and implementation of the use of 21 to 23bp siRNAs, which can be prepared by chemical synthesis, in vitro transcription, or expressed in cells using siRNA expression systems, allows specific and effective gene silencing in mammalian cells to occur without activation of PKR.

Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice

PubMed Central

Overman, Jeroen; Fontaine, Frank; Moustaqil, Mehdi; Mittal, Deepak; Sierecki, Emma; Sacilotto, Natalia; Zuegg, Johannes; Robertson, Avril AB; Holmes, Kelly; Salim, Angela A; Mamidyala, Sreeman; Butler, Mark S; Robinson, Ashley S; Lesieur, Emmanuelle; Johnston, Wayne; Alexandrov, Kirill; Black, Brian L; Hogan, Benjamin M; De Val, Sarah; Capon, Robert J; Carroll, Jason S; Bailey, Timothy L; Koopman, Peter; Jauch, Ralf; Smyth, Mark J; Cooper, Matthew A; Gambin, Yann; Francois, Mathias

2017-01-01

Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics. DOI: http://dx.doi.org/10.7554/eLife.21221.001 PMID:28137359

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

NASA Astrophysics Data System (ADS)

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

2016-05-01

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

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

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

Skupin, Michalina; Sobczak, Krzysztof; Zieliński, Ryszard

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

Deconvolution of seed and RNA-binding protein crosstalk in RNAi-based functional genomics.

PubMed

Suzuki, Hiroshi I; Spengler, Ryan M; Grigelioniene, Giedre; Kobayashi, Tatsuya; Sharp, Phillip A

2018-05-01

RNA interference (RNAi) is a major, powerful platform for gene perturbations, but is restricted by off-target mechanisms. Communication between RNAs, small RNAs, and RNA-binding proteins (RBPs) is a pervasive feature of cellular RNA networks. We present a crosstalk scenario, designated as crosstalk with endogenous RBPs' (ceRBP), in which small interfering RNAs or microRNAs with seed sequences that overlap RBP motifs have extended biological effects by perturbing endogenous RBP activity. Systematic analysis of small interfering RNA (siRNA) off-target data and genome-wide RNAi cancer lethality screens using 501 human cancer cell lines, a cancer dependency map, identified that seed-to-RBP crosstalk is widespread, contributes to off-target activity, and affects RNAi performance. Specifically, deconvolution of the interactions between gene knockdown and seed-mediated silencing effects in the cancer dependency map showed widespread contributions of seed-to-RBP crosstalk to growth-phenotype modulation. These findings suggest a novel aspect of microRNA biology and offer a basis for improvement of RNAi agents and RNAi-based functional genomics.

Prognostic psychosocial factors for disabling low back pain in Japanese hospital workers.

PubMed

Yoshimoto, Takahiko; Oka, Hiroyuki; Katsuhira, Junji; Fujii, Tomoko; Masuda, Katsuhiko; Tanaka, Sakae; Matsudaira, Ko

2017-01-01

Although the occupational health field has identified psychosocial factors as risk factors for low back pain that causes disability, the association between disabling low back pain and psychosocial factors has not been examined adequately in Japanese hospital workers. Therefore, this study examined the association between low back pain, which interfered with work, and psychosocial factors in Japanese hospital workers. This cross-sectional study was conducted at a hospital in Japan. In total, 280 hospital workers were recruited from various occupational settings. Of these, 203 completed a self-administered questionnaire that included items concerning individual characteristics, severity of low back pain, fear-avoidance beliefs (Fear-Avoidance Beliefs Questionnaire), somatic symptoms (Somatic Symptom Scale-8), psychological distress (K6), workaholism, and work-related psychosocial factors (response rate: 72.5%). Logistic regression was used to explore risk factors associated with disabling low back pain. Of the 203 participants who completed questionnaires, 36 (17.7%) reported low back pain that interfered with their work. Multivariate analyses with individual factors and occupations adjusted for showed statistically significant associations between disabling low back pain and fear-avoidance beliefs (adjusted odds ratio [OR]: 2.619, 95% confidence interval [CI]: 1.003-6.538], somatic symptoms (OR: 4.034, 95% CI: 1.819-9.337), and interpersonal stress at work (OR: 2.619, 95% CI: 1.067-6.224). Psychosocial factors, such as fear-avoidance beliefs, somatic symptoms, and interpersonal relationships at work, were important risk factors in low back pain that interfered with work in Japanese hospital workers. With respect to occupational health, consideration of psychosocial factors is required to reduce disability related to low back pain.

Prognostic psychosocial factors for disabling low back pain in Japanese hospital workers

PubMed Central

Yoshimoto, Takahiko; Oka, Hiroyuki; Katsuhira, Junji; Fujii, Tomoko; Masuda, Katsuhiko; Tanaka, Sakae; Matsudaira, Ko

2017-01-01

Background Although the occupational health field has identified psychosocial factors as risk factors for low back pain that causes disability, the association between disabling low back pain and psychosocial factors has not been examined adequately in Japanese hospital workers. Therefore, this study examined the association between low back pain, which interfered with work, and psychosocial factors in Japanese hospital workers. Method This cross-sectional study was conducted at a hospital in Japan. In total, 280 hospital workers were recruited from various occupational settings. Of these, 203 completed a self-administered questionnaire that included items concerning individual characteristics, severity of low back pain, fear-avoidance beliefs (Fear-Avoidance Beliefs Questionnaire), somatic symptoms (Somatic Symptom Scale-8), psychological distress (K6), workaholism, and work-related psychosocial factors (response rate: 72.5%). Logistic regression was used to explore risk factors associated with disabling low back pain. Results Of the 203 participants who completed questionnaires, 36 (17.7%) reported low back pain that interfered with their work. Multivariate analyses with individual factors and occupations adjusted for showed statistically significant associations between disabling low back pain and fear-avoidance beliefs (adjusted odds ratio [OR]: 2.619, 95% confidence interval [CI]: 1.003–6.538], somatic symptoms (OR: 4.034, 95% CI: 1.819–9.337), and interpersonal stress at work (OR: 2.619, 95% CI: 1.067–6.224). Conclusions Psychosocial factors, such as fear-avoidance beliefs, somatic symptoms, and interpersonal relationships at work, were important risk factors in low back pain that interfered with work in Japanese hospital workers. With respect to occupational health, consideration of psychosocial factors is required to reduce disability related to low back pain. PMID:28531194

[Effect of qinghuobaiduyin on the expression of high mobility group box chromosomal protein 1 in macrophage].

PubMed

Li, Ping; Xu, Dan; Luo, Chengqun

2010-07-01

To observe the expression of high mobility group box chromosomal protein 1(HMGB1) in RAW264.7 macrophages after interfering with burning serum and qinghuobaidu-yin (QHBDY), and to find out the endogenous protection mechanism of QHBDY resisting inflammation reaction. RT-PCR was used to detect the expression of HMGB1 in RAW264.7 macrophages after interfering RAW264.7 macrophages with normal SD rat serum, burning SD rat serum, and QHBDY feeding SD rat serum. Small quantity of HMGB1 mRNA was expressed in RAW264.7. The expression of HMGB1 mRNA fluctuated around the standard level after interfering with normal serum of SD rats. The expression of HMGB1 mRNA rose at 3 h, and then decreased to the standard level; at 18 h, it rose rapidly; at 36 h, it reached the peak; and at 48 h, it remained at the high level after interfering with burning serum. The expression of HMGB1 mRNA increased at 3 h, and then decreased to the standard level. At 24 h, it started to rise after interfering with herb serum, and was lower than that of; the burning serum group (P<0.05). Burning serum can increase the expression of HMGB1 mRNA in RAW264.7. QHBDY can decrease the high expression of HMGB1 mRNA in RAW264.7 caused by burning serum.

Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1

PubMed Central

Schalk, Catherine; Cognat, Valérie; Graindorge, Stéfanie; Vincent, Timothée; Voinnet, Olivier; Molinier, Jean

2017-01-01

As photosynthetic organisms, plants need to prevent irreversible UV-induced DNA lesions. Through an unbiased, genome-wide approach, we have uncovered a previously unrecognized interplay between Global Genome Repair and small interfering RNAs (siRNAs) in the recognition of DNA photoproducts, prevalently in intergenic regions. Genetic and biochemical approaches indicate that, upon UV irradiation, the DNA DAMAGE-BINDING PROTEIN 2 (DDB2) and ARGONAUTE 1 (AGO1) of Arabidopsis thaliana form a chromatin-bound complex together with 21-nt siRNAs, which likely facilitates recognition of DNA damages in an RNA/DNA complementary strand-specific manner. The biogenesis of photoproduct-associated siRNAs involves the noncanonical, concerted action of RNA POLYMERASE IV, RNA-DEPENDENT RNA POLYMERASE-2, and DICER-LIKE-4. Furthermore, the chromatin association/dissociation of the DDB2-AGO1 complex is under the control of siRNA abundance and DNA damage signaling. These findings reveal unexpected nuclear functions for DCL4 and AGO1, and shed light on the interplay between small RNAs and DNA repair recognition factors at damaged sites. PMID:28325872

Riding in silence: a little snowboarding, a lot of small RNAs

PubMed Central

2010-01-01

The recent symposium, RNA silencing: Mechanism, Biology and Applications, organized by Phillip D. Zamore (University of Massachusetts Medical School) and Beverly Davidson (University of Iowa), and held in Keystone, Colorado, brought together scientists working on diverse aspects of RNA silencing, a field that comprises a multitude of gene regulatory pathways guided by microRNAs, small interfering RNAs and PIWI-interacting RNAs. PMID:20230614

Ultrathin epidermal strain sensor based on an elastomer nanosheet with an inkjet-printed conductive polymer

NASA Astrophysics Data System (ADS)

Tetsu, Yuma; Yamagishi, Kento; Kato, Akira; Matsumoto, Yuya; Tsukune, Mariko; Kobayashi, Yo; Fujie, Masakatsu G.; Takeoka, Shinji; Fujie, Toshinori

2017-08-01

To minimize the interference that skin-contact strain sensors cause natural skin deformation, physical conformability to the epidermal structure is critical. Here, we developed an ultrathin strain sensor made from poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) inkjet-printed on a polystyrene-polybutadiene-polystyrene (SBS) nanosheet. The sensor, whose total thickness and gauge factor were ˜1 µm and 0.73 ± 0.10, respectively, deeply conformed to the epidermal structure and successfully detected the small skin strain (˜2%) while interfering minimally with the natural deformation of the skin. Such an epidermal strain sensor will open a new avenue for precisely detecting the motion of human skin and artificial soft-robotic skin.

Small interfering RNA targeting nuclear factor kappa B to prevent vein graft stenosis in rat models.

PubMed

Meng, X B; Bi, X L; Zhao, H L; Feng, J B; Zhang, J P; Song, G M; Sun, W Y; Bi, Y W

2013-01-01

Intimal hyperplasia plays an important role in vein graft stenosis. Inflammatory injury, especially nuclear factor kappaB (NF-κB) gene activation, is highly involved in stenosis progression. We examined whether neointimal hyperplasia and vein graft stenosis could be inhibited by silencing the NF-κB gene with small interference RNA (siRNA). Sixty adult male Sprague-Dawley rats were randomly divided into a normal vein group, a vein graft group, a scrambled siRNA group, and an NF-κB siRNA group. We performed reverse interpositional grafting of the autologous external jugular vein to the abdominal aorta. Vein grafts were treated with liposome and gel complexes containing NF-κB siRNA or scrambled siRNA. The levels of monocyte chemoattractant protein -1, tumor necrosis factor-α, and NF-κB p65 in vessel tissues were evaluated after surgery for content of proliferating cell nuclear antigen (PCNA) and vascular wall thickness. NF-κB siRNA treated vein graft showed less neointimal formation and fewer positive PCNA cells (P < .05). In addition there were lower levels of, NF-κB p65 protein and of inflammatory mediators (P < .05) compared with the vein graft group. Our study suggested that siRNA transfection suppressed NF-κB expression, reduced inflammatory factors, lessened neointimal proliferation, and suppressed PCNA. Copyright © 2013 Elsevier Inc. All rights reserved.

Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2.

PubMed

Wyhs, Nicolas; Walker, David; Giovinazzo, Hugh; Yegnasubramanian, Srinivasan; Nelson, William G

2014-08-01

Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z' factor = 0.58) emerged as a superior screening strategy compared with FP (Z' factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET-based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions. © 2014 Society for Laboratory Automation and Screening.

Rapid and Efficient Isolation of High-Quality Small RNAs from Recalcitrant Plant Species Rich in Polyphenols and Polysaccharides

PubMed Central

Pu, Jinji; Guo, Jianrong; Fan, Zaifeng

2014-01-01

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are important regulators of plant development and gene expression. The acquisition of high-quality small RNAs is the first step in the study of its expression and function analysis, yet the extraction method of small RNAs in recalcitrant plant tissues with various secondary metabolites is not well established, especially for tropical and subtropical plant species rich in polysaccharides and polyphenols. Here, we developed a simple and efficient method for high quality small RNAs extraction from recalcitrant plant species. Prior to RNA isolation, a precursory step with a CTAB-PVPP buffer system could efficiently remove compounds and secondary metabolites interfering with RNAs from homogenized lysates. Then, total RNAs were extracted by Trizol reagents followed by a differential precipitation of high-molecular-weight (HMW) RNAs using polyethylene glycol (PEG) 8000. Finally, small RNAs could be easily recovered from supernatant by ethanol precipitation without extra elimination steps. The isolated small RNAs from papaya showed high quality through a clear background on gel and a distinct northern blotting signal with miR159a probe, compared with other published protocols. Additionally, the small RNAs extracted from papaya were successfully used for validation of both predicted miRNAs and the putative conserved tasiARFs. Furthermore, the extraction method described here was also tested with several other subtropical and tropical plant tissues. The purity of the isolated small RNAs was sufficient for such applications as end-point stem-loop RT-PCR and northern blotting analysis, respectively. The simple and feasible extraction method reported here is expected to have excellent potential for isolation of small RNAs from recalcitrant plant tissues rich in polyphenols and polysaccharides. PMID:24787387

Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC

PubMed Central

Castanotto, Daniela; Sakurai, Kumi; Lingeman, Robert; Li, Haitang; Shively, Louise; Aagaard, Lars; Soifer, Harris; Gatignol, Anne; Riggs, Arthur; Rossi, John J.

2007-01-01

Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs. PMID:17660190

Podocalyxin EBP50 Ezrin Molecular Complex Enhances the Metastatic Potential of Renal Cell Carcinoma Through Recruiting Rac1 Guanine Nucleotide Exchange Factor ARHGEF7

PubMed Central

Hsu, Yung-Ho; Lin, Wei-Ling; Hou, Yi-Ting; Pu, Yeong-Shiau; Shun, Chia-Tung; Chen, Chi-Ling; Wu, Yih-Yiing; Chen, Jen-Yau; Chen, Tso-Hsiao; Jou, Tzuu-Shuh

2010-01-01

Podocalyxin was initially identified in glomerular podocytes to critically maintain the structural and functional integrity of the glomerular ultrafiltrative apparatus. Lately, it has emerged as a malignant marker in tumors arising from a variety of tissue origins. By immunohistochemistry, we identified that 9.6% of renal cell carcinoma patients overexpress this protein. This subset of patients had significantly shorter disease-specific and overall survivals, and, importantly, we established podocalyxin overexpression as an independent prognostic factor for latent distant metastasis with multivariate analysis. Podocalyxin down-regulation by small interfering RNA led to defective migration in model renal tubular cells, which was corrected by re-expression of podocalyxin. The activity of the small GTPase Rac1, a well-characterized modulator of cell migration, was diminished by podocalyxin knock-down. Conversely, podocalyxin overexpression in human embryonic kidney cells up-regulated Rac1 activity, which depended on a complex formed by podocalyxin, ERM-binding phosphoprotein 50, ezrin, and ARHGEF7, a Rac1 activator. Therefore, podocalyxin can serve as a biomarker to identify renal cell carcinoma patients with higher metastatic potential for more aggressive intervention at earlier clinical stages. PMID:20395446

Small interfering RNA-mediated down-regulation of caveolin-1 differentially modulates signaling pathways in endothelial cells.

PubMed

Gonzalez, Eva; Nagiel, Aaron; Lin, Alison J; Golan, David E; Michel, Thomas

2004-09-24

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules in endothelial cells. To explore the role of this protein in receptor-modulated signaling pathways, we transfected bovine aortic endothelial cells (BAEC) with small interfering RNA (siRNA) duplexes to down-regulate caveolin-1 expression. Transfection of BAEC with duplex siRNA targeted against caveolin-1 mRNA selectively "knocked-down" the expression of caveolin-1 by approximately 90%, as demonstrated by immunoblot analyses of BAEC lysates. We used discontinuous sucrose gradients to purify caveolin-containing lipid rafts from siRNA-treated endothelial cells. Despite the near-total down-regulation of caveolin-1 expression, the lipid raft targeting of diverse signaling proteins (including the endothelial isoform of nitric-oxide synthase, Src-family tyrosine kinases, Galphaq and the insulin receptor) was unchanged. We explored the consequences of caveolin-1 knockdown on kinase pathways modulated by the agonists sphingosine-1 phosphate (S1P) and vascular endothelial growth factor (VEGF). siRNA-mediated caveolin-1 knockdown enhanced basal as well as S1P- and VEGF-induced phosphorylation of the protein kinase Akt and did not modify the basal or agonist-induced phosphorylation of extracellular signal-regulated kinases 1/2. Caveolin-1 knock-down also significantly enhanced the basal and agonist-induced activity of the small GTPase Rac. We used siRNA to down-regulate Rac expression in BAEC, and we observed that Rac knockdown significantly reduced basal, S1P-, and VEGF-induced Akt phosphorylation, suggesting a role for Rac activation in the caveolin siRNA-mediated increase in Akt phosphorylation. By using siRNA to knockdown caveolin-1 and Rac expression in cultured endothelial cells, we have found that caveolin-1 does not seem to be required for the targeting of signaling molecules to caveolae/lipid rafts and that caveolin-1 differentially modulates specific kinase pathways in endothelial cells. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.

Class I ADP-Ribosylation Factors Are Involved in Enterovirus 71 Replication

PubMed Central

Wang, Jianmin; Du, Jiang; Jin, Qi

2014-01-01

Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies. PMID:24911624

[Atelocollagen-mediated small interfering RNA delivery for effective gene silencing in rat vein grafts].

PubMed

Qiu, Xue-feng; Dong, Nian-guo; Sun, Zong-quan; Su, Wei; Shi, Jia-wei

2009-07-01

To evaluate the efficacy of using small interfering RNA targeting TF as a therapy for vein graft failure. External jugular vein to carotid artery interposition vein grafts, which were applied to a low flow condition, were made in 120 Sprague-Dawley rats weighing 260 to 300 g. These rats were randomly divided into 4 groups, 30 rats each group. Group A was atelocollagen-TF Stealth Select RNAi group. Group B was atelocollagen-TF Stealth RNAi group. Group C was atelocollagen group. Group D was control group. Small interfering RNA mixed with atelocollagen was administrated to the external wall of grafted veins. The TF protein expression of vein grafts was analyzed by Western blot at 1, 3, 7, 14, and 28 d postoperatively, and by immunochemistry at 3 d postoperatively. The proliferation index was determined at 14 d postoperatively. Neointimal hyperplasia was evaluated at 28 d postoperatively. BLOCK-iT fluorescent oligo was used to confirm its stability and successful transfer into the vein graft wall at 3 and 7 d postoperatively for another group (n=12). Fluorescence of BLOCK-iT fluorescent oligo could be detected in the graft wall even at 7 d postoperatively. Knockdown of the TF expression was achieved by perivascular application of siRNA using atelocollagen. Compared with control group, the intima thickness at 28 d after grafting was significantly reduced (P < 0.05). This phenomenon was preceded by significant reduction of cell proliferation in siRNA-treated grafts at 14 d postoperatively (P < 0.05). The expression of TF in vein grafts can be effectively inhibited by specific siRNAs using a atelocollagen-based nonviral delivery approach in vivo, so that the neointimal thickening can be prevented. Transplants;

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-05-01

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

Inhibition of U snRNP assembly by a virus-encoded proteinase.

PubMed

Almstead, Laura L; Sarnow, Peter

2007-05-01

It has been proposed that defects in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes could account for the death of motor neurons in spinal muscular atrophy (SMA). We discovered that infection of cultured cells with poliovirus results in the specific cleavage of the host factor Gemin3 by a virus-encoded proteinase, 2A(pro). Gemin3 is a component of the macromolecular SMN complex that mediates assembly of U snRNP complexes by aiding the heptameric oligomerization of Sm proteins onto U snRNAs. Using in vitro Sm core assembly assays, we found that lowering the intracellular amounts of Gemin3 by either poliovirus infection or small interfering RNA (siRNA)-mediated knockdown of Gemin3 resulted in reduced assembly of U snRNPs. Immunofluorescence analyses revealed a specific redistribution of Sm proteins from the nucleoplasm to the cytoplasmic periphery of the nucleus in poliovirus-infected cells. We propose that defects in U snRNP assembly may be shared features of SMA and poliomyelitis.

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

PubMed Central

de Wilde, Adriaan H.; Wannee, Kazimier F.; Scholte, Florine E. M.; Goeman, Jelle J.; ten Dijke, Peter; Snijder, Eric J.

2015-01-01

ABSTRACT To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although substantial progress has been made in dissecting the coronavirus replicative cycle, our understanding of the host factors that stimulate (proviral factors) or restrict (antiviral factors) infection remains far from complete. To study the role of host proteins in SARS-CoV infection, we set out to systematically identify kinase-regulated processes that influence virus replication. Protein kinases are key regulators in signal transduction, controlling a wide variety of cellular processes, and many of them are targets of approved drugs and other compounds. Our screen identified a variety of hits and will form the basis for more detailed follow-up studies that should contribute to a better understanding of SARS-CoV replication and coronavirus-host interactions in general. The identified factors could be interesting targets for the development of host-directed antiviral therapy to treat infections with SARS-CoV or other pathogenic coronaviruses. PMID:26041291

Poly(alkylene oxide) Copolymers for Nucleic Acid Delivery

DTIC Science & Technology

2012-07-17

biofilm infection treatments, pain control and cancer chemotherapy. Charles M. Roth is an Associate Professor in the Department of Chemical and...technology and engineering approaches to cancer . REFERENCES 1. Aigner A. Nonviral in vivo delivery of therapeutic small interfering RNAs. Curr Opin Mol Ther

Effective mRNA Inhibition in PANC-1 Cells in Vitro Mediated via an mPEG-SeSe-PEI Delivery System.

PubMed

Zhang, Yuefeng; Yang, Bin; Liu, Yajie; Qin, Wenjie; Li, Chao; Wang, Lantian; Zheng, Wen; Wu, Yulian

2016-05-01

RNA interference (RNAi)-mediated gene therapy is a promising approach to cure various diseases. However, developing an effective, safe, specific RNAi delivery system remains a major challenge. In this study, a novel redox-responsive polyetherimide (PEI)-based nanovector, mPEG-SeSe-PEI, was developed and its efficacy evaluated. We prepared three mPEG-SeSe-PEI vector candidates for small interfering glyceraldehyde-3-phosphate dehydrogenase (siGADPH) and determined their physiochemical properties and transfection efficiency using flow cytometry and PEG11.6-SeSe-PEI polymer. We investigated the silencing efficacy of GADPH mRNA expression in PANC-1 cells and observed that PEG11.6-SeSe-PEI/siGADPH (N/P ratio=10) polyplexes possessed the appropriate size and zeta-potential and exhibited excellent in vitro gene silencing effects with the least cytotoxicity in PANC-1 cells. In conclusion, we present PEG11.6-SeSe-PEI as a potential therapeutic gene delivery system for small interfering RNA (siRNA).

Silencing of cytosolic NADP(+)-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine.

PubMed

Lee, Su-Min; Park, Sin Young; Shin, Seoung Woo; Kil, In Sup; Yang, Eun Sun; Park, Jeen-Woo

2009-02-01

Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.

Functionalization of carbon nanotubes enables non-covalent binding and intracellular delivery of small interfering RNA for efficient knock-down of genes.

PubMed

Krajcik, Rasti; Jung, Adrian; Hirsch, Andreas; Neuhuber, Winfried; Zolk, Oliver

2008-05-02

The lipophilic nature of biological membranes restricts the direct intracellular delivery of potential drugs and molecular probes and makes intracellular transport one of the key problems in gene therapy. Because of their ability to cross cell membranes, single walled carbon nanotubes (SWNTs) are of interest as carriers of biologically active molecules, such as small interfering RNAs (siRNAs). We developed a strategy for chemical functionalization of SWNTs with hexamethylenediamine (HMDA) and poly(diallyldimethylammonium)chloride (PDDA) to obtain a material that was able to bind negatively charged siRNA by electrostatic interactions. PDDA-HMDA-SWNTs exhibited negligible cytotoxic effects on isolated rat heart cells at concentrations up to 10mg/l. PDDA-HMDA-SWNTs loaded with extracellular signal-regulated kinase (ERK) siRNA were able to cross the cell membrane and to suppress expression of the ERK target proteins in primary cardiomyocytes by about 75%. PDDA-functionalized SWNTs thus present an effective carrier system for applications in siRNA-mediated gene silencing.

Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA

NASA Astrophysics Data System (ADS)

Woodrow, Kim A.; Cu, Yen; Booth, Carmen J.; Saucier-Sawyer, Jennifer K.; Wood, Monica J.; Mark Saltzman, W.

2009-06-01

Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protection against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternative approach using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing, we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA to the vaginal mucosa.

A novel dendritic nanocarrier of polyamidoamine-polyethylene glycol-cyclic RGD for “smart” small interfering RNA delivery and in vitro antitumor effects by human ether-à-go-go-related gene silencing in anaplastic thyroid carcinoma cells

PubMed Central

Li, Guanhua; Hu, Zuojun; Yin, Henghui; Zhang, Yunjian; Huang, Xueling; Wang, Shenming; Li, Wen

2013-01-01

The application of RNA interference techniques is promising in gene therapeutic approaches, especially for cancers. To improve safety and efficiency of small interfering RNA (siRNA) delivery, a triblock dendritic nanocarrier, polyamidoamine-polyethylene glycol-cyclic RGD (PAMAM-PEG-cRGD), was developed and studied as an siRNA vector targeting the human ether-à-go-go-related gene (hERG) in human anaplastic thyroid carcinoma cells. Structure characterization, particle size, zeta potential, and gel retardation assay confirmed that complete triblock components were successfully synthesized with effective binding capacity of siRNA in this triblock nanocarrier. Cytotoxicity data indicated that conjugation of PEG significantly alleviated cytotoxicity when compared with unmodified PAMAM. PAMAM-PEG-cRGD exerted potent siRNA cellular internalization in which transfection efficiency measured by flow cytometry was up to 68% when the charge ratio (N/P ratio) was 3.5. Ligand-receptor affinity together with electrostatic interaction should be involved in the nano-siRNA endocytosis mechanism and we then proved that attachment of cRGD enhanced cellular uptake via RGD-integrin recognition. Gene silencing was evaluated by reverse transcription polymerase chain reaction and PAMAM-PEG-cRGD-siRNA complex downregulated the expression of hERG to 26.3% of the control value. Furthermore, gene knockdown of hERG elicited growth suppression as well as activated apoptosis by means of abolishing vascular endothelial growth factor secretion and triggering caspase-3 cascade in anaplastic thyroid carcinoma cells. Our study demonstrates that this novel triblock polymer, PAMAM-PEG-cRGD, exhibits negligible cytotoxicity, effective transfection, “smart” cancer targeting, and therefore is a promising siRNA nanocarrier. PMID:23569377

Radiation-induced interleukin-6 expression through MAPK/p38/NF-kappaB signaling pathway and the resultant antiapoptotic effect on endothelial cells through Mcl-1 expression with sIL6-Ralpha.

PubMed

Chou, Chia-Hung; Chen, Shee-Uan; Cheng, Jason Chia-Hsien

2009-12-01

To investigate the mechanism of interleukin-6 (IL-6) activity induced by ionizing radiation. Human umbilical vascular endothelial cells (HUVECs) were irradiated with different doses to induce IL-6. The IL-6 promoter assay and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to examine transcriptional regulation. Specific chemical inhibitors, decoy double-stranded oligodeoxynucleotides, and Western blotting were conducted to investigate the signal transduction pathway. Recombinant soluble human IL-6 receptor alpha-chain (sIL6-Ralpha) and specific small interfering RNA were used to evaluate the biologic function of radiation-induced IL-6. Four grays of radiation induced the highest level of IL-6 protein. The promoter assay and RT-PCR data revealed that the induction of IL-6 was mediated through transcriptional regulation. The p38 inhibitor SB203580, by blocking nuclear factor-kappaB (NF-kappaB) activation, prevented both the transcriptional and translational regulation of radiation-induced IL-6 expression. The addition of sIL6-Ralpha rescued HUVECs from radiation-induced death in an IL-6 concentratio-dependent manner. The antiapoptotic effect of combined sIL6-Ralpha and radiation-induced IL-6 was inhibited by mcl-1-specific small interfering RNA. Radiation transcriptionally induces IL-6 expression in endothelial cells through mitogen-activated protein kinase/p38-mediated NF-kappaB/IkappaB (inhibitor of NF-kappaB) complex activation. In the presence of sIL6-Ralpha, radiation-induced IL-6 expression acts through Mcl-1 expression to rescue endothelial cells from radiation-induced death.

Targets of small interfering RNA restriction during human immunodeficiency virus type 1 replication.

PubMed

Gao, Yong; Lobritz, Michael A; Roth, Justin; Abreha, Measho; Nelson, Kenneth N; Nankya, Immaculate; Moore-Dudley, Dawn M; Abraha, Awet; Gerson, Stanton L; Arts, Eric J

2008-03-01

Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5alpha did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5' of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3'-to-5' siRNA amplification and spreading. In contrast, HIV-1 RNA 3' of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively "naked" cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5alpha, or other host restriction factors.

Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

PubMed

Zhang, Jia-Hua; He, Yan-Li; Zhu, Rui; Du, Wen; Xiao, Jun-Hua

2017-06-01

Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.

Inhibition of Midkine Suppresses Prostate Cancer CD133+ Stem Cell Growth and Migration.

PubMed

Erdogan, Suat; Doganlar, Zeynep B; Doganlar, Oguzhan; Turkekul, Kader; Serttas, Riza

2017-09-01

Midkine (MDK) is a tumor-promoting factor that is often overexpressed in various human carcinomas, and the role of MDK has not yet been fully investigated in prostate cancer stem cells. Prostate cancer CD133 + stem cells (PCSCs) were isolated from human castration-resistant PC3 cells. PCSCs were treated with different concentrations of MDK inhibitor, iMDK, for 24-72 hours. The IC 50 values were determined by the MTT test. Endogenous MDK messenger RNA expression was knocked down by small interfering RNA. Quantitative reverse transcription polymerase chain reaction, Western blot analyses and image-based cytometry were used to investigate apoptosis and cell cycle progression as well as their underlying molecular mechanisms. Cell migration was evaluated by the wound healing test. iMDK caused dose- and time-dependent inhibition of PCSC survival. Similar growth inhibition was also obtained by small interfering RNA-mediated knockdown of endogenous MDK expression. iMDK was shown to preferentially induce cell cycle arrest at the S and G2/M phases. Suppressed PCSC growth was also accompanied by increases in p53 and the cell cycle inhibitor p21 genes. Combinatorial treatment of iMDK with docetaxel significantly inhibited cell proliferation versus either of the agents used alone. Inhibition of MDK expression strongly suppressed the migration of PCSCs compared to untreated and docetaxel-treated cells. iMDK and the knockdown of MDK decreased p-Akt and significantly upregulated the expression of PI3K/phosphatase/tensin homolog. Our data indicate that MDK plays a crucial role in controlling PCSC proliferation and migration. Therefore, suppression of endogenous expression of MDK would, in combination with traditional chemotherapy drugs, be a potential treatment for PCSCs. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth muscle cell migration.

PubMed

Ashino, Takashi; Sudhahar, Varadarajan; Urao, Norifumi; Oshikawa, Jin; Chen, Gin-Fu; Wang, Huan; Huo, Yuqing; Finney, Lydia; Vogt, Stefan; McKinney, Ronald D; Maryon, Edward B; Kaplan, Jack H; Ushio-Fukai, Masuko; Fukai, Tohru

2010-09-17

Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

PubMed

Odemuyiwa, Solomon O; Ilarraza, Ramses; Davoine, Francis; Logan, Michael R; Shayeganpour, Anooshirvan; Wu, Yingqi; Majaesic, Carina; Adamko, Darryl J; Moqbel, Redwan; Lacy, Paige

2015-04-01

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation. © 2014 John Wiley & Sons Ltd.

The Use of MS-based Metabolomics to Determine Markers Associated with Endocrine Disruption in Small Fish Species

EPA Science Inventory

Endocrine disrupting chemicals (EDCs) are exogenous substances that disrupt the physiological function of endogenous hormones. In fish, these xenobiotics are capable of interfering with the dynamic equilibrium of the hypothalamic-pituitary-gonadal (HPG) axis resulting in adverse ...

RNAi control of aflatoxins in peanut plants, a multifactorial system

USDA-ARS?s Scientific Manuscript database

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

Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs

PubMed Central

Park, Jong-Hyeon; Lee, Kwang-Nyeong; Kim, Se-Kyung; You, Su-Hwa; Kim, Taeseong; Tark, Dongseob; Lee, Hyang-Sim; Seo, Min-Goo; Kim, Byounghan

2015-01-01

ABSTRACT Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-αγ and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-αγ and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD. IMPORTANCE The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against FMDV, although the virus has associated mechanisms of resistance to type I interferons and siRNAs. We have developed recombinant adenoviruses for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) to enhance the inhibitory effects of these antiviral agents observed in previous studies. Here, we show enhanced antiviral effects against FMDV by combination treatment with Ad-porcine IFN-αγ and Ad-3siRNA to overcome the mechanisms of resistance of FMDV in swine. PMID:26041279

Innate and intrinsic antiviral immunity in Drosophila

PubMed Central

Mussabekova, Assel; Daeffler, Laurent; Imler, Jean-Luc

2017-01-01

The fruitfly Drosophila melanogaster has been a valuable model to investigate the genetic mechanisms of innate immunity. Initially focused on the resistance to bacteria and fungi, these studies have been extended to include antiviral immunity over the last decade. Like all living organisms, insects are continually exposed to viruses and have developed efficient defense mechanisms. We review here our current understanding on antiviral host-defense in fruit flies. A major antiviral defense in Drosophila is RNA interference, in particular the small interfering (si) RNA pathway. In addition, complex inducible responses and restriction factors contribute to the control of infections. Some of the genes involved in these pathways have been conserved through evolution, highlighting loci that may account for susceptibility to viral infections in humans. Other genes are not conserved and represent species-specific innovations. PMID:28102430

Innate and intrinsic antiviral immunity in Drosophila.

PubMed

Mussabekova, Assel; Daeffler, Laurent; Imler, Jean-Luc

2017-06-01

The fruit fly Drosophila melanogaster has been a valuable model to investigate the genetic mechanisms of innate immunity. Initially focused on the resistance to bacteria and fungi, these studies have been extended to include antiviral immunity over the last decade. Like all living organisms, insects are continually exposed to viruses and have developed efficient defense mechanisms. We review here our current understanding on antiviral host defense in fruit flies. A major antiviral defense in Drosophila is RNA interference, in particular the small interfering (si) RNA pathway. In addition, complex inducible responses and restriction factors contribute to the control of infections. Some of the genes involved in these pathways have been conserved through evolution, highlighting loci that may account for susceptibility to viral infections in humans. Other genes are not conserved and represent species-specific innovations.

The gene for human U2 snRNP auxiliary factor small 35-kDa subunit (U2AF1) maps to the progressive myoclonus epilepsy (EPM1) critical region on chromosome 21q22.3

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

Lalioti, M.D.; Rossier, C.; Antonarakis, S.E.

1996-04-15

We used targeted exon trapping to clone portions of genes from human chromosome 21q22.3. One trapped sequence showed complete homology with the cDNA of human U2AF{sup 35} (M96982; HGM-approved nomenclature U2AF1), which encodes for the small 35-kDa subunit of the U2 snRNP auxiliary factor. Using the U2AF1 cDNA as a probe, we mapped this gene to cosmid Q15D2, a P1, and YAC 350F7 of the Chumakov et al. contig, close to the cystathionine-{beta}-synthase gene (CBS) on 21q22.3. This localization was confirmed by PCR using oligonucleotides from the 3{prime} UTR and by FISH. As U2AF1 associated with a number of differentmore » factors during mRNA splicing, overexpression in trisomy 21 individuals could contribute to some Down syndrome phenotypes by interfering with the splicing process. Furthermore, because this gene maps in the critical region for the progressive myoclonus epilepsy I locus (EPM1), mutation analysis will be carried out in patients to evaluate the potential role of U2AF1 as a candidate for EPM1. 24 refs., 1 fig.« less

Selenium is a Chemotherapeutic Agent for the Treatment of Prostate Cancer

DTIC Science & Technology

2006-02-01

receptor activity by a hammerhead ribozyme . Mol Endrocrinol 1998;12:1558-1566. 10. Ko YJ, Devi GR, London CA, et al. Androgen receptor down-regulation...Strategic targeting of the AR with ribozymes , antisense oligomers, and small interfering RNAs has been shown to significantly inhibit prostate cancer

Selenium is a Chemotherapeutic Agent for the Treatment of Prostate Cancer

DTIC Science & Technology

2007-12-01

to the AR without decreasing AR levels. Strategic targeting of the AR with ribozymes , antisense oligomers, and small interfering RNAs has been shown... ribozyme . Mol Endrocrinol 1998;12:1558-1566. 20 10. Ko YJ, Devi GR, London CA, et al. Androgen receptor down-regulation in prostate cancer with

California mild CTV strains that break resistance in Trifoliate Orange

USDA-ARS?s Scientific Manuscript database

This is the final report of a project to characterize California isolates of Citrus tristeza virus (CTV) that replicate in Poncirus trifoliata (trifoliate orange). Next Generation Sequencing (NGS) of viral small interfering RNAs (siRNAs) and assembly of full-length sequences of mild California CTV i...

Genetic and Functional Diversification of Small RNA Pathways in Plants

PubMed Central

Gustafson, Adam M; Kasschau, Kristin D; Lellis, Andrew D; Zilberman, Daniel; Jacobsen, Steven E

2004-01-01

Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense. PMID:15024409

Computational Prediction and Validation of BAHD1 as a Novel Molecule for Ulcerative Colitis

NASA Astrophysics Data System (ADS)

Zhu, Huatuo; Wan, Xingyong; Li, Jing; Han, Lu; Bo, Xiaochen; Chen, Wenguo; Lu, Chao; Shen, Zhe; Xu, Chenfu; Chen, Lihua; Yu, Chaohui; Xu, Guoqiang

2015-07-01

Ulcerative colitis (UC) is a common inflammatory bowel disease (IBD) producing intestinal inflammation and tissue damage. The precise aetiology of UC remains unknown. In this study, we applied a rank-based expression profile comparative algorithm, gene set enrichment analysis (GSEA), to evaluate the expression profiles of UC patients and small interfering RNA (siRNA)-perturbed cells to predict proteins that might be essential in UC from publicly available expression profiles. We used quantitative PCR (qPCR) to characterize the expression levels of those genes predicted to be the most important for UC in dextran sodium sulphate (DSS)-induced colitic mice. We found that bromo-adjacent homology domain (BAHD1), a novel heterochromatinization factor in vertebrates, was the most downregulated gene. We further validated a potential role of BAHD1 as a regulatory factor for inflammation through the TNF signalling pathway in vitro. Our findings indicate that computational approaches leveraging public gene expression data can be used to infer potential genes or proteins for diseases, and BAHD1 might act as an indispensable factor in regulating the cellular inflammatory response in UC.

Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons.

PubMed

Welsbie, Derek S; Mitchell, Katherine L; Jaskula-Ranga, Vinod; Sluch, Valentin M; Yang, Zhiyong; Kim, Jessica; Buehler, Eugen; Patel, Amit; Martin, Scott E; Zhang, Ping-Wu; Ge, Yan; Duan, Yukan; Fuller, John; Kim, Byung-Jin; Hamed, Eman; Chamling, Xitiz; Lei, Lei; Fraser, Iain D C; Ronai, Ze'ev A; Berlinicke, Cynthia A; Zack, Donald J

2017-06-21

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthetic Lethal Therapy for KRAS Mutant Non-small-cell Lung Carcinoma with Nanoparticle-mediated CDK4 siRNA Delivery

PubMed Central

Mao, Cheng-Qiong; Xiong, Meng-Hua; Liu, Yang; Shen, Song; Du, Xiao-Jiao; Yang, Xian-Zhu; Dou, Shuang; Zhang, Pei-Zhuo; Wang, Jun

2014-01-01

The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in non-small-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4. PMID:24496383

Unsteady transonic flow calculations for two-dimensional canard-wing configurations with aeroelastic applications

NASA Technical Reports Server (NTRS)

Batina, J. T.

1985-01-01

Unsteady transonic flow calculations for aerodynamically interfering airfoil configurations are performed as a first step toward solving the three dimensional canard wing interaction problem. These calculations are performed by extending the XTRAN2L two dimensional unsteady transonic small disturbance code to include an additional airfoil. Unsteady transonic forces due to plunge and pitch motions of a two dimensional canard and wing are presented. Results for a variety of canard wing separation distances reveal the effects of aerodynamic interference on unsteady transonic airloads. Aeroelastic analyses employing these unsteady airloads demonstrate the effects of aerodynamic interference on aeroelastic stability and flutter. For the configurations studied, increases in wing flutter speed result with the inclusion of the aerodynamically interfering canard.

AIP1 recruits phosphatase PP2A to ASK1 in tumor necrosis factor-induced ASK1-JNK activation.

PubMed

Min, Wang; Lin, Yan; Tang, Shibo; Yu, Luyang; Zhang, Haifeng; Wan, Ting; Luhn, Tricia; Fu, Haian; Chen, Hong

2008-04-11

Previously we have shown that AIP1 (apoptosis signal-regulating kinase [ASK]1-interacting protein 1), a novel member of the Ras-GAP protein family, facilitates dephosphorylation of ASK1 at pSer967 and subsequently 14-3-3 release from ASK1, leading to enhanced ASK1-JNK signaling. However, the phosphatase(s) responsible for ASK1 dephosphorylation at pSer967 has not been identified. In the present study, we identified protein phosphatase (PP)2A as a potential phosphatase in vascular endothelial cells (ECs). Tumor necrosis factor (TNF)-induced dephosphorylation of ASK1 pSer967 in ECs was blocked by PP2A inhibitor okadaic acid. Overexpression of PP2A catalytic subunit induced dephosphorylation of ASK1 pSer967 and activation of c-Jun N-terminal kinase (JNK). In contrast, a catalytic inactive form of PP2A or PP2A small interfering RNA blunted TNF-induced dephosphorylation of ASK1 pSer967 and activation of JNK without effects on NF-kappaB activation. Whereas AIP1, via its C2 domain, binds to ASK1, PP2A binds to the GAP domain of AIP1. Endogenous AIP1-PP2A complex can be detected in the resting state, and TNF induces a complex formation of AIP1-PP2A with ASK1. Furthermore, TNF-induced association of PP2A with ASK1 was diminished in AIP1-knockdown ECs, suggesting a critical role of AIP1 in recruiting PP2A to ASK1. TNF-signaling molecules TRAF2 and RIP1, known to be in complex with AIP1 and activate AIP1 by phosphorylating AIP1 at Ser604, are critical for TNF-induced ASK1 dephosphorylation. Finally, PP2A and AIP1 cooperatively induce activation of ASK1-JNK signaling and EC apoptosis, as demonstrated by both overexpression and small interfering RNA knockdown approaches. Taken together, our data support a critical role of PP2A-AIP1 complex in TNF-induced activation of ASK1-JNK apoptotic signaling.

Engineering of small interfering RNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles for highly efficient and safe gene silencing: A quality by design-based approach.

PubMed

Thanki, Kaushik; Zeng, Xianghui; Justesen, Sarah; Tejlmann, Sarah; Falkenberg, Emily; Van Driessche, Elize; Mørck Nielsen, Hanne; Franzyk, Henrik; Foged, Camilla

2017-11-01

Safety and efficacy of therapeutics based on RNA interference, e.g., small interfering RNA (siRNA), are dependent on the optimal engineering of the delivery technology, which is used for intracellular delivery of siRNA to the cytosol of target cells. We investigated the hypothesis that commonly used and poorly tolerated cationic lipids might be replaced with more efficacious and safe lipidoids as the lipid component of siRNA-loaded lipid-polymer hybrid nanoparticles (LPNs) for achieving more efficient gene silencing at lower and safer doses. However, formulation design of such a complex formulation is highly challenging due to a strong interplay between several contributing factors. Hence, critical formulation variables, i.e. the lipidoid content and siRNA:lipidoid ratio, were initially identified, followed by a systematic quality-by-design approach to define the optimal operating space (OOS), eventually resulting in the identification of a robust, highly efficacious and safe formulation. A 17-run design of experiment with an I-optimal approach was performed to systematically assess the effect of selected variables on critical quality attributes (CQAs), i.e. physicochemical properties (hydrodynamic size, zeta potential, siRNA encapsulation/loading) and the biological performance (in vitro gene silencing and cell viability). Model fitting of the obtained data to construct predictive models revealed non-linear relationships for all CQAs, which can be readily overlooked in one-factor-at-a-time optimization approaches. The response surface methodology further enabled the identification of an OOS that met the desired quality target product profile. The optimized lipidoid-modified LPNs revealed more than 50-fold higher in vitro gene silencing at well-tolerated doses and approx. a twofold increase in siRNA loading as compared to reference LPNs modified with the commonly used cationic lipid dioleyltrimethylammonium propane (DOTAP). Thus, lipidoid-modified LPNs show highly promising prospects for efficient and safe intracellular delivery of siRNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Making RISC.

PubMed

Kawamata, Tomoko; Tomari, Yukihide

2010-07-01

It is well established that 20- to 30-nt small RNAs, including small interfering RNAs, microRNAs and Piwi-interacting RNAs, play crucial roles in regulating gene expression and control a surprisingly diverse array of biological processes. These small RNAs cannot work alone: they must form effector ribonucleoprotein complexes - RNA-induced silencing complexes (RISCs) - to exert their function. Thus, RISC assembly is a key process in small RNA-mediated silencing. Recent biochemical analyses of RISC assembly, together with new structural studies of Argonaute, the core protein component of RISC, suggest a revised view of how mature RISC, which contains single-stranded guide RNA, is built from small RNAs that are born double-stranded. Copyright 2010 Elsevier Ltd. All rights reserved.

Glucose-dependent downregulation of glucagon gene expression mediated by selective interactions between ALX3 and PAX6 in mouse alpha cells.

PubMed

Mirasierra, Mercedes; Vallejo, Mario

2016-04-01

The stimulation of glucagon secretion in response to decreased glucose levels has been studied extensively. In contrast, little is known about the regulation of glucagon gene expression in response to fluctuations in glucose concentration. Paired box 6 (PAX6) is a key transcription factor that regulates the glucagon promoter by binding to the G1 and G3 elements. Here, we investigated the role of the transcription factor aristaless-like homeobox 3 (ALX3) as a glucose-dependent modulator of PAX6 activity in alpha cells. Experiments were performed in wild-type or Alx3-deficient islets and alphaTC1 cells. We used chromatin immunoprecipitations and electrophoretic mobility shift assays for DNA binding, immunoprecipitations and pull-down assays for protein interactions, transfected cells for promoter activity, and small interfering RNA and quantitative RT-PCR for gene expression. Elevated glucose concentration resulted in stimulated expression of Alx3 and decreased glucagon gene expression in wild-type islets. In ALX3-deficient islets, basal glucagon levels were non-responsive to changes in glucose concentration. In basal conditions ALX3 bound to the glucagon promoter at G3, but not at G1. ALX3 could form heterodimers with PAX6 that were permissive for binding to G3 but not to G1. Thus, increasing the levels of ALX3 in response to glucose resulted in the sequestration of PAX6 by ALX3 for binding to G1, thus reducing glucagon promoter activation and glucagon gene expression. Glucose-stimulated expression of ALX3 in alpha cells provides a regulatory mechanism for the downregulation of glucagon gene expression by interfering with PAX6-mediated transactivation on the glucagon G1 promoter element.

The Vasa homolog RDE-12 engages target mRNA and multiple Argonaute proteins to promote RNAi in C. elegans

PubMed Central

Shirayama, Masaki; Stanney, William; Gu, Weifeng; Seth, Meetu; Mello, Craig C.

2014-01-01

Summary Argonaute proteins (AGOs) are key nuclease effectors of RNA interference (RNAi) [1]. Although purified AGOs can mediate a single round of target-RNA cleavage in vitro, accessory factors are required for short-interfering (si)RNAs loading and to achieve multiple-target turnover [2, 3]. To identify AGO co-factors we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi [4]. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 co-localizes with WAGO-1 in germline P-granules and in cytoplasmic and peri-nuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1) where it promotes small-RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing. PMID:24684931

Astaxanthin Induces the Nrf2/HO-1 Antioxidant Pathway in Human Umbilical Vein Endothelial Cells by Generating Trace Amounts of ROS.

PubMed

Niu, Tingting; Xuan, Rongrong; Jiang, Ligang; Wu, Wei; Zhen, Zhanghe; Song, Yuling; Hong, Lili; Zheng, Kaiqin; Zhang, Jiaxing; Xu, Qingshan; Tan, Yinghong; Yan, Xiaojun; Chen, Haimin

2018-02-14

Astaxanthin is a powerful antioxidant that possesses potent protective effects against various human diseases and physiological disorders. However, the mechanisms underlying its antioxidant functions in cells are not fully understood. In the present study, the effects of astaxanthin on reactive oxygen species (ROS) production and antioxidant enzyme activity, as well as mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K)/Akt, and the nuclear factor erythroid 2-related factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) pathways in human umbilical vein endothelial cells (HUVECs), were examined. It was shown that astaxanthin (0.1, 1, and 10 μM) induced ROS production by 9.35%, 14.8%, and 18.06% compared to control, respectively, in HUVECs. In addition, astaxanthin increased the mRNA levels of phase II enzymes HO-1 and also promoted GSH-Px enzyme activity. Furthermore, we observed ERK phosphorylation, nuclear translocation of Nrf-2, and activation of antioxidant response element-driven luciferase activity upon astaxanthin treatment. Knockdown of Nrf-2 by small interfering RNA inhibited HO-1 mRNA expression by 60%, indicating that the Nrf-2/ARE signaling pathway is activated by astaxanthin. Our results suggest that astaxanthin activates the Nrf-2/HO-1 antioxidant pathway by generating small amounts of ROS.

Small RNA sorting: matchmaking for Argonautes

PubMed Central

Czech, Benjamin; Hannon, Gregory J.

2013-01-01

Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes — microRNAs (miRNAs) and small interfering RNAs (siRNAs) — in plants and animals. PMID:21116305

Interactions between insulin-like growth factor-I, estrogen receptor-α (ERα) and ERβ in regulating growth/apoptosis of MCF-7 human breast cancer cells

PubMed Central

Mendoza, Rhone A.; Enriquez, Marlene I; Mejia, Sylvia M; Moody, Emily E; Thordarson, Gudmundur

2011-01-01

Understanding of the interactions between estradiol (E2) and insulin-like growth factor-I (IGF-I) is still incomplete. Cell lines derived from the MCF-7 breast cancer cells were generated with suppressed expression of the IGF-I receptor (IGF-IR), termed IGF-IR.low cells, by stable transfection using small interfering RNA (siRNA) expression vector. Vector for control cells carried sequence generating non-interfering RNA. Concomitant with reduction in the IGF-IR levels, the IGF-IR.low cells also showed a reduction in estrogen receptor α (ERα) and progesterone receptor expressions and an elevation in the expression of ERβ. The number of the IGF-IR.low cells was reduced in response to IGF-I and human growth hormone plus epidermal growth factor, but E2 did not cause increase in the number of the IGF-IR.low cells compared to controls. Proliferation rate of IGF-IR.low cells was only reduced in response to E2 compared to controls, whereas their basal and hormone stimulated apoptosis rate was increased. Phosphorylation of p38 mitogen activated protein kinase (p38 MAPK) was increased in the IGF-IR.low cells after treatment with E2, without affecting control cells. Further, phosphorylation of the tumor suppressor protein p53 was elevated in the IGF-IR.low cells compared to the controls. Summary, suppressing the IGF-IR expression decreased the level of ERα but increased the level of ERβ. Overall growth rate of the IGF-IR.low cells was reduced mostly through an increase in apoptosis without affecting proliferation substantially. We hypothesize that a decreased ERα:ERβ ratio triggered a rapid phosphorylation of p38 MAPK which in turn phosphorylated the p53 tumor suppressor and accelerated apoptosis rate. PMID:20974640

Interfering with DNA Damage Signals: Radiosensitizing Prostate Cancer Using Small Peptides

DTIC Science & Technology

2009-05-01

25-l sample of the supernatant was analyzed for free phosphate in the malachite green assay by dilution with 100 l of a developing solution... malachite green). After incubation for 15 min, the release of phosphate was quantified by measuring the absorbance at 650 nm in a microtiter plate reader

Interfering with DNA Damage Signals: Radiosensitizing Prostate Cancer using Small Peptides

DTIC Science & Technology

2007-11-01

were pelleted, and a 25-l sample of the supernatant was analyzed for free phosphate in the malachite green assay by dilution with 100 l of a...developing solution ( malachite green). After incubation for 15 min, the release of phosphate was quantified by measuring the absorbance at 650 nm in a

Plum pox virus (PPV) genome expression in genetically engineered RNAi plants

USDA-ARS?s Scientific Manuscript database

An important approach to controlling sharka disease caused by Plum pox virus (PPV) is the development of PPV resistant plants using small interfering RNAs (siRNA) technology. In order to evaluate siRNA induced gene silencing, we studied, based on knowledge of the PPV genome sequence, virus genome t...

Functional Nanostructures for Effective Delivery of Small Interfering RNA Therapeutics

PubMed Central

Hong, Cheol Am; Nam, Yoon Sung

2014-01-01

Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA. PMID:25285170

Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA

PubMed Central

Woodrow, Kim A.; Cu, Yen; Booth, Carmen J.; Saucier-Sawyer, Jennifer K.; Wood, Monica J.; Saltzman, W. Mark

2009-01-01

Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protected against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternate approach, using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA in the vaginal mucosa. PMID:19404239

Contributions of 3'-overhang to the dissociation of small interfering RNAs from the PAZ domain: molecular dynamics simulation study.

PubMed

Lee, Hui Sun; Lee, Soo Nam; Joo, Chul Hyun; Lee, Heuiran; Lee, Han Saem; Yoon, Seung Yong; Kim, Yoo Kyum; Choe, Han

2007-03-01

RNA interference (RNAi) is a 'knock-down' reaction to reduce expression of a specific gene through highly regulated, enzyme-mediated processes. Small interfering RNAs (siRNAs) are RNA molecules that play an effector role in RNAi and can bind the PAZ domains present in Dicer and RISC. We investigated the interaction between the PAZ domain and the siRNA-like duplexes through dissociation molecular dynamics (DMD) simulations. Specifically, we focused on the response of the PAZ domain to various 3'-overhang structures of the siRNA-like duplexes. We found that the siRNA-like duplex with the 3' UU-overhang made relatively more stable complex with the PAZ domain compared to those with 3' CC-, AA-, and GG-overhangs. The siRNA-like duplex with UU-overhang was easily dissociated from the PAZ domain once the structural stability of the complex is impaired. Interestingly, the 3' UU-overhang spent the least time at the periphery region of the binding pocket during the dissociation process, which can be mainly attributable to UU-overhang's smallest number of hydrogen bonds.

A Glu-urea-Lys Ligand-conjugated Lipid Nanoparticle/siRNA System Inhibits Androgen Receptor Expression In Vivo

PubMed Central

Lee, Justin B; Zhang, Kaixin; Tam, Yuen Yi C; Quick, Joslyn; Tam, Ying K; Lin, Paulo JC; Chen, Sam; Liu, Yan; Nair, Jayaprakash K; Zlatev, Ivan; Rajeev, Kallanthottathil G; Manoharan, Muthiah; Rennie, Paul S; Cullis, Pieter R

2016-01-01

The androgen receptor plays a critical role in the progression of prostate cancer. Here, we describe targeting the prostate-specific membrane antigen using a lipid nanoparticle formulation containing small interfering RNA designed to silence expression of the messenger RNA encoding the androgen receptor. Specifically, a Glu-urea-Lys PSMA-targeting ligand was incorporated into the lipid nanoparticle system formulated with a long alkyl chain polyethylene glycol-lipid to enhance accumulation at tumor sites and facilitate intracellular uptake into tumor cells following systemic administration. Through these features, and by using a structurally refined cationic lipid and an optimized small interfering RNA payload, a lipid nanoparticle system with improved potency and significant therapeutic potential against prostate cancer and potentially other solid tumors was developed. Decreases in serum prostate-specific antigen, tumor cellular proliferation, and androgen receptor levels were observed in a mouse xenograft model following intravenous injection. These results support the potential clinical utility of a prostate-specific membrane antigen–targeted lipid nanoparticle system to silence the androgen receptor in advanced prostate cancer. PMID:28131285

Sign phase transition in the problem of interfering directed paths

NASA Astrophysics Data System (ADS)

Baldwin, C. L.; Laumann, C. R.; Spivak, B.

2018-01-01

We investigate the statistical properties of interfering directed paths in disordered media. At long distance, the average sign of the sum over paths may tend to zero (sign disordered) or remain finite (sign ordered) depending on dimensionality and the concentration of negative scattering sites x . We show that in two dimensions the sign-ordered phase is unstable even for arbitrarily small x by identifying rare destabilizing events. In three dimensions, we present strong evidence that there is a sign phase transition at a finite xc>0 . These results have consequences for several different physical systems. In two-dimensional insulators at low temperature, the variable-range-hopping magnetoresistance is always negative, while in three dimensions, it changes sign at the point of the sign phase transition. We also show that in the sign-disordered regime a small magnetic field may enhance superconductivity in a random system of D -wave superconducting grains embedded in a metallic matrix. Finally, the existence of the sign phase transition in three dimensions implies new features in the spin-glass phase diagram at high temperature.

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Ra, Hyejun; Gonzalez-Gonzalez, Emilio; Smith, Bryan R.; Gambhir, Sanjiv S.; Kino, Gordon S.; Solgaard, Olav; Kaspar, Roger L.; Contag, Christopher H.

2010-05-01

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

psRNATarget: a plant small RNA target analysis server

PubMed Central

Dai, Xinbin; Zhao, Patrick Xuechun

2011-01-01

Plant endogenous non-coding short small RNAs (20–24 nt), including microRNAs (miRNAs) and a subset of small interfering RNAs (ta-siRNAs), play important role in gene expression regulatory networks (GRNs). For example, many transcription factors and development-related genes have been reported as targets of these regulatory small RNAs. Although a number of miRNA target prediction algorithms and programs have been developed, most of them were designed for animal miRNAs which are significantly different from plant miRNAs in the target recognition process. These differences demand the development of separate plant miRNA (and ta-siRNA) target analysis tool(s). We present psRNATarget, a plant small RNA target analysis server, which features two important analysis functions: (i) reverse complementary matching between small RNA and target transcript using a proven scoring schema, and (ii) target-site accessibility evaluation by calculating unpaired energy (UPE) required to ‘open’ secondary structure around small RNA’s target site on mRNA. The psRNATarget incorporates recent discoveries in plant miRNA target recognition, e.g. it distinguishes translational and post-transcriptional inhibition, and it reports the number of small RNA/target site pairs that may affect small RNA binding activity to target transcript. The psRNATarget server is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster. The server front-end integrates three simplified user-friendly interfaces to accept user-submitted or preloaded small RNAs and transcript sequences; and outputs a comprehensive list of small RNA/target pairs along with the online tools for batch downloading, key word searching and results sorting. The psRNATarget server is freely available at http://plantgrn.noble.org/psRNATarget/. PMID:21622958

Taiwan: growing, growing, gone.

PubMed

Hanson, R

1979-10-01

Accommodation between Taiwan and the People's Republic of China may not be inconceivable as trade contacts (though officially disallowed) grow. Because of Taiwan's well-established success and the pressing need in China to industrialize, it appears, however, that such an accommodation will occur only after China becomes more like Taiwan. Taiwan owes its success, first, to land reform and then, in the 1960s, to steady industrialization. Besides broad controls over money supply and capital designed to ward off inflationary pressures when needed, and the grand outlines for development, another factor in the island's economic success is that the government has interfered little with private enterprise. The economy has an underpinning of small to medium size businesses. There are more than 10,000 trading companies. This diverse foundation has given the economy as a whole a flexible buffer on which more sophisticated industires can be formed.

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

Sun, Zhen; Department of Biochemistry and Molecular Biology, Program in Molecular Cell Biology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058; Xiang, Wenqing

Highlights: {yields} LNA-modified oligonucleotides can pass through the plasma membrane of cultured cells even without using transfection machinery. {yields} LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. {yields} LNA-oligonucleotide designed to target nuclear HBV DNA efficiently suppresses HBV replication and transcription in cultured hepatic cells. -- Abstract: Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently, triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry ofmore » oligonucleotides and enhancement of their affinity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modified oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more efficiently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modified oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-modified oligonucleotides has strong potential as a new strategy for HBV inhibition.« less

An Analysis of Factors Affecting Teacher Attrition in High Performing and Low Performing Elementary Rural Schools in South Carolina

ERIC Educational Resources Information Center

Carter-Blocker, Vickie R.

2012-01-01

The purpose of this study was to examine the factors impacting teacher attrition in high-performing and low-performing elementary rural schools in South Carolina. Several factors were identified that interfered with teachers returning to the teaching profession. School districts in rural areas need to be better informed of the factors that affect…

Targeting Germinal Matrix Hemorrhage-Induced Overexpression of Sodium-Coupled Bicarbonate Exchanger Reduces Posthemorrhagic Hydrocephalus Formation in Neonatal Rats.

PubMed

Li, Qian; Ding, Yan; Krafft, Paul; Wan, Weifeng; Yan, Feng; Wu, Guangyong; Zhang, Yixin; Zhan, Qunling; Zhang, John H

2018-01-31

Germinal matrix hemorrhage (GMH) is a leading cause of mortality and lifelong morbidity in preterm infants. Posthemorrhagic hydrocephalus (PHH) is a common complication of GMH. A sodium-coupled bicarbonate exchanger (NCBE) encoded by solute carrier family 4 member 10 gene is expressed on the choroid plexus basolateral membrane and may play a role in cerebrospinal fluid production and the development of PHH. Following GMH, iron degraded from hemoglobin has been linked to PHH. Choroid plexus epithelial cells also contain iron-responsive element-binding proteins (IRPs), IRP1, and IRP2 that bind to mRNA iron-responsive elements. The present study aims to resolve the following issues: (1) whether the expression of NCBE is regulated by IRPs; (2) whether NCBE regulates the formation of GMH-induced hydrocephalus; and (3) whether inhibition of NCBE reduces PHH development. GMH model was established in P7 rat pups by injecting bacterial collagenase into the right ganglionic eminence. Another group received iron trichloride injections instead of collagenase. Deferoxamine was administered intraperitoneally for 3 consecutive days after GMH/iron trichloride. Solute carrier family 4 member 10 small interfering RNA or scrambled small interfering RNA was administered by intracerebroventricular injection 24 hours before GMH and followed with an injection every 7 days over 21 days. NCBE expression increased while IRP2 expression decreased after GMH/iron trichloride. Deferoxamine ameliorated both the GMH-induced and iron trichloride-induced decrease of IRP2 and decreased NCBE expressions. Deferoxamine and solute carrier family 4 member 10 small interfering RNA improved cognitive and motor functions at 21 to 28 days post GMH and reduced cerebrospinal fluid production as well as the degree of hydrocephalus at 28 days after GMH. Targeting iron-induced overexpression of NCBE may be a translatable therapeutic strategy for the treatment of PHH following GMH. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Netrin-1 Preserves Blood-Brain Barrier Integrity Through Deleted in Colorectal Cancer/Focal Adhesion Kinase/RhoA Signaling Pathway Following Subarachnoid Hemorrhage in Rats.

PubMed

Xie, Zongyi; Enkhjargal, Budbazar; Reis, Cesar; Huang, Lei; Wan, Weifeng; Tang, Jiping; Cheng, Yuan; Zhang, John H

2017-05-19

Netrin-1 (NTN-1) has been established to be a novel intrinsic regulator of blood-brain barrier (BBB) maintenance. This study was carried out to investigate the potential roles of exogenous NTN-1 in preserving BBB integrity after experimental subarachnoid hemorrhage (SAH) as well as the underlying mechanisms of its protective effects. A total of 309 male Sprague-Dawley rats were subjected to an endovascular perforation model of SAH. Recombinant NTN-1 was administered intravenously 1 hour after SAH induction. NTN-1 small interfering RNA or Deleted in Colorectal Cancer small interfering RNA was administered intracerebroventricular at 48 hours before SAH. Focal adhesion kinase inhibitor was administered by intraperitoneal injection at 1 hour prior to SAH. Neurological scores, brain water content, BBB permeability, RhoA activity, Western blot, and immunofluorescence staining were evaluated. The expression of endogenous NTN-1 and its receptor Deleted in Colorectal Cancer were increased after SAH. Administration of exogenous NTN-1 significantly reduced brain water content and BBB permeability and ameliorated neurological deficits at 24 and 72 hours after SAH. Exogenous NTN-1 treatment significantly promoted phosphorylated focal adhesion kinase activation and inhibited RhoA activity, as well as upregulated the expression of ZO-1 and Occludin. Conversely, depletion of endogenous NTN-1 aggravated BBB breakdown and neurological impairments at 24 hours after SAH. The protective effects of NTN-1 at 24 hours after SAH were also abolished by pretreatment with Deleted in Colorectal Cancer small interfering RNA and focal adhesion kinase inhibitor. NTN-1 treatment preserved BBB integrity and improved neurological functions through a Deleted in Colorectal Cancer/focal adhesion kinase/RhoA signaling pathway after SAH. Thus, NTN-1 may serve as a promising treatment to alleviate early brain injury following SAH. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

A reverse signaling pathway downstream of Sema4A controls cell migration via Scrib

PubMed Central

Yang, Lida; Kaur, Harmandeep; Pestel, Jenny; Looso, Mario; Nolte, Hendrik; Krishnan, Ramesh K.; Bünemann, Moritz; Offermanns, Stefan; Swiercz, Jakub M.

2017-01-01

Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor βPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration. PMID:28007914

Streptococcus pyogenes CAMP factor promotes bacterial adhesion and invasion in pharyngeal epithelial cells without serum via PI3K/Akt signaling pathway.

PubMed

Kurosawa, Mie; Oda, Masataka; Domon, Hisanori; Isono, Toshihito; Nakamura, Yuki; Saitoh, Issei; Hayasaki, Haruaki; Yamaguchi, Masaya; Kawabata, Shigetada; Terao, Yutaka

2018-01-01

Streptococcus pyogenes is a bacterium that causes systemic diseases, such as pharyngitis and toxic shock syndrome, via oral- or nasal-cavity infection. S. pyogenes produces various molecules known to function with serum components that lead to bacterial adhesion and invasion in human tissues. In this study, we identified a novel S. pyogenes adhesin/invasin. Our results revealed that CAMP factor promoted streptococcal adhesion and invasion in pharyngeal epithelial Detroit562 cells without serum. Recombinant CAMP factor initially localized on the membranes of cells and then became internalized in the cytosol following S. pyogenes infection. Additionally, CAMP factor phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in the cells. ELISA results demonstrate that CAMP factor affected the amount of phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in Detroit562 cells. Furthermore, CAMP factor did not reverse the effect of phosphoinositide 3-kinase knockdown by small interfering RNA in reducing the level of adhesion and invasion of S. pyogenes isogenic cfa-deficient mutant. These results suggested that S. pyogenes CAMP factor activated the phosphoinositide 3-kinase/serine-threonine kinase signaling pathway, promoting S. pyogenes invasion of Detroit562 cells without serum. Our findings suggested that CAMP factor played an important role on adhesion and invasion in pharyngeal epithelial cells. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Roles of small RNAs in the immune defense mechanisms of crustaceans.

PubMed

He, Yaodong; Ju, Chenyu; Zhang, Xiaobo

2015-12-01

Small RNAs, 21-24 nucleotides in length, are non-coding RNAs found in most multicellular organisms, as well as in some viruses. There are three main types of small RNAs including microRNA (miRNA), small-interfering RNA (siRNA), and piwi-interacting RNA (piRNA). Small RNAs play key roles in the genetic regulation of eukaryotes; at least 50% of all eukaryote genes are the targets of small RNAs. In recent years, studies have shown that some unique small RNAs are involved in the immune response of crustaceans, leading to lower or higher immune responses to infections and diseases. SiRNAs could be used as therapy for virus infection. In this review, we provide an overview of the diverse roles of small RNAs in the immune defense mechanisms of crustaceans. Copyright © 2015 Elsevier Ltd. All rights reserved.

Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1β.

PubMed

Choi, Yun-Hee; McNally, Brian T; Igarashi, Peter

2013-07-01

Hepatocyte nuclear factor-1β (HNF-1β) is an epithelial tissue-specific transcription factor that regulates gene expression in the kidney, liver, pancreas, intestine, and other organs. Mutations of HNF-1β in humans produce renal cysts and congenital kidney anomalies. Here, we identify the LIM-domain protein zyxin as a novel binding partner of HNF-1β in renal epithelial cells. Zyxin shuttles to the nucleus where it colocalizes with HNF-1β. Immunoprecipitation of zyxin in leptomycin B-treated cells results in coprecipitation of HNF-1β. The protein interaction requires the second LIM domain of zyxin and two distinct domains of HNF-1β. Overexpression of zyxin stimulates the transcriptional activity of HNF-1β, whereas small interfering RNA silencing of zyxin inhibits HNF-1β-dependent transcription. Epidermal growth factor (EGF) induces translocation of zyxin into the nucleus and stimulates HNF-1β-dependent promoter activity. The EGF-mediated nuclear translocation of zyxin requires activation of Akt. Expression of dominant-negative mutant HNF-1β, knockdown of zyxin, or inhibition of Akt inhibits EGF-stimulated cell migration. These findings reveal a novel pathway by which extracellular signals are transmitted to the nucleus to regulate the activity of a transcription factor that is essential for renal epithelial differentiation.

Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway

PubMed Central

René, Céline; Taulan, Magali; Iral, Florence; Doudement, Julien; L'Honoré, Aurore; Gerbon, Catherine; Demaille, Jacques; Claustres, Mireille; Romey, Marie-Catherine

2005-01-01

CFTR expression is tightly controlled by a complex network of ubiquitous and tissue-specific cis-elements and trans-factors. To better understand mechanisms that regulate transcription of CFTR, we examined transcription factors that specifically bind a CFTR CArG-like motif we have previously shown to modulate CFTR expression. Gel mobility shift assays and chromatin immunoprecipitation analyses demonstrated the CFTR CArG-like motif binds serum response factor both in vitro and in vivo. Transient co-transfections with various SRF expression vector, including dominant-negative forms and small interfering RNA, demonstrated that SRF significantly increases CFTR transcriptional activity in bronchial epithelial cells. Mutagenesis studies suggested that in addition to SRF other co-factors, such as Yin Yang 1 (YY1) previously shown to bind the CFTR promoter, are potentially involved in the CFTR regulation. Here, we show that functional interplay between SRF and YY1 might provide interesting perspectives to further characterize the underlying molecular mechanism of the basal CFTR transcriptional activity. Furthermore, the identification of multiple CArG binding sites in highly conserved CFTR untranslated regions, which form specific SRF complexes, provides direct evidence for a considerable role of SRF in the CFTR transcriptional regulation into specialized epithelial lung cells. PMID:16170155

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

PubMed Central

Hocker, Harrison J.; Cho, Kwang-Jin; Chen, Chung-Ying K.; Rambahal, Nandini; Sagineedu, Sreenivasa Rao; Shaari, Khozirah; Stanslas, Johnson; Hancock, John F.; Gorfe, Alemayehu A.

2013-01-01

Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteins occurs in ∼15% of all human tumors, yet direct inhibition of Ras by small molecules has remained elusive. Recently, several small-molecule ligands have been discovered that directly bind Ras and inhibit its function by interfering with exchange factor binding. However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active oncogenic mutant Ras. Using a dynamics-based pocket identification scheme, ensemble docking, and innovative cell-based assays, here we show that andrographolide (AGP)—a bicyclic diterpenoid lactone isolated from Andrographis paniculata—and its benzylidene derivatives bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP–GTP exchange. As expected for inhibitors of exchange factor binding, AGP derivatives reduced GTP loading of wild-type K-Ras in response to acute EGF stimulation with a concomitant reduction in MAPK activation. Remarkably, however, prolonged treatment with AGP derivatives also reduced GTP loading of, and signal transmission by, oncogenic mutant K-RasG12V. In sum, the combined analysis of our computational and cell biology results show that AGP derivatives directly bind Ras, block GDP–GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling. Importantly, our findings not only show that nucleotide exchange factors are required for oncogenic Ras signaling but also demonstrate that inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras. PMID:23737504

Bromodomains: Are Readers Right for Epigenetic Therapy?

PubMed Central

2012-01-01

There is intense interest in the development of small molecule inhibitors of the acetyl-lysine-reading bromodomain protein module. These inhibitors represent a way of interfering therapeutically in epigenetic processes, and there are currently two bromodomain inhibitors in clinical trials. The success of these compounds rests on safety aspects of epigenetic target modulation being addressed. PMID:24900532

The Poor Little Rich District: The Effects of Suburbanization on a Rural School and Community

ERIC Educational Resources Information Center

Howley, Aimee; Carnes, Marilyn; Eldridge, Anita; Huber, Donna; Lado, Longun Moses; Kotler, Ruth; Turner, Maryalice

2005-01-01

Contextualized in relationship to other case studies about rural districts that have experienced population growth and decline as well as in relationship to the small sociological literature on "boom towns," this study considered the dynamics that seem to be interfering with one previously rural and now suburbanizing district's ability to address…

Homologous interference mediated by defective interfering influenza virus derived from a temperature-sensitive mutant of influenza virus.

PubMed Central

Nayak, D P; Tobita, K; Janda, J M; Davis, A R; De, B K

1978-01-01

A temperature-sensitive group II mutant of influenza virus, ts-52, with a presumed defect in viral RNA synthesis, readily produced von Magnus-type defective interfering virus (DI virus) when passed serially (four times) at high multiplicity in MDBK cells. The defective virus (ts-52 DI virus) had a high hemagglutinin and a low infectivity titer, and strongly interfered with the replication of standard infectious viruses (both ts-52 and wild-type ts+) in co-infected cells. Progeny virus particles produced by co-infection of DI virus and infectious virus were also defective and also had low infectivity, high hemagglutinating activity, and a strong interfering property. Infectious viruses ts+ and ts-52 were indistinguishable from ts-52 DI viruses by sucrose velocity or density gradient analysis. Additionally, these viruses all possessed similar morphology. However, when the RNA of DI viruses was analyzed by use of polyacrylamide gels containing 6 M urea, there was a reduction in the amount of large RNA species (V1 to V4), and a number of new smaller RNA species (D1 to D6) with molecular weights ranging from 2.9 X 10(5) to 1.05 X 10(5) appeared. Since these smaller RNA species (D1 to D6) were absent in some clones of infectious viruses, but were consistently associated with DI viruses and increased during undiluted passages and during co-infection of ts-52 with DI virus, they appeared to be a characteristic of DI viruses. Additionally, the UV target size of interfering activity and infectivity of DI virus indicated that interfering activity was 40 times more resistant to UV irradiation than was infectivity, further implicating small RNA molecules in interference. Our data suggest that the loss of infectivity observed among DI viruses may be due to nonspecific loss of a viral RNA segment(s), and the interfering property of DI viruses may be due to interfering RNA segments (DIRNA, D1 to D6). ts-52 DI virus interfered with the replication of standard virus (ts+) at both permissive (34 degrees C) and nonpermissive temperatures. The infectivity of the progeny virus was reduced to 0.2% for ts+ and 0.05% for ts-52 virus without a reduction in hemagglutinin titer. Interference was dependent on the concentration of DI virus. A particle ratio of 1 between DI virus (0.001 PFU/cell) and infectious virus (1.0 PFU/cell) produced a maximal amount of interference. Infectious virus yield was reduced 99.9% without any reduction of the yield of DI viruses Interference was also dependent on the time of addition of DI virus. Interference was most effective within the first 3 h of infection by infectious virus, indicating interference with an early function during viral replication. Images PMID:702654

N-Myc Interactor Inhibits Prototype Foamy Virus by Sequestering Viral Tas Protein in the Cytoplasm

PubMed Central

Hu, Xiaomei; Yang, Wei; Liu, Ruikang; Geng, Yunqi; Qiao, Wentao

2014-01-01

ABSTRACT Foamy viruses (FVs) are complex retroviruses that establish lifelong persistent infection without evident pathology. However, the roles of cellular factors in FV latency are poorly understood. This study revealed that N-Myc interactor (Nmi) could inhibit the replication of prototype foamy virus (PFV). Overexpression of Nmi reduced PFV replication, whereas its depletion by small interfering RNA increased PFV replication. The Nmi-mediated impairment of PFV replication resulted from the diminished transactivation by PFV Tas of the viral long terminal repeat (LTR) and an internal promoter (IP). Nmi was determined to interact with Tas and abrogate its function by sequestration in the cytoplasm. In addition, human and bovine Nmi proteins were found to inhibit the replication of bovine foamy virus (BFV) and PFV. Together, these results indicate that Nmi inhibits both human and bovine FVs by interfering with the transactivation function of Tas and may have a role in the host defense against FV infection. IMPORTANCE From this study, we report that the N-Myc interactor (Nmi), an interferon-induced protein, can interact with the regulatory protein Tas of the prototype foamy virus and sequester it in the cytoplasm. The results of this study suggest that Nmi plays an important role in maintaining foamy virus latency and may reveal a new pathway in the interferon-mediated antiviral barrier against viruses. These findings are important for understanding virus-host relationships not only with FVs but potentially for other retroviruses as well. PMID:24719420

Small interfering RNAs from bidirectional transcripts of GhMML3_A12 regulate cotton fiber development.

PubMed

Wan, Qun; Guan, Xueying; Yang, Nannan; Wu, Huaitong; Pan, Mengqiao; Liu, Bingliang; Fang, Lei; Yang, Shouping; Hu, Yan; Ye, Wenxue; Zhang, Hua; Ma, Peiyong; Chen, Jiedan; Wang, Qiong; Mei, Gaofu; Cai, Caiping; Yang, Donglei; Wang, Jiawei; Guo, Wangzhen; Zhang, Wenhua; Chen, Xiaoya; Zhang, Tianzhen

2016-06-01

Natural antisense transcripts (NATs) are commonly observed in eukaryotic genomes, but only a limited number of such genes have been identified as being involved in gene regulation in plants. In this research, we investigated the function of small RNA derived from a NAT in fiber cell development. Using a map-based cloning strategy for the first time in tetraploid cotton, we cloned a naked seed mutant gene (N1 ) encoding a MYBMIXTA-like transcription factor 3 (MML3)/GhMYB25-like in chromosome A12, GhMML3_A12, that is associated with fuzz fiber development. The extremely low expression of GhMML3_A12 in N1 is associated with NAT production, driven by its 3' antisense promoter, as indicated by the promoter-driven histochemical staining assay. In addition, small RNA deep sequencing analysis suggested that the bidirectional transcriptions of GhMML3_A12 form double-stranded RNAs and generate 21-22 nt small RNAs. Therefore, in a fiber-specific manner, small RNA derived from the GhMML3_A12 locus can mediate GhMML3_A12 mRNA self-cleavage and result in the production of naked seeds followed by lint fiber inhibition in N1 plants. The present research reports the first observation of gene-mediated NATs and siRNA directly controlling fiber development in cotton. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

The Vasa Homolog RDE-12 engages target mRNA and multiple argonaute proteins to promote RNAi in C. elegans.

PubMed

Shirayama, Masaki; Stanney, William; Gu, Weifeng; Seth, Meetu; Mello, Craig C

2014-04-14

Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Extrinsic Factors Influencing Fetal Deformations and Intrauterine Growth Restriction

PubMed Central

Moh, Wendy; Graham, John M.; Wadhawan, Isha; Sanchez-Lara, Pedro A.

2012-01-01

The causes of intrauterine growth restriction (IUGR) are multifactorial with both intrinsic and extrinsic influences. While many studies focus on the intrinsic pathological causes, the possible long-term consequences resulting from extrinsic intrauterine physiological constraints merit additional consideration and further investigation. Infants with IUGR can exhibit early symmetric or late asymmetric growth abnormality patterns depending on the fetal stage of development, of which the latter is most common occurring in 70–80% of growth-restricted infants. Deformation is the consequence of extrinsic biomechanical factors interfering with normal growth, functioning, or positioning of the fetus in utero, typically arising during late gestation. Biomechanical forces play a critical role in the normal morphogenesis of most tissues. The magnitude and direction of force impact the form of the developing fetus, with a specific tissue response depending on its pliability and stage of development. Major uterine constraining factors include primigravida, small maternal size, uterine malformation, uterine fibromata, early pelvic engagement of the fetal head, aberrant fetal position, oligohydramnios, and multifetal gestation. Corrective mechanical forces similar to those that gave rise to the deformation to reshape the deformed structures are often used and should take advantage of the rapid postnatal growth to correct form. PMID:22888434

PKB/Akt modulates TGF-beta signalling through a direct interaction with Smad3.

PubMed

Remy, Ingrid; Montmarquette, Annie; Michnick, Stephen W

2004-04-01

Transforming growth factor beta (TGF-beta) has a major role in cell proliferation, differentiation and apoptosis in many cell types. Integration of the TGF-beta pathway with other signalling cascades that control the same cellular processes may modulate TGF-beta responses. Here we report the discovery of a new functional link between TGF-beta and growth factor signalling pathways, mediated by a physical interaction between the serine-threonine kinase PKB (protein kinase B)/Akt and the transcriptional activator Smad3. Formation of the complex is induced by insulin, but inhibited by TGF-beta stimulation, placing PKB-Smad3 at a point of convergence between these two pathways. PKB inhibits Smad3 by preventing its phosphorylation, binding to Smad4 and nuclear translocation. In contrast, Smad3 does not inhibit PKB. Inhibition of Smad3 by PKB occurs through a kinase-activity-independent mechanism, resulting in a decrease in Smad3-mediated transcription and protection of cells against TGF-beta-induced apoptosis. Consistently, knockdown of the endogenous PKB gene with small-interfering RNA (siRNA) has the opposite effect. Our results suggest a very simple mechanism for the integration of signals arising from growth-factor- and TGF-beta-mediated pathways.

Budding Capability of the Influenza Virus Neuraminidase Can Be Modulated by Tetherin▿

PubMed Central

Yondola, Mark A.; Fernandes, Fiona; Belicha-Villanueva, Alan; Uccelini, Melissa; Gao, Qinshan; Carter, Carol; Palese, Peter

2011-01-01

We have determined that, in addition to its receptor-destroying activity, the influenza virus neuraminidase is capable of efficiently forming virus-like particles (VLPs) when expressed individually from plasmid DNA. This observation applies to both human subtypes of neuraminidase, N1 and N2. However, it is not found with every strain of influenza virus. Through gain-of-function and loss-of-function analyses, a critical determinant within the neuraminidase ectodomain was identified that contributes to VLP formation but is not sufficient to accomplish release of plasmid-derived VLPs. This sequence lies on the plasma membrane-proximal side of the neuraminidase globular head. Most importantly, we demonstrate that the antiviral restriction factor tetherin plays a role in determining the strain-specific limitations of release competency. If tetherin is counteracted by small interfering RNA knockdown or expression of the HIV anti-tetherin factor vpu, budding and release capability is bestowed upon an otherwise budding-deficient neuraminidase. These data suggest that budding-competent neuraminidase proteins possess an as-yet-unidentified means of counteracting the antiviral restriction factor tetherin and identify a novel way in which the influenza virus neuraminidase can contribute to virus release. PMID:21209114

Splicing Factor 2-Associated Protein p32 Participates in Ribosome Biogenesis by Regulating the Binding of Nop52 and Fibrillarin to Preribosome Particles*

PubMed Central

Yoshikawa, Harunori; Komatsu, Wataru; Hayano, Toshiya; Miura, Yutaka; Homma, Keiichi; Izumikawa, Keiichi; Ishikawa, Hideaki; Miyazawa, Naoki; Tachikawa, Hiroyuki; Yamauchi, Yoshio; Isobe, Toshiaki; Takahashi, Nobuhiro

2011-01-01

Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S pre-ribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52. PMID:21536856

Hypoxia induces mucin expression and secretion in human bronchial epithelial cells.

PubMed

Zhou, Xiangdong; Tu, Jing; Li, Qi; Kolosov, Victor P; Perelman, Juliy M

2012-12-01

The study objective was to investigate the role of hypoxia-inducible factor 1 (HIF-1) in the transcriptional activation of MUC5AC in human bronchial epithelial (HBE) 16 cells under hypoxia conditions and the effect of hypoxia on expression and secretion of MUC5AC. Cells were incubated in hypoxia medium. Serial deletions or mutations of the MUC5AC promoter were cloned in the reporter pGL3-basic plasmid (Promega Biotech Co, Ltd, Beijing, China). These reporter plasmids were cotransfected with HIF-1α small interfering RNA. Hypoxia markedly increased the level of MUC5AC secretion and the transcriptional activity of MUC5AC promoters. Western blot analysis showed that HIF-1α and MUC5AC proteins were strongly increased after HBE16 cells were exposed to hypoxic conditions. Treatment of HBE16 cells with HIF-1α inhibitor (YC-1) or HIF-1α small interfering RNA significantly inhibited the expression of HIF-1α and MUC5AC, and the secretion of MUC5AC. Depletion of the promoter sequence did not reduce the MUC5AC promoter activity to hypoxia. Luciferase assay indicated that HRE in the MUC5AC promoter was in the region from -120 to +54. Promoter sequence analysis showed that 1 HRE site at -65 plays an important role in hypoxia activation of the MUC5AC. The inactivation of the HRE site using site-directed mutagenesis led to the complete loss of induction by hypoxia, which further confirmed the key role of the HRE site. MUC5AC expression and secretion are upregulated in response to hypoxia. The HRE site at -65 in the MUC5AC promoter and the HIF-1α are the major regulators for the cellular response against hypoxia in human bronchial epithelial cells. Copyright © 2012 Mosby, Inc. All rights reserved.

Molecular-Targeted Immunotherapeutic Strategy for Melanoma via Dual-Targeting Nanoparticles Delivering Small Interfering RNA to Tumor-Associated Macrophages.

PubMed

Qian, Yuan; Qiao, Sha; Dai, Yanfeng; Xu, Guoqiang; Dai, Bolei; Lu, Lisen; Yu, Xiang; Luo, Qingming; Zhang, Zhihong

2017-09-26

Tumor-associated macrophages (TAMs) are a promising therapeutic target for cancer immunotherapy. Targeted delivery of therapeutic drugs to the tumor-promoting M2-like TAMs is challenging. Here, we developed M2-like TAM dual-targeting nanoparticles (M2NPs), whose structure and function were controlled by α-peptide (a scavenger receptor B type 1 (SR-B1) targeting peptide) linked with M2pep (an M2 macrophage binding peptide). By loading anti-colony stimulating factor-1 receptor (anti-CSF-1R) small interfering RNA (siRNA) on the M2NPs, we developed a molecular-targeted immunotherapeutic approach to specifically block the survival signal of M2-like TAMs and deplete them from melanoma tumors. We confirmed the validity of SR-B1 for M2-like TAM targeting and demonstrated the synergistic effect of the two targeting units (α-peptide and M2pep) in the fusion peptide (α-M2pep). After being administered to tumor-bearing mice, M2NPs had higher affinity to M2-like TAMs than to tissue-resident macrophages in liver, spleen, and lung. Compared with control treatment groups, M2NP-based siRNA delivery resulted in a dramatic elimination of M2-like TAMs (52%), decreased tumor size (87%), and prolonged survival. Additionally, this molecular-targeted strategy inhibited immunosuppressive IL-10 and TGF-β production and increased immunostimulatory cytokines (IL-12 and IFN-γ) expression and CD8 + T cell infiltration (2.9-fold) in the tumor microenvironment. Moreover, the siRNA-carrying M2NPs down-regulated expression of the exhaustion markers (PD-1 and Tim-3) on the infiltrating CD8 + T cells and stimulated their IFN-γ secretion (6.2-fold), indicating the restoration of T cell immune function. Thus, the dual-targeting property of M2NPs combined with RNA interference provides a potential strategy of molecular-targeted cancer immunotherapy for clinical application.

ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans.

PubMed

Subramaniam, Selvakumar; Ozdener, Mehmet Hakan; Abdoul-Azize, Souleymane; Saito, Katsuyoshi; Malik, Bilal; Maquart, Guillaume; Hashimoto, Toshihiro; Marambaud, Philippe; Aribi, Mourad; Tordoff, Michael G; Besnard, Philippe; Khan, Naim Akhtar

2016-10-01

Obesity is a major public health problem. An in-depth knowledge of the molecular mechanisms of oro-sensory detection of dietary lipids may help fight it. Humans and rodents can detect fatty acids via lipido-receptors, such as CD36 and GPR120. We studied the implication of the MAPK pathways, in particular, ERK1/2, in the gustatory detection of fatty acids. Linoleic acid, a dietary fatty acid, induced via CD36 the phosphorylation of MEK1/2-ERK1/2-ETS-like transcription factor-1 cascade, which requires Fyn-Src kinase and lipid rafts in human taste bud cells (TBCs). ERK1/2 cascade was activated by Ca 2+ signaling via opening of the calcium-homeostasis modulator-1 (CALHM1) channel. Furthermore, fatty acid-evoked Ca 2+ signaling and ERK1/2 phosphorylation were decreased in both human TBCs after small interfering RNA knockdown of CALHM1 channel and in TBCs from Calhm1 -/- mice. Targeted knockdown of ERK1/2 by small interfering RNA or PD0325901 (MEK1/2 inhibitor) in the tongue and genetic ablation of Erk1 or Calhm1 genes impaired preference for dietary fat in mice. Lingual inhibition of ERK1/2 in healthy volunteers also decreased orogustatory sensitivity for linoleic acid. Our data demonstrate that ERK1/2-MAPK cascade is regulated by the opening of CALHM1 Ca 2+ channel in TBCs to modulate orogustatory detection of dietary lipids in mice and humans.-Subramaniam, S., Ozdener, M. H., Abdoul-Azize, S., Saito, K., Malik, B., Maquart, G., Hashimoto, T., Marambaud, P., Aribi, M., Tordoff, M. G., Besnard, P., Khan, N. A. ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans. © FASEB.

A Novel Role for SIRT3 in Regulating Mediators Involved in the Terminal Pathways of Human Labor and Delivery.

PubMed

Lim, Ratana; Barker, Gillian; Menon, Ramkumar; Lappas, Martha

2016-11-01

Preterm birth remains the major cause of neonatal mortality and morbidity, mediated largely by an inflammatory process. The sirtuin (SIRT) family of cellular regulators has been implicated as key inhibitors of inflammation. We have previously reported a role for SIRT1, SIRT2, and SIRT6 in regulating inflammation-induced prolabor mediators. In this study, we determined the effect of term labor and pro-inflammatory cytokines on SIRT3, SIRT4, SIRT5, and SIRT7 expression in human myometrium. Functional studies were also used to investigate the effect of small interfering RNA (siRNA) knockdown of SIRTs in regulating inflammation-induced prolabor mediators. Western blot analysis and qRT-PCR were used to determine SIRT3, SIRT4, SIRT5, and SIRT7 mRNA and protein expression in human myometrium. Small interfering RNA knockdown of SIRT3 in myometrial primary cells determined its role in response to inflammatory stimuli IL1B and TNF. SIRT3 mRNA and protein expression levels were significantly lower in term laboring myometrium compared with term nonlaboring myometrium. There was no effect of labor on SIRT4, SIRT5 or SIRT7 protein expression. The pro-inflammatory cytokines IL1B and TNF significantly decreased levels of SIRT3 mRNA and protein expression. SIRT3 knockdown by siRNA significantly augmented IL1B- and TNF-stimulated IL6, CXCL8, and CCL2 mRNA expression and release; PTGS2 mRNA expression and subsequent PGF 2alpha release; the mRNA expression and secretion of the adhesion molecule ICAM1 and the extracellular matrix remodeling enzyme MMP9; and nuclear factor kappa B1 (NFkappaB1) transcriptional activity. In human myometrium, SIRT3 expression decreases with term labor and regulates the mediators involved in the terminal effector pathways of human labor and delivery through the NFkappaB1 pathway. © 2016 by the Society for the Study of Reproduction, Inc.

Intranasally delivered small interfering RNA-mediated suppression of scavenger receptor Mac-1 attenuates microglial phenotype switching and working memory impairment following hypoxia.

PubMed

Das, Sudeshna; Mishra, K P; Ganju, Lilly; Singh, S B

2018-05-05

Brain, being the highest consumer of oxygen, is prone to increased risk of hypoxia-induced neurological insults. In response to hypoxia, microglia, the major resident immune cells of brain switches to an activated phenotype and promote inflammatory responses leading to tissue damage and loss of cognitive functions including working memory impairment. Till date, no proven clinical therapeutics is available to retard the progression of neurodegenerative memory impairment. In the present study, we investigated the therapeutic potential of intranasal small interfering RNA (siRNA) delivery in a mouse model of hypoxia-induced working memory impairment using microglial receptor, Mac-1 as a target gene. Here, we implicate Mac-1 scavenger receptor in microglial phenotype switching, neurodegeneration in prefrontal cortex, hippocampus and working memory impairment. RNA mediated silencing of Mac-1 in both in vitro and in vivo model showed significant impact of it on hypoxia induced altered expression of Mac-1 endogenous ligand, signaling cascade proteins, transcription factors and NADPH oxidase pathway. Efficient degradation of Mac-1 mRNA suppressed expression of M1 phenotypic markers, inflammatory chemokines, and cytokines, but on the other hand, it upregulated M2 phenotypic markers and anti-inflammatory cytokines. Neuronal viability and synaptic plasticity markers were also modulated significantly by this strategy. Behavioral study revealed significant downregulation in the number of working memory errors at a time-dependent manner after silencing the Mac-1 gene during continuous hypoxic exposure. The novel findings of this study for the very first time, unmasked the role of Mac-1 receptor in neurodegenerative disease progression under hypoxic condition and at the same time indicated the potential therapeutic value of this non-invasive siRNA delivery approach for treating working memory loss. Copyright © 2018 Elsevier Ltd. All rights reserved.

Interleukin-18 Enhances Vascular Calcification and Osteogenic Differentiation of Vascular Smooth Muscle Cells Through TRPM7 Activation.

PubMed

Zhang, Kun; Zhang, Yinyin; Feng, Weijing; Chen, Renhua; Chen, Jie; Touyz, Rhian M; Wang, Jingfeng; Huang, Hui

2017-10-01

Vascular calcification (VC) is an important predictor of cardiovascular morbidity and mortality. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) is a key mechanism of VC. Recent studies show that IL-18 (interleukin-18) favors VC while TRPM7 (transient receptor potential melastatin 7) channel upregulation inhibits VC. However, the relationship between IL-18 and TRPM7 is unclear. We questioned whether IL-18 enhances VC and osteogenic differentiation of VSMCs through TRPM7 channel activation. Coronary artery calcification and serum IL-18 were measured in patients by computed tomographic scanning and enzyme-linked immunosorbent assay, respectively. Primary rat VSMCs calcification were induced by high inorganic phosphate and exposed to IL-18. VSMCs were also treated with TRPM7 antagonist 2-aminoethoxy-diphenylborate or TRPM7 small interfering RNA to block TRPM7 channel activity and expression. TRPM7 currents were recorded by patch-clamp. Human studies showed that serum IL-18 levels were positively associated with coronary artery calcium scores ( r =0.91; P <0.001). In VSMCs, IL-18 significantly decreased expression of contractile markers α-smooth muscle actin, smooth muscle 22 α, and increased calcium deposition, alkaline phosphatase activity, and expression of osteogenic differentiation markers bone morphogenetic protein-2, Runx2 (runt-related transcription factor 2), and osteocalcin ( P <0.05). IL-18 increased TRPM7 expression through ERK1/2 (extracellular signal-regulated kinase 1/2) signaling activation, and TRPM7 currents were augmented by IL-18 treatment. Inhibition of TRPM7 channel by 2-aminoethoxy-diphenylborate or TRPM7 small interfering RNA prevented IL-18-enhanced osteogenic differentiation and VSMCs calcification. These findings suggest that coronary artery calcification is associated with increased IL-18 levels. IL-18 enhances VSMCs osteogenic differentiation and subsequent VC induced by β-glycerophosphate via TRPM7 channel activation. Accordingly, IL-18 may contribute to VC in proinflammatory conditions. © 2017 American Heart Association, Inc.

Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex, C3PO, Determines Their Regulation by Phospholipase Cβ*

PubMed Central

Sahu, Shriya; Philip, Finly; Scarlata, Suzanne

2014-01-01

C3PO plays a key role in promoting RNA-induced gene silencing. C3PO consists of two subunits of the endonuclease translin-associated factor X (TRAX) and six subunits of the nucleotide-binding protein translin. We have found that TRAX binds strongly to phospholipase Cβ (PLCβ), which transmits G protein signals from many hormones and sensory inputs. The association between PLCβ and TRAX is thought to underlie the ability of PLCβ to reverse gene silencing by small interfering RNAs. However, this reversal only occurs for some genes (e.g. GAPDH and LDH) but not others (e.g. Hsp90 and cyclophilin A). To understand this specificity, we carried out studies using fluorescence-based methods. In cells, we find that PLCβ, TRAX, and their complexes are identically distributed through the cytosol suggesting that selectivity is not due to large scale sequestration of either the free or complexed proteins. Using purified proteins, we find that PLCβ binds ∼5-fold more weakly to translin than to TRAX but ∼2-fold more strongly to C3PO. PLCβ does not alter TRAX-translin assembly to C3PO, and brightness studies suggest one PLCβ binds to one C3PO octamer without a change in the number of TRAX/translin molecules suggesting that PLCβ binds to an external site. Functionally, we find that C3PO hydrolyzes siRNA(GAPDH) at a faster rate than siRNA(Hsp90). However, when PLCβ is bound to C3PO, the hydrolysis rate of siRNA(GAPDH) becomes comparable with siRNA(Hsp90). Our results show that the selectivity of PLCβ toward certain genes lies in the rate at which the RNA is hydrolyzed by C3PO. PMID:24338081

Minireview: The Roles of Small RNA Pathways in Reproductive Medicine

PubMed Central

Buchold, Gregory M.

2011-01-01

The discovery of small noncoding RNA, including P-element-induced wimpy testis-interacting RNA, small interfering RNA, and microRNA, has energized research in reproductive medicine. In the two decades since the identification of small RNA, first in Caenorhabditis elegans and then in other animals, scientists in many disciplines have made significant progress in elucidating their biology. A powerful battery of tools, including knockout mice and small RNA mimics and antagonists, has facilitated investigation into the functional roles and therapeutic potential of these small RNA pathways. Current data indicate that small RNA play significant roles in normal development and physiology and pathological conditions of the reproductive tracts of females and males. Biologically plausible mRNA targets for these microRNA are aggressively being discovered. The next phase of research will focus on elucidating the clinical utility of small RNA-selective agonists and antagonists. PMID:21546411

Endogenous small RNAs and antibacterial immunity in plants.

PubMed

Jin, Hailing

2008-08-06

Small RNAs are non-coding regulatory RNA molecules that control gene expression by mediating mRNA degradation, translational inhibition, or chromatin modification. Virus-derived small RNAs induce silencing of viral RNAs and are essential for antiviral defense in both animal and plant systems. The role of host endogenous small RNAs on antibacterial immunity has only recently been recognized. Host disease resistance and defense responses are achieved by activation and repression of a large array of genes. Certain endogenous small RNAs in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are induced or repressed in response to pathogen attack and subsequently regulate the expression of genes involved in disease resistance and defense responses by mediating transcriptional or post-transcriptional gene silencing. Thus, these small RNAs play an important role in gene expression reprogramming in plant disease resistance and defense responses. This review focuses on the recent findings of plant endogenous small RNAs in antibacterial immunity.

Deletion of Cytoplasmic Double-Stranded RNA Sensors Does Not Uncover Viral Small Interfering RNA Production in Human Cells.

PubMed

Schuster, Susan; Tholen, Lotte E; Overheul, Gijs J; van Kuppeveld, Frank J M; van Rij, Ronald P

2017-01-01

Antiviral immunity in insects and plants is mediated by the RNA interference (RNAi) pathway in which viral long double-stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer enzymes. Although this pathway is evolutionarily conserved, its involvement in antiviral defense in mammals is the subject of debate. In vertebrates, recognition of viral RNA induces a sophisticated type I interferon (IFN)-based immune response, and it has been proposed that this response masks or inhibits antiviral RNAi. To test this hypothesis, we analyzed viral small RNA production in differentiated cells deficient in the cytoplasmic RNA sensors RIG-I and MDA5. We did not detect 22-nucleotide (nt) viral siRNAs upon infection with three different positive-sense RNA viruses. Our data suggest that the depletion of cytoplasmic RIG-I-like sensors is not sufficient to uncover viral siRNAs in differentiated cells. IMPORTANCE The contribution of the RNA interference (RNAi) pathway in antiviral immunity in vertebrates has been widely debated. It has been proposed that RNAi possesses antiviral activity in mammalian systems but that its antiviral effect is masked by the potent antiviral interferon response in differentiated mammalian cells. In this study, we show that inactivation of the interferon response is not sufficient to uncover antiviral activity of RNAi in human epithelial cells infected with three wild-type positive-sense RNA viruses.

Analysis of RDR1/RDR2/RDR6-independent small RNAs in Arabidopsis thaliana improves MIRNA annotations and reveals unexplained types of short interfering RNA loci.

PubMed

Polydore, Seth; Axtell, Michael J

2018-06-01

Plant small RNAs (sRNAs) modulate key physiological mechanisms through post-transcriptional and transcriptional silencing of gene expression. Small RNAs fall into two major categories: those are reliant on RNA-dependent RNA polymerases (RDRs) for biogenesis and those that are not. Known RDR1/2/6-dependent sRNAs include phased and repeat-associated short interfering RNAs, while known RDR1/2/6-independent sRNAs are primarily microRNAs (miRNA) and other hairpin-derived sRNAs. In this study we produced and analyzed sRNA-seq libraries from rdr1/rdr2/rdr6 triple mutant plants. We found 58 previously annotated miRNA loci that were reliant on RDR1, -2, or -6 function, casting doubt on their classification. We also found 38 RDR1/2/6-independent sRNA loci that are not MIRNAs or otherwise hairpin-derived, and did not fit into other known paradigms for sRNA biogenesis. These 38 sRNA-producing loci have as-yet-undescribed biogenesis mechanisms, and are frequently located in the vicinity of protein-coding genes. Altogether, our analysis suggests that these 38 loci represent one or more undescribed types of sRNA in Arabidopsis thaliana. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Fabry-Perot Interferometry in the Integer and Fractional Quantum Hall Regimes

NASA Astrophysics Data System (ADS)

McClure, Douglas; Chang, Willy; Kou, Angela; Marcus, Charles; Pfeiffer, Loren; West, Ken

2011-03-01

We present measurements of electronic Fabry-Perot interferometers in the integer and fractional quantum Hall regimes. Two classes of resistance oscillations may be seen as a function of magnetic field and gate voltage, as we have previously reported. In small interferometers in the integer regime, oscillations of the type associated with Coulomb interaction are ubiquitous, while those consistent with single-particle Aharonov-Bohm interference are seen to co-exist in some configurations. The amplitude scaling of both types with temperature and device size is consistent with a theoretical model. Oscillations are further observed in the fractional quantum Hall regime. Here the dependence of the period on the filling factors in the constrictions and bulk of the interferometer can shed light on the effective charge of the interfering quasiparticles, but care is needed to distinguish these oscillations from those associated with integer quantum Hall states. We acknowledge funding from Microsoft Project Q and IBM.

Mobile Transcripts and Intercellular Communication in Plants.

PubMed

Saplaoura, E; Kragler, F

2016-01-01

Phloem serves as a highway for mobile signals in plants. Apart from sugars and hormones, proteins and RNAs are transported via the phloem and contribute to the intercellular communication coordinating growth and development. Different classes of RNAs have been found mobile and in the phloem exudate such as viral RNAs, small interfering RNAs (siRNAs), microRNAs, transfer RNAs, and messenger RNAs (mRNAs). Their transport is considered to be mediated via ribonucleoprotein complexes formed between phloem RNA-binding proteins and mobile RNA molecules. Recent advances in the analysis of the mobile transcriptome indicate that thousands of transcripts move along the plant axis. Although potential RNA mobility motifs were identified, research is still in progress on the factors triggering siRNA and mRNA mobility. In this review, we discuss the approaches used to identify putative mobile mRNAs, the transport mechanism, and the significance of mRNA trafficking. © 2016 Elsevier Inc. All rights reserved.

Troyer Syndrome Protein Spartin Is Mono-Ubiquitinated and Functions in EGF Receptor Trafficking

PubMed Central

Jupille, Henri; Fatheddin, Parvin; Puertollano, Rosa

2007-01-01

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia caused by mutation in the spartin (SPG20) gene, which encodes a widely expressed protein of unknown function. This mutation results in premature protein truncation and thus might signify a loss-of-function disease mechanism. In this study, we have found that spartin is mono-ubiquitinated and functions in degradation of the epidermal growth factor receptor (EGFR). Upon EGF stimulation, spartin translocates from the cytoplasm to the plasma membrane and colocalizes with internalized EGF-Alexa. Knockdown of spartin by small interfering RNA decreases the rate of EGFR degradation and also affects EGFR internalization, recycling, or both. Furthermore, overexpression of spartin results in a prominent decrease in EGFR degradation. Taken together, our data suggest that spartin is involved in the intracellular trafficking of EGFR and that impaired endocytosis may underlie the pathogenesis of Troyer syndrome. PMID:17332501

[siRNA-mediated tissue factor knockdown in porcine neonatal islet cell clusters in vitro].

PubMed

Ji, Ming; Yi, Shounan; Yu, Deling; Wang, Wei

2011-12-01

To determine the genetic modification on neonatal porcine islet cell clusters (NICC) by small interfering RNA (siRNA)-mediated tissue factor (TF) knockdown in vitro. Porcine NICC were transfected with 5 pairs of designed siRNA respectively or in different combinations with lipofectamine 2000. Transfected NICC were analyzed for TF gene by real-time PCR to select the siRNA which worked best. Meanwhile, the viability of NICC after the TF siRNA transfection was examined by FACS. The efficiency of TF gene and protein suppression was measured by real-time PCR and and FACS respectively. Real-time PCR and FACS showed that a 60% reduction in the TF gene expression and a 50% reduction in the protien level of TF on NICC were achieved by transfecting 3 pairs of selected siRNA. The siRNA transfection had no significant effect on the viability of NICC which was analyzed by FACS. The expression of TF on porcine NICC is efficiently suppressed by 3 pairs of designed siRNA in vitro.

Xenon Preconditioning Protects against Renal Ischemic-Reperfusion Injury via HIF-1α Activation

PubMed Central

Ma, Daqing; Lim, Ta; Xu, Jing; Tang, Haidy; Wan, Yanjie; Zhao, Hailin; Hossain, Mahmuda; Maxwell, Patrick H.; Maze, Mervyn

2009-01-01

The mortality rate from acute kidney injury after major cardiovascular operations can be as high as 60%, and no therapies have been proved to prevent acute kidney injury in this setting. Here, we show that preconditioning with the anesthetic gas xenon activates hypoxia-inducible factor 1α (HIF-1α) and its downstream effectors erythropoietin and vascular endothelial growth factor in a time-dependent manner in the kidneys of adult mice. Xenon increased the efficiency of HIF-1α translation via modulation of the mammalian target of rapamycin pathway. In a model of renal ischemia-reperfusion injury, xenon provided morphologic and functional renoprotection; hydrodynamic injection of HIF-1α small interfering RNA demonstrated that this protection is HIF-1α dependent. These results suggest that xenon preconditioning is a natural inducer of HIF-1α and that administration of xenon before renal ischemia can prevent acute renal failure. If these data are confirmed in the clinical setting, then preconditioning with xenon may be beneficial before procedures that temporarily interrupt renal perfusion. PMID:19144758

Intercomparisons of nine sky brightness detectors.

PubMed

den Outer, Peter; Lolkema, Dorien; Haaima, Marty; van der Hoff, Rene; Spoelstra, Henk; Schmidt, Wim

2011-01-01

Nine Sky Quality Meters (SQMs) have been intercompared during a night time measurement campaign held in the Netherlands in April 2011. Since then the nine SQMs have been distributed across The Netherlands and form the Dutch network for monitoring night sky brightness. The goal of the intercomparison was to infer mutual calibration factors and obtain insight into the variability of the SQMs under different meteorological situations. An ensemble average is built from the individual measurements and used as a reference to infer the mutual calibration factors. Data required additional synchronization prior to the calibration determination, because the effect of moving clouds combined with small misalignments emerges as time jitter in the measurements. Initial scatter of the individual instruments lies between ±14%. Individual night time sums range from -16% to +20%. Intercalibration reduces this to 0.5%, and -7% to +9%, respectively. During the campaign the smallest luminance measured was 0.657 ± 0.003 mcd/m(2) on 12 April, and the largest value was 5.94 ± 0.03 mcd/m(2) on 2 April. During both occurrences interfering circumstances like snow cover or moonlight were absent.

CaMKII inhibition promotes neuronal apoptosis by transcriptionally upregulating Bim expression.

PubMed

Zhao, Yiwei; Zhu, Lin; Yu, Shaojun; Zhu, Jing; Wang, Chong

2016-09-28

The effects of Ca/calmodulin-dependent protein kinase II (CaMKII) on neuronal apoptosis are complex and contradictory, and the underlying mechanisms remain unclear. Bcl-2-interacting mediator of cell death (Bim) is an important proapoptotic protein under many physiological and pathophysiological conditions. However, there is no evidence that CaMKII and Bim are mechanistically linked in neuronal apoptosis. In this study, we showed that CaMKII inhibition by the inhibitors KN-62 and myristoylated autocamtide-2-related inhibitory peptide promoted apoptosis in cerebellar granule neurons in a dose-dependent manner. CaMKII inhibition increased Bim protein and messenger RNA levels. The expression of early growth response factor-1, a transcription factor of Bim, was also induced by CaMKII inhibitors. These data suggested that CaMKII repressed the transcriptional expression of Bim. Moreover, knockdown of Bim using small interfering RNAs attenuated the proapoptotic effects of CaMKII inhibition. Taken together, this is the first report to show that CaMKII inhibition transcriptionally upregulates Bim expression to promote neuronal apoptosis, providing new insights into the proapoptotic mechanism of CaMKII inhibition.

Constitutive activation of alternative nuclear factor kappa B pathway in canine diffuse large B-cell lymphoma contributes to tumor cell survival and is a target of new adjuvant therapies.

PubMed

Seelig, Davis M; Ito, Daisuke; Forster, Colleen L; Yoon, Una A; Breen, Matthew; Burns, Linda J; Bachanova, Veronika; Lindblad-Toh, Kerstin; O'Brien, Timothy D; Schmechel, Stephen C; Rizzardi, Anthony E; Modiano, Jaime F; Linden, Michael A

2017-07-01

Activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway is a common molecular event observed in both human and canine diffuse large B-cell lymphoma (DLBCL). Although the oncogenic potential of the alternative NFκB pathway (ANFκBP) has also been recently identified in DLBCL, its precise role in tumor pathogenesis and potential as a treatment target is understudied. We hypothesized that up-regulation of the ANFκBP plays an important role in the proliferation and survival of canine DLBCL cells, and we demonstrate that the ANFκBP is constitutively active in primary canine DLBCL samples and a cell line (CLBL1). We further demonstrate that a small interfering RNA inhibits the activation of the NFκB pathway and induces apoptosis in canine DLBCL cells. In conclusion, the ANFκBP facilitates survival of canine DLBCL cells, and thus, dogs with spontaneous DLBCL can provide a useful large animal model to study therapies targeting the ANFκBP.

Hook1 inhibits malignancy and epithelial-mesenchymal transition in hepatocellular carcinoma.

PubMed

Sun, Xu; Zhang, Qi; Chen, Wei; Hu, Qida; Lou, Yu; Fu, Qi-Han; Zhang, Jing-Ying; Chen, Yi-Wen; Ye, Long-Yun; Wang, Yi; Xie, Shang-Zhi; Hu, Li-Qiang; Liang, Ting-Bo; Bai, Xue-Li

2017-07-01

Hook1 is a member of the hook family of coiled-coil proteins, which is recently found to be associated with malignant tumors. However, its biological function in hepatocellular carcinoma is yet unknown. Here, we evaluated the Hook1 levels in human hepatocellular carcinoma samples and matched peritumoral tissues by real-time polymerase chain reaction. Small interfering RNA knockdown and a transforming growth factor-β-induced epithelial-mesenchymal transition model were employed to investigate the biological effects of Hook1 in hepatocellular carcinoma. Our results indicated that Hook1 levels were significantly lower in hepatocellular carcinoma tissues than in the peritumoral tissues. In addition, Hook1 expression was significantly associated with hepatocellular carcinoma malignancy. Hook1 was downregulated after transforming growth factor-β-induced epithelial-mesenchymal transition. Moreover, Hook1 knockdown promoted epithelial-mesenchymal transition and attenuated the sensitivity of hepatocellular carcinoma cells to doxorubicin. In summary, our results indicate that downregulation of Hook1 plays a pivotal role in hepatocellular carcinoma progression via epithelial-mesenchymal transition. Hook1 may be used as a novel marker and therapeutic molecular target in hepatocellular carcinoma.

Mesoporous silica nanorods toward efficient loading and intracellular delivery of siRNA

NASA Astrophysics Data System (ADS)

Chen, Lijue; She, Xiaodong; Wang, Tao; Shigdar, Sarah; Duan, Wei; Kong, Lingxue

2018-02-01

The technology of RNA interference (RNAi) that uses small interfering RNA (siRNA) to silence the gene expression with complementary messenger RNA (mRNA) sequence has great potential for the treatment of cancer in which certain genes were usually found overexpressed. However, the carry and delivery of siRNA to the target site in the human body can be challenging for this technology to be used clinically to silence the cancer-related gene expression. In this work, rod shaped mesoporous silica nanoparticles (MSNs) were developed as siRNA delivery system for specific intracellular delivery. The rod MSNs with an aspect ratio of 1.5 had a high surface area of 934.28 m2/g and achieved a siRNA loading of more than 80 mg/g. With the epidermal growth factor (EGF) grafted on the surface of the MSNs, siRNA can be delivered to the epidermal growth factor receptor (EGFR) overexpressed colorectal cancer cells with high intracellular concentration compared to MSNs without EGF and lead to survivin gene knocking down to less than 30%.

Genome-wide piRNA profiles of virus transmitting whitefly Bemisia tabaci during feeding on TYLCV-infected tomato

USDA-ARS?s Scientific Manuscript database

Small RNAs (sRNAs) are 20-31 nucleotide (nt) non-coding regulatory elements commonly found in plants and animals, which are classified as short interfering RNA (siRNA), microRNA (miRNA) and Piwi-interacting RNA (piRNA). The whitefly Bemisia tabaci MEAM1 is a vector capable of transmitting many devas...

High-Throughput RNA Interference Screening: Tricks of the Trade

PubMed Central

Nebane, N. Miranda; Coric, Tatjana; Whig, Kanupriya; McKellip, Sara; Woods, LaKeisha; Sosa, Melinda; Sheppard, Russell; Rasmussen, Lynn; Bjornsti, Mary-Ann; White, E. Lucile

2016-01-01

The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z′ factor, which incorporates both standard deviation and signal window. A Z′ factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z′ factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z′ factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality. PMID:23616418

Ambulatory blood pressure and adherence monitoring: diagnosing pseudoresistant hypertension.

PubMed

Burnier, Michel; Wuerzner, Gregoire

2014-01-01

A small proportion of the treated hypertensive population consistently has a blood pressure greater than 140/90 mm Hg despite a triple therapy including a diuretic, a calcium channel blocker, and a blocker of the renin-angiotensin system. According to guidelines, these patients have so-called resistant hypertension. The prevalence of this clinical condition is higher in tertiary than primary care centers and often is associated with chronic kidney disease, diabetes, obesity, and sleep apnea syndrome. Exclusion of pseudoresistant hypertension using ambulatory or home blood pressure monitoring is a crucial step in the investigation of patients with resistant hypertension. Thus, among the multiple factors to consider when investigating patients with resistant hypertension, ambulatory blood pressure monitoring should be performed very early. Among other factors to consider, physicians should investigate patient adherence to therapy, assess the adequacy of treatment, exclude interfering factors, and, finally, look for secondary forms of hypertension. Poor adherence to therapy accounts for 30% to 50% of cases of resistance to therapy depending on the methodology used to diagnose adherence problems. This review discusses the clinical factors implicated in the pathogenesis of resistant hypertension with a particular emphasis on pseudoresistance, drug adherence, and the use of ambulatory blood pressure monitoring for the diagnosis and management of resistant hypertension.

Downregulation of SS18-SSX1 expression in synovial sarcoma by small interfering RNA enhances the focal adhesion pathway and inhibits anchorage-independent growth in vitro and tumor growth in vivo.

PubMed

Takenaka, Satoshi; Naka, Norifumi; Araki, Nobuhito; Hashimoto, Nobuyuki; Ueda, Takafumi; Yoshioka, Kiyoko; Yoshikawa, Hideki; Itoh, Kazuyuki

2010-04-01

Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by a unique t(X;18) translocation resulting in expression of SS18-SSX fusion protein. In order to investigate the biological function of this fusion protein and to develop a novel therapeutic option, we examined downregulation of SS18-SSX1 expression by small interfering RNA targeting SS18-SSX1 in three human SS cell lines. Microarray analysis comparing SS18-SSX1-silenced cells with control cells in three SS cell lines showed that SS18-SSX1 mainly affected the focal adhesion pathway. In accord with the array data, silencing of SS18-SSX1 enhances adhesion to the extracellular matrix through the induction of expression of myosin light-chain kinase. Furthermore, the silencing of SS18-SSX1 inhibits anchorage-independent growth in vitro and systemic delivery of siRNA against SS18-SSX1 using a nanoparticle system inhibited tumor growth in a nude mouse xenograft model. Our results demonstrate that siRNA targeting of SS18-SSX1 has therapeutic potential for the treatment of SS.

Targeted Delivery of Small Interfering RNA Using Reconstituted High-Density Lipoprotein Nanoparticles12

PubMed Central

Shahzad, Mian MK; Mangala, Lingegowda S; Han, Hee Dong; Lu, Chunhua; Bottsford-Miller, Justin; Nishimura, Masato; Mora, Edna M; Lee, Jeong-Won; Stone, Rebecca L; Pecot, Chad V; Thanapprapasr, Duangmani; Roh, Ju-Won; Gaur, Puja; Nair, Maya P; Park, Yun-Yong; Sabnis, Nirupama; Deavers, Michael T; Lee, Ju-Seog; Ellis, Lee M; Lopez-Berestein, Gabriel; McConathy, Walter J; Prokai, Laszlo; Lacko, Andras G; Sood, Anil K

2011-01-01

RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches. PMID:21472135

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

PubMed

Zhu, Kai; Lai, Hao; Guo, Changfa; Li, Jun; Wang, Yulin; Wang, Lingyan; Wang, Chunsheng

2014-01-01

Mesenchymal stem cell (MSC) transplantation has attracted much attention in myocardial infarction therapy. One of the limitations is the poor survival of grafted cells in the ischemic microenvironment. Small interfering RNA-mediated prolyl hydroxylase domain protein 2 (PHD2) silencing in MSCs holds tremendous potential to enhance their survival and paracrine effect after transplantation. However, an efficient and biocompatible PHD2 silencing system for clinical application is lacking. Herein, we developed a novel PHD2 silencing system based on arginine-terminated generation 4 poly(amidoamine) (Arg-G4) nanoparticles. The system exhibited effective and biocompatible small interfering RNA delivery and PHD2 silencing in MSCs in vitro. After genetically modified MSC transplantation in myocardial infarction models, MSC survival and paracrine function of IGF-1 were enhanced significantly in vivo. As a result, we observed decreased cardiomyocyte apoptosis, scar size, and interstitial fibrosis, and increased angiogenesis in the diseased myocardium, which ultimately attenuated ventricular remodeling and improved heart function. This work demonstrated that an Arg-G4 nanovector-based PHD2 silencing system could enhance the efficiency of MSC transplantation for infarcted myocardium repair.

Effects of downregulation of S100A8 protein expression on cell cycle and apoptosis of fibroblasts derived from hypertrophic scars.

PubMed

Yaundong, Lv; Dongyan, Wang; Lijun, Hao; Zhibo, Xiao

2014-01-01

Uncontrolled growth and lack of apoptosis in fibroblasts derived from a hypertrophic scar play an important role in pathology. The authors explore the contribution of S100A8 overexpression to the phenotype of cells and discuss how the downregulation of S100A8 could inhibit the growth and induce apoptosis of fibroblasts derived from hypertrophic scars. Fibroblasts were harvested from hypertrophic scar tissue in 8 patients treated with small interfering RNA against S100A8 in an in vitro culture. The effects of silencing S100A8 were analyzed by Western blot. Cellular proliferation and apoptosis were detected by flow cytometry. Fibroblasts treated with small interfering RNA targeting S100A8 showed a significant decrease in S100A8 protein 48 hours after treatment. They also proliferated significantly slower and showed more apoptosis than control fibroblasts. Inhibition of S100A8 resulted in significant growth reduction and apoptosis acceleration in fibroblasts derived from hypertrophic scars. Manipulation of S100A8 protein expression by gene silencing may represent something new in the treatment of hypertrophic scarring.

The Expression and Significance of Neuronal Iconic Proteins in Podocytes

PubMed Central

Sun, Yu; Zhang, Hongxia; Hu, Ruimin; Sun, Jianyong; Mao, Xing; Zhao, Zhonghua; Chen, Qi; Zhang, Zhigang

2014-01-01

Growing evidence suggests that there are many common cell biological features shared by neurons and podocytes; however, the mechanism of podocyte foot process formation remains unclear. Comparing the mechanisms of process formation between two cell types should provide useful guidance from the progress of neuron research. Studies have shown that some mature proteins of podocytes, such as podocin, nephrin, and synaptopodin, were also expressed in neurons. In this study, using cell biological experiments and immunohistochemical techniques, we showed that some neuronal iconic molecules, such as Neuron-specific enolase, nestin and Neuron-specific nuclear protein, were also expressed in podocytes. We further inhibited the expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 by Small interfering RNA in cultured mouse podocytes and observed the significant morphological changes in treated podocytes. When podocytes were treated with Adriamycin, the protein expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 decreased over time. Meanwhile, the morphological changes in the podocytes were consistent with results of the Small interfering RNA treatment of these proteins. The data demonstrated that neuronal iconic proteins play important roles in maintaining and regulating the formation and function of podocyte processes. PMID:24699703

Protection against lethal Marburg virus infection mediated by lipid encapsulated small interfering RNA.

PubMed

Ursic-Bedoya, Raul; Mire, Chad E; Robbins, Marjorie; Geisbert, Joan B; Judge, Adam; MacLachlan, Ian; Geisbert, Thomas W

2014-02-15

Marburg virus (MARV) infection causes severe morbidity and mortality in humans and nonhuman primates. Currently, there are no licensed therapeutics available for treating MARV infection. Here, we present the in vitro development and in vivo evaluation of lipid-encapsulated small interfering RNA (siRNA) as a potential therapeutic for the treatment of MARV infection. The activity of anti-MARV siRNAs was assessed using dual luciferase reporter assays followed by in vitro testing against live virus. Lead candidates were tested in lethal guinea pig models of 3 different MARV strains (Angola, Ci67, Ravn). Treatment resulted in 60%-100% survival of guinea pigs infected with MARV. Although treatment with siRNA targeting other MARV messenger RNA (mRNA) had a beneficial effect, targeting the MARV NP mRNA resulted in the highest survival rates. NP-718m siRNA in lipid nanoparticles provided 100% protection against MARV strains Angola and Ci67, and 60% against Ravn. A cocktail containing NP-718m and NP-143m provided 100% protection against MARV Ravn. These data show protective efficacy against the most pathogenic Angola strain of MARV. Further development of the lipid nanoparticle technology has the potential to yield effective treatments for MARV infection.

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.

PubMed

Song, Erwei; Zhu, Pengcheng; Lee, Sang-Kyung; Chowdhury, Dipanjan; Kussman, Steven; Dykxhoorn, Derek M; Feng, Yi; Palliser, Deborah; Weiner, David B; Shankar, Premlata; Marasco, Wayne A; Lieberman, Judy

2005-06-01

Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

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

PubMed

Runo, Steven

2011-01-01

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

Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations.

PubMed

Sakaguchi, Aisa; Sarkies, Peter; Simon, Matt; Doebley, Anna-Lisa; Goldstein, Leonard D; Hedges, Ashley; Ikegami, Kohta; Alvares, Stacy M; Yang, Liwei; LaRocque, Jeannine R; Hall, Julie; Miska, Eric A; Ahmed, Shawn

2014-10-14

Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of late-generation rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing.

Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations

PubMed Central

Sakaguchi, Aisa; Sarkies, Peter; Simon, Matt; Doebley, Anna-Lisa; Goldstein, Leonard D.; Hedges, Ashley; Ikegami, Kohta; Alvares, Stacy M.; Yang, Liwei; LaRocque, Jeannine R.; Hall, Julie; Miska, Eric A.; Ahmed, Shawn

2014-01-01

Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of late-generation rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing. PMID:25258416

Schools as Sanctuaries: A Systematic Review of Contextual Factors Which Contribute to Student Retention in Alternative Education

ERIC Educational Resources Information Center

O'Gorman, Eva; Salmon, Nancy; Murphy, Carol-Anne

2016-01-01

Early school leaving is an international concern. Previous research indicates that the school context contributes to early school leaving. This systematic review is aimed to gather marginalised young peoples' perceptions concerning contextual factors that contributed to and interfered with their decisions to stay in alternative education.…

Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

PubMed

Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

2010-07-30

Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.

Gene regulation by noncoding RNAs

PubMed Central

Patil, Veena S.; Zhou, Rui; Rana, Tariq M.

2015-01-01

The past two decades have seen an explosion in research on noncoding RNAs and their physiological and pathological functions. Several classes of small (20–30 nucleotides) and long (>200 nucleotides) noncoding RNAs have been firmly established as key regulators of gene expression in myriad processes ranging from embryonic development to innate immunity. In this review, we focus on our current understanding of the molecular mechanisms underlying the biogenesis and function of small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). In addition, we briefly review the relevance of small and long noncoding RNAs to human physiology and pathology and their potential to be exploited as therapeutic agents. PMID:24164576

Wind interference effect on an octagonal plan shaped tall building due to square plan shaped tall buildings

NASA Astrophysics Data System (ADS)

Kar, Rony; Dalui, Sujit Kumar

2016-03-01

The variation of pressure at the faces of the octagonal plan shaped tall building due to interference of three square plan shaped tall building of same height is analysed by computational fluid dynamics module, namely ANSYS CFX for 0° wind incidence angle only. All the buildings are closely spaced (distance between two buildings varies from 0.4 h to 2 h, where h is the height of the building). Different cases depending upon the various positions of the square plan shaped buildings are analysed and compared with the octagonal plan shaped building in isolated condition. The comparison is presented in the form of interference factors (IF) and IF contours. Abnormal pressure distribution is observed in some cases. Shielding and channelling effect on the octagonal plan shaped building due to the presence of the interfering buildings are also noted. In the interfering condition the pressure distribution at the faces of the octagonal plan shaped building is not predictable. As the distance between the principal octagonal plan shaped building and the third square plan shaped interfering building increases the behaviour of faces becomes more systematic. The coefficient of pressure (C p) for each face of the octagonal plan shaped building in each interfering case can be easily found if we multiply the IF with the C p in the isolated case.

A steady and oscillatory kernel function method for interfering surfaces in subsonic, transonic and supersonic flow. [prediction analysis techniques for airfoils

NASA Technical Reports Server (NTRS)

Cunningham, A. M., Jr.

1976-01-01

The theory, results and user instructions for an aerodynamic computer program are presented. The theory is based on linear lifting surface theory, and the method is the kernel function. The program is applicable to multiple interfering surfaces which may be coplanar or noncoplanar. Local linearization was used to treat nonuniform flow problems without shocks. For cases with imbedded shocks, the appropriate boundary conditions were added to account for the flow discontinuities. The data describing nonuniform flow fields must be input from some other source such as an experiment or a finite difference solution. The results are in the form of small linear perturbations about nonlinear flow fields. The method was applied to a wide variety of problems for which it is demonstrated to be significantly superior to the uniform flow method. Program user instructions are given for easy access.

A reverse signaling pathway downstream of Sema4A controls cell migration via Scrib.

PubMed

Sun, Tianliang; Yang, Lida; Kaur, Harmandeep; Pestel, Jenny; Looso, Mario; Nolte, Hendrik; Krasel, Cornelius; Heil, Daniel; Krishnan, Ramesh K; Santoni, Marie-Josée; Borg, Jean-Paul; Bünemann, Moritz; Offermanns, Stefan; Swiercz, Jakub M; Worzfeld, Thomas

2017-01-02

Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor βPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration. © 2017 Sun et al.

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

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

Smialowska, Agata, E-mail: smialowskaa@gmail.com; School of Life Sciences, Södertörn Högskola, Huddinge 141-89; Djupedal, Ingela

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

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib.

PubMed

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-05-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC.

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib

PubMed Central

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-01-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC. PMID:28521430

Development of siRNA Technology to Prevent Scar Formation in Tendon Repair

DTIC Science & Technology

2013-12-01

Anti-sense RNA technologies: Under normal conditions cells produce small interfering (si) RNAs that inhibit protein synthesis and stimulate...stimulation of fibroblast proliferation and migration, collagen and fibronectin synthesis , and altered tissue remodeling through regulation of MMPs...expression by an antisense oligonucleotide protects mice from fulminant hepatitis. Nat Biotechnol 2000;18:862-7. 7. Guha M, Xu ZG, Tung D, Lanting L

Small-interfering RNA (siRNA)-based functional micro- and nanostructures for efficient and selective gene silencing.

PubMed

Lee, Soo Hyeon; Chung, Bong Hyun; Park, Tae Gwan; Nam, Yoon Sung; Mok, Hyejung

2012-07-17

Because of RNA's ability to encode structure and functional information, researchers have fabricated diverse geometric structures from this polymer at the micro- and nanoscale. With their tunable structures, rigidity, and biocompatibility, novel two-dimensional and three-dimensional RNA structures can serve as a fundamental platform for biomedical applications, including engineered tissues, biosensors, and drug delivery vehicles. The discovery of the potential of small-interfering RNA (siRNA) has underscored the applications of RNA-based micro- and nanostructures in medicine. Small-interfering RNA (siRNA), synthetic double-stranded RNA consisting of approximately 21 base pairs, suppresses problematic target genes in a sequence-specific manner via inherent RNA interference (RNAi) processing. As a result, siRNA offers a potential strategy for treatment of many human diseases. However, due to inefficient delivery to cells and off-target effects, the clinical application of therapeutic siRNA has been very challenging. To address these issues, researchers have studied a variety of nanocarrier systems for siRNA delivery. In this Account, we describe several strategies for efficient siRNA delivery and selective gene silencing. We took advantage of facile chemical conjugation and complementary hybridization to design novel siRNA-based micro- and nanostructures. Using chemical crosslinkers and hydrophobic/hydrophilic polymers at the end of siRNA, we produced various RNA-based structures, including siRNA block copolymers, micelles, linear siRNA homopolymers, and microhydrogels. Because of their increased charge density and flexibility compared with conventional siRNA, these micro- and nanostructures can form polyelectrolyte complexes with poorly charged and biocompatible cationic carriers that are both more condensed and more homogenous than the complexes formed in other carrier systems. In addition, the fabricated siRNA-based structures are linked by cleavable disulfide bonds for facile generation of original siRNA in the cytosol and for target-specific gene silencing. These newly developed siRNA-based structures greatly enhance intracellular uptake and gene silencing both in vitro and in vivo, making them promising biomaterials for siRNA therapeutics.

Targeted Gene Silencing of Tumor Necrosis Factor Attenuates the Negative Inotropic Effects of Lipopolysaccharide in Isolated Contracting Cardiac Myocytes

PubMed Central

Ramabadran, R. S.; Chancey, Amanda; Vallejo, Jesus G.; Barger, Philip M.; Sivasubramanian, Natarajan; Mann, Douglas L.

2008-01-01

Bacterial endotoxin (lipopolysaccharide) depresses cardiovascular function; however, the mediators and signaling pathways that are responsible for the negative inotropic effects of lipopolysaccharide are not fully known. We used RNA interference to determine the relative role of tumor necrosis factor with respect to mediating the negative inotropic effects of lipopolysaccharide in isolated cardiac myocytes. Cardiac myocyte cultures were treated with lipopolysaccharide in the presence or absence of small interfering RNAs (siRNA) for tumor necrosis factor. We examined the effects of tumor necrosis factor siRNA on lipopolysaccharide-induced tumor necrosis factor messenger RNA (mRNA) and protein biosynthesis, as well as the negative inotropic effects of lipopolysaccharide in isolated contracting cardiac myocytes. Treatment of adult cardiac myocyte cultures with tumor necrosis factor siRNA significantly attenuated lipopolysaccharide-induced tumor necrosis factor mRNA and protein biosynthesis, whereas transfection with a double-stranded RNA that does not target mammalian mRNA had no effect. Pretreatment with tumor necrosis factor siRNA significantly attenuated, but did not abrogate, the lipopolysaccharide-induced decrease in sarcomere shortening in isolated contracting cardiac myocytes. In contrast, tumor necrosis factor siRNA had a comparatively smaller effect on improving sarcomere shortening once the negative inotropic effects of lipopolysaccharide were fully established. These results suggest that tumor necrosis factor plays an important upstream role in lipopolysaccharide-induced negative inotropic effects in isolated contracting cardiac myocytes and that other molecular mechanisms are responsible for the decrease in sarcomere shortening after sustained lipopolysaccharide signaling. PMID:18427645

Revisiting Criteria for Plant MicroRNA Annotation in the Era of Big Data[OPEN

PubMed Central

2018-01-01

MicroRNAs (miRNAs) are ∼21-nucleotide-long regulatory RNAs that arise from endonucleolytic processing of hairpin precursors. Many function as essential posttranscriptional regulators of target mRNAs and long noncoding RNAs. Alongside miRNAs, plants also produce large numbers of short interfering RNAs (siRNAs), which are distinguished from miRNAs primarily by their biogenesis (typically processed from long double-stranded RNA instead of single-stranded hairpins) and functions (typically via roles in transcriptional regulation instead of posttranscriptional regulation). Next-generation DNA sequencing methods have yielded extensive data sets of plant small RNAs, resulting in many miRNA annotations. However, it has become clear that many miRNA annotations are questionable. The sheer number of endogenous siRNAs compared with miRNAs has been a major factor in the erroneous annotation of siRNAs as miRNAs. Here, we provide updated criteria for the confident annotation of plant miRNAs, suitable for the era of “big data” from DNA sequencing. The updated criteria emphasize replication and the minimization of false positives, and they require next-generation sequencing of small RNAs. We argue that improved annotation systems are needed for miRNAs and all other classes of plant small RNAs. Finally, to illustrate the complexities of miRNA and siRNA annotation, we review the evolution and functions of miRNAs and siRNAs in plants. PMID:29343505

Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom expression for citrus huanglongbing disease.

PubMed

Zhao, Hongwei; Sun, Ruobai; Albrecht, Ute; Padmanabhan, Chellappan; Wang, Airong; Coffey, Michael D; Girke, Thomas; Wang, Zonghua; Close, Timothy J; Roose, Mikeal; Yokomi, Raymond K; Folimonova, Svetlana; Vidalakis, Georgios; Rouse, Robert; Bowman, Kim D; Jin, Hailing

2013-03-01

Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus 'Candidatus Liberibacter' (Ca. L.). Powerful diagnostic tools and management strategies are desired to control HLB. Host small RNAs (sRNA) play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases, including cancers. To determine whether citrus sRNAs regulate host responses to HLB, sRNAs were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca. L. asiaticus (Las)-positive or healthy tissue. Ten new microRNAs (miRNAs), 76 conserved miRNAs, and many small interfering RNAs (siRNAs) were discovered. Several miRNAs and siRNAs were highly induced by Las infection, and can be potentially developed into early diagnosis markers of HLB. miR399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn-a disease with symptoms similar to HLB. We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida. Our molecular, physiological, and field data suggest that phosphorus deficiency is linked to HLB disease symptomology.

Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing.

PubMed

Xu, Chenxi; Sun, Xuepeng; Taylor, Angela; Jiao, Chen; Xu, Yimin; Cai, Xiaofeng; Wang, Xiaoli; Ge, Chenhui; Pan, Guanghui; Wang, Quanxi; Fei, Zhangjun; Wang, Quanhua

2017-06-01

Tomato is a major vegetable crop that has tremendous popularity. However, viral disease is still a major factor limiting tomato production. Here, we report the tomato virome identified through sequencing small RNAs of 170 field-grown samples collected in China. A total of 22 viruses were identified, including both well-documented and newly detected viruses. The tomato viral community is dominated by a few species, and they exhibit polymorphisms and recombination in the genomes with cold spots and hot spots. Most samples were coinfected by multiple viruses, and the majority of identified viruses are positive-sense single-stranded RNA viruses. Evolutionary analysis of one of the most dominant tomato viruses, Tomato yellow leaf curl virus (TYLCV), predicts its origin and the time back to its most recent common ancestor. The broadly sampled data have enabled us to identify several unreported viruses in tomato, including a completely new virus, which has a genome of ∼13.4 kb and groups with aphid-transmitted viruses in the genus Cytorhabdovirus Although both DNA and RNA viruses can trigger the biogenesis of virus-derived small interfering RNAs (vsiRNAs), we show that features such as length distribution, paired distance, and base selection bias of vsiRNA sequences reflect different plant Dicer-like proteins and Argonautes involved in vsiRNA biogenesis. Collectively, this study offers insights into host-virus interaction in tomato and provides valuable information to facilitate the management of viral diseases. IMPORTANCE Tomato is an important source of micronutrients in the human diet and is extensively consumed around the world. Virus is among the major constraints on tomato production. Categorizing virus species that are capable of infecting tomato and understanding their diversity and evolution are challenging due to difficulties in detecting such fast-evolving biological entities. Here, we report the landscape of the tomato virome in China, the leading country in tomato production. We identified dozens of viruses present in tomato, including both well-documented and completely new viruses. Some newly emerged viruses in tomato were found to spread fast, and therefore, prompt attention is needed to control them. Moreover, we show that the virus genomes exhibit considerable degree of polymorphisms and recombination, and the virus-derived small interfering RNA (vsiRNA) sequences indicate distinct vsiRNA biogenesis mechanisms for different viruses. The Chinese tomato virome that we developed provides valuable information to facilitate the management of tomato viral diseases. Copyright © 2017 American Society for Microbiology.

Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing

PubMed Central

Xu, Chenxi; Taylor, Angela; Jiao, Chen; Xu, Yimin; Cai, Xiaofeng; Wang, Xiaoli; Ge, Chenhui; Pan, Guanghui; Wang, Quanxi

2017-01-01

ABSTRACT Tomato is a major vegetable crop that has tremendous popularity. However, viral disease is still a major factor limiting tomato production. Here, we report the tomato virome identified through sequencing small RNAs of 170 field-grown samples collected in China. A total of 22 viruses were identified, including both well-documented and newly detected viruses. The tomato viral community is dominated by a few species, and they exhibit polymorphisms and recombination in the genomes with cold spots and hot spots. Most samples were coinfected by multiple viruses, and the majority of identified viruses are positive-sense single-stranded RNA viruses. Evolutionary analysis of one of the most dominant tomato viruses, Tomato yellow leaf curl virus (TYLCV), predicts its origin and the time back to its most recent common ancestor. The broadly sampled data have enabled us to identify several unreported viruses in tomato, including a completely new virus, which has a genome of ∼13.4 kb and groups with aphid-transmitted viruses in the genus Cytorhabdovirus. Although both DNA and RNA viruses can trigger the biogenesis of virus-derived small interfering RNAs (vsiRNAs), we show that features such as length distribution, paired distance, and base selection bias of vsiRNA sequences reflect different plant Dicer-like proteins and Argonautes involved in vsiRNA biogenesis. Collectively, this study offers insights into host-virus interaction in tomato and provides valuable information to facilitate the management of viral diseases. IMPORTANCE Tomato is an important source of micronutrients in the human diet and is extensively consumed around the world. Virus is among the major constraints on tomato production. Categorizing virus species that are capable of infecting tomato and understanding their diversity and evolution are challenging due to difficulties in detecting such fast-evolving biological entities. Here, we report the landscape of the tomato virome in China, the leading country in tomato production. We identified dozens of viruses present in tomato, including both well-documented and completely new viruses. Some newly emerged viruses in tomato were found to spread fast, and therefore, prompt attention is needed to control them. Moreover, we show that the virus genomes exhibit considerable degree of polymorphisms and recombination, and the virus-derived small interfering RNA (vsiRNA) sequences indicate distinct vsiRNA biogenesis mechanisms for different viruses. The Chinese tomato virome that we developed provides valuable information to facilitate the management of tomato viral diseases. PMID:28331089

Centrosomal CK1delta Promotes Neurite Outgrowth | Center for Cancer Research

Cancer.gov

Previously we determined that Dishevelled-2/3 (Dvl) mediate Wnt-3a–dependent neurite outgrowth in Ewing sarcoma family tumor cells. Here we report that neurite extension was associated with Dvl phosphorylation and that both were inhibited by the casein kinase 1 (CK1) δ/ε inhibitor IC261. Small interfering RNAs targeting either CK1δ or CK1ε decreased Dvl phosphorylation, but

Raman properties of gold nanoparticle-decorated individual carbon nanotubes

NASA Astrophysics Data System (ADS)

Assmus, Tilman; Balasubramanian, Kannan; Burghard, Marko; Kern, Klaus; Scolari, Matteo; Fu, Nan; Myalitsin, Anton; Mews, Alf

2007-04-01

Single-wall carbon nanotubes decorated by gold nanoparticles with sizes of a few tens of nanometers were investigated by confocal Raman microscopy. It was found that individual nanoparticles impart a sizable Raman enhancement exceeding one order of magnitude, without appreciably interfering with polarization dependent Raman measurements. By contrast, cavity effects within small nanoparticle agglomerates resulted in a 20-fold stronger enhancement and significant distortions of the polarization characteristic.

MiR-21 suppresses the anticancer activities of curcumin by targeting PTEN gene in human non-small cell lung cancer A549 cells.

PubMed

Zhang, W; Bai, W; Zhang, W

2014-08-01

Curcumin, a natural phytochemical, exhibits potent anticancer activities. Here, we sought to determine the molecular mechanisms underlying the cytotoxic effects of curcumin against human non-small cell lung cancer (NSCLC) cells. MTT assay and annexin-V/PI staining were used to analyze the effects of curcumin on the proliferation and apoptosis of A549 cells. The expression of microRNA-21 in curcumin-treated A549 cells was measured by quantitative real-time polymerase chain reaction assay. The protein level of phosphatase and tensin homolog (PTEN), a putative target of microRNA-21, was determined by Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN small interfering RNA was performed to modulate the expression of microRNA-21 and PTEN under the treatment of curcumin. Curcumin at 20-40 μM inhibited cell proliferation and induced apoptosis in A549 cells. Curcumin treatment produced a dose-dependent and significant (P < 0.05) suppression of microRNA-21 expression, compared to untreated A549 cells. Moreover, the protein level of PTEN, a putative target of microRNA-21, was significantly elevated in curcumin-treated A549 cells, as determined by Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN small interfering RNA significantly (P < 0.05) reversed the growth suppression and apoptosis induction by curcumin, compared to corresponding controls. Our data suggest a novel molecular mechanism in which inhibition of microRNA-21 and upregulation of PTEN mediate the anticancer activities of curcumin in NSCLC cells. Suppression of microRNA-21 may thus have therapeutic benefits against this malignancy.

DNA enzymes as potential therapeutics: towards clinical application of 10-23 DNAzymes.

PubMed

Fokina, Alesya A; Stetsenko, Dmitry A; François, Jean-Christophe

2015-05-01

Ongoing studies on the inhibition of gene expression at the mRNA level have identified several types of specific inhibitors such as antisense oligonucleotides, small interfering RNA, ribozymes and DNAzymes (Dz). After its discovery in 1997, the 10-23 Dz (which can cleave RNA efficiently and site-specifically, has flexible design, is independent from cell mechanisms, does not require expensive chemical modifications for effective use in vivo) has been employed to downregulate a range of therapeutically important genes. Recently, 10-23 Dzs have taken their first steps into clinical trials. This review focuses predominantly on Dz applications as potential antiviral, antibacterial, anti-cancer and anti-inflammatory agents as well as for the treatment of cardiovascular disease and diseases of CNS, summarizing results of their clinical trials up to the present day. In comparison with antisense oligonucleotides and small interfering RNAs, Dzs do not usually show off-target effects due to their high specificity and lack of immunogenicity in vivo. As more results of clinical trials carried out so far are gradually becoming available, Dzs may turn out to be safe and well-tolerated therapeutics in humans. Therefore, there is a good chance that we may witness a deoxyribozyme drug reaching the clinic in the near future.

Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway.

PubMed

Fabozzi, Giulia; Nabel, Christopher S; Dolan, Michael A; Sullivan, Nancy J

2011-03-01

Cellular RNA interference (RNAi) provides a natural response against viral infection, but some viruses have evolved mechanisms to antagonize this form of antiviral immunity. To determine whether Ebolavirus (EBOV) counters RNAi by encoding suppressors of RNA silencing (SRSs), we screened all EBOV proteins using an RNAi assay initiated by exogenously delivered small interfering RNAs (siRNAs) against either an EBOV or a reporter gene. In addition to viral protein 35 (VP35), we found that VP30 and VP40 independently act as SRSs. Here, we present the molecular mechanisms of VP30 and VP35. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of siRNA. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). Taken together, our findings elucidate a new mechanism of RNAi suppression that extends beyond the role of SRSs in double-stranded RNA (dsRNA) binding and IFN antagonism. The presence of three suppressors highlights the relevance of host RNAi-dependent antiviral immunity in EBOV infection and illustrates the importance of RNAi in shaping the evolution of RNA viruses.

Protection Against Lethal Marburg Virus Infection Mediated by Lipid Encapsulated Small Interfering RNA

PubMed Central

Ursic-Bedoya, Raul; Mire, Chad E.; Robbins, Marjorie; Geisbert, Joan B.; Judge, Adam; MacLachlan, Ian; Geisbert, Thomas W.

2014-01-01

Background. Marburg virus (MARV) infection causes severe morbidity and mortality in humans and nonhuman primates. Currently, there are no licensed therapeutics available for treating MARV infection. Here, we present the in vitro development and in vivo evaluation of lipid-encapsulated small interfering RNA (siRNA) as a potential therapeutic for the treatment of MARV infection. Methods. The activity of anti-MARV siRNAs was assessed using dual luciferase reporter assays followed by in vitro testing against live virus. Lead candidates were tested in lethal guinea pig models of 3 different MARV strains (Angola, Ci67, Ravn). Results. Treatment resulted in 60%–100% survival of guinea pigs infected with MARV. Although treatment with siRNA targeting other MARV messenger RNA (mRNA) had a beneficial effect, targeting the MARV NP mRNA resulted in the highest survival rates. NP-718m siRNA in lipid nanoparticles provided 100% protection against MARV strains Angola and Ci67, and 60% against Ravn. A cocktail containing NP-718m and NP-143m provided 100% protection against MARV Ravn. Conclusions. These data show protective efficacy against the most pathogenic Angola strain of MARV. Further development of the lipid nanoparticle technology has the potential to yield effective treatments for MARV infection. PMID:23990568

Systematic Evaluation of Promising Clinical Trials-Gene Silencing for the Treatment of Glioblastoma.

PubMed

Karaarslan, Numan; Yilmaz, Ibrahim; Ozbek, Hanefi; Caliskan, Tezcan; Topuk, Savas; Sirin, Duygu Yasar; Ates, Ozkan

2018-04-06

The aim of this study was to systematically investigate the role of artificial small interfering RNA (siRNA) molecules in glioblastoma treatment and to give a detailed overview of the literature concerning studies performed in this field worldwide in the last 31 years. Articles about clinical trials conducted between December 1, 1949 and November 8, 2017, were identified from the Cochrane Collaboration, the Cochrane Library, Ovid MEDLINE, ProQuest, the National Library of Medicine, and PubMed electronic databases, using the terms "post transcriptional gene silencing," "small interfering RNA," "siRNA," and "glioblastoma," either individually or combined (\\"OR\\" and \\"AND"), without language and country restrictions. Articles that met the examination criteria were included in the study. After descriptive statistical evaluation, the results were reported in frequency (%). After scanning 2.752 articles, five articles were found that met the research criteria. Examination of full texts of the five identified articles provided no sufficient evidence for research conducted with regard to the use of gene silencing via siRNAs in glioblastoma treatment. To be able to evaluate the clinical use of siRNAs, there is an urgent need for in-vivo studies and for trials with randomized, controlled, and clinical designs that provide long-term functional outcomes.

Sensitization of human carcinoma cells to alkylating agents by small interfering RNA suppression of 3-alkyladenine-DNA glycosylase.

PubMed

Paik, Johanna; Duncan, Tod; Lindahl, Tomas; Sedgwick, Barbara

2005-11-15

One of the major cytotoxic lesions generated by alkylating agents is DNA 3-alkyladenine, which can be excised by 3-alkyladenine DNA glycosylase (AAG). Inhibition of AAG may therefore result in increased cellular sensitivity to chemotherapeutic alkylating agents. To investigate this possibility, we have examined the role of AAG in protecting human tumor cells against such agents. Plasmids that express small interfering RNAs targeted to two different regions of AAG mRNA were transfected into HeLa cervical carcinoma cells and A2780-SCA ovarian carcinoma cells. Stable derivatives of both cell types with low AAG protein levels were sensitized to alkylating agents. Two HeLa cell lines with AAG protein levels reduced by at least 80% to 90% displayed a 5- to 10-fold increase in sensitivity to methyl methanesulfonate, N-methyl-N-nitrosourea, and the chemotherapeutic drugs temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea. These cells showed no increase in sensitivity to UV light or ionizing radiation. After treatment with methyl methanesulfonate, AAG knockdown HeLa cells were delayed in S phase but accumulated in G2-M. Our data support the hypothesis that ablation of AAG activity in human tumor cells may provide a useful strategy to enhance the efficacy of current chemotherapeutic regimens that include alkylating agents.

Zwitterionic poly(carboxybetaine)-based cationic liposomes for effective delivery of small interfering RNA therapeutics without accelerated blood clearance phenomenon.

PubMed

Li, Yan; Liu, Ruiyuan; Shi, Yuanjie; Zhang, Zhenzhong; Zhang, Xin

2015-01-01

For efficient delivery of small interfering RNA (siRNA) to the target diseased site in vivo, it is important to design suitable vehicles to control the blood circulation of siRNA. It has been shown that surface modification of cationic liposome/siRNA complexes (lipoplexes) with polyethylene glycol (PEG) could enhance the circulation time of lipoplexes. However, the first injection of PEGylated lipoplexes in vivo induces accelerated blood clearance and enhances hepatic accumulation of the following injected PEGylated lipoplexes, which is known as the accelerated blood clearance (ABC) phenomenon. Herein, we developed zwitterionic poly(carboxybetaine) (PCB) modified lipoplexes for the delivery of siRNA therapeutics, which could avoid protein adsorption and enhance the stability of lipoplexes as that for PEG. Quite different from the PEGylation, the PCBylated lipoplexes could avoid ABC phenomenon, which extended the blood circulation time and enhanced the tumor accumulation of lipoplexes in vivo. After accumulation in tumor site, the PCBylation could promote the cellular uptake and endosomal/lysosomal escape of lipoplexes due to its unique chemical structure and pH-sensitive ability. With excellent tumor accumulation, cellular uptake and endosomal/lysosomal escape abilities, the PCBylated lipoplexes significantly inhibited tumor growth and induced tumor cell apoptosis.

Essential role of mitochondrial antiviral signaling, IFN regulatory factor (IRF)3, and IRF7 in Chlamydophila pneumoniae-mediated IFN-beta response and control of bacterial replication in human endothelial cells.

PubMed

Buss, Claudia; Opitz, Bastian; Hocke, Andreas C; Lippmann, Juliane; van Laak, Vincent; Hippenstiel, Stefan; Krüll, Matthias; Suttorp, Norbert; Eitel, Julia

2010-03-15

Chlamydophila pneumoniae infection of the vascular wall as well as activation of the transcription factor IFN regulatory factor (IRF)3 have been linked to development of chronic vascular lesions and atherosclerosis. The innate immune system detects invading pathogens by use of pattern recognition receptors, some of which are able to stimulate IRF3/7 activation and subsequent type I IFN production (e. g., IFN-beta). In this study, we show that infection of human endothelial cells with C. pneumoniae-induced production of IFN-beta, a cytokine that so far has been mainly associated with antiviral immunity. Moreover, C. pneumoniae infection led to IRF3 and IRF7 nuclear translocation in HUVECs and RNA interference experiments showed that IRF3 and IRF7 as well as the mitochondrial antiviral signaling (MAVS) were essential for IFN-beta induction. Finally, C. pneumoniae replication was enhanced in endothelial cells in which IRF3, IRF7, or MAVS expression was inhibited by small interfering RNA and attenuated by IFN-beta treatment. In conclusion, C. pneumoniae infection of endothelial cells activates an MAVS-, IRF3-, and IRF7-dependent signaling, which controls bacterial growth and might modulate development of vascular lesions.

Mediator of DNA damage checkpoint 1 (MDC1) contributes to high NaCl-induced activation of the osmoprotective transcription factor TonEBP/OREBP.

PubMed

Kunin, Margarita; Dmitrieva, Natalia I; Gallazzini, Morgan; Shen, Rong-Fong; Wang, Guanghui; Burg, Maurice B; Ferraris, Joan D

2010-08-11

Hypertonicity, such as induced by high NaCl, increases the activity of the transcription factor TonEBP/OREBP whose target genes increase osmoprotective organic osmolytes and heat shock proteins. We used mass spectrometry to analyze proteins that coimmunoprecipitate with TonEBP/OREBP in order to identify ones that might contribute to its high NaCl-induced activation. We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in TonEBP/OREBP transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with TonEBP/OREBP and contribute to its activation by hypertonicity. MDC1 associates with TonEBP/OREBP and contributes to high NaCl-induced increase of that factor's transcriptional activity.

Vector-based RNA interference against vascular endothelial growth factor-A significantly limits vascularization and growth of prostate cancer in vivo.

PubMed

Wannenes, Francesca; Ciafré, Silvia Anna; Niola, Francesco; Frajese, Gaetano; Farace, Maria Giulia

2005-12-01

RNA interference technology is emerging as a very potent tool to obtain a cellular knockdown of a desired gene. In this work we used vector-based RNA interference to inhibit vascular endothelial growth factor (VEGF) expression in prostate cancer in vitro and in vivo. We demonstrated that transduction with a plasmid carrying a small interfering RNA targeting all isoforms of VEGF, dramatically impairs the expression of this growth factor in the human prostate cancer cell line PC3. As a consequence, PC3 cells loose their ability to induce one of the fundamental steps of angiogenesis, namely the formation of a tube-like network in vitro. Most importantly, our "therapeutic" vector is able to impair tumor growth rate and vascularization in vivo. We show that a single injection of naked plasmid in developing neoplastic mass significantly decreases microvessel density in an androgen-refractory prostate xenograft and is able to sustain a long-term slowing down of tumor growth. In conclusion, our results confirm the basic role of VEGF in the angiogenic development of prostate carcinoma, and suggest that the use of our vector-based RNA interference approach to inhibit angiogenesis could be an effective tool in view of future gene therapy applications for prostate cancer.

Emodin suppresses maintenance of stemness by augmenting proteosomal degradation of epidermal growth factor receptor/epidermal growth factor receptor variant III in glioma stem cells.

PubMed

Kim, Jeongyub; Lee, Jong-Seon; Jung, Jieun; Lim, Inhye; Lee, Ji-Yun; Park, Myung-Jin

2015-02-01

There is a growing body of evidence that small subpopulations of cells with stem cell-like characteristics within most solid tumors are responsible for the malignancy of aggressive cancer cells and that targeting these cells might be a good therapeutic strategy to reduce the risk of tumor relapse after therapy. Here, we examined the effects of emodin (1,3,8-trihydroxy-6-methylanthraquinone), an active component of the root and rhizome of Rheum palmatum that has several biological activities, including antitumor effects, on primary cultured glioma stem cells (GSCs). Emodin inhibited the self-renewal activity of GSCs in vitro as evidenced by neurosphere formation, limiting dilution, and soft agar clonogenic assays. Emodin inhibited the maintenance of stemness by suppressing the expression of Notch intracellular domain, nonphosphorylated β-catenin, and phosphorylated STAT3 proteins. In addition, treatment with emodin partially induced apoptosis, reduced cell invasiveness, and sensitized GSCs to ionizing radiation. Intriguingly, emodin induced proteosomal degradation of epidermal growth factor receptor (EGFR)/EGFR variant III (EGFRvIII) by interfering with the association of EGFR/EGFRvIII with heat shock protein 90, resulting in the suppression of stemness pathways. Based on these data, we propose that emodin could be considered as a potent therapeutic adjuvant that targets GSCs.

Resveratrol induces mitochondrial biogenesis in endothelial cells.

PubMed

Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

2009-07-01

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Resveratrol induces mitochondrial biogenesis in endothelial cells

PubMed Central

Csiszar, Anna; Labinskyy, Nazar; Pinto, John T.; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

2009-01-01

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1α, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases. PMID:19429820

Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

PubMed

Croci, Stefania; Landuzzi, Lorena; Astolfi, Annalisa; Nicoletti, Giordano; Rosolen, Angelo; Sartori, Francesca; Follo, Matilde Y; Oliver, Noelynn; De Giovanni, Carla; Nanni, Patrizia; Lollini, Pier-Luigi

2004-03-01

Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity.

PubMed

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

2017-09-11

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

Orphan nuclear receptor small heterodimer partner inhibits transforming growth factor-beta signaling by repressing SMAD3 transactivation.

PubMed

Suh, Ji Ho; Huang, Jiansheng; Park, Yun-Yong; Seong, Hyun-A; Kim, Dongwook; Shong, Minho; Ha, Hyunjung; Lee, In-Kyu; Lee, Keesook; Wang, Li; Choi, Hueng-Sik

2006-12-22

Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling.

New basic approach to treat non-small cell lung cancer based on RNA-interference.

PubMed

Makowiecki, Christina; Nolte, Andrea; Sutaj, Besmire; Keller, Timea; Avci-Adali, Meltem; Stoll, Heidi; Schlensak, Christian; Wendel, Hans Peter; Walker, Tobias

2014-03-01

To date the therapy for non-small cell lung cancer (NSCLC) is associated with severe side effects, frustrating outcomes, and does not consider different tumor characteristics. The RNA-interference (RNAi) pathway represents a potential new approach to treat NSCLC. With small interfering ribonucleic acids (siRNAs), it is possible to reduce the expression of proliferation-dependent proteins in tumor cells, leading to their apoptosis. We propose that siRNAs could be adapted to the tumor type and may cause fewer side effects than current therapy. Four NSCLC cell lines were cultured under standard conditions and transfected with three different concentrations of siRNAs targeted against the hypoxia-inducible factors 1α and 2α (HIF1α and HIF2α) and signal transducer and activator of transcription 3 (STAT3). The expression was observed by quantitative real-time polymerase chain reaction and western blots. For the analysis of cell growth three days after transfection, the cell number was detected using a CASY cell counter system. The results of the silencing of the analyzed factors differ in each cell line. Cell growth was significantly reduced in all cell lines after transfection with HIF1α- and STAT3-siRNA. The silencing of HIF2α resulted in a significant effect on cell growth in squamous, and large-cell lung cancer. This study shows that the knockdown and viability to siRNA transfection differ in each tumor type according to the used siRNA. This implies that the tumor types differ among themselves and should be treated differently. Therefore, the authors suggest a possible approach to a more personalized treatment of NSCLC.

Screening of Small Molecule Interactor Library by Using In-Cell NMR Spectroscopy (SMILI-NMR)

PubMed Central

Xie, Jingjing; Thapa, Rajiv; Reverdatto, Sergey; Burz, David S.; Shekhtman, Alexander

2011-01-01

We developed an in-cell NMR assay for screening small molecule interactor libraries (SMILI-NMR) for compounds capable of disrupting or enhancing specific interactions between two or more components of a biomolecular complex. The method relies on the formation of a well-defined biocomplex and utilizes in-cell NMR spectroscopy to identify the molecular surfaces involved in the interaction at atomic scale resolution. Changes in the interaction surface caused by a small molecule interfering with complex formation are used as a read-out of the assay. The in-cell nature of the experimental protocol insures that the small molecule is capable of penetrating the cell membrane and specifically engaging the target molecule(s). Utility of the method was demonstrated by screening a small dipeptide library against the FKBP–FRB protein complex involved in cell cycle arrest. The dipeptide identified by SMILI-NMR showed biological activity in a functional assay in yeast. PMID:19422228

Platelet receptors as therapeutic targets: Past, present and future.

PubMed

Jamasbi, Janina; Ayabe, Keng; Goto, Shinya; Nieswandt, Bernhard; Peter, Karlheinz; Siess, Wolfgang

2017-06-28

Anti-platelet drugs reduce arterial thrombosis after plaque rupture and erosion, prevent stent thrombosis and are used to prevent and treat myocardial infarction and ischaemic stroke. Some of them may also be helpful in treating less frequent diseases such as thrombotic thrombocytopenic purpura. The present concise review aims to cover current and future developments of anti-platelet drugs interfering with the interaction of von Willebrand factor (VWF) with glycoprotein (GP) Ibα, and directed against GPVI, GPIIb/IIIa (integrin α IIb β 3 ), the thrombin receptor PAR-1, and the ADP receptor P2Y 12 . The high expectations of having novel antiplatelet drugs which selectively inhibit arterial thrombosis without interfering with normal haemostasis could possibly be met in the near future.

Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes

PubMed Central

Wong, Hui Hui; Kumar, Pankaj; Tay, Felicia Pei Ling; Moreau, Dimitri

2015-01-01

ABSTRACT Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. IMPORTANCE Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics. PMID:26311884

Allosteric analysis of glucocorticoid receptor-DNA interface induced by cyclic Py-Im polyamide: a molecular dynamics simulation study.

PubMed

Wang, Yaru; Ma, Na; Wang, Yan; Chen, Guangju

2012-01-01

It has been extensively developed in recent years that cell-permeable small molecules, such as polyamide, can be programmed to disrupt transcription factor-DNA interfaces and can silence aberrant gene expression. For example, cyclic pyrrole-imidazole polyamide that competes with glucocorticoid receptor (GR) for binding to glucocorticoid response elements could be expected to affect the DNA dependent binding by interfering with the protein-DNA interface. However, how such small molecules affect the transcription factor-DNA interfaces and gene regulatory pathways through DNA structure distortion is not fully understood so far. In the present work, we have constructed some models, especially the ternary model of polyamides+DNA+GR DNA-binding domain (GRDBD) dimer, and carried out molecular dynamics simulations and free energy calculations for them to address how polyamide molecules disrupt the GRDBD and DNA interface when polyamide and protein bind at the same sites on opposite grooves of DNA. We found that the cyclic polyamide binding in minor groove of DNA can induce a large structural perturbation of DNA, i.e. a >4 Å widening of the DNA minor groove and a compression of the major groove by more than 4 Å as compared with the DNA molecule in the GRDBD dimer+DNA complex. Further investigations for the ternary system of polyamides+DNA+GRDBD dimer and the binary system of allosteric DNA+GRDBD dimer revealed that the compression of DNA major groove surface causes GRDBD to move away from the DNA major groove with the initial average distance of ∼4 Å to the final average distance of ∼10 Å during 40 ns simulation course. Therefore, this study straightforward explores how small molecule targeting specific sites in the DNA minor groove disrupts the transcription factor-DNA interface in DNA major groove, and consequently modulates gene expression.

RISC-Target Interaction: Cleavage and Translational Suppression

PubMed Central

van den Berg, Arjen; Mols, Johann; Han, Jiahuai

2008-01-01

Summary Small RNA molecules have been known and utilized to suppress gene expression for more than a decade. The discovery that these small RNA molecules are endogenously expressed in many organisms and have a critical role in controlling gene expression have led to the arising of a whole new field of research. Termed small interfering RNA (siRNA) or microRNA (miRNA) these ~22 nt RNA molecules have the capability to suppress gene expression through various mechanisms once they are incorporated in the multi-protein RNA-Induced Silencing Complex (RISC) and interact with their target mRNA. This review introduces siRNAs and microRNAs in a historical perspective and focuses on the key molecules in RISC, structural properties and mechanisms underlying the process of small RNA regulated post-transcriptional suppression of gene expression. PMID:18692607

ATP-dependent human RISC assembly pathways.

PubMed

Yoda, Mayuko; Kawamata, Tomoko; Paroo, Zain; Ye, Xuecheng; Iwasaki, Shintaro; Liu, Qinghua; Tomari, Yukihide

2010-01-01

The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1-4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3'-mid (guide position 12-15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.

Whole genome siRNA cell-based screen links mitochondria to Akt signaling network through uncoupling of electron transport chain

PubMed Central

Senapedis, William T.; Kennedy, Caleb J.; Boyle, Patrick M.; Silver, Pamela A.

2011-01-01

Forkhead transcription factors (FOXOs) alter a diverse array of cellular processes including the cell cycle, oxidative stress resistance, and aging. Insulin/Akt activation directs phosphorylation and cytoplasmic sequestration of FOXO away from its target genes and serves as an endpoint of a complex signaling network. Using a human genome small interfering RNA (siRNA) library in a cell-based assay, we identified an extensive network of proteins involved in nuclear export, focal adhesion, and mitochondrial respiration not previously implicated in FOXO localization. Furthermore, a detailed examination of mitochondrial factors revealed that loss of uncoupling protein 5 (UCP5) modifies the energy balance and increases free radicals through up-regulation of uncoupling protein 3 (UCP3). The increased superoxide content induces c-Jun N-terminal kinase 1 (JNK1) kinase activity, which in turn affects FOXO localization through a compensatory dephosphorylation of Akt. The resulting nuclear FOXO increases expression of target genes, including mitochondrial superoxide dismutase. By connecting free radical defense and mitochondrial uncoupling to Akt/FOXO signaling, these results have implications in obesity and type 2 diabetes development and the potential for therapeutic intervention. PMID:21460183

Whole genome siRNA cell-based screen links mitochondria to Akt signaling network through uncoupling of electron transport chain.

PubMed

Senapedis, William T; Kennedy, Caleb J; Boyle, Patrick M; Silver, Pamela A

2011-05-15

Forkhead transcription factors (FOXOs) alter a diverse array of cellular processes including the cell cycle, oxidative stress resistance, and aging. Insulin/Akt activation directs phosphorylation and cytoplasmic sequestration of FOXO away from its target genes and serves as an endpoint of a complex signaling network. Using a human genome small interfering RNA (siRNA) library in a cell-based assay, we identified an extensive network of proteins involved in nuclear export, focal adhesion, and mitochondrial respiration not previously implicated in FOXO localization. Furthermore, a detailed examination of mitochondrial factors revealed that loss of uncoupling protein 5 (UCP5) modifies the energy balance and increases free radicals through up-regulation of uncoupling protein 3 (UCP3). The increased superoxide content induces c-Jun N-terminal kinase 1 (JNK1) kinase activity, which in turn affects FOXO localization through a compensatory dephosphorylation of Akt. The resulting nuclear FOXO increases expression of target genes, including mitochondrial superoxide dismutase. By connecting free radical defense and mitochondrial uncoupling to Akt/FOXO signaling, these results have implications in obesity and type 2 diabetes development and the potential for therapeutic intervention.

Downregulation of connective tissue growth factor reduces migration and invasiveness of osteosarcoma cells.

PubMed

Huang, Yinjun; Zhao, Shichang; Zhang, Changqing; Li, Xiaolin

2016-02-01

As one of the most serious types of primary bone tumor, osteosarcoma (OSA) features metastatic lesions, and resistance to chemotherapy is common. The underlying mechanisms of these characteristics may account for the failure of treatments and the poor prognosis of patients with OSA. It has been reported that inhibition of Cyr61 suppresses OSA cell proliferation as it represents a target of statins. In addition to cystein‑rich protein 61 (Cyr61) and nephroblastoma overexpression, connective tissue growth factor (CTGF) is a member of the CCN family and may therefore exhibit effects on human OSA cells similar to those of Cyr61. In the current study, acridine orange/ethidium bromide staining were used to determine the rate of apoptosis. The present study demonstrated that small interfering RNA‑mediated silencing of CTGF promoted cell death and suppressed OSA cell migration and invasion, as indicated by wound healing and Transwell assays, while lentivirus‑mediated overexpression of CTGF reversed these effects. Furthermore, a colorimetric caspase assay demonstrated that CTGF knockdown enhanced the efficacy of chemotherapeutic drugs. The results of the present study provided a novel molecular target which may be utilized for the treatment of metastatic OSA.

Androstenediol inhibits the trauma-hemorrhage-induced increase in caspase-3 by downregulating the inducible nitric oxide synthase pathway.

PubMed

Kiang, Juliann G; Peckham, Russell M; Duke, Leah E; Shimizu, Tomoharu; Chaudry, Irshad H; Tsokos, George C

2007-03-01

Soft tissue trauma and hemorrhage (T-H) diminishes various aspects of liver function, while it increases hepatic nitrate/nitrite, inducible nitric oxide synthase (iNOS), and endothelin-1 levels. Treatment with androstenediol (AED) inhibits the T-H-induced alterations of the above parameters. We sought to identify the molecular events underlying the beneficial effect of AED. Exposure of rats to T-H significantly increased the caspase-3 activity and protein, whereas treatment with AED significantly limited these increases. AED treatment also suppressed the T-H-induced increase in iNOS by effectively altering the levels of key transcription factors involved in the regulation of iNOS expression. Immunoprecipitation and immunoblotting analyses indicate that T-H increased apoptosome formation, and AED treatment significantly decreased it. Modulating the iNOS protein by transfecting cells with iNOS gene or small interfering RNA further confirmed the correlation between iNOS and caspase-3. Our data indicate that AED limits caspase-3 expression by suppressing the expression of transcription factors involved in the production of iNOS, resulting in decreased apoptosome. AED can potentially be a useful adjuvant for limiting liver apoptosis following T-H shock.

Enhanced HIV-1 replication in ex vivo ectocervical tissues from post-menopausal women correlates with increased inflammatory responses.

PubMed

Rollenhagen, C; Asin, S N

2011-11-01

Knowledge about early innate immune responses at the mucosal surfaces of the female genital tract is important in understanding the pathogenesis of heterosexual transmission of human immunodeficiency virus type-1 (HIV-1). As estradiol decreases inflammatory responses, we postulated that an estradiol-deficient state such as post-menopause could enhance expression of inflammatory factors that stimulate HIV-1 replication. We compare HIV-1 integration, transcription, and viral p24 release levels among ectocervical tissues obtained from pre- and post-menopausal donors. We detected enhanced HIV-1 p24 release levels in post- compared with pre-menopausal tissues (P<0.0001), but saw no difference in HIV-1 integration. Overall, 100% of post-menopausal tissues exhibited levels of HIV-1 transcription above background compared with only 60% of pre-menopausal tissues. Increased HIV-1 transcription was associated with enhanced interleukin (IL)-1β, IL-6, monocyte chemotactic protein-1, growth-regulated oncogene-α, and interferon-γ-inducible protein-10 expression. Neutralization and nuclear factor-κB-targeting small-interfering RNA experiments both decreased HIV-1 transcription, suggesting that the early inflammatory response may facilitate HIV-1 replication in ex vivo ectocervical tissues from post-menopausal women.

Requirement of kinesin-mediated membrane transport of WAVE2 along microtubules for lamellipodia formation promoted by hepatocyte growth factor.

PubMed

Takahashi, Kazuhide; Suzuki, Katsuo

2008-07-01

Lamellipodia formation necessary for epithelial cell migration and invasion is accomplished by rearrangement of the actin cytoskeleton at the leading edge through membrane transport of WAVE2. However, how WAVE2 is transported to the cell periphery where lamellipodia are formed remains to be established. We report here that hepatocyte growth factor (HGF) promoted lamellipodia formation and intracellular transport of WAVE2 to the cell periphery, depending on Rac1 activity, in MDA-MB-231 human breast cancer cells. Immunoblot analyses indicating the coimmunoprecipitation of WAVE2 with kinesin heavy chain KIF5B, one of the motor proteins, and IQGAP1 suggest that KIF5B and IQGAP1 formed a complex with WAVE2 in serum-starved cells and increased in their amount after HGF stimulation. Both downregulation of KIF5B by the small interfering RNA and depolymerization of microtubules with nocodazole abrogated the HGF-induced lamellipodia formation and WAVE2 transport. Therefore, we propose here that the promotion of lamellipodia formation by HGF in MDA-MB-231 cells is Rac1-dependent and requires KIF5B-mediated transport of WAVE2 and IQGAP1 to the cell periphery along microtubules.

5-Aminoimidazole-4-Carboxamide Riboside Enhances Effect of Ionizing Radiation in PC3 Prostate Cancer Cells

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

Isebaert, Sofie F., E-mail: sofie.isebaert@med.kuleuven.be; Swinnen, Johannes V.; McBride, William H.

2011-12-01

Purpose: The nucleoside 5-aminoimidazole-4-carboxamide riboside (AICAR) is a low-energy mimetic and adenosine monophosphate (AMP)-activated protein kinase (AMPK) agonist that can affect the phenotype of malignant cells by diminishing their anabolism. It does this by being converted to 5-aminoimidazole-4-carboxamide ribotide (ZMP), an AMP analog. We combined this promising antineoplastic agent with ionizing radiation in an attempt to increase its efficacy. Methods and Materials: The effect of AICAR on cell proliferation, cell viability, apoptosis, reactive oxygen species production, radiosensitivity, and AMPK activation was determined in the human prostate cancer cell line PC3. To elucidate the radiosensitizing mechanism, clonogenic survival assays in themore » presence of a drug agonist or antagonist or with small interfering RNA targeting AMPK were done, as well as measurements of ZMP production and double strand break repair. Moreover, immunoblot analysis of the radiation response signaling pathways after AICAR treatment was performed. Results: The incubation of human PC3 prostate cancer cells with AICAR-activated AMPK inhibited cell proliferation, decreased viability, increased apoptosis, and generated reactive oxygen species in a dose- and time-dependent manner. None of these endpoints gave more than additive effects when radiation was added. Radiosensitization was observed but only after 72 hours of treatment with 250 {mu}M AICAR, suggesting that it was independent of AMPK activation. This finding was confirmed by small interfering RNA knockdown of AMPK. The mechanism of radiosensitization was associated with imbalanced deoxynucleotide pools owing to ZMP accumulation after AICAR administration that interfered with DNA repair. Conclusions: Our findings on the favorable interaction between low doses of AICAR and ionizing radiation in PC3 cells could open new perspectives for the clinical use of this or similar compounds. However, additional research is still required to establish the ZMP pathway as being of general applicability.« less

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

PubMed

Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian; Wei, Taiyun

2016-01-15

Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus

PubMed Central

Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian

2015-01-01

ABSTRACT Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. IMPORTANCE Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector. PMID:26537672

A Small Stem Loop Structure Of The Ebola Virus Trailer Is Essential For Replication And Interacts With Heat Shock Protein A8

DTIC Science & Technology

2016-12-02

agarose gel electrophoresis TR-16-205 Nucleic Acids Research, 2016 3 (Seakem GTG , Sigma-Aldrich) and purified using the QI- Aquick Gel Extraction Kit... gtg +cga 1923-1938 3′ LNA2 +caa+aaa+ga+aa+gaa+gaa 3E-5E eGFP, 3E-5E plasmid containing enhanced green fluorescent protein; aiSHAPE, antisense-interfered

Formation of algae growth constitutive relations for improved algae modeling.

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

Gharagozloo, Patricia E.; Drewry, Jessica Louise.

This SAND report summarizes research conducted as a part of a two year Laboratory Directed Research and Development (LDRD) project to improve our abilities to model algal cultivation. Algae-based biofuels have generated much excitement due to their potentially large oil yield from relatively small land use and without interfering with the food or water supply. Algae mitigate atmospheric CO2 through metabolism. Efficient production of algal biofuels could reduce dependence on foreign oil by providing a domestic renewable energy source. Important factors controlling algal productivity include temperature, nutrient concentrations, salinity, pH, and the light-to-biomass conversion rate. Computational models allow for inexpensivemore » predictions of algae growth kinetics in these non-ideal conditions for various bioreactor sizes and geometries without the need for multiple expensive measurement setups. However, these models need to be calibrated for each algal strain. In this work, we conduct a parametric study of key marine algae strains and apply the findings to a computational model.« less

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

PubMed Central

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

2012-01-01

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

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

PubMed Central

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

2014-01-01

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

Design and development of C-arm based cone-beam CT for image-guided interventions: initial results

NASA Astrophysics Data System (ADS)

Chen, Guang-Hong; Zambelli, Joseph; Nett, Brian E.; Supanich, Mark; Riddell, Cyril; Belanger, Barry; Mistretta, Charles A.

2006-03-01

X-ray cone-beam computed tomography (CBCT) is of importance in image-guided intervention (IGI) and image-guided radiation therapy (IGRT). In this paper, we present a cone-beam CT data acquisition system using a GE INNOVA 4100 (GE Healthcare Technologies, Waukesha, Wisconsin) clinical system. This new cone-beam data acquisition mode was developed for research purposes without interfering with any clinical function of the system. It provides us a basic imaging pipeline for more advanced cone-beam data acquisition methods. It also provides us a platform to study and overcome the limiting factors such as cone-beam artifacts and limiting low contrast resolution in current C-arm based cone-beam CT systems. A geometrical calibration method was developed to experimentally determine parameters of the scanning geometry to correct the image reconstruction for geometric non-idealities. Extensive phantom studies and some small animal studies have been conducted to evaluate the performance of our cone-beam CT data acquisition system.

Viral Mimicry to Usurp Ubiquitin and SUMO Host Pathways

PubMed Central

Wimmer, Peter; Schreiner, Sabrina

2015-01-01

Posttranslational modifications (PTMs) of proteins include enzymatic changes by covalent addition of cellular regulatory determinants such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) moieties. These modifications are widely used by eukaryotic cells to control the functional repertoire of proteins. Over the last decade, it became apparent that the repertoire of ubiquitiylation and SUMOylation regulating various biological functions is not restricted to eukaryotic cells, but is also a feature of human virus families, used to extensively exploit complex host-cell networks and homeostasis. Intriguingly, besides binding to host SUMO/Ub control proteins and interfering with the respective enzymatic cascade, many viral proteins mimic key regulatory factors to usurp this host machinery and promote efficient viral outcomes. Advanced detection methods and functional studies of ubiquitiylation and SUMOylation during virus-host interplay have revealed that human viruses have evolved a large arsenal of strategies to exploit these specific PTM processes. In this review, we highlight the known viral analogs orchestrating ubiquitin and SUMO conjugation events to subvert and utilize basic enzymatic pathways. PMID:26343706

The efficacy of rituximab in the treatment of inhibitor-associated hemostatic disorders.

PubMed

Franchini, Massimo; Veneri, Dino; Lippi, Giuseppe; Stenner, Rachel

2006-08-01

Rituximab is a chimeric anti-CD20 monoclonal antibody active against normal and malignant B cells which has proven to be effective in the therapy of CD-20 positive lymphomas. Its B-cell cytotoxic action has also been exploited in many non-malignant autoimmune disorders in which it has been used with the aim of interfering with the production of pathologic antibodies. The present knowledge regarding the use of rituximab in antibody-associated disorders of hemostasis (i.e. idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, acquired hemophilia A, congenital hemophilia with inhibitors, acquired inhibitors against coagulation factors) is presented briefly in this review. The results suggest that rituximab can be useful in the treatment of disorders of hemostasis associated with inhibitor formation. Although collectively the number of patients treated is now quite substantial, most of the data are drawn from isolated case reports or descriptions of small, uncontrolled series. Large, prospective, randomized trials are, therefore, needed to confirm the positive, preliminary results.

Feature Matching of Historical Images Based on Geometry of Quadrilaterals

NASA Astrophysics Data System (ADS)

Maiwald, F.; Schneider, D.; Henze, F.; Münster, S.; Niebling, F.

2018-05-01

This contribution shows an approach to match historical images from the photo library of the Saxon State and University Library Dresden (SLUB) in the context of a historical three-dimensional city model of Dresden. In comparison to recent images, historical photography provides diverse factors which make an automatical image analysis (feature detection, feature matching and relative orientation of images) difficult. Due to e.g. film grain, dust particles or the digitalization process, historical images are often covered by noise interfering with the image signal needed for a robust feature matching. The presented approach uses quadrilaterals in image space as these are commonly available in man-made structures and façade images (windows, stones, claddings). It is explained how to generally detect quadrilaterals in images. Consequently, the properties of the quadrilaterals as well as the relationship to neighbouring quadrilaterals are used for the description and matching of feature points. The results show that most of the matches are robust and correct but still small in numbers.

A close relative of the nuclear, chromosomal high-mobility group protein HMG1 in yeast mitochondria.

PubMed Central

Diffley, J F; Stillman, B

1991-01-01

ABF2 (ARS-binding factor 2), a small, basic DNA-binding protein that binds specifically to the autonomously replicating sequence ARS1, is located primarily in the mitochondria of the yeast Saccharomyces cerevisiae. The abundance of ABF2 and the phenotype of abf2- null mutants argue that this protein plays a key role in the structure, maintenance, and expression of the yeast mitochondrial genome. The predicted amino acid sequence of ABF2 is closely related to the high-mobility group proteins HMG1 and HMG2 from vertebrate cell nuclei and to several other DNA-binding proteins. Additionally, ABF2 and the other HMG-related proteins are related to a globular domain from the heat shock protein hsp70 family. ABF2 interacts with DNA both nonspecifically and in a specific manner within regulatory regions, suggesting a mechanism whereby it may aid in compacting the mitochondrial genome without interfering with expression. Images PMID:1881919

Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

PubMed Central

Yang, Jae-Seong; Kwon, Oh Sung; Kim, Sanguk; Jang, Sung Key

2013-01-01

Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets. PMID:23593195

The Caenorhabditis elegans RDE-10/RDE-11 complex regulates RNAi by promoting secondary siRNA amplification.

PubMed

Zhang, Chi; Montgomery, Taiowa A; Fischer, Sylvia E J; Garcia, Susana M D A; Riedel, Christian G; Fahlgren, Noah; Sullivan, Christopher M; Carrington, James C; Ruvkun, Gary

2012-05-22

In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans (C. elegans), the interaction between the RNA-induced silencing complex (RISC) loaded with primary small interfering RNAs (siRNAs) and the target messenger RNA (mRNA) leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template. The mechanism and genetic requirements for secondary siRNA accumulation are not well understood. From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10, and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, and other candidate RNAi factors. The RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and other dosage-sensitive RNAi-defective genes rsd-2, rsd-6, and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways. The RDE-10/RDE-11 complex is essential for the amplification of RNAi in C. elegans by promoting secondary siRNA accumulation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors.

PubMed

Taniguchi, Hirokazu; Takeuchi, Shinji; Fukuda, Koji; Nakagawa, Takayuki; Arai, Sachiko; Nanjo, Shigeki; Yamada, Tadaaki; Yamaguchi, Hiroyuki; Mukae, Hiroshi; Yano, Seiji

2017-01-01

Crizotinib, a first-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, is known to be effective against echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive non-small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib-resistant cells (A925LPE3-CR) via long-term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4-ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib-resistant cells, and these cells were cross-resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3-CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine-kinase inhibitor (erlotinib) or an anti-EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4-ALK lung cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Am I Interfering? Preschool Teacher Participation in Children Play

ERIC Educational Resources Information Center

Tsai, Chia-Yen

2015-01-01

This study aimed to explore the timing and strategies of teacher participation in children's play and the factors which have a bearing on teacher participation. This study used qualitative research and conducted observation of natural situations. The samples were preschool teachers in an elementary school's affiliated kindergarten in Hualien. The…

Deer and diversity in Allegheny hardwood forests: managing an unlikely challenge

Treesearch

David S. deCalesta

1994-01-01

High white-tailed deer (Odocoileus virginianus) density and interfering vegetation were identified as factors affecting the regeneration of hardwood forests in the Allegheny National Forest and surrounding forests in northwestern Pennsylvania. Research was designed by Forest Service scientists to quantify these effects. A high degree of interest in...

Metabolic factors, adipose tissue, and plasminogen activator inhibitor-1 levels in Type 2 diabetes

USDA-ARS?s Scientific Manuscript database

Plasminogen activator inhibitor-1 (PAI-1) production by adipose tissue is increased in obesity, and its circulating levels are high in type 2 diabetes. PAI-1 increases cardiovascular risk by favoring clot stability, interfering with vascular remodeling, or both. We investigated in obese diabetic per...

Predictive Factors of Anxiety and Depression in Patients with Acute Coronary Syndrome.

PubMed

Altino, Denise Meira; Nogueira-Martins, Luiz Antônio; de Barros, Alba Lucia Bottura Leite; Lopes, Juliana de Lima

2017-12-01

To identify the predictive factors of anxiety and depression in patients with acute coronary syndrome. Cross-sectional and retrospective study conducted with 120 patients hospitalized with acute coronary syndrome. Factors interfering with anxiety and depression were assessed. Anxiety was related to sex, stress, years of education, and depression, while depression was related to sex, diabetes mellitus, obesity, years of education, and trait-anxiety. Obesity and anxiety were considered predictive factors for depression, while depression and fewer years of education were considered predictive factors for anxiety. Copyright © 2017. Published by Elsevier Inc.

Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitize tumor cells for TRAIL-induced apoptosis.

PubMed Central

Siegmund, Daniela; Hadwiger, Philipp; Pfizenmaier, Klaus; Vornlocher, Hans-Peter; Wajant, Harald

2002-01-01

BACKGROUND: Most tumors express death receptors and their activation represents a potential selective approach in cancer treatment. The most promising candidate for tumor selective death receptor-activation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L, which activates the death receptors TRAIL-R1 and TRAIL-R2, and induces apoptosis preferentially in tumor cells but not in normal tissues. However, many cancer cells are not or only moderately sensitive towards TRAIL and require cotreatment with irradiation or chemotherapy to yield a therapeutically reasonable apoptotic response. Because chemotherapy can have a broad range of unwanted side effects, more specific means for sensitizing tumor cells for TRAIL are desirable. The expression of the cellular FLICE-like inhibitory protein (cFLIP) is regarded as a major cause of TRAIL resistance. We therefore analyzed the usefulness of targeting FLIP to sensitize tumor cells for TRAIL-induced apoptosis. MATERIALS AND METHODS: To selectively interfere with expression of cFLIP short double-stranded RNA oligonucleotides (small interfering RNAs [siRNAs]) were introduced in the human cell lines SV80 and KB by electroporation. Effects of siRNA on FLIP expression were analyzed by Western blotting and RNase protection assay and correlated with TRAIL sensitivity upon stimulation with recombinant soluble TRAIL and TRAIL-R1- and TRAIL-R2-specific agonistic antibodies. RESULTS: FLIP expression can be inhibited by RNA interference using siRNAs, evident from reduced levels of FLIP-mRNA and FLIP protein. Inhibition of cFLIP expression sensitizes cells for apoptosis induction by TRAIL and other death ligands. In accordance with the presumed function of FLIP as an inhibitor of death receptor-induced caspase-8 activation, down-regulation of FLIP by siRNAs enhanced TRAIL-induced caspase-8 activation. CONCLUSION: Inhibition of FLIP expression was sufficient to sensitize tumor cells for TRAIL-induced apoptosis. The combination of TRAIL and FLIP-targeting siRNA could therefore be a useful strategy to attack cancer cells, which are resistant to TRAIL alone. PMID:12520089

Nanoparticles for targeted delivery of therapeutics and small interfering RNAs in hepatocellular carcinoma

PubMed Central

Varshosaz, Jaleh; Farzan, Maryam

2015-01-01

Hepatocellular carcinoma (HCC) is the 5th most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorable systemic side-effects of chemotherapeutic agents and susceptibility to the degradation of small interfering RNAs (siRNAs), which can knock down a specific gene involved in the disease, have hampered their clinical application. So, it could be beneficial to develop an efficient carrier for the stabilization and specific delivery of drugs and siRNA to cells. Targeted nanoparticles have gained considerable attention as an efficient drug and gene delivery system, which is due to their capability in achieving the highest accumulation of cytotoxic agents in tumor tissue, modifiable drug pharmacokinetic- and bio-distribution, improved effectiveness of treatment, and limited side-effects. Recent studies have shed more light on the advantages of novel drug loaded carrier systems vs free drugs. Most of the animal studies have reported improvement in treatment efficacy and survival rate using novel carrier systems. Targeted delivery may be achieved passively or actively. In passive targeting, no ligand as homing device is used, while targeting is achieved by incorporating the therapeutic agent into a macromolecule or nanoparticle that passively reaches the target organ. However, in active targeting, the therapeutic agent or carrier system is conjugated to a tissue or cell-specific receptor which is over-expressed in a special malignancy using a ligand called a homing device. This review covers a broad spectrum of targeted nanoparticles as therapeutic and non-viral siRNA delivery systems, which are developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and their characteristics and opportunities for the clinical applications of drugs and therapeutic siRNA are discussed in this article. Asialoglycoprotein receptors, low-density lipoprotein, ganglioside GM1 cell surface ligand, epidermal growth factor receptor receptors, monoclonal antibodies, retinoic acid receptors, integrin receptors targeted by Arg-Gly-Asp peptide, folate, and transferrin receptors are the most widely studied cell surface receptors which are used for the site specific delivery of drugs and siRNA-based therapeutics in HCC and discussed in detail in this article. PMID:26576089

Reduced transforming growth factor β1 (TGF-β1) in the repair of airway epithelial cells of children with asthma.

PubMed

Ling, Kak-Ming; Sutanto, Erika N; Iosifidis, Thomas; Kicic-Starcevich, Elizabeth; Looi, Kevin; Garratt, Luke W; Martinovich, Kelly M; Lannigan, Francis J; Knight, Darryl A; Stick, Stephen M; Kicic, Anthony

2016-10-01

Evidence into the role of TGF-β1 in airway epithelial repair in asthma is still controversial. This study tested the hypothesis that the reduced TGF-β1 levels previously observed in paediatric asthmatic airway epithelial cells directly contribute to the dysregulated repair seen in these cells. Primary airway epithelial cells (pAEC) from children with asthma (n = 16) and non-asthmatic subjects (n = 20) were isolated, and subcultured for investigation of TGF-β1 gene and protein via quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression of other associated genes such as integrins αvβ6, αvβ8 and MT1-MMP were also tested. Small interfering RNA (siRNA) was employed to assess the role of TGF-β1 during wound repair. TGF-β1 gene and protein expression were significantly downregulated in asthmatic pAEC over the course of repair, compared with cells from non-asthmatic children. Messenger RNA (mRNA) expression of TGF-β1 was also directly implicated in non-asthmatic and asthmatic pAEC proliferation over their quiescent counterparts. Small interfering RNA-mediated knockdown of TGF-β1 compromised repair in non-asthmatic pAEC and exacerbated the dysregulated repair seen in asthmatic pAEC. Expression of major TGF-β1 activators of epithelial cells, integrin αvβ6 and αvβ8 was also measured and there was no difference in αvβ6 gene expression between the two cohorts. Although integrin αvβ8 gene expression was significantly higher in asthmatic pAEC, the expression of MT1-MMP (MMP14) which facilitates the αvβ8 mediated TGF-β1 activation was significantly downregulated. Our data has highlighted the importance of TGF-β1 in pAEC wound repair in vitro. The significantly lower levels seen in asthmatic pAEC subsequently contributes to the dysregulated repair observed in these cells. © 2016 Asian Pacific Society of Respirology.

Rootstock-to-scion transfer of transgene-derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry.

PubMed

Zhao, Dongyan; Song, Guo-qing

2014-12-01

Small interfering RNAs (siRNAs) are silencing signals in plants. Virus-resistant transgenic rootstocks developed through siRNA-mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock-to-scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV-hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV-hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long-distance (1.2 m) transfer of PNRSV-hpRNA-derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for 'using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes.

PubMed

Zhao, Ying-Tao; Wang, Meng; Fu, San-Xiong; Yang, Wei-Cai; Qi, Cun-Kou; Wang, Xiu-Jie

2012-02-01

MicroRNAs (miRNAs) and small interfering RNAs are important regulators of plant development and seed formation, yet their population and abundance in the oil crop Brassica napus are still not well understood, especially at different developmental stages and among cultivars with varied seed oil contents. Here, we systematically analyzed the small RNA expression profiles of Brassica napus seeds at early embryonic developmental stages in high-oil-content and low-oil-content B. napus cultivars, both cultured in two environments. A total of 50 conserved miRNAs and 9 new miRNAs were identified, together with some new miRNA targets. Expression analysis revealed some miRNAs with varied expression levels in different seed oil content cultivars or at different embryonic developmental stages. A large number of 23-nucleotide small RNAs with specific nucleotide composition preferences were also identified, which may present new classes of functional small RNAs.

Small RNAs as important regulators for the hybrid vigour of super-hybrid rice.

PubMed

Zhang, Lei; Peng, Yonggang; Wei, Xiaoli; Dai, Yan; Yuan, Dawei; Lu, Yufei; Pan, Yangyang; Zhu, Zhen

2014-11-01

Heterosis is an important biological phenomenon; however, the role of small RNA (sRNA) in heterosis of hybrid rice remains poorly described. Here, we performed sRNA profiling of F1 super-hybrid rice LYP9 and its parents using high-throughput sequencing technology, and identified 355 distinct mature microRNAs and trans-acting small interfering RNAs, 69 of which were differentially expressed sRNAs (DES) between the hybrid and the mid-parental value. Among these, 34 DES were predicted to target 176 transcripts, of which 112 encoded 94 transcription factors. Further analysis showed that 67.6% of DES expression levels were negatively correlated with their target mRNAs either in flag leaves or panicles. The target genes of DES were significantly enriched in some important biological processes, including the auxin signalling pathway, in which existed a regulatory network mediated by DES and their targets, closely associated with plant growth and development. Overall, 20.8% of DES and their target genes were significantly enriched in quantitative trait loci of small intervals related to important rice agronomic traits including growth vigour, grain yield, and plant architecture, suggesting that the interaction between sRNAs and their targets contributes to the heterotic phenotypes of hybrid rice. Our findings revealed that sRNAs might play important roles in hybrid vigour of super-hybrid rice by regulating their target genes, especially in controlling the auxin signalling pathway. The above finding provides a novel insight into the molecular mechanism of heterosis. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1

PubMed Central

2014-01-01

Background Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor specific stem cell marker in colorectal and pancreatic cancer. Previous in vitro and in vivo studies have demonstrated the therapeutic effects of inhibiting DCLK1 with small interfering RNA (siRNA) as well as genetically targeting the DCLK1+ cell for deletion. However, the effects of inhibiting DCLK1 kinase activity have not been studied directly. Therefore, we assessed the effects of inhibiting DCLK1 kinase activity using the novel small molecule kinase inhibitor, LRRK2-IN-1, which demonstrates significant affinity for DCLK1. Results Here we report that LRRK2-IN-1 demonstrates potent anti-cancer activity including inhibition of cancer cell proliferation, migration, and invasion as well as induction of apoptosis and cell cycle arrest. Additionally we found that it regulates stemness, epithelial-mesenchymal transition, and oncogenic targets on the molecular level. Moreover, we show that LRRK2-IN-1 suppresses DCLK1 kinase activity and downstream DCLK1 effector c-MYC, and demonstrate that DCLK1 kinase activity is a significant factor in resistance to LRRK2-IN-1. Conclusions Given DCLK1’s tumor stem cell marker status, a strong understanding of its biological role and interactions in gastrointestinal tumors may lead to discoveries that improve patient outcomes. The results of this study suggest that small molecule inhibitors of DCLK1 kinase should be further investigated as they may hold promise as anti-tumor stem cell drugs. PMID:24885928

Brothers in Arms

PubMed Central

Bhindi, Ravinay; Fahmy, Roger G.; Lowe, Harry C.; Chesterman, Colin N.; Dass, Crispin R.; Cairns, Murray J.; Saravolac, Edward G.; Sun, Lun-Quan; Khachigian, Levon M.

2007-01-01

The past decade has seen the rapid evolution of small-molecule gene-silencing strategies, driven largely by enhanced understanding of gene function in the pathogenesis of disease. Over this time, many genes have been targeted by specifically engineered agents from different classes of nucleic acid-based drugs in experimental models of disease to probe, dissect, and characterize further the complex processes that underpin molecular signaling. Arising from this, a number of molecules have been examined in the setting of clinical trials, and several have recently made the successful transition from the bench to the clinic, heralding an exciting era of gene-specific treatments. This is particularly important because clear inadequacies in present therapies account for significant morbidity, mortality, and cost. The broad umbrella of gene-silencing therapeutics encompasses a range of agents that include DNA enzymes, short interfering RNA, antisense oligonucleotides, decoys, ribozymes, and aptamers. This review tracks current movements in these technologies, focusing mainly on DNA enzymes and short interfering RNA, because these are poised to play an integral role in antigene therapies in the future. PMID:17717148

Infection of Hepatocytes With HCV Increases Cell Surface Levels of Heparan Sulfate Proteoglycans, Uptake of Cholesterol and Lipoprotein, and Virus Entry by Up-regulating SMAD6 and SMAD7.

PubMed

Zhang, Fang; Sodroski, Catherine; Cha, Helen; Li, Qisheng; Liang, T Jake

2017-01-01

The signaling molecule and transcriptional regulator SMAD6, which inhibits the transforming growth factor β signaling pathway, is required for infection of hepatocytes by hepatitis C virus (HCV). We investigated the mechanisms by which SMAD6 and another inhibitory SMAD (SMAD7) promote HCV infection in human hepatoma cells and hepatocytes. We infected Huh7 and Huh7.5.1 cells and primary human hepatocytes with Japanese fulminant hepatitis-1 (JFH1) HCV cell culture system (HCVcc). We then measured HCV binding, intracellular levels of HCV RNA, and expression of target genes. We examined HCV entry in HepG2/microRNA (miR) 122/CD81 cells, which support entry and replication of HCV, were transfected these cells with small interfering RNAs targeting inhibitory SMADs to analyze gene expression profiles. Uptake of labeled low-density lipoprotein (LDL) and cholesterol was measured. Cell surface proteins were quantified by flow cytometry. We obtained liver biopsy samples from 69 patients with chronic HCV infection and 19 uninfected individuals (controls) and measured levels of syndecan 1 (SDC1), SMAD7, and SMAD6 messenger RNAs (mRNAs). Small interfering RNA knockdown of SMAD6 blocked the binding and infection of hepatoma cell lines and primary human hepatocytes by HCV, whereas SMAD6 overexpression increased HCV infection. We found levels of mRNAs encoding heparan sulfate proteoglycans (HSPGs), particularly SDC1 mRNA, and cell surface levels of heparan sulfate to be reduced in cells after SMAD6 knockdown. SMAD6 knockdown also reduced transcription of genes encoding lipoprotein and cholesterol uptake receptors, including the LDL receptor (LDLR), the very LDLR, and the scavenger receptor class B member 1 in hepatocytes; knockdown of SMAD6 also inhibited cell uptake of cholesterol and lipoprotein. Overexpression of SMAD6 increased the expression of these genes. Similar effects were observed with knockdown and overexpression of SMAD7. In addition, HCV infection of cells increased the expression of SMAD6, which required the activity of nuclear factor-κB, but not transforming growth factor β. Liver tissues from patients with chronic HCV infection had significantly higher levels of SMAD6, SMAD7, and HSPG mRNAs than controls. In studies of hepatoma cell lines and primary human hepatocytes, we found that infection with HCV leads to activation of nuclear factor-κB, resulting in increased expression of SMAD6 and SMAD7. Up-regulation of SMAD6 and SMAD7 induces the expression of HSPGs, such as SDC1, as well as LDLR, very LDLR, and the scavenger receptor class B member 1, which promote HCV entry and propagation, as well as cellular uptake of cholesterol and lipoprotein. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Tim-3 facilitates osteosarcoma proliferation and metastasis through the NF-κB pathway and epithelial-mesenchymal transition.

PubMed

Feng, Z M; Guo, S M

2016-09-02

The aim of this study was to investigate the expression of T-cell immunoglobulin mucin domain molecule-3 (Tim-3) in osteosarcoma tissues, and analyze its effect on cell proliferation and metastasis in an osteosarcoma cell line. Tim-3 mRNA and protein expression in osteosarcoma tissue was detected by reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Additionally, the cell viability, apoptosis rate, and invasive ability of the osteosarcoma cell line MG-63 were tested using the methyl thiazolyl tetrazolium assay, Annexin V-propidium iodide flow cytometry, and a Transwell assay, respectively, following Tim-3 interference using small interfering RNA (siRNA). We also analyzed the expression of Snail, E-cadherin, vimentin, and nuclear factor (NF)-kB in the cells by western blot. We observed that Tim-3 mRNA and protein was significantly overexpressed in osteosarcoma tissues, compared to the adjacent normal tissue (P < 0.01). Moreover, MG-63 cells transfected with the Tim-3 siRNA presented lower cell viability, a greater number of apoptotic cells, and decreased invasive ability (P < 0.01), compared to control cells. Additionally, we observed a decrease in Snail and vimentin expression, an increase in the E-cadherin level, and an increase in NF-kB p65 phosphorylation (P < 0.01) in Tim-3 siRNA-transfected MG-63 cells. Based on these results, we concluded that Tim-3 is highly expressed in osteosarcoma tissue. Moreover, we speculated that interfering in Tim-3 expression could significantly suppress osteosarcoma cell (MG-63) proliferation and metastasis via the NF-kB/Snail signaling pathway and epithelial-mesenchymal transition.

Release of Infectious Hepatitis C Virus from Huh7 Cells Occurs via a trans-Golgi Network-to-Endosome Pathway Independent of Very-Low-Density Lipoprotein Secretion

PubMed Central

Mankouri, Jamel; Walter, Cheryl; Stewart, Hazel; Bentham, Matthew; Park, Wei Sun; Heo, Won Do; Fukuda, Mitsunori

2016-01-01

ABSTRACT The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosomal sorting complex required for transport (ESCRT). Here, we show a critical role of trans-Golgi network (TGN)-endosome trafficking during the assembly, but principally the secretion, of infectious virus. This was demonstrated by both small interfering RNA (siRNA)-mediated silencing of TGN-associated adaptor proteins and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of triglycerides, ApoB, or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct from that of very-low-density lipoprotein (VLDL). Finally, we show that HCV NS2 perturbs TGN architecture, redistributing TGN membranes to closely associate with HCV core protein residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, although essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins. IMPORTANCE The mechanisms by which infectious hepatitis C virus particles are assembled and released from the cell are poorly understood. We show that the virus subverts host cell trafficking pathways to effect the release of virus particles and disrupts the structure of the Golgi apparatus, a key cellular organelle involved in secretion. In addition, we demonstrate that the mechanisms used by the virus to exit the cell are distinct from those used by the cell to release lipoproteins, suggesting that the virus effects a unique modification to cellular trafficking pathways. PMID:27226379

Tumorigenicity of MCF-7 human breast cancer cells lacking the p38α mitogen-activated protein kinase

PubMed Central

Mendoza, Rhone A; Moody, Emily E; Enriquez, Marlene I; Mejia, Sylvia M; Thordarson, Gudmundur

2011-01-01

We have generated cell lines with significantly reduced expression of the p38 mitogen-activated protein kinase (p38 MAPK), Min-p38 MAPK cells, and used these cells to investigate its role in tumorigenesis of breast cancer cells. MCF-7 cells were stably transfected with a plasmid producing small interfering RNA that inhibited the expression of p38 MAPK. Control cells were stably transfected with the same plasmid producing non-interfering RNA. The reduction in the p38 MAPK activity caused a significant increase in the expressions of the estrogen receptor-α (ERα) and the progesterone receptor, but eliminated the expression of the ERβ. Min-p38 MAPK cells showed an enhanced overall growth response to 17β-estradiol (E2), whereas growth hormone plus epidermal growth factor were largely ineffective growth stimulators in these cells compared to controls. Although the long-term net growth rate of the Min-p38 MAPK cells was increased in response to E2, their proliferation rate was not different from controls in short-term cultures. However, the Min-p38 MAPK cells did show a significant decreased rate of apoptosis after E2 treatment and a reduction in the basal phosphorylation of p53 tumor suppressor protein compared to controls. When the Min-p38 MAPK cells were xenografted into E2-treated athymic nude mice, their tumorigenicity was enhanced compared to control cells. Conclusions: increased tumorigenicity of Min-p38 MAPK cells was caused mainly by a decrease in apoptosis rate indicating that the lack of the p38 MAPK caused an imbalance to increase the ERα:ERβ ratio and a reduction in the activity of the p53 tumor suppressor protein. PMID:20974639

Tumorigenicity of MCF-7 human breast cancer cells lacking the p38α mitogen-activated protein kinase.

PubMed

Mendoza, Rhone A; Moody, Emily E; Enriquez, Marlene I; Mejia, Sylvia M; Thordarson, Gudmundur

2011-01-01

We have generated cell lines with significantly reduced expression of the p38 mitogen-activated protein kinase (p38 MAPK), Min-p38 MAPK cells, and used these cells to investigate p38 MAPK's role in tumorigenesis of breast cancer cells. MCF-7 cells were stably transfected with a plasmid producing small interfering RNA that inhibited the expression of p38 MAPK. Control cells were stably transfected with the same plasmid producing non-interfering RNA. The reduction in the p38 MAPK activity caused a significant increase in the expressions of estrogen receptor-α (ERα) and the progesterone receptor, but eliminated the expression of ERβ. Min-p38 MAPK cells showed an enhanced overall growth response to 17β-estradiol (E₂), whereas GH plus epidermal growth factor were largely ineffective growth stimulators in these cells compared to controls. Although the long-term net growth rate of the Min-p38 MAPK cells was increased in response to E₂, their proliferation rate was lower compared to controls in short-term cultures. However, the Min-p38 MAPK cells did show a significant decreased rate of apoptosis after E₂ treatment and a reduction in the basal phosphorylation of p53 tumor suppressor protein compared to controls. When the Min-p38 MAPK cells were xenografted into E₂-treated athymic nude mice, their tumorigenicity was enhanced compared to control cells. Increased tumorigenicity of Min-p38 MAPK cells was caused mainly by a decrease in the apoptosis rate indicating that the lack of the p38 MAPK caused an imbalance to increase the ERα:ERβ ratio and a reduction in the activity of the p53 tumor suppressor protein.

The Practical Implementation of Non-Contacting Laser Strain Measurements Systems

NASA Technical Reports Server (NTRS)

Yunis, Isam; Quinn, roger D.; Kadambi, Jaikrishnan R.

2000-01-01

Measurement of stress and strain in rotating turbomachinery is critical to many industries. The search for a non-contacting, non-interfering, non-degrading measurement system is on going and extensive. While several methods seem promising in theory, implementation has proven troublesome. This work uncovers and quantifies these implementation issues in the context of a laser measurement system. Both a Laser Doppler Velocimeter system and a displacement laser system are utilized. It is found that the key issues are signal to noise ratio, rigid body compensation, measurement location, and conversion of intermittent measurements to a continuous signal. Accounting for these factors leads to successful measurements. These results should lead to better ideas and more practical solutions to the non-contacting, non-degrading, non-interfering strain measurement system problem.

Micelle-like Nanoparticles as Carriers for DNA and siRNA

PubMed Central

Navarro, Gemma; Pan, Jiayi; Torchilin, Vladimir P.

2015-01-01

Gene therapy represents a potential efficient approach of disease prevention and therapy. However, due to their poor in vivo stability, gene molecules need to be associated with delivery systems to overcome extracellular and intracellular barriers and allow access to the site of action. Cationic polymeric nanoparticles are popular carriers for small interfering RNA (siRNA) and DNA-based therapeutics for which efficient and safe delivery are important factors that need to be optimized. Micelle-like nanoparticles (MNP) (half micelles, half polymeric nanoparticles) can overcome some of the disadvantages of such cationic carriers by unifying in one single carrier the best of both delivery systems. In this review, we will discuss how the unique properties of MNP including self-assembly, condensation and protection of nucleic acids, improved cell association and gene transfection, and low toxicity may contribute to the successful application of siRNA- and DNA-based therapeutics into the clinic. Recent developments of MNP involving the addition of stimulus-sensitive functions to respond specifically to pathological or externally applied “triggers” (e.g., temperature, pH or enzymatic catalysis, light, or magnetic fields) will be discussed. Finally, we will overview the use of MNP as two-in-one carriers for the simultaneous delivery of different agents (small molecules, imaging agents) and nucleic acid combinations. PMID:25557580

Silencing of AtRAP, a target gene of a bacteria-induced small RNA, triggers antibacterial defense responses through activation of LSU2 and down-regulation of GLK1

PubMed Central

Wang, Huan; Seo, Jang-Kyun; Gao, Shang; Cui, Xinping; Jin, Hailing

2017-01-01

Summary Plants fine-tune their sophisticated immunity systems in response to pathogen infections. We previously showed that AtlsiRNA-1, a bacteria-induced plant endogenous small interfering RNA, silences the AtRAP gene, which encodes a putative RNA binding protein.In this study, we demonstrate that AtRAP functions as a negative regulator in plant immunity by characterizing molecular and biological responses of the knockout mutant and overexpression lines of AtRAP upon bacterial infection.AtRAP is localized in chloroplasts and physically interacts with Low Sulfur Upregulated 2 (LSU2), which positively regulates plant defense. Our results suggest that AtRAP negatively regulates defense responses by suppressing LSU2 through physical interaction. We also detected downregulation of the transcription factor GOLDEN2-LIKE 1 (GLK1) in atrap-1 using microarray analysis. The glk1 glk2 double mutant showed enhanced resistance to Pseudomonas syringae pv. tomato, which is consistent with a previous study showing enhanced resistance of a glk1 glk2 double mutant to Hyaloperonospora arabidopsidis.Taken together, our data suggest that silencing of AtRAP by AtlsiRNA-1 upon bacterial infection triggers defense responses through regulation of LSU2 and GLK1. PMID:28656601

The co-chaperones Fkbp4/5 control Argonaute2 expression and facilitate RISC assembly.

PubMed

Martinez, Natalia J; Chang, Hao-Ming; Borrajo, Jacob de Riba; Gregory, Richard I

2013-11-01

Argonaute2 (Ago2) protein and associated microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC) for target messenger RNA cleavage and post-transcriptional gene silencing. Although Ago2 is essential for RISC activity, the mechanism of RISC assembly is not well understood, and factors controlling Ago2 protein expression are largely unknown. A role for the Hsc70/Hsp90 chaperone complex in loading small RNA duplexes into the RISC has been demonstrated in cell extracts, and unloaded Ago2 is unstable and degraded by the lysosome in mammalian cells. Here we identify the co-chaperones Fkbp4 and Fkbp5 as Ago2-associated proteins in mouse embryonic stem cells. Pharmacological inhibition of this interaction using FK506 or siRNA-mediated Fkbp4/5 depletion leads to decreased Ago2 protein levels. We find FK506 treatment inhibits, whereas Fkbp4/5 overexpression promotes, miRNA-mediated stabilization of Ago2 expression. Simultaneous treatment with a lysosome inhibitor revealed the accumulation of unloaded Ago2 complexes in FK506-treated cells. We find that, consistent with unloaded miRNAs being unstable, FK506 treatment also affects miRNA abundance, particularly nascent miRNAs. Our results support a role for Fkbp4/5 in RISC assembly.

Antinutritive effects of wheat-germ agglutinin and other N-acetylglucosamine-specific lectins.

PubMed

Pusztai, A; Ewen, S W; Grant, G; Brown, D S; Stewart, J C; Peumans, W J; Van Damme, E J; Bardocz, S

1993-07-01

Incorporation of N-acetylglucosamine-specific agglutinins from wheat germ (Triticum aestivum; WGA), thorn apple (Datura stramonium) or nettle (Urtica dioica) rhizomes in the diet at the level of 7 g/kg reduced the apparent digestibility and utilization of dietary proteins and the growth of rats, with WGA being the most damaging. As a result of their binding and endocytosis by the epithelial cells of the small intestine, all three lectins were growth factors for the gut and interfered with its metabolism and function to varying degrees. WGA was particularly effective; it induced extensive polyamine-dependent hyperplastic and hypertrophic growth of the small bowel by increasing its content of proteins, RNA and DNA. Furthermore, an appreciable portion of the endocytosed WGA was transported across the gut wall into the systemic circulation, where it was deposited in the walls of the blood and lymphatic vessels. WGA also induced the hypertrophic growth of the pancreas and caused thymus atrophy. Although the transfer of the gene of WGA into crop plants has been advocated to increase their insect resistance, as the presence of this lectin in the diet may harm higher animals at the concentrations required to be effective against most pests, its use in plants as natural insecticide is not without health risks for man.

miR-137 inhibits the invasion of melanoma cells through downregulation of multiple oncogenic target genes.

PubMed

Luo, Chonglin; Tetteh, Paul W; Merz, Patrick R; Dickes, Elke; Abukiwan, Alia; Hotz-Wagenblatt, Agnes; Holland-Cunz, Stefan; Sinnberg, Tobias; Schittek, Birgit; Schadendorf, Dirk; Diederichs, Sven; Eichmüller, Stefan B

2013-03-01

MicroRNAs are small noncoding RNAs that regulate gene expression and have important roles in various types of cancer. Previously, miR-137 was reported to act as a tumor suppressor in different cancers, including malignant melanoma. In this study, we show that low miR-137 expression is correlated with poor survival in stage IV melanoma patients. We identified and validated two genes (c-Met and YB1) as direct targets of miR-137 and confirmed two previously known targets, namely enhancer of zeste homolog 2 (EZH2) and microphthalmia-associated transcription factor (MITF). Functional studies showed that miR-137 suppressed melanoma cell invasion through the downregulation of multiple target genes. The decreased invasion caused by miR-137 overexpression could be phenocopied by small interfering RNA knockdown of EZH2, c-Met, or Y box-binding protein 1 (YB1). Furthermore, miR-137 inhibited melanoma cell migration and proliferation. Finally, miR-137 induced apoptosis in melanoma cell lines and decreased BCL2 levels. In summary, our study confirms that miR-137 acts as a tumor suppressor in malignant melanoma and reveals that miR-137 regulates multiple targets including c-Met, YB1, EZH2, and MITF.

IBM1, a JmjC domain-containing histone demethylase, is involved in the regulation of RNA-directed DNA methylation through the epigenetic control of RDR2 and DCL3 expression in Arabidopsis

PubMed Central

Fan, Di; Dai, Yan; Wang, Xuncheng; Wang, Zhenjie; He, Hang; Yang, Hongchun; Cao, Ying; Deng, Xing Wang; Ma, Ligeng

2012-01-01

Small RNA-directed DNA methylation (RdDM) is an important epigenetic pathway in Arabidopsis that controls the expression of multiple genes and several developmental processes. RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3) are necessary factors in 24-nt small interfering RNA (siRNA) biogenesis, which is part of the RdDM pathway. Here, we found that Increase in BONSAI Methylation 1 (IBM1), a conserved JmjC family histone demethylase, is directly associated with RDR2 and DCL3 chromatin. The mutation of IBM1 induced the hypermethylation of H3K9 and DNA non-CG sites within RDR2 and DCL3, which repressed their expression. A genome-wide analysis suggested that the reduction in RDR2 and DCL3 expression affected siRNA biogenesis in a locus-specific manner and disrupted RdDM-directed gene repression. Together, our results suggest that IBM1 regulates gene expression through two distinct pathways: direct association to protect genes from silencing by preventing the coupling of histone and DNA methylation, and indirect silencing of gene expression through RdDM-directed repression. PMID:22772985

Evasion of Short Interfering RNA-Directed Antiviral Silencing in Musa acuminata Persistently Infected with Six Distinct Banana Streak Pararetroviruses

PubMed Central

Rajeswaran, Rajendran; Seguin, Jonathan; Chabannes, Matthieu; Duroy, Pierre-Olivier; Laboureau, Nathalie; Farinelli, Laurent; Iskra-Caruana, Marie-Line

2014-01-01

ABSTRACT Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes posttranscriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt) endogenous small RNAs with 5′-terminal guanosine was identified. In all virus-infected plants, 21- to 24-nt viral siRNAs accumulated at relatively high levels (up to 22% of the total small RNA population) and covered the entire circular viral DNA genomes in both orientations. The hotspots of 21-nt and 22-nt siRNAs occurred within open reading frame (ORF) I and II and the 5′ portion of ORF III, while 24-nt siRNAs were more evenly distributed along the viral genome. Despite the presence of abundant viral siRNAs of different size classes, the viral DNA was largely free of cytosine methylation. Thus, the virus is able to evade siRNA-directed DNA methylation and thereby avoid transcriptional silencing. This evasion of silencing likely contributes to the persistence of pararetroviruses in banana plants. IMPORTANCE We report that DNA pararetroviruses in Musa acuminata banana plants are able to evade DNA cytosine methylation and transcriptional gene silencing, despite being targeted by the host silencing machinery generating abundant 21- to 24-nucleotide short interfering RNAs. At the same time, the banana genomic DNA is extensively methylated in both healthy and virus-infected plants. Our findings shed light on the siRNA-generating gene silencing machinery of banana and provide a possible explanation why episomal pararetroviruses can persist in plants whereas true retroviruses with an obligatory genome-integration step in their replication cycle do not exist in plants. PMID:25056897

Evasion of short interfering RNA-directed antiviral silencing in Musa acuminata persistently infected with six distinct banana streak pararetroviruses.

PubMed

Rajeswaran, Rajendran; Seguin, Jonathan; Chabannes, Matthieu; Duroy, Pierre-Olivier; Laboureau, Nathalie; Farinelli, Laurent; Iskra-Caruana, Marie-Line; Pooggin, Mikhail M

2014-10-01

Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes posttranscriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt) endogenous small RNAs with 5'-terminal guanosine was identified. In all virus-infected plants, 21- to 24-nt viral siRNAs accumulated at relatively high levels (up to 22% of the total small RNA population) and covered the entire circular viral DNA genomes in both orientations. The hotspots of 21-nt and 22-nt siRNAs occurred within open reading frame (ORF) I and II and the 5' portion of ORF III, while 24-nt siRNAs were more evenly distributed along the viral genome. Despite the presence of abundant viral siRNAs of different size classes, the viral DNA was largely free of cytosine methylation. Thus, the virus is able to evade siRNA-directed DNA methylation and thereby avoid transcriptional silencing. This evasion of silencing likely contributes to the persistence of pararetroviruses in banana plants. We report that DNA pararetroviruses in Musa acuminata banana plants are able to evade DNA cytosine methylation and transcriptional gene silencing, despite being targeted by the host silencing machinery generating abundant 21- to 24-nucleotide short interfering RNAs. At the same time, the banana genomic DNA is extensively methylated in both healthy and virus-infected plants. Our findings shed light on the siRNA-generating gene silencing machinery of banana and provide a possible explanation why episomal pararetroviruses can persist in plants whereas true retroviruses with an obligatory genome-integration step in their replication cycle do not exist in plants. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Emotional responses to work-family conflict: an examination of gender role orientation working men and women.

PubMed

Livingston, Beth A; Judge, Timothy A

2008-01-01

The present study tested the effect of work-family conflict on emotions and the moderating effects of gender role orientation. On the basis of a multilevel design, the authors found that family-interfering-with- work was positively related to guilt, and gender role orientation interacted with both types of conflict (work-interfering-with-family and family-interfering-with-work) to predict guilt. Specifically, in general, traditional individuals experienced more guilt from family-interfering-with-work, and egalitarian individuals experienced more guilt from work-interfering-with-family. Additionally, a higher level interaction indicated that traditional men tended to experience a stronger relationship between family-interfering-with-work and guilt than did egalitarian men or women of either gender role orientation. 2008 APA

Mechanism of Telomerase Inhibition Using Small Inibitory RNAs and Induction of Breast Tumor Cell Sensitivity

DTIC Science & Technology

2007-03-01

RTb motif mutants hTERT Senescence Apoptosis Long lag period [20,25] Ribozymes Hairpin hTR, hTERT Apoptosis Incomplete knockdown of target [26...O-(2-Methoxyethyl) oligomers. b Reverse transcriptase motif.the growth and viability of cancer cells (Table 1). Ribozymes and short-interfering RNA...recent studies indicate that complete knockdown is not essential for efficient and rapid apoptosis in reference to siRNA against hTR and ribozymes

Postexposure Application of Fas Receptor Small-Interfering RNA to Suppress Sulfur Mustard-Induced Apoptosis in Human Airway Epithelial Cells: Implication for a Therapeutic Approach

DTIC Science & Technology

2013-01-01

bronchiolitis, bronchopneumo- nia, chronic obstructive pulmonary disease, bronchiectasis, asthma, large airway narrowing, and pulmonary fibrosis ... pulmonary fibrosis , acute lung injury [ALI], acute respiratory distress syndrome, etc.) (Beheshti et al., 2006; Emmler et al., 2007; Kuwano, 2008...cytokine in bronchoalveolar lavage (BAL) fluid in patients with pulmonary fibrosis due to sulfur mustard gas inhalation. J In- terferon Cytokine Res 27:38

Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

PubMed

Hecke, Anneke; Brooks, Hilary; Meryet-Figuière, Matthieu; Minne, Stephanie; Konstantinides, Stavros; Hasenfuss, Gerd; Lebleu, Bernard; Schäfer, Katrin

2006-05-01

Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

Development of a functionalized polymer for stent coating in the arterial delivery of small interfering RNA.

PubMed

San Juan, Aurélie; Bala, Madiha; Hlawaty, Hanna; Portes, Patrick; Vranckx, Roger; Feldman, Laurent J; Letourneur, Didier

2009-11-09

In patients receiving drug eluting stents, there is a growing concern about both the long-term toxicity/degradability of the polymers used for the coating, and the nature of the therapeutic agents. We hypothesized that the use of a functionalized biocompatible polymer for a stent coating could be appropriate for local arterial therapy. A cationized pullulan hydrogel was thus prepared to cover bare metal stents that could be further loaded with small interfering RNA (siRNA) targeted at MMP2 for gene silencing in vascular cells. The efficient coverage of the stent struts by a smooth polymeric layer, which can withstand the crimping of the stent on a balloon-catheter and its deployment, was demonstrated by fluorescence microscopy, scanning electron microscopy, and atomic force microscopy. The release of siRNA from the stents was modulated by the presence of the cationic groups, as compared to noncationized pullulan hydrogel. In vivo implantation of coated stents was successful and cationized pullulan-based hydrogels loaded with siRNA in rabbit balloon-injured carotid arteries induced an uptake of siRNA into the arterial wall and a decrease of pro-MMP2 activity. These results suggest that cationized pullulan-based hydrogel could be used as a new biocompatible and biodegradable stent coating for local gene therapy in the arterial wall.

Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases.

PubMed

Takahashi, Yuki; Nishikawa, Makiya; Kobayashi, Naoki; Takakura, Yoshinobu

2005-07-20

Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference (RNAi) offers a promising approach to treating tumor patients. To achieve RNAi-based tumor therapy, a small interfering RNA (siRNA) or siRNA-expressing vector needs to be delivered to tumor cells, but little information about its in vivo delivery has been reported. In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. To quantitatively evaluate the RNAi effects in tumor cells, mouse melanoma B16-BL6 cells were stably transfected with both firefly (a model target gene) and sea pansy (an internal standard gene) luciferase genes to obtain B16-BL6/dual Luc cells. The target gene expression in subcutaneous primary tumors of B16-BL6/dual Luc cells was significantly suppressed by direct injection of the RNAi effectors followed by electroporation. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure. These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells.

The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells

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

Xu, Y.; Mirmalek-Sani, S.-H.; Yang, X.

2006-06-10

RNA interference (RNAi) has been used in functional genomics and offers innovative approaches in the development of novel therapeutics. Human mesenchymal stem cells offer a unique cell source for tissue engineering/regeneration strategies. The current study examined the potential of small interfering RNAs (siRNA) against human peroxisome proliferator activated receptor gamma (PPAR{gamma}) to suppress adipocyte differentiation (adipogenesis) in human preadipocytes and fetal-femur-derived mesenchymal cells. Adipogenesis was investigated using cellular and biochemical analysis. Transient transfection with PPAR{gamma}-siRNA using a liposomal-based strategy resulted in a significant inhibition of adipogenesis in human preadipocytes and fetal-femur-derived mesenchymal cells, compared to controls (cell, liposomal and negativemore » siRNA). The inhibitory effect of PPAR{gamma}-siRNA was supported by testing human PPAR{gamma} mRNA and adipogenic associated genes using reverse transcription polymerase chain reaction (RT-PCR) to adiponectin receptor 1 and 2 as well as examination of fatty acid binding protein 3 (FABP{sub 3}) expression, an adipocyte-specific marker. The current studies indicate that PPAR{gamma}-siRNA is a useful tool to study adipogenesis in human cells, with potential applications both therapeutic and in the elucidation of mesenchymal cell differentiation in the modulation of cell differentiation in human mesenchymal cells.« less

Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

NASA Technical Reports Server (NTRS)

Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

2002-01-01

Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

Modulation of the response of prostate cancer cell lines to cisplatin treatment using small interfering RNA.

PubMed

Parra, Eduardo; Ferreira, Jorge

2013-10-01

Cisplatin is one of the most effective and widely used chemotherapeutic agents against several types of human cancers. However, the underlying mechanisms of action are not fully understood. We aimed to investigate the possible molecular mechanism(s) of acquired chemoresistance observed in prostate cancer cells treated with cisplatin. Human LNCaP cells (bearing wild-type p53) and PC-3 cells (lacking p53) were used. The expression levels of protein were determined by western blotting, and the mRNA levels were determined by reverse transcription-polymerase chain reaction (RT-PCR). Cell viability was measured by MTT assay, and the transcriptional effect of small interfering RNA (siRNA) was measured by luciferase reporter gene. We showed that cisplatin treatment increased JNK-1 and JNK-2 activity and expression in both LNCaP and PC-3 cells. In addition, the knockdown of JNK-1 expression by siRNA-JNK-1 or siRNA-JNK-2 significantly impaired the upregulation of AP-1 luciferase reporter gene, but failed to decrease the levels of AP-1 reporter gene expression induced by TPA treatment. Our observations indicate that JNK-1 and JNK-2 may be involved in the chemoresistance observed in prostate cancer cells treated with cisplatin and that blocking the stimulation of Jun kinase (JNK) signaling may be important for regulating the susceptibility to cisplatin of prostate cancer.

RNA-induced silencing complex-bound small interfering RNA is a determinant of RNA interference-mediated gene silencing in mice.

PubMed

Wei, Jie; Jones, Jeffrey; Kang, Jing; Card, Ananda; Krimm, Michael; Hancock, Paula; Pei, Yi; Ason, Brandon; Payson, Elmer; Dubinina, Natalya; Cancilla, Mark; Stroh, Mark; Burchard, Julja; Sachs, Alan B; Hochman, Jerome H; Flanagan, W Michael; Kuklin, Nelly A

2011-06-01

Deeper knowledge of pharmacokinetic and pharmacodynamic (PK/PD) concepts for RNA therapeutics is important to streamline the drug development process and for rigorous selection of best performing drug candidates. Here we characterized the PK/PD relationship for small interfering RNAs (siRNAs) targeting luciferase by examining siRNA concentration in plasma and liver, the temporal RNA-induced silencing complex binding profiles, mRNA reduction, and protein inhibition measured by noninvasive bioluminescent imaging. A dose-dependent and time-related decrease in bioluminescence was detected over 25 days after a single treatment of a lipid nanoparticle-formulated siRNA targeting luciferase messenger RNA. A direct relationship was observed between the degree of in vivo mRNA and protein reduction and the Argonaute2 (Ago2)-bound siRNA fraction but not with the total amount of siRNA found in the liver, suggesting that the Ago2-siRNA complex is the key determinant of target inhibition. These observations were confirmed for an additional siRNA that targets endogenously expressed Sjögren syndrome antigen B (Ssb) mRNA, indicating that our observations are not limited to a transgenic mouse system. Our data provide detailed information of the temporal regulation of siRNA liver delivery, Ago2 loading, mRNA reduction, and protein inhibition that are essential for the rapid and cost-effective clinical development of siRNAs therapeutics.

A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics.

PubMed

Alterman, Julia F; Coles, Andrew H; Hall, Lauren M; Aronin, Neil; Khvorova, Anastasia; Didiot, Marie-Cécile

2017-08-20

Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo . We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, and accurately quantify the mRNA silencing of hundreds of hsiRNAs, we use the QuantiGene 2.0 quantitative gene expression assay. This high-throughput, 96-well plate-based assay can quantify mRNA levels directly from sample lysate. Here, we describe a method to prepare short-term cultures of mouse primary cortical neurons in a 96-well plate format for high-throughput testing of oligonucleotide therapeutics. This method supports the testing of hsiRNA libraries and the identification of potential therapeutics within just two weeks. We detail methodologies of our high throughput assay workflow from primary neuron preparation to data analysis. This method can help identify oligonucleotide therapeutics for treatment of various neurological diseases.

Small interfering RNA-producing loci in the ancient parasitic eukaryote Trypanosoma brucei

PubMed Central

2012-01-01

Background At the core of the RNA interference (RNAi) pathway in Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in controlling retroposon and repeat transcripts. Recent evidence from higher eukaryotes suggests that a variety of genomic sequences with the potential to produce double-stranded RNA are sources for small interfering RNAs (siRNAs). Results To test whether such endogenous siRNAs are present in T. brucei and to probe the individual role of the two Dicer-like enzymes, we affinity purified TbAGO1 from wild-type procyclic trypanosomes, as well as from cells deficient in the cytoplasmic (TbDCL1) or nuclear (TbDCL2) Dicer, and subjected the bound RNAs to Illumina high-throughput sequencing. In wild-type cells the majority of reads originated from two classes of retroposons. We also considerably expanded the repertoire of trypanosome siRNAs to encompass a family of 147-bp satellite-like repeats, many of the regions where RNA polymerase II transcription converges, large inverted repeats and two pseudogenes. Production of these newly described siRNAs is strictly dependent on the nuclear DCL2. Notably, our data indicate that putative centromeric regions, excluding the CIR147 repeats, are not a significant source for endogenous siRNAs. Conclusions Our data suggest that endogenous RNAi targets may be as evolutionarily old as the mechanism itself. PMID:22925482

Delivery of Small Interfering RNA by Peptide-Targeted Mesoporous Silica Nanoparticle-Supported Lipid Bilayers

PubMed Central

Ashley, Carlee E.; Carnes, Eric C.; Epler, Katharine E.; Padilla, David P.; Phillips, Genevieve K.; Castillo, Robert E.; Wilkinson, Dan C.; Wilkinson, Brian S.; Burgard, Cameron A.; Sewell, Robin M.; Townson, Jason L.; Chackerian, Bryce; Willman, Cheryl L.; Peabody, David S.; Wharton, Walker; Brinker, C. Jeffrey

2012-01-01

The therapeutic potential of small interfering RNAs (siRNAs) is severely limited by the availability of delivery platforms that protect siRNA from degradation, deliver it to the target cell with high specificity and efficiency, and promote its endosomal escape and cytosolic dispersion. Here we report that mesoporous silica nanoparticle-supported lipid bilayers (or ‘protocells’), exhibit multiple properties that overcome many of the limitations of existing delivery platforms. Protocells have a 10- to 100-fold greater capacity for siRNA than corresponding lipid nanoparticles and are markedly more stable when incubated under physiological conditions. Protocells loaded with a cocktail of siRNAs bind to cells in a manner dependent on the presence of an appropriate targeting peptide and, through an endocytic pathway followed by endosomal disruption, promote delivery of the silencing nucleotides to the cytoplasm. The expression of each of the genes targeted by the siRNAs was shown to be repressed at the protein level, resulting in a potent induction of growth arrest and apoptosis. Incubation of control cells that lack expression of the antigen recognized by the targeting peptide with siRNA-loaded protocells induced neither repression of protein expression nor apoptosis, indicating the precise specificity of cytotoxic activity. In terms of loading capacity, targeting capabilities, and potency of action, protocells provide unique attributes as a delivery platform for therapeutic oligonucleotides. PMID:22309035

Histone Deacetylase-1 Is Enriched at the Platelet-derived Growth Factor-D Promoter in Response to Interleukin-1β and Forms a Cytokine-inducible Gene-silencing Complex with NF-κB p65 and Interferon Regulatory Factor-1*

PubMed Central

Liu, Mary Y.; Khachigian, Levon M.

2009-01-01

Understanding the mechanisms governing cytokine control of growth factor expression in smooth muscle cells would provide invaluable insight into the molecular regulation of vascular phenotypes and create future opportunities for therapeutic intervention. Here, we report that the proinflammatory cytokine interleukin (IL)-1β suppresses platelet-derived growth factor (PDGF)-D promoter activity and mRNA and protein expression in smooth muscle cells. NF-κB p65, induced by IL-1β, interacts with a novel element in the PDGF-D promoter and inhibits PDGF-D transcription. Interferon regulatory factor-1 (IRF-1) is also induced by IL-1β and binds to a different element upstream in the promoter. Immunoprecipitation and chromatin immunoprecipitation experiments showed that IL-1β stimulates p65 interaction with IRF-1 and the accumulation of both factors at the PDGF-D promoter. Mutation of the IRF-1 and p65 DNA-binding elements relieved the promoter from IL-1β-mediated repression. PDGF-D repression by IL-1β involves histone deacetylation and interaction of HDAC-1 with IRF-1 and p65. HDAC-1 small interfering RNA ablates complex formation with IRF-1 and p65 and abrogates IRF-1 and p65 occupancy of the PDGF-D promoter. Thus, HDAC-1 is enriched at the PDGF-D promoter in cells exposed to IL-1β and forms a cytokine-inducible gene-silencing complex with p65 and IRF-1. PMID:19843519

Small RNA Profiling Reveals Phosphorus Deficiency as a Contributing Factor in Symptom Expression for Citrus Huanglongbing Disease

PubMed Central

Zhao, Hongwei; Sun, Ruobai; Jin, Hailing

2013-01-01

Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus ‘Candidatus Liberibacter’ (Ca. L.). Powerful diagnostic tools and management strategies are desired to control HLB. Host small RNAs (sRNA) play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases, including cancers. To determine whether citrus sRNAs regulate host responses to HLB, sRNAs were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca. L. asiaticus (Las)-positive or healthy tissue. Ten new microRNAs (miRNAs), 76 conserved miRNAs, and many small interfering RNAs (siRNAs) were discovered. Several miRNAs and siRNAs were highly induced by Las infection, and can be potentially developed into early diagnosis markers of HLB. miR399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn—a disease with symptoms similar to HLB. We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida. Our molecular, physiological, and field data suggest that phosphorus deficiency is linked to HLB disease symptomology. PMID:23292880

Bio-inspired materials in drug delivery: Exploring the role of pulmonary surfactant in siRNA inhalation therapy.

PubMed

De Backer, Lynn; Cerrada, Alejandro; Pérez-Gil, Jesús; De Smedt, Stefaan C; Raemdonck, Koen

2015-12-28

Many pathologies of the respiratory tract are inadequately treated with existing small molecule-based therapies. The emergence of RNA interference (RNAi) enables the post-transcriptional silencing of key molecular disease factors that cannot readily be targeted with conventional small molecule drugs. Pulmonary administration of RNAi effectors, such as small interfering RNA (siRNA), allows direct delivery into the lung tissue, hence reducing systemic exposure. Unfortunately, the clinical translation of RNAi is severely hampered by inefficient delivery of siRNA therapeutics towards the cytoplasm of the target cells. In order to have a better control of the siRNA delivery process, both extra- and intracellular, siRNAs are typically formulated in nanosized delivery vehicles (nanoparticles, NPs). In the lower airways, which are the targeted sites of action for multiple pulmonary disorders, these siRNA-loaded NPs will encounter the pulmonary surfactant (PS) layer, covering the entire alveolar surface. The interaction between the instilled siRNA-loaded NPs and the PS at this nano-bio interface results in the adsorption of PS components onto the surface of the NPs. The formation of this so-called biomolecular corona conceals the original NP surface and will therefore profoundly determine the biological efficacy of the NP. Though this interplay has initially been regarded as a barrier towards efficient siRNA delivery to the respiratory target cell, recent reports have illustrated that the interaction with PS might also be beneficial for local pulmonary siRNA delivery.

De novo assembly of honey bee RNA viral genomes by tapping into the innate insect antiviral response pathway.

PubMed

Fung, Elisabeth; Hill, Kelly; Hogendoorn, Katja; Glatz, Richard V; Napier, Kathryn R; Bellgard, Matthew I; Barrero, Roberto A

2018-02-01

Bee pollination is critical for improving productivity of one third of all plants or plant products consumed by humans. The health of honey bees is in decline in many countries worldwide, and RNA viruses together with other biological, environmental and anthropogenic factors have been identified as the main causes. The rapid genetic variation of viruses represents a challenge for diagnosis. Thus, application of deep sequencing methods for detection and analysis of viruses has increased over the last years. In this study, we leverage from the innate Dicer-2 mediated antiviral response against viruses to reconstruct complete viral genomes using virus-derived small interfering RNAs (vsiRNAs). Symptomatic A. mellifera larvae collected from hives free of Colony Collapse Disorder (CCD) and the parasitic Varroa mite (Varroa destructor) were used to generate more than 107 million small RNA reads. We show that de novo assembly of insect viral sequences is less fragmented using only 22 nt long vsiRNAs rather than a combination of 21-22 nt small RNAs. Our results show that A. mellifera larvae activate the RNAi immune response in the presence of Sacbrood virus (SBV). We assembled three SBV genomes from three individual larvae from different hives in a single apiary, with 1-2% nucleotide sequence variability among them. We found 3-4% variability between SBV genomes generated in this study and earlier published Australian variants suggesting the presence of different SBV quasispecies within the country. Copyright © 2018. Published by Elsevier Inc.

Role of Bruton's tyrosine kinase inhibitors in HIV-1-infected cells.

PubMed

Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

2015-06-01

Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high-throughput proteomic assays, we have previously identified Bruton's tyrosine kinase (BTK) as a host protein that was uniquely upregulated in the plasma membrane of human immunodeficiency virus (HIV-1)-infected T cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant upregulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells; however, new BTK protein complexes were identified and distributed in both high molecular weight (∼600 kDa) and a small molecular weight complex (∼60-120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1-infected cells using small interfering RNA (siRNA) resulted in selective death of infected, but not uninfected, cells. Using BTK-specific antibody and small-molecule inhibitors including LFM-A13 and a FDA-approved compound, ibrutinib (PCI-32765), we have found that HIV-1-infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1-infected cells are sensitive to treatments targeting BTK expressed in infected cells.

A Transcriptional Regulatory Network Containing Nuclear Receptors and Long Noncoding RNAs Controls Basal and Drug-Induced Expression of Cytochrome P450s in HepaRG Cells.

PubMed

Chen, Liming; Bao, Yifan; Piekos, Stephanie C; Zhu, Kexin; Zhang, Lirong; Zhong, Xiao-Bo

2018-07-01

Cytochrome P450 (P450) enzymes are responsible for metabolizing drugs. Expression of P450s can directly affect drug metabolism, resulting in various outcomes in therapeutic efficacy and adverse effects. Several nuclear receptors are transcription factors that can regulate expression of P450s at both basal and drug-induced levels. Some long noncoding RNAs (lncRNAs) near a transcription factor are found to participate in the regulatory functions of the transcription factors. The aim of this study is to determine whether there is a transcriptional regulatory network containing nuclear receptors and lncRNAs controlling both basal and drug-induced expression of P450s in HepaRG cells. Small interfering RNAs or small hairpin RNAs were applied to knock down four nuclear receptors [hepatocyte nuclear factor 1 α (HNF1 α ), hepatocyte nuclear factor 4 α (HNF4 α ), pregnane X receptor (PXR), and constitutive androstane receptor (CAR)] as well as two lncRNAs [HNF1 α antisense RNA 1 (HNF1 α -AS1) and HNF4 α antisense RNA 1 (HNF4 α -AS1)] in HepaRG cells with or without treatment of phenobarbital or rifampicin. Expression of eight P450 enzymes was examined in both basal and drug-induced levels. CAR and PXR mainly regulated expression of specific P450s. HNF1 α and HNF4 α affected expression of a wide range of P450s as well as other transcription factors. HNF1 α and HNF4 α controlled the expression of their neighborhood lncRNAs, HNF1 α -AS1 and HNF4 α -AS1, respectively. HNF1 α -AS1 and HNF4 α -AS1 was also involved in the regulation of P450s and transcription factors in diverse manners. Altogether, our study concludes that a transcription regulatory network containing the nuclear receptors and lncRNAs controls both basal and drug-induced expression of P450s in HepaRG cells. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Developmental Differences in the Impact of Contextual Factors on Susceptibility to Retroactive Interference

ERIC Educational Resources Information Center

Levy-Gigi, Einat; Vakil, Eli

2010-01-01

The influence of contextual similarity on children's recognition memory performance was examined using a retroactive interference paradigm. In the study, 9- and 12-year-olds were randomly assigned to one of two contextual conditions. In both conditions, target and interfering information were presented in distinctive contexts by using different…

Is Fatigue a Common Crohn's Disease Symptom? What Can Be Done about It?

MedlinePlus

... to fatigue — either directly or by interfering with sleep. Seek psychological therapy. Consider talking to a doctor or a counselor about ways to manage fatigue and other psychological factors that can impact fatigue, ... foods and get adequate sleep. Be sure to share your concerns about fatigue ...

The 3Ps of oak regeneration: planning, patience and persistence

Treesearch

Dale R. Weigel; Daniel C. Dey; John. Kabrick

2012-01-01

Oak regeneration research in the United States has been ongoing in earnest since the late 1950s. Most research has focused on specific silvicultural practices, regeneration processes, site characteristics, and local limiting factors such as deer browsing or interfering species. Research has evaluated the effects of thinning on regeneration development, methods for oak...

Rethinking District Budgets to Unify and Sustain a Critical Mass of Student and Learning Supports at Schools. A Center Brief

ERIC Educational Resources Information Center

Center for Mental Health in Schools at UCLA, 2012

2012-01-01

imperative for policy makers to reverse trends toward lopsided cutbacks that decimate efforts to address factors interfering with learning and teaching. Furthermore, it is essential to move forward in more cost-effective ways by unifying student and learning supports and…

The 3 Ps of oak regeneration: planning, persistence, and patience

Treesearch

Dale R. Weigel; Daniel C. Dey; John Kabrick

2012-01-01

Oak regeneration research in the United States has been ongoing in earnest since the late 1950s. Most research has focused on specific silvicultural practices, regeneration processes, site characteristics, and local limiting factors such as deer browsing or interfering species. Research has evaluated the effects of thinning on regeneration development, methods for oak...

A first approach to the distortion analysis of nonlinear analog circuits utilizing X-parameters

NASA Astrophysics Data System (ADS)

Weber, H.; Widemann, C.; Mathis, W.

2013-07-01

In this contribution a first approach to the distortion analysis of nonlinear 2-port-networks with X-parameters1 is presented. The X-parameters introduced by Verspecht and Root (2006) offer the possibility to describe nonlinear microwave 2-port-networks under large signal conditions. On the basis of X-parameter measurements with a nonlinear network analyzer (NVNA) behavioral models can be extracted for the networks. These models can be used to consider the nonlinear behavior during the design process of microwave circuits. The idea of the present work is to extract the behavioral models in order to describe the influence of interfering signals on the output behavior of the nonlinear circuits. Hereby, a simulator is used instead of a NVNA to extract the X-parameters. Assuming that the interfering signals are relatively small compared to the nominal input signal, the output signal can be described as a superposition of the effects of each input signal. In order to determine the functional correlation between the scattering variables, a polynomial dependency is assumed. The required datasets for the approximation of the describing functions are simulated by a directional coupler model in Cadence Design Framework. The polynomial coefficients are obtained by a least-square method. The resulting describing functions can be used to predict the system's behavior under certain conditions as well as the effects of the interfering signal on the output signal. 1 X-parameter is a registered trademark of Agilent Technologies, Inc.

MEK, p38, and PI-3K mediate cross talk between EGFR and TNFR in enhancing hepatocyte growth factor production from human mesenchymal stem cells

PubMed Central

Wang, Yue; Weil, Brent R.; Herrmann, Jeremy L.; Abarbanell, Aaron M.; Tan, Jiangning; Markel, Troy A.; Kelly, Megan L.

2009-01-01

Human bone marrow mesenchymal stem cells (MSCs) are a potent source of growth factors, which are partly responsible for their beneficial paracrine effects. We reported previously that transforming growth factor-α (TGF-α), a putative mediator of wound healing and the injury response, increases the release of vascular endothelial growth factor (VEGF), augments tumor necrosis factor-α (TNF-α)-stimulated VEGF production, and activates mitogen-activated protein kinases and phosphatidylinositol 3-kinase (PI-3K) pathway in human MSCs. The experiments described in this report indicate that TGF-α increases MSC-derived hepatocyte growth factor (HGF) production. TGF-α-stimulated HGF production was abolished by inhibition of MEK, p38, PI-3K, or by small interfering RNA (siRNA) targeting TNF receptor 2 (TNFR2), but was not attenuated by siRNA targeting TNF receptor 1 (TNFR1). Ablation of TNFR1 significantly increased basal and stimulated HGF. A potent synergy between TGF-α and TNF-α was noted in MSC HGF production. This synergistic effect was abolished by MEK, P38, PI-3K inhibition, or by ablation of both TNF receptors using siRNA. We conclude that 1) novel cross talk occurs between tumor necrosis factor receptor and TGF-α/epidermal growth factor receptor in stimulating MSC HGF production; 2) this cross talk is mediated, at least partially, via activation of MEK, p38, and PI-3K; 3) TGF-α stimulates MSCs to produce HGF by MEK, p38, PI-3K, and TNFR2-dependent mechanisms; and 4) TNFR1 acts to decrease basal TGF-α and TNF-α-stimulated HGF. PMID:19692652

Emodin Suppresses Maintenance of Stemness by Augmenting Proteosomal Degradation of Epidermal Growth Factor Receptor/Epidermal Growth Factor Receptor Variant III in Glioma Stem Cells

PubMed Central

Kim, Jeongyub; Lee, Jong-Seon; Jung, Jieun; Lim, Inhye; Lee, Ji-Yun

2015-01-01

There is a growing body of evidence that small subpopulations of cells with stem cell-like characteristics within most solid tumors are responsible for the malignancy of aggressive cancer cells and that targeting these cells might be a good therapeutic strategy to reduce the risk of tumor relapse after therapy. Here, we examined the effects of emodin (1,3,8-trihydroxy-6-methylanthraquinone), an active component of the root and rhizome of Rheum palmatum that has several biological activities, including antitumor effects, on primary cultured glioma stem cells (GSCs). Emodin inhibited the self-renewal activity of GSCs in vitro as evidenced by neurosphere formation, limiting dilution, and soft agar clonogenic assays. Emodin inhibited the maintenance of stemness by suppressing the expression of Notch intracellular domain, nonphosphorylated β-catenin, and phosphorylated STAT3 proteins. In addition, treatment with emodin partially induced apoptosis, reduced cell invasiveness, and sensitized GSCs to ionizing radiation. Intriguingly, emodin induced proteosomal degradation of epidermal growth factor receptor (EGFR)/EGFR variant III (EGFRvIII) by interfering with the association of EGFR/EGFRvIII with heat shock protein 90, resulting in the suppression of stemness pathways. Based on these data, we propose that emodin could be considered as a potent therapeutic adjuvant that targets GSCs. PMID:25229646

Effect of siRNA silencing of inducible co-stimulatory molecule on myocardial cell hypertrophy after cardiac infarction in rats.

PubMed

Wang, W M; Liu, Z; Chen, G

2016-05-20

As the most common cardiac disease, myocardial infarction is followed by hypertrophy of cardiac myocytes and reconstruction of ventricular structure. The up-regulation of a series of factors including metalloproteinases, inflammatory factors, and growth factors after primary infarction lead to the hypertrophy, apoptosis, necrosis, and fibroblast proliferation in cardiac muscle tissues. Recent studies have reported on the potency of small interfering RNA (siRNA) in treating cardiac diseases. We thus investigated the efficacy of inducible co-stimulatory molecule (ICOS)-specific siRNA silencing in myocardial hypertrophy in a cardiac infarction rat model. This cardiac infarction model was prepared by ligating the left anterior descending coronary artery. ICOS-siRNA treatment was administered in parallel with non-sense siRNA. After 18 days, the cross-sectional area of cardiac muscle tissues and the left ventricle weight index were measured, along with ICOS mRNA and protein expression levels, and pathological staining. Compared to those in the control groups, in myocardial infarcted rats, the application of ICOS-siRNA effectively decreased the left ventricle weight index, as well as the surface area of cardiac myocytes. Both mRNA and protein levels of ICOS were also significantly decreased. HE staining was consistent with these results. In conclusion, ICOS-targeted siRNA can effectively silence gene expression of ICOS, and provided satisfactory treatment efficacy for myocardial cell hypertrophy after infarction.

Luteolin, a novel natural inhibitor of tumor progression locus 2 serine/threonine kinase, inhibits tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in JB6 mouse epidermis cells.

PubMed

Kim, Jong-Eun; Son, Joe Eun; Jang, Young Jin; Lee, Dong Eun; Kang, Nam Joo; Jung, Sung Keun; Heo, Yong-Seok; Lee, Ki Won; Lee, Hyong Joo

2011-09-01

Targeting tumor necrosis factor (TNF)-α-mediated signal pathways may be a promising strategy for developing chemopreventive agents, because TNF-α-mediated cyclooxygenase (COX)-2 expression plays a key role in inflammation and carcinogenesis. Luteolin [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone] exerts anticarcinogenic effects, although little is known about the underlying molecular mechanisms and specific targets of this compound. In the present study, we found that luteolin inhibited TNF-α-induced COX-2 expression by down-regulating the transactivation of nuclear factor-κB and activator protein-1. Furthermore, luteolin inhibited TNF-α-induced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/ERK/p90(RSK), mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase/c-Jun, and Akt/p70(S6K). However, it had no effect on the phosphorylation of p38. These effects of luteolin on TNF-α-mediated signaling pathways and COX-2 expression are similar to those achieved by blocking tumor progression locus 2 serine/threonine kinase (TPL2) using pharmacologic inhibitors and small interfering RNAs. Luteolin inhibited TPL2 activity in vitro and in TPL2 immunoprecipitation kinase assays by binding directly in an ATP-competitive manner. Overall, these results indicate that luteolin exerts potent chemopreventive activities, which primarily target TPL2.

[TLR2 modulates Staphylococcus aureus-induced inflammatory response and autophagy in macrophages through PI3K signaling pathway].

PubMed

Li, Shuai; Fang, Lei; Wang, Jiong; Liu, Rongyu

2017-09-01

Objective To investigate the molecular mechanisms of Toll-like receptor 2 (TLR2) taking part in inflammatory response in Staphylococcus aureus (SA)-induced asthma. Methods We established the cell inflammatory response model through stimulating mouse RAW264.7 macrophages with SA. The TLR2, myeloid differentiation factor 88 (MyD88), phosphoinositide-3 kinase (PI3K), nuclear factor κBp65 (NF-κBp65), phospho-NF-κBp65, beclin-1 and microtubule-associated protein 1 light chain 3B (LC3B) were detected by Western blot analysis after treatment with TLR2 small interfering RNA (siRNA) and 3-methyladenine (3-MA), and the tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) were determined by ELISA. In addition, the number of autolysosomes was observed by the laser scanning confocal microscope. Results SA-stimulated macrophages activated various signaling pathways including TLR2. TLR2 siRNA markedly repressed the expressions of PI3K, phospho-NF-κBp65, the autophagy protein beclin-1 and LC3B as well as the number of autolysosomes and the production of TNF- and IL-6. We also demonstrated that 3-MA had the same effect on autophagy and inflammation as TLR2 siRNA did. Conclusion TLR2 modulates SA-induced inflammatory response and autophagy in macrophages through PI3K signaling pathway.

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

PubMed Central

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

2014-01-01

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

Genome-Wide siRNA Screen Identifies Complementary Signaling Pathways Involved in Listeria Infection and Reveals Different Actin Nucleation Mechanisms during Listeria Cell Invasion and Actin Comet Tail Formation

PubMed Central

Kühbacher, Andreas; Emmenlauer, Mario; Rämo, Pauli; Kafai, Natasha; Dehio, Christoph

2015-01-01

ABSTRACT Listeria monocytogenes enters nonphagocytic cells by a receptor-mediated mechanism that is dependent on a clathrin-based molecular machinery and actin rearrangements. Bacterial intra- and intercellular movements are also actin dependent and rely on the actin nucleating Arp2/3 complex, which is activated by host-derived nucleation-promoting factors downstream of the cell receptor Met during entry and by the bacterial nucleation-promoting factor ActA during comet tail formation. By genome-wide small interfering RNA (siRNA) screening for host factors involved in bacterial infection, we identified diverse cellular signaling networks and protein complexes that support or limit these processes. In addition, we could precise previously described molecular pathways involved in Listeria invasion. In particular our results show that the requirements for actin nucleators during Listeria entry and actin comet tail formation are different. Knockdown of several actin nucleators, including SPIRE2, reduced bacterial invasion while not affecting the generation of comet tails. Most interestingly, we observed that in contrast to our expectations, not all of the seven subunits of the Arp2/3 complex are required for Listeria entry into cells or actin tail formation and that the subunit requirements for each of these processes differ, highlighting a previously unsuspected versatility in Arp2/3 complex composition and function. PMID:25991686

Cx43 in mesenchymal stem cells promotes angiogenesis of the infarcted heart independent of gap junctions.

PubMed

Wang, De-Guo; Zhang, Feng-Xiang; Chen, Ming-Long; Zhu, Hong-Jun; Yang, Bing; Cao, Ke-Jiang

2014-04-01

Mesenchymal stem cells (MSCs) with elevated levels of connexin 43 (Cx43) have been shown to exhibit improved protection for ischemic hearts. However, it remains unclear whether Cx43 is involved in the paracrine actions of angiogenesis, the major mechanism of cell therapy. In the present study, an in vitro model with deprivation of oxygen and a murine myocardial infarction model with permanent ligation of the left anterior‑descending (LAD) coronary artery were used to determine whether gap junctions in MSCs promote angiogenesis. It was observed that MSCs that overexpressed Cx43 (MSCs‑Cx43), improve the cardiac function of infarcted myocardium as compared with control MSCs (MSCs‑vector) and MSCs with Cx43 knocked down by small interfering RNA (MSCs‑siCx43), accompanied with a reduction of infarct size and an increase in the vascular density and maturity. Increased levels of representative angiogenic factors [vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)] were produced by MSCs‑Cx43 compared with MSCs‑siCx43 in vivo and in vitro. However, neither Cx43 formed gap junction specific inhibitor (Cx43 mimetic peptide) or gap junction opener (antiarrhythmic peptide) affected the production of VEGF and bFGF in MSCs under hypoxic stress. These data support the hypothesis that Cx43 in MSCs promotes angiogenesis in the infarcted heart, independent of gap junction formation.

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs.

PubMed

Khan, Aly A; Betel, Doron; Miller, Martin L; Sander, Chris; Leslie, Christina S; Marks, Debora S

2009-06-01

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

Regulation of B7.1 costimulatory molecule is mediated by the IFN regulatory factor-7 through the activation of JNK in lipopolysaccharide-stimulated human monocytic cells.

PubMed

Lim, Wilfred; Gee, Katrina; Mishra, Sasmita; Kumar, Ashok

2005-11-01

The engagement of CD28 or CTLA-4 with B7.1 provides the essential second costimulatory signal that regulates the development of immune responses, including T cell activation, differentiation, and induction of peripheral tolerance. The signaling molecules and the transcription factors involved in B7.1 regulation are poorly understood. In this study we investigated the role of MAPKs in the regulation of LPS-induced B7.1 expression in human monocytes and the promonocytic THP-1 cells. Our results show that LPS-induced B7.1 expression in monocytic cells did not involve the activation of either p38 or ERKs. Using the JNK-specific inhibitor SP600125, small interfering RNAs specific for JNK1 and JNK2, and agents such as dexamethasone that inhibit JNK activation, we determined that LPS-induced B7.1 expression was regulated by JNK MAPK in both monocytes and THP-1 cells. In addition, we identified a distinct B7.1-responsive element corresponding to the IFN regulatory factor-7 (IRF-7) binding site in the B7.1 promoter responsible for the regulation of LPS-induced B7.1 transcription. Furthermore, SP600125 and dexamethasone inhibited LPS-induced IRF-7 activity. Taken together, these results suggest that LPS-induced B7.1 transcription in human monocytic cells may be regulated by JNK-mediated activation of the IRF-7 transcription factor.

Nicotine promotes vascular endothelial growth factor secretion by human trophoblast cells under hypoxic conditions and improves the proliferation and tube formation capacity of human umbilical endothelial cells.

PubMed

Zhao, Hongbo; Wu, Lanxiang; Wang, Yahui; Zhou, Jiayi; Li, Ruixia; Zhou, Jiabing; Wang, Zehua; Xu, Congjian

2017-04-01

Pre-eclampsia, characterized as defective uteroplacental vascularization, remains the major cause of maternal and fetal mortality and morbidity. Previous epidemiological studies demonstrated that cigarette smoking reduced the risk of pre-eclampsia. However, the molecular mechanism remains elusive. In the present study, it is demonstrated that a low dose of nicotine decreased soluble vascular endothelial growth factor receptor 1 (sFlt1) secretion in human trophoblast cells under hypoxic conditions. Nicotine was then observed to promote vascular endothelial growth factor (VEGF) secretion by reducing sFlt1 secretion and increasing VEGF mRNA transcription. Further data showed that nicotine enhanced hypoxia-mediated hypoxia-inducible factor-1α (HIF-1α) expression and HIF-1α small interfering RNA abrogated nicotine-induced VEGF secretion, indicating that HIF-1α may be responsible for nicotine-mediated VEGF transcription under hypoxic conditions. Moreover, conditioned medium from human trophoblast cells treated with nicotine under hypoxic conditions promoted the proliferation and tube formation capacity of human umbilical endothelial cells (HUVEC) by promoting VEGF secretion. These findings indicate that nicotine may promote VEGF secretion in human trophoblast cells under hypoxic conditions by reducing sFlt1 secretion and up-regulating VEGF transcription and improve the proliferation and tube formation of HUVEC cells, which may contribute to elucidate the protective effect of cigarette smoking against pre-eclampsia. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

The TAM-family receptor Mer mediates production of HGF through the RhoA-dependent pathway in response to apoptotic cells.

PubMed

Park, Hyun-Jung; Baen, Ji-Yeon; Lee, Ye-Ji; Choi, Youn-Hee; Kang, Jihee Lee

2012-08-01

The TAM receptor protein tyrosine kinases Tyro3, Axl, and Mer play important roles in macrophage function. We investigated the roles of the TAM receptors in mediating the induction of hepatocyte growth factor (HGF) during the interaction of macrophages with apoptotic cells. Mer-specific neutralizing antibody, small interfering RNA (siRNA), and a recombinant Mer protein (Mer/Fc) inhibited HGF mRNA and protein expression, as well as activation of RhoA, Akt, and specific mitogen-activated protein (MAP) kinases in response to apoptotic cells. Inhibition of Axl or Tyro3 with specific antibodies, siRNA, or Fc-fusion proteins did not prevent apoptotic cell-induced HGF mRNA and protein expression and did not inhibit activation of the postreceptor signaling molecules RhoA and certain MAP kinases, including extracellular signal-regulated protein kinase and c-Jun NH(2)-terminal kinase. However, Axl- and Tyro3-specific blockers did inhibit the activation of Akt and p38 MAP kinase in response to apoptotic cells. In addition, none of the TAM receptors mediated the effects of apoptotic cells on transforming growth factor-β or epidermal growth factor mRNA expression. However, they were involved in the induction of vascular endothelial growth factor mRNA expression. Our data provide evidence that when macrophages interact with apoptotic cells, only Mer of the TAM-family receptors is responsible for mediating transcriptional HGF production through a RhoA-dependent pathway.

Impact of Implantable Transvenous Device Lead Location on Severity of Tricuspid Regurgitation

PubMed Central

Addetia, Karima; Maffessanti, Francesco; Mediratta, Anuj; Yamat, Megan; Weinert, Lynn; Moss, Joshua D.; Nayak, Hemal M.; Burke, Martin C.; Patel, Amit R.; Kruse, Eric; Jeevanandam, Valluvan; Mor-Avi, Victor; Lang, Roberto M.

2015-01-01

Background Implantable device leads can cause tricuspid regurgitation (TR) when they interfere with leaflet motion. The aim of this study was to determine whether lead-leaflet interference is associated with TR severity, independent of other causative factors of functional TR. Methods A total of 100 patients who underwent transthoracic two-dimensional and three-dimensional (3D) echocardiography of the tricuspid valve before and after lead placement were studied. Lead position was classified on 3D echocardiography as leaflet-interfering or noninterfering. TR severity was estimated by vena contracta (VC) width. Logistic regression analysis was used to identify factors associated with postdevice TR, including predevice VC width, right ventricular end-diastolic and end-systolic areas, fractional area change, right atrial size, tricuspid annular diameter, TR gradient, device lead age, and presence or absence of lead interference. Odds ratios were used to describe the association with moderate (VC width ≥ 0.5 cm) or severe (VC width ≥ 0.7 cm) TR, separately, using bivariate and stepwise multivariate logistic regression analysis. Results Forty-five of 100 patients showed device lead tricuspid valve leaflet interference. The septal leaflet was the most commonly affected (23 patients). On bivariate analysis, preimplantation VC width, right atrial size, tricuspid annular diameter, and lead-leaflet interference were significantly associated with postdevice TR. On multivariate analysis, preimplantation VC width and the presence of an interfering lead were independently associated with postdevice TR. Furthermore, the presence of an interfering lead was the only factor associated with TR worsening, increasing the likelihood of developing moderate or severe TR by 15- and 11-fold, respectively. Conclusion Lead-leaflet interference as seen on 3D echocardiography is associated with TR after device lead placement, suggesting that 3D echocardiography should be used to assess for lead interference in patients with significant TR. PMID:25129393

[The fragmentation of representational space in schizophrenia].

PubMed

Plagnol, A; Oïta, M; Montreuil, M; Granger, B; Lubart, T

2003-01-01

Existent neurocognitive models of schizophrenia converge towards a core of impairments involving working memory, context processing, action planning, controlled and intentional processing. However, the emergence of this core remains itself difficult to explain and more specific hypotheses do not explain the heterogeneity of schizophrenia. To overcome these limits, we propose a new paradigm based on representational theory from cognitive science. Some recent developments of this theory enable us to describe a subjective universe as a representational space which is displayed from memory. We outline a conceptual framework to construct such a representational space from analogical -representations that can be activated in working memory and are connected to a network of symbolic structures. These connections are notably made through an analytic process of the analogical fragments, which involves the attentional focus. This framework allows us to define rigorously some defense processes in response to traumatic tensions that are expressed on the representational space. The fragmentation of representational space is a consequence of a defensive denial based on an impairment of the analytic process. The fragmentation forms some parasitic areas in memory which are excluded from the main part of the representational space and disturb information processing. The key clinical concepts of paranoid syndromes can be defined in this conceptual framework: mental automatism, delusional intuition, acute destructuration, psychotic dissociation, and autistic withdrawal. We show that these syndromes imply each other, which in return increases the fragmentation of the representational space. Some new concepts emerge naturally in this framework, such as the concept of "suture" which is defined as a link between a parasitic area and the main representational space. Schizophrenia appears as a borderline case of fragmentation of the representational space. This conceptual framework is compatible with numerous etiological factors. Multiple clinical forms can be differentiated in accordance with the persistence of parasitic areas, the degree of fragmentation, and the formation of sutures. We use this approach to account for an empirical study concerning the analysis of analogical representations in schizophrenia. We used the Parallel Visual Information Processing Test (PVIPT) which assesses the analysis of interfering visual information. Subjects were asked to connect several small geometric figures printed on a transparency. The transparency was displayed above four photographs which were the interfering material. Then, subjects completed three tasks concerning the photographs: a recognition task, a recall task, and an affective qualification task. Using a case-by-case study, this test allows us to access the defense processes of the subjects, which is not possible with the usual methods in cognitive psychopathology. Twelve clinically-stable schizophrenic subjects participated in the study which also included a self-assessment of alexithymia by the Toronto Alexithymia Scale. We obtained 2 main results: (a) creation of items in recall or false recognition by 8 subjects, and (b) lack of the usual -negative correlations between the alexithymia score and the recall, recognition and affective qualification scores in the PVIPT. These 2 results contrast with what has been previously observed for alexithymia using the same methodology. The result (a) confirms an interfering activation in schizophrenic memory, which can be interpreted in our framework as indicative of parasitic areas. The creation of items suggests the formation of sutures between the semantic content of photographs and some delusional fragments. The result (b) suggests that the apparent alexithymia in schizophrenia is a defense against interfering activation in parasitic areas. We underline the interest of individual protocols to exhibit the dynamic interplay between an interfering activity in memory and a defensive flattening of affects.

Interfering Effect of Black Tea Consumption on Diagnosis of Pancreatic Cancer by CA 19-9.

PubMed

Al-Janabi, Ali Abdul Hussein S; Tawfeeq, Ekhlas F

2017-06-01

The study aims to determine the possible effects of black tea consumption on the level of CA 19-9 antigen in the human body. The level of CA 19-9 was measured in 270 healthy individuals who consumed heavy amounts of black tea. About 43.3 % of involved individuals were revealed to have elevated levels of CA 19-9. Males with high values of CA 19-9 represented the greatest number of involved individuals. The cutoff value of high levels of CA 19-9 in all individuals was ranged 69-105 U/ml. Consuming heavy amounts of black tea could be considered an important interfering factor that affects the levels of CA 19-9. The cutoff or predictive value of CA 19-9 in heavy-consuming people of black tea was determined.

Argonaute: The executor of small RNA function.

PubMed

Azlan, Azali; Dzaki, Najat; Azzam, Ghows

2016-08-20

The discovery of small non-coding RNAs - microRNA (miRNA), short interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) - represents one of the most exciting frontiers in biology specifically on the mechanism of gene regulation. In order to execute their functions, these small RNAs require physical interactions with their protein partners, the Argonaute (AGO) family proteins. Over the years, numerous studies have made tremendous progress on understanding the roles of AGO in gene silencing in various organisms. In this review, we summarize recent progress of AGO-mediated gene silencing and other cellular processes in which AGO proteins have been implicated with a particular focus on progress made in flies, humans and other model organisms as compliment. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

POZ domain transcription factor, FBI-1, represses transcription of ADH5/FDH by interacting with the zinc finger and interfering with DNA binding activity of Sp1.

PubMed

Lee, Dong-Kee; Suh, Dongchul; Edenberg, Howard J; Hur, Man-Wook

2002-07-26

The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

Plant Atrium System for Food Production in NASA's Deep Space Habitat Tests

NASA Technical Reports Server (NTRS)

Massa, Gioia D.; Simpson, Morgan; Wheeler, Raymond M.; Newsham, Gary; Stutte, Gary W.

2013-01-01

Future human space exploration missions will need functional habitat systems. Possible concepts are assessed for integration issues, power requirements, crew operations, technology, and system performance. A food production system concept was analyzed at NASA Desert Research and Technology Studies (DRATS) in 2011, and at NASA JSC in 2012. System utilizes fresh foods (vegetables and small fruits) which are harvested on a continuous basis. Designed to improve crew's diet and quality of life without interfering with other components or operations.

The Contribution of Prohibitin 1 to Prostate Cancer Chemoresistance

DTIC Science & Technology

2015-10-01

ceramide-PEG5K in normal BALB/c mice after i.v. injection. The siRNA was labeled with near infrared (NIR) dye DY647. (B) Ex vivo fluorescence image of...ceramide-PEG5K (Right) siRNA NPs at 18 h. The siRNA was labeled with dye DY547. (Scale bar, 10 μm.) Zhu et al. PNAS | June 23, 2015 | vol. 112 | no...Woodrow KA, et al. (2009) Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA. Nat Mater

Impact of epidermal growth factor receptor gene expression level on clinical outcomes in epidermal growth factor receptor mutant lung adenocarcinoma patients taking first-line epidermal growth factor receptor-tyrosine kinase inhibitors.

PubMed

Chang, Huang-Chih; Chen, Yu-Mu; Tseng, Chia-Cheng; Huang, Kuo-Tung; Wang, Chin-Chou; Chen, Yung-Che; Lai, Chien-Hao; Fang, Wen-Feng; Kao, Hsu-Ching; Lin, Meng-Chih

2017-03-01

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are first-choice treatments for advanced non-small-cell lung cancer patients harboring EGFR mutations. Although EGFR mutations are strongly predictive of patients' outcomes and their response to treatment with EGFR-TKIs, early failure of first-line therapy with EGFR-TKIs in patients with EGFR mutations is not rare. Besides several clinical factors influencing EGFR-TKI efficacies studied earlier such as the Eastern Cooperative Oncology Group performance status or uncommon mutation, we would like to see whether semi-quantify EGFR mutation gene expression calculated by 2 -ΔΔct was a prognostic factor in EGFR-mutant non-small cell lung cancer patients receiving first-line EGFR-TKIs. This retrospective study reviews 926 lung cancer patients diagnosed from January 2011 to October 2013 at the Kaohsiung Chang Gung Memorial Hospital in Taiwan. Of 224 EGFR-mutant adenocarcinoma patients, 148 patients who had 2 -ΔΔct data were included. The best cutoff values of 2 -ΔΔct for in-frame deletions in exon 19 (19 deletion) and a position 858 substituted from leucine (L) to an arginine (R) in exon 21 (L858R) were determined using receiver operating characteristic curves. Patients were divided into high and low 2 -ΔΔct expression based on the above cutoff level. The best cutoff point of 2 -ΔΔct value of 19 deletion and L858R was 31.1 and 104.7, respectively. In all, 92 (62.1%) patients showed high 2 -ΔΔct expression and 56 patients (37.9%) low 2 -ΔΔct expression. The mean age was 65.6 years. Progression-free survival of 19 deletion mutant patients with low versus high expression level was 17.07 versus 12.04 months (P = 0.004), respectively. Progression-free survival of L858 mutant patients was 13.75 and 7.96 months (P = 0.008), respectively. EGFR-mutant lung adenocarcinoma patients with lower EGFR gene expression had longer progression-free survival duration without interfering overall survival.

Selection of peptides interfering with protein-protein interaction.

PubMed

Gaida, Annette; Hagemann, Urs B; Mattay, Dinah; Räuber, Christina; Müller, Kristian M; Arndt, Katja M

2009-01-01

Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing suitable selection techniques and ending with purification for further down-stream characterization. Such generated peptides will function as versatile tools to interfere with the natural function of their targets thereby illuminating their down-stream signaling and, in general, promoting understanding of factors leading to specificity and stability in protein-protein interactions. Furthermore, peptides interfering with medically relevant proteins might become important diagnostics and therapeutics.

HIF-2α-ILK Is Involved in Mesenchymal Stromal Cell Angiogenesis in Multiple Myeloma Under Hypoxic Conditions

PubMed Central

Zhang, Xiaoying; Xu, Yinhui; Liu, Hongbo; Zhao, Pan; Chen, Yafang; Yue, Zhijie; Zhang, Zhiqing; Wang, Xiaofang

2018-01-01

Mesenchymal stromal cells are proven to be likely induce the angiogenic response in multiple myeloma and thus represent an enticing target for antiangiogenesis therapies for multiple myeloma. Substantial evidence indicates that angiogenesis in multiple myeloma is complex and involves direct production of angiogenic cytokines by abnormal plasma cells and these B-cell neoplasia generated pathophysiology change within the microenvironment. In this study, we demonstrated that mesenchymal stromal cells cultured with U266/Lp-1 under hypoxic conditions resulted in an increased α-smooth muscle actin expression and high productive levels of both hypoxia-inducible factor-2α and integrin-linked kinase proteins. Moreover, inhibition of hypoxia-inducible factor-2α by Small interfering RNA (siRNA) in mesenchymal stromal cells decreased the protein levels of both α-smooth muscle actin and integrin-linked kinase after mesenchymal stromal cells cultured with U266 under hypoxic conditions. We further demonstrated that transfection of integrin-linked kinase-siRNA reduced the protein level of α-smooth muscle actin and attenuated angiogenesis in vitro by decreasing the attachment of Q-dot labeled cells and secretion of angiogenic factors. In conclusion, our research showed that mesenchymal stromal cells cultured with myeloma cells under hypoxia participated in the angiogenesis of multiple myeloma, which is regulated by the hypoxia-inducible factor-2α-integrin-linked kinase pathway. Thus, targeting integrin-linked kinase may represent an effective strategy to block hypoxia-inducible factor-2α-induced angiogenesis in the treatment of multiple myeloma. PMID:29656700

The gene expression program of prostate fibroblast senescence modulates neoplastic epithelial cell proliferation through paracrine mechanisms.

PubMed

Bavik, Claes; Coleman, Ilsa; Dean, James P; Knudsen, Beatrice; Plymate, Steven; Nelson, Peter S

2006-01-15

The greatest risk factor for developing carcinoma of the prostate is advanced age. Potential molecular and physiologic contributors to the frequency of cancer occurrence in older individuals include the accumulation of somatic mutations through defects in genome maintenance, epigenetic gene silencing, oxidative stress, loss of immune surveillance, telomere dysfunction, chronic inflammation, and alterations in tissue microenvironment. In this context, the process of prostate carcinogenesis can be influenced through interactions between intrinsic cellular alterations and the extrinsic microenvironment and macroenvironment, both of which change substantially as a consequence of aging. In this study, we sought to characterize the molecular alterations that occur during the process of prostate fibroblast senescence to identify factors in the aged tissue microenvironment capable of promoting the proliferation and potentially the neoplastic progression of prostate epithelium. We evaluated three mechanisms leading to cell senescence: oxidative stress, DNA damage, and replicative exhaustion. We identified a consistent program of gene expression that includes a subset of paracrine factors capable of influencing adjacent prostate epithelial growth. Both direct coculture and conditioned medium from senescent prostate fibroblasts stimulated epithelial cell proliferation, 3-fold and 2-fold, respectively. The paracrine-acting proteins fibroblast growth factor 7, hepatocyte growth factor, and amphiregulin (AREG) were elevated in the extracellular environment of senescent prostate fibroblasts. Exogenous AREG alone stimulated prostate epithelial cell growth, and neutralizing antibodies and small interfering RNA targeting AREG attenuated, but did not completely abrogate the growth-promoting effects of senescent fibroblast conditioned medium. These results support the concept that aging-related changes in the prostate microenvironment may contribute to the progression of prostate neoplasia.

Impact of wastewater derived dissolved interfering compounds on growth, enzymatic activity and trace organic contaminant removal of white rot fungi - A critical review.

PubMed

Asif, Muhammad B; Hai, Faisal I; Hou, Jingwei; Price, William E; Nghiem, Long D

2017-10-01

White-rot fungi (WRF) and their ligninolytic enzymes have been investigated for the removal of a broad spectrum of trace organic contaminants (TrOCs) mostly from synthetic wastewater in lab-scale experiments. Only a few studies have reported the efficiency of such systems for the removal of TrOCs from real wastewater. Wastewater derived organic and inorganic compounds can inhibit: (i) WRF growth and their enzyme production capacity; (ii) enzymatic activity of ligninolytic enzymes; and (iii) catalytic efficiency of both WRF and enzymes. It is observed that essential metals such as Cu, Mn and Co at trace concertation (up to 1 mM) can improve the growth of WRF species, whereas non-essential metal such as Pb, Cd and Hg at 1 mM concentration can inhibit WRF growth and their enzyme production. In the case of purified enzymes, most of the tested metals at 1-5 mM concentration do not significantly inhibit the activity of laccases. Organic interfering compounds such as oxalic acid and ethylenediaminetetraacetic acid (EDTA) at 1 mM concentration are potent inhibitors of WRF and their extracellular enzymes. However, inhibitory effects induced by interfering compounds are strongly influenced by the type of WRF species as well as experimental conditions (e.g., incubation time and TrOC type). In this review, mechanisms and factors governing the interactions of interfering compounds with WRF and their ligninolytic enzymes are reviewed and elucidated. In addition, the performance of WRF and their ligninolytic enzymes for the removal of TrOCs from synthetic and real wastewater is critically summarized. Copyright © 2017 Elsevier Ltd. All rights reserved.

US Army Software Symposium (2nd) Held at Williamsburg, Virginia on 25-27 October 1978.

DTIC Science & Technology

1978-01-01

Requirement6 Generation - An Evat~uation 55 Carl G. Davis iii TABLE OF CONTENTS Cont’d TITLE PAGE An Approach to Requitementb De ~in tion6 For ReaL Tij e...Interf ace Concept. 3. Essential to architectural development is the inclusion of certain operational design criteria, the most important of which is...such systems by the Army makes a review of human factors considerations in de - velopment of these systems highly desirable. Human Factors

Selenium deficiency aggravates T-2 toxin-induced injury of primary neonatal rat cardiomyocytes through ER stress.

PubMed

Xu, Jing; Pan, Shengchi; Gan, Fang; Hao, Shu; Liu, Dandan; Xu, Haibin; Huang, Kehe

2018-04-01

Keshan disease is a potentially fatal cardiomyopathy in humans. Selenium deficiency, T-2 toxin, and myocarditis virus are thought to be the major factors contributing to Keshan disease. But the relationship among these three factors is poorly described. This study aims to explore whether selenium deficiency aggravates T-2 toxin-induced cardiomyocyte injury and its underlying mechanism. Cardiomyocytes were isolated from neonatal rat and cultured at the physiological (2.0 μM) or lower concentrations of selenium with different concentrations of T-2 toxin. Our results showed that selenium deficiencies aggravated T-2 toxin-induced cardiomyocyte injury in a concentration-dependent manner as demonstrated by MTT bioassay, LDH activity, reactive oxygen species levels and caspase 3 protein expressions. T-2 toxin treatment significantly increased mRNA expressions for stress proteins GRP78 and CHOP in cardiomyocytes compared with the control. Selenium deficiencies further promoted GRP78, CHOP and p-eIF2α expressions. Knockdown of CHOP by the specific small interfering RNA eliminated the effect of selenium deficiencies on T-2 toxin-induced injury. It could be concluded that selenium deficiency aggravates T-2 toxin-induced cardiomyocyte injury through initiating more aggressive endoplasmic reticulum stress. Copyright © 2018 Elsevier B.V. All rights reserved.

Modulating the tumor microenvironment with RNA interference as a cancer treatment strategy.

PubMed

Zins, Karin; Sioud, Mouldy; Aharinejad, Seyedhossein; Lucas, Trevor; Abraham, Dietmar

2015-01-01

The tumor microenvironment is composed of accessory cells and immune cells in addition to extracellular matrix (ECM) components. The stromal compartment interacts with cancer cells in a complex crosstalk to support tumor development. Growth factors and cytokines produced by stromal cells support the growth of tumor cells and promote interaction with the vasculature to enhance tumor progression and invasion. The activation of autocrine and paracrine oncogenic signaling pathways by growth factors, cytokines, and proteases derived from both tumor cells and the stromal compartment is thought to play a major role in assisting tumor cells during metastasis. Consequently, targeting tumor-stroma interactions by RNA interference (RNAi)-based approaches is a promising strategy in the search for novel treatment modalities in human cancer. Recent advances in packaging technology including the use of polymers, peptides, liposomes, and nanoparticles to deliver small interfering RNAs (siRNAs) into target cells may overcome limitations associated with potential RNAi-based therapeutics. Newly developed nonviral gene delivery approaches have shown improved anticancer efficacy suggesting that RNAi-based therapeutics provide novel opportunities to elicit significant gene silencing and induce regression of tumor growth. This chapter summarizes our current understanding of the tumor microenvironment and highlights some potential targets for therapeutic intervention with RNAi-based cancer therapeutics.

Microtubule actin crosslinking factor 1b: a novel plakin that localizes to the Golgi complex.

PubMed

Lin, Chung-Ming; Chen, Hui-Jye; Leung, Conrad L; Parry, David A D; Liem, Ronald K H

2005-08-15

MACF1 (microtubule actin crosslinking factor), also called ACF7 (actin crosslinking family 7) is a cytoskeletal linker protein that can associate with both actin filaments and microtubules. We have identified a novel alternatively spliced isoform of MACF1. We named this isoform MACF1b and renamed the original isoform MACF1a. MACF1b is identical to MACF1a, except that it has a region containing plakin (or plectin) repeats in the middle of the molecule. MACF1b is ubiquitously expressed in adult tissues with especially high levels in the lung. We studied the subcellular localization of MACF1b proteins in mammalian cell lines. In two lung cell lines, MACF1b was chiefly localized to the Golgi complex. Upon treatments that disrupt the Golgi complex, MACF1b redistributed into the cytosol, but remained co-localized with the dispersed Golgi ministacks. MACF1b proteins can be detected in the enriched Golgi fraction by western blotting. The domain of MACF1b that targets it to the Golgi was found at the N-terminal part of the region that contains the plakin repeats. Reducing the level of MACF1 proteins by small-interfering RNA resulted in the dispersal of the Golgi complex.

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a host factor required for dengue virus and Junín virus multiplication.

PubMed

Brunetti, Jesús E; Scolaro, Luis A; Castilla, Viviana

2015-05-04

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are cellular factors involved in the replication of several viruses. In this study we analyzed the expression and intracellular localization of hnRNP A2 and hnRNP K in cell cultures infected with two viruses that cause human hemorrhagic fevers: dengue virus type 2 (DENV-2) and Junín virus (JUNV). We determined that DENV-2 promoted the cytoplasmic translocation of hnRNP K and to a lesser extent of hnRNP A2, meanwhile, JUNV infection induced an increase in hnRNP K cytoplasmic localization whereas hnRNP A2 remained mainly in the nucleus of infected cells. Both hnRNP K and hnRNP A2 were localized predominantly in the nucleus of JUNV persistently-infected cells even after superinfection with JUNV indicating that persistent infection does not alter nucleo-cytoplasmic transport of these hnRNPs. Total levels of hnRNP K expression were unaffected by DENV-2 or JUNV infection. In addition we determined, using small interfering RNAs, that hnRNP K knockout inhibits DENV-2 and JUNV multiplication. Our results indicate that DENV-2 and JUNV induce hnRNP K cytoplasmic translocation to favor viral multiplication. Copyright © 2015 Elsevier B.V. All rights reserved.

LBH589, a deacetylase inhibitor, induces apoptosis in adult T-cell leukemia/lymphoma cells via activation of a novel RAIDD-caspase-2 pathway

PubMed Central

Hasegawa, H; Yamada, Y; Tsukasaki, K; Mori, N; Tsuruda, K; Sasaki, D; Usui, T; Osaka, A; Atogami, S; Ishikawa, C; Machijima, Y; Sawada, S; Hayashi, T; Miyazaki, Y; Kamihira, S

2011-01-01

Adult T-cell leukemia/lymphoma (ATLL), an aggressive neoplasm etiologically associated with human T-lymphotropic virus type-1 (HTLV-1), is resistant to treatment. In this study, we examined the effects of a new inhibitor of deacetylase enzymes, LBH589, on ATLL cells. LBH589 effectively induced apoptosis in ATLL-related cell lines and primary ATLL cells and reduced the size of tumors inoculated in SCID mice. Analyses, including with a DNA microarray, revealed that neither death receptors nor p53 pathways contributed to the apoptosis. Instead, LBH589 activated an intrinsic pathway through the activation of caspase-2. Furthermore, small interfering RNA experiments targeting caspase-2, caspase-9, RAIDD, p53-induced protein with a death domain (PIDD) and RIPK1 (RIP) indicated that activation of RAIDD is crucial and an event initiating this pathway. In addition, LBH589 caused a marked decrease in levels of factors involved in ATLL cell proliferation and invasion such as CCR4, IL-2R and HTLV-1 HBZ-SI, a spliced form of the HTLV-1 basic zipper factor HBZ. In conclusion, we showed that LBH589 is a strong inducer of apoptosis in ATLL cells and uncovered a novel apoptotic pathway initiated by activation of RAIDD. PMID:21242994

Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3.

PubMed

Yoneda-Kato, Noriko; Tomoda, Kiichiro; Umehara, Mari; Arata, Yukinobu; Kato, Jun-ya

2005-05-04

Myeloid leukemia factor 1 (MLF1) was first identified as the leukemic fusion protein NPM-MLF1 generated by the t(3;5)(q25.1;q34) chromosomal translocation. Although MLF1 expresses normally in a variety of tissues including hematopoietic stem cells and the overexpression of MLF1 correlates with malignant transformation in human cancer, little is known about how MLF1 is involved in the regulation of cell growth. Here we show that MLF1 is a negative regulator of cell cycle progression functioning upstream of the tumor suppressor p53. MLF1 induces p53-dependent cell cycle arrest in murine embryonic fibroblasts. This action requires a novel binding partner, subunit 3 of the COP9 signalosome (CSN3). A reduction in the level of CSN3 protein with small interfering RNA abrogated MLF1-induced G1 arrest and impaired the activation of p53 by genotoxic stress. Furthermore, ectopic MLF1 expression and CSN3 knockdown inversely affect the endogenous level of COP1, a ubiquitin ligase for p53. Exogenous expression of COP1 overcomes MLF1-induced growth arrest. These results indicate that MLF1 is a critical regulator of p53 and suggest its involvement in leukemogenesis through a novel CSN3-COP1 pathway.

Notch signaling sustains the expression of Mcl-1 and the activity of eIF4E to promote cell survival in CLL

PubMed Central

De Falco, Filomena; Sabatini, Rita; Del Papa, Beatrice; Falzetti, Franca; Di Ianni, Mauro; Sportoletti, Paolo; Baldoni, Stefano; Screpanti, Isabella; Marconi, Pierfrancesco; Rosati, Emanuela

2015-01-01

In chronic lymphocytic leukemia (CLL), Notch1 and Notch2 signaling is constitutively activated and contributes to apoptosis resistance. We show that genetic inhibition of either Notch1 or Notch2, through small-interfering RNA, increases apoptosis of CLL cells and is associated with decreased levels of the anti-apoptotic protein Mcl-1. Thus, Notch signaling promotes CLL cell survival at least in part by sustaining Mcl-1 expression. In CLL cells, an enhanced Notch activation also contributes to the increase in Mcl-1 expression and cell survival induced by IL-4. Mcl-1 downregulation by Notch targeting is not due to reduced transcription or degradation by caspases, but in part, to increased degradation by the proteasome. Mcl-1 downregulation by Notch targeting is also accompanied by reduced phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), suggesting that this protein is another target of Notch signaling in CLL cells. Overall, we show that Notch signaling sustains CLL cell survival by promoting Mcl-1 expression and eIF4E activity, and given the oncogenic role of these factors, we underscore the therapeutic potential of Notch inhibition in CLL. PMID:26041884

HOXA9 Methylation by PRMT5 Is Essential for Endothelial Cell Expression of Leukocyte Adhesion Molecules

PubMed Central

Bandyopadhyay, Smarajit; Harris, Daniel P.; Adams, Gregory N.; Lause, Gregory E.; McHugh, Anne; Tillmaand, Emily G.; Money, Angela; Willard, Belinda; Fox, Paul L.

2012-01-01

The induction of proinflammatory proteins in stimulated endothelial cells (EC) requires activation of multiple transcription programs. The homeobox transcription factor HOXA9 has an important regulatory role in cytokine induction of the EC-leukocyte adhesion molecules (ELAM) E-selectin and vascular cell adhesion molecule 1 (VCAM-1). However, the mechanism underlying stimulus-dependent activation of HOXA9 is completely unknown. Here, we elucidate the molecular mechanism of HOXA9 activation by tumor necrosis factor alpha (TNF-α) and show an unexpected requirement for arginine methylation by protein arginine methyltransferase 5 (PRMT5). PRMT5 was identified as a TNF-α-dependent binding partner of HOXA9 by mass spectrometry. Small interfering RNA (siRNA)-mediated depletion of PRMT5 abrogated stimulus-dependent HOXA9 methylation with concomitant loss in E-selectin or VCAM-1 induction. Chromatin immunoprecipitation analysis revealed that PRMT5 is recruited to the E-selectin promoter following transient HOXA9 binding to its cognate recognition sequence. PRMT5 induces symmetric dimethylation of Arg140 on HOXA9, an event essential for E-selectin induction. In summary, PRMT5 is a critical coactivator component in a newly defined, HOXA9-containing transcription complex. Moreover, stimulus-dependent methylation of HOXA9 is essential for ELAM expression during the EC inflammatory response. PMID:22269951

Novel and general approach to linear filter design for contrast-to-noise ratio enhancement of magnetic resonance images with multiple interfering features in the scene

NASA Astrophysics Data System (ADS)

Soltanian-Zadeh, Hamid; Windham, Joe P.

1992-04-01

Maximizing the minimum absolute contrast-to-noise ratios (CNRs) between a desired feature and multiple interfering processes, by linear combination of images in a magnetic resonance imaging (MRI) scene sequence, is attractive for MRI analysis and interpretation. A general formulation of the problem is presented, along with a novel solution utilizing the simple and numerically stable method of Gram-Schmidt orthogonalization. We derive explicit solutions for the case of two interfering features first, then for three interfering features, and, finally, using a typical example, for an arbitrary number of interfering feature. For the case of two interfering features, we also provide simplified analytical expressions for the signal-to-noise ratios (SNRs) and CNRs of the filtered images. The technique is demonstrated through its applications to simulated and acquired MRI scene sequences of a human brain with a cerebral infarction. For these applications, a 50 to 100% improvement for the smallest absolute CNR is obtained.

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

Ma, Xusheng, E-mail: maxushengtt@163.com; Yang, Xing; Nian, Xiaofeng

Peste des petits ruminants virus (PPRV) causes a fatal disease in small ruminants. V protein of PPRV plays a pivotal role in interfering with host innate immunity by blocking IFNs signaling through interacting with STAT1 and STAT2. In the present study, the results demonstrated that PPRV V protein blocks IFN actions in a dose dependent manner and restrains the translocation of STAT1/2 proteins. We speculate that the translocation inhibition might be caused by the interfering of the downstream of STAT protein. Mutagenesis defines that Cys cluster and Trp motif of PPRV V protein are essential for STAT-mediated IFN signaling. Thesemore » findings give a new sight for the further studies to understand the delicate mechanism of PPRV to escape the IFN signaling. - Highlights: • PPRV V protein inhibits type I IFN production and blocks its activation. • PPRV V protein negatively regulates activation of ISRE and GAS promoter. • PPRV V protein inhibits nuclear translocation of STAT protein by non-degradation. • PNT and VCT domain of PPRV V protein inhibit IFN transduction. • PPRV V protein binds with STAT protein via some conserved motifs.« less

32 CFR 1903.8 - Interfering with Agency functions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 6 2011-07-01 2011-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are prohibited...

32 CFR 1903.8 - Interfering with Agency functions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 6 2010-07-01 2010-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are prohibited...

32 CFR 1903.8 - Interfering with Agency functions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 6 2012-07-01 2012-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are prohibited...

32 CFR 1903.8 - Interfering with Agency functions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 6 2014-07-01 2014-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are prohibited...

32 CFR 1903.8 - Interfering with Agency functions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 32 National Defense 6 2013-07-01 2013-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are prohibited...

Biodistribution of small interfering RNA at the organ and cellular levels after lipid nanoparticle-mediated delivery.

PubMed

Shi, Bin; Keough, Ed; Matter, Andrea; Leander, Karen; Young, Stephanie; Carlini, Ed; Sachs, Alan B; Tao, Weikang; Abrams, Marc; Howell, Bonnie; Sepp-Lorenzino, Laura

2011-08-01

Chemically stabilized small interfering RNA (siRNA) can be delivered systemically by intravenous injection of lipid nanoparticles (LNPs) in rodents and primates. The biodistribution and kinetics of LNP-siRNA delivery in mice at organ and cellular resolution have been studied using immunofluorescence (IF) staining and quantitative polymerase chain reaction (qPCR). At 0.5 and 2 hr post tail vein injection of Cy5-labeled siRNA encapsulated in LNP, the organ rank-order of siRNA levels is liver > spleen > kidney, with only negligible accumulation in duodenum, lung, heart, and brain. Similar conclusions were drawn by using qPCR to measure tissue siRNA levels as a secondary end point. siRNA levels in these tissues decreased by more than 10-fold after 24 hr. Within the liver, LNPs delivered siRNA to hepatocytes, Kupffer cells, and sinusoids in a time-dependent manner, as revealed by IF staining and signal quantitation methods established using OPERA/Columbus software. siRNA first accumulated in liver sinusoids and trafficked to hepatocytes by 2 hr post dose, corresponding to the onset of target mRNA silencing. Fluorescence in situ hybridization methods were used to detect both strands of siRNA in fixed tissues. Collectively, the authors have implemented a platform to evaluate biodistribution of siRNA across cell types and across tissues in vivo, with the objective of elucidating the pharmacokinetic and pharmacodynamic relationship to guide optimization of delivery vehicles. © The Author(s) 2011

Biodistribution of Small Interfering RNA at the Organ and Cellular Levels after Lipid Nanoparticle-mediated Delivery

PubMed Central

Shi, Bin; Keough, Ed; Matter, Andrea; Leander, Karen; Young, Stephanie; Carlini, Ed; Sachs, Alan B.; Tao, Weikang; Abrams, Marc; Howell, Bonnie; Sepp-Lorenzino, Laura

2011-01-01

Chemically stabilized small interfering RNA (siRNA) can be delivered systemically by intravenous injection of lipid nanoparticles (LNPs) in rodents and primates. The biodistribution and kinetics of LNP–siRNA delivery in mice at organ and cellular resolution have been studied using immunofluorescence (IF) staining and quantitative polymerase chain reaction (qPCR). At 0.5 and 2 hr post tail vein injection of Cy5-labeled siRNA encapsulated in LNP, the organ rank-order of siRNA levels is liver > spleen > kidney, with only negligible accumulation in duodenum, lung, heart, and brain. Similar conclusions were drawn by using qPCR to measure tissue siRNA levels as a secondary end point. siRNA levels in these tissues decreased by more than 10-fold after 24 hr. Within the liver, LNPs delivered siRNA to hepatocytes, Kupffer cells, and sinusoids in a time-dependent manner, as revealed by IF staining and signal quantitation methods established using OPERA/Columbus software. siRNA first accumulated in liver sinusoids and trafficked to hepatocytes by 2 hr post dose, corresponding to the onset of target mRNA silencing. Fluorescence in situ hybridization methods were used to detect both strands of siRNA in fixed tissues. Collectively, the authors have implemented a platform to evaluate biodistribution of siRNA across cell types and across tissues in vivo, with the objective of elucidating the pharmacokinetic and pharmacodynamic relationship to guide optimization of delivery vehicles. PMID:21804077

Small interfering RNA targeting focal adhesion kinase prevents cardiac dysfunction in endotoxemia.

PubMed

Guido, Maria C; Clemente, Carolina F; Moretti, Ana I; Barbeiro, Hermes V; Debbas, Victor; Caldini, Elia G; Franchini, Kleber G; Soriano, Francisco G

2012-01-01

Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.

Inhibition of MMP-2 gene expression with small interfering RNA in rabbit vascular smooth muscle cells.

PubMed

Hlawaty, Hanna; San Juan, Aurélie; Jacob, Marie-Paule; Vranckx, Roger; Letourneur, Didier; Feldman, Laurent J

2007-12-01

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs). Using small interfering RNA (siRNA), we evaluated the effect of MMP-2 inhibition in VSMCs in vitro and ex vivo. Rabbit VSMCs were transfected in vitro with 50 nmol/l MMP-2 siRNA or scramble siRNA. Flow cytometry and confocal microscopy showed cellular uptake of siRNA in approximately 80% of VSMCs. MMP-2 mRNA levels evaluated by real-time RT-PCR, pro-MMP-2 activity from conditioned culture media evaluated by gelatin zymography, and VSMC migration were reduced by 44 +/- 19%, 43 +/- 14%, and 36 +/- 14%, respectively, in MMP-2 siRNA-transfected compared with scramble siRNA-transfected VSMCs (P < 0.005 for all). Ex vivo MMP-2 siRNA transfection was performed 2 wk after balloon injury of hypercholesterolemic rabbit carotid arteries. Fluorescence microscopy showed circumferential siRNA uptake in neointimal cells. Gelatin zymography of carotid artery culture medium demonstrated a significant decrease of pro-MMP-2 activity in MMP-2 siRNA-transfected compared with scramble siRNA-transfected arteries (P < 0.01). Overall, our results demonstrate that in vitro MMP-2 siRNA transfection in VSMCs markedly inhibits MMP-2 gene expression and VSMC migration and that ex vivo delivery of MMP-2 siRNA in balloon-injured arteries reduces pro-MMP-2 activity in neointimal cells, suggesting that siRNA could be used to modify arterial biology in vivo.

BMPR2 inhibition induced apoptosis and autophagy via destabilization of XIAP in human chondrosarcoma cells

PubMed Central

Jiao, G; Guo, W; Ren, T; Lu, Q; Sun, Y; Liang, W; Ren, C; Yang, K; Sun, K

2014-01-01

Bone morphogenetic proteins (BMPs) are multifunctional proteins, and their receptors (BMPRs) have crucial roles in the process of signaling. However, their function in cancer is somewhat inconsistent. It has been demonstrated that more prevalent expression of bone morphogenetic protein receptor 2 (BMPR2) has been detected in dedifferentiated chondrosarcomas than conventional chondrosarcomas. Here, we find that BMPR2 inhibition induces apoptosis and autophagy of chondrosarcoma. We found that BMPR2 expression was correlated with the clinicopathological features of chondrosarcomas, and could predict the treatment outcome. Knockdown of BMPR2 by small interfering RNA results in growth inhibition in chondrosarcoma cells. Silencing BMPR2 promoted G2/M cell cycle arrest, induced chondrosarcoma cell apoptosis through caspase-3-dependent pathway via repression of X-linked inhibitor of apoptosis protein (XIAP) and induced autophagy of chondrosarcoma cells via XIAP-Mdm2-p53 pathway. Inhibition of autophagy induced by BMPR2 small interfering RNA (siBMPR2) sensitized chondrosarcoma cells to siBMPR2-induced apoptotic cell death, suggesting that autophagy has a protective role for chondrosarcoma cells in context of siBMPR2-induced apoptotic cell death. In vivo tumorigenicity assay in mice indicated that inhibition of BMPR2 reduced tumor growth. Taken together, our results suggest that BMPR2 has a significant role in the tumorigenesis of chondrosarcoma, and could be an important prognostic marker for chondrosarcoma. BMPR2 inhibition could eventually provide a promising therapy for chondrosarcoma treatment. PMID:25501832

Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families

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

Carbonell, Alberto; Martinez de Alba, Angel-Emilio; Flores, Ricardo

2008-02-05

Infection by viroids, non-protein-coding circular RNAs, occurs with the accumulation of 21-24 nt viroid-derived small RNAs (vd-sRNAs) with characteristic properties of small interfering RNAs (siRNAs) associated to RNA silencing. The vd-sRNAs most likely derive from dicer-like (DCL) enzymes acting on viroid-specific dsRNA, the key elicitor of RNA silencing, or on the highly structured genomic RNA. Previously, viral dsRNAs delivered mechanically or agroinoculated have been shown to interfere with virus infection in a sequence-specific manner. Here, we report similar results with members of the two families of nuclear- and chloroplast-replicating viroids. Moreover, homologous vd-sRNAs co-delivered mechanically also interfered with one ofmore » the viroids examined. The interference was sequence-specific, temperature-dependent and, in some cases, also dependent on the dose of the co-inoculated dsRNA or vd-sRNAs. The sequence-specific nature of these effects suggests the involvement of the RNA induced silencing complex (RISC), which provides sequence specificity to RNA silencing machinery. Therefore, viroid titer in natural infections might be regulated by the concerted action of DCL and RISC. Viroids could have evolved their secondary structure as a compromise between resistance to DCL and RISC, which act preferentially against RNAs with compact and relaxed secondary structures, respectively. In addition, compartmentation, association with proteins or active replication might also help viroids to elude their host RNA silencing machinery.« less

Peroxisome proliferator-activated receptor δ inhibits Porphyromonas gingivalis lipopolysaccharide-induced activation of matrix metalloproteinase-2 by downregulating NADPH oxidase 4 in human gingival fibroblasts.

PubMed

Yoo, T; Ham, S A; Hwang, J S; Lee, W J; Paek, K S; Oh, J W; Kim, J H; Do, J T; Han, C W; Kim, J H; Seo, H G

2016-10-01

We investigated the roles of peroxisome proliferator-activated receptor δ (PPARδ) in Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced activation of matrix metalloproteinase 2 (MMP-2). In human gingival fibroblasts (HGFs), activation of PPARδ by GW501516, a specific ligand of PPARδ, inhibited Pg-LPS-induced activation of MMP-2 and generation of reactive oxygen species (ROS), which was associated with reduced expression of NADPH oxidase 4 (Nox4). These effects were significantly smaller in the presence of small interfering RNA targeting PPARδ or the specific PPARδ inhibitor GSK0660, indicating that PPARδ is involved in these events. In addition, modulation of Nox4 expression by small interfering RNA influenced the effect of PPARδ on MMP-2 activity, suggesting a mechanism in which Nox4-derived ROS modulates MMP-2 activity. Furthermore, c-Jun N-terminal kinase and p38, but not extracellular signal-regulated kinase, mediated PPARδ-dependent inhibition of MMP-2 activity in HGFs treated with Pg-LPS. Concomitantly, PPARδ-mediated inhibition of MMP-2 activity was associated with the restoration of types I and III collagen to levels approaching those in HGFs not treated with Pg-LPS. These results indicate that PPARδ-mediated downregulation of Nox4 modulates cellular redox status, which in turn plays a critical role in extracellular matrix homeostasis through ROS-dependent regulation of MMP-2 activity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Anti-CD22 Antibody Targeting of pH-responsive Micelles Enhances Small Interfering RNA Delivery and Gene Silencing in Lymphoma Cells

PubMed Central

Palanca-Wessels, Maria C; Convertine, Anthony J; Cutler-Strom, Richelle; Booth, Garrett C; Lee, Fan; Berguig, Geoffrey Y; Stayton, Patrick S; Press, Oliver W

2011-01-01

The application of small interfering RNA (siRNA) for cancer treatment is a promising strategy currently being explored in early phase clinical trials. However, efficient systemic delivery limits clinical implementation. We developed and tested a novel delivery system comprised of (i) an internalizing streptavidin-conjugated monoclonal antibody (mAb-SA) directed against CD22 and (ii) a biotinylated diblock copolymer containing both a positively charged siRNA condensing block and a pH-responsive block to facilitate endosome release. The modular design of the carrier facilitates the exchange of different targeting moieties and siRNAs to permit its usage in a variety of tumor types. The polymer was synthesized using the reversible addition fragmentation chain transfer (RAFT) technique and formed micelles capable of binding siRNA and mAb-SA. A hemolysis assay confirmed the predicted membrane destabilizing activity of the polymer under acidic conditions typical of the endosomal compartment. Enhanced siRNA uptake was demonstrated in DoHH2 lymphoma and transduced HeLa-R cells expressing CD22 but not in CD22 negative HeLa-R cells. Gene knockdown was significantly improved with CD22-targeted vs. nontargeted polymeric micelles. Treatment of DoHH2 cells with CD22-targeted polymeric micelles containing 15 nmol/l siRNA produced 70% reduction of gene expression. This CD22-targeted polymer carrier may be useful for siRNA delivery to lymphoma cells. PMID:21629223

Anti-CD22 antibody targeting of pH-responsive micelles enhances small interfering RNA delivery and gene silencing in lymphoma cells.

PubMed

Palanca-Wessels, Maria C; Convertine, Anthony J; Cutler-Strom, Richelle; Booth, Garrett C; Lee, Fan; Berguig, Geoffrey Y; Stayton, Patrick S; Press, Oliver W

2011-08-01

The application of small interfering RNA (siRNA) for cancer treatment is a promising strategy currently being explored in early phase clinical trials. However, efficient systemic delivery limits clinical implementation. We developed and tested a novel delivery system comprised of (i) an internalizing streptavidin-conjugated monoclonal antibody (mAb-SA) directed against CD22 and (ii) a biotinylated diblock copolymer containing both a positively charged siRNA condensing block and a pH-responsive block to facilitate endosome release. The modular design of the carrier facilitates the exchange of different targeting moieties and siRNAs to permit its usage in a variety of tumor types. The polymer was synthesized using the reversible addition fragmentation chain transfer (RAFT) technique and formed micelles capable of binding siRNA and mAb-SA. A hemolysis assay confirmed the predicted membrane destabilizing activity of the polymer under acidic conditions typical of the endosomal compartment. Enhanced siRNA uptake was demonstrated in DoHH2 lymphoma and transduced HeLa-R cells expressing CD22 but not in CD22 negative HeLa-R cells. Gene knockdown was significantly improved with CD22-targeted vs. nontargeted polymeric micelles. Treatment of DoHH2 cells with CD22-targeted polymeric micelles containing 15 nmol/l siRNA produced 70% reduction of gene expression. This CD22-targeted polymer carrier may be useful for siRNA delivery to lymphoma cells.

Antifibrotic effects of Smad4 small interfering RNAs in injured skeletal muscle after acute contusion.

PubMed

Li, H; Chen, J; Chen, S; Zhang, Q; Chen, S

2011-10-01

Muscle injuries are common musculoskeletal problems encountered in sports medicine clinics. In this study, we examined the effect of lentivirus-mediated small interfering RNA (siRNA) targeting Smad4 on the suppression of the fibrosis in injured skeletal muscles. We found that Smad4-siRNA could efficiently knock down the expression of Smad4 in the C2C12 myoblast cells and in the contunded mice gastrocnemius muscle. The expression of mRNA level of Smad4 decreased to 11% and 49% compared to the control group, respectively, and the expression of protein level decreased to 13% and 57% respectively. Moreover, the lentivirus-mediated siRNA was stably transfected only into the skeletal muscle and not into the liver of the animals. In contunded mice gastrocnemius, the collagenous and vimentin-positive area in the Smad4 siRNA group reduced to 36% and 37% compared to the control group, respectively. Furthermore, compared to the scrambled Smad4 siRNA-injected mice and PBS control-injected mice, the muscle function of the mice injected with lentivirus-mediated Smad4 siRNA improved in terms of both fast-twitch and tetanic strength (P<0.05). The results suggest that the gene therapy of inhibiting Smad4 by lentivirus-mediated siRNA could be a useful approach to prevent scar tissue formation and improve the function of injured skeletal muscle. © Georg Thieme Verlag KG Stuttgart · New York.

Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation.

PubMed

Kubota, Kohei; Onishi, Kohei; Sawaki, Kazuaki; Li, Tianshu; Mitsuoka, Kaoru; Sato, Takaaki; Takeoka, Shinji

2017-01-01

Two lipid-based nanoformulations have been used to date in clinical studies: lipoplexes and lipid nanoparticles (LNPs). In this study, we prepared small interfering RNA (siRNA)-loaded carriers using lipid components of the same composition to form molecular assemblies of differing structures, and evaluated the impact of structure on cellular uptake and immune stimulation. Lipoplexes are electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic siRNA in an aqueous environment, whereas LNPs are nanoparticles embedding siRNA prepared by mixing an alcoholic lipid solution with an aqueous siRNA solution in one step. Although the physicochemical properties of lipoplexes and LNPs were similar except for small increases in apparent size of lipoplexes and zeta potential of LNPs, siRNA uptake efficiency of LNPs was significantly higher than that of lipoplexes. Furthermore, in the case of LNPs, both siRNA and lipid were effectively incorporated into cells in a co-assembled state; however, in the case of lipoplexes, the amount of siRNA internalized into cells was small in comparison with lipid. siRNAs in lipoplexes were thought to be more likely to localize on the particle surface and thereby undergo dissociation into the medium. Inflammatory cytokine responses also appeared to differ between lipoplexes and LNPs. For tumor necrosis factor-α, release was mainly caused by siRNA. On the other hand, the release of interleukin-1β was mainly due to the cationic nature of particles. LNPs released lower amounts of tumor necrosis factor-α and interleukin-1β than lipoplexes and were thus considered to be better tolerated with respect to cytokine release. In conclusion, siRNA-loaded nanoformulations effect their cellular uptake and immune stimulation in a manner that depends on the structure of the molecular assembly; therefore, nanoformulations should be optimized before extending studies into the in vivo environment.

Long non‑coding RNA AK001796 contributes to cisplatin resistance of non‑small cell lung cancer.

PubMed

Liu, Bin; Pan, Chun-Feng; Ma, Teng; Wang, Jun; Yao, Guo-Liang; Wei, Ke; Chen, Yi-Jiang

2017-10-01

Cisplatin (DDP)‑based chemotherapy is the most widely used therapy for non‑small cell lung cancer (NSCLC). However, the existence of chemoresistance has become a major limitation in its efficacy. Long non‑coding RNAs (lncRNAs) have been shown to be involved in chemotherapy drug resistance. The aim of the present study was to investigate the biological role of lncRNA AK001796 in cisplatin‑resistant NSCLC A549/DDP cells. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis was performed to monitor the differences in the expression of AK001796 in cisplatin-resistant (A549/DDP) cells and parental A549 cells. Cellular sensitivity to cisplatin and cell viability were examined using an MTT assay. Cell apoptosis and cell cycle distribution were measured using flow cytometry. The expression levels of cell cycle proteins cyclin C (CCNC), baculoviral IAP repeat containing 5 (BIRC5), cyclin‑dependent kinase 1 (CDK1) and G2 and S phase‑expressed 1 (GTSE1) were assessed using RT‑qPCR and western blot analyses. It was found that the expression of AK001796 was increased in A549/DDP cells, compared with that in A549 cells. The knockdown of AK001796 by small interfering RNA reduced cellular cisplatin resistance and cell viability, and resulted in cell‑cycle arrest, with a marked increase in the proportion of A549/DDP cells in the G0/G1 phase. By contrast, the knockdown of AK001796 increased the number of apoptotic cancer cells during cisplatin treatment. It was also shown that the knockdown of AK001796 positively induced the expression of cell apoptosis‑associated factors, CCNC and BIRC5, and suppressed the expression of cell cycle‑associated factors, CDK1 and GTSE5. Taken together, these findings indicated that lncRNA AK001796 increased the resistance of NSCLC cells to cisplatin through regulating cell apoptosis and cell proliferation, and thus provides an attractive therapeutic target for NSCLC.

A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5’UTRs of Selected Tumor Suppressors

PubMed Central

Master, Adam; Wójcicka, Anna; Giżewska, Kamilla; Popławski, Piotr; Williams, Graham R.; Nauman, Alicja

2016-01-01

Background Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. Findings We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5’-untranslated region (5’UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5’UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5’UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5’UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5’UTRs of complex structure. Significance This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5’UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene and may have future application in gene therapy strategies to enhance expression of proteins including tumor suppressors. PMID:27171412

A Transgenic Transcription Factor (TaDREB3) in Barley Affects the Expression of MicroRNAs and Other Small Non-Coding RNAs

PubMed Central

Hackenberg, Michael; Shi, Bu-Jun; Gustafson, Perry; Langridge, Peter

2012-01-01

Transcription factors (TFs), microRNAs (miRNAs), small interfering RNAs (siRNAs) and other functional non-coding small RNAs (sRNAs) are important gene regulators. Comparison of sRNA expression profiles between transgenic barley over-expressing a drought tolerant TF (TaDREB3) and non-transgenic control barley revealed many group-specific sRNAs. In addition, 42% of the shared sRNAs were differentially expressed between the two groups (|log2| >1). Furthermore, TaDREB3-derived sRNAs were only detected in transgenic barley despite the existence of homologous genes in non-transgenic barley. These results demonstrate that the TF strongly affects the expression of sRNAs and siRNAs could in turn affect the TF stability. The TF also affects size distribution and abundance of sRNAs including miRNAs. About half of the sRNAs in each group were derived from chloroplast. A sRNA derived from tRNA-His(GUG) encoded by the chloroplast genome is the most abundant sRNA, accounting for 42.2% of the total sRNAs in transgenic barley and 28.9% in non-transgenic barley. This sRNA, which targets a gene (TC245676) involved in biological processes, was only present in barley leaves but not roots. 124 and 136 miRNAs were detected in transgenic and non-transgenic barley, respectively. miR156 was the most abundant miRNA and up-regulated in transgenic barley, while miR168 was the most abundant miRNA and up-regulated in non-transgenic barley. Eight out of 20 predicted novel miRNAs were differentially expressed between the two groups. All the predicted novel miRNA targets were validated using a degradome library. Our data provide an insight into the effect of TF on the expression of sRNAs in barley. PMID:22870277

Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation

PubMed Central

Kubota, Kohei; Onishi, Kohei; Sawaki, Kazuaki; Li, Tianshu; Mitsuoka, Kaoru; Sato, Takaaki; Takeoka, Shinji

2017-01-01

Two lipid-based nanoformulations have been used to date in clinical studies: lipoplexes and lipid nanoparticles (LNPs). In this study, we prepared small interfering RNA (siRNA)-loaded carriers using lipid components of the same composition to form molecular assemblies of differing structures, and evaluated the impact of structure on cellular uptake and immune stimulation. Lipoplexes are electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic siRNA in an aqueous environment, whereas LNPs are nanoparticles embedding siRNA prepared by mixing an alcoholic lipid solution with an aqueous siRNA solution in one step. Although the physicochemical properties of lipoplexes and LNPs were similar except for small increases in apparent size of lipoplexes and zeta potential of LNPs, siRNA uptake efficiency of LNPs was significantly higher than that of lipoplexes. Furthermore, in the case of LNPs, both siRNA and lipid were effectively incorporated into cells in a co-assembled state; however, in the case of lipoplexes, the amount of siRNA internalized into cells was small in comparison with lipid. siRNAs in lipoplexes were thought to be more likely to localize on the particle surface and thereby undergo dissociation into the medium. Inflammatory cytokine responses also appeared to differ between lipoplexes and LNPs. For tumor necrosis factor-α, release was mainly caused by siRNA. On the other hand, the release of interleukin-1β was mainly due to the cationic nature of particles. LNPs released lower amounts of tumor necrosis factor-α and interleukin-1β than lipoplexes and were thus considered to be better tolerated with respect to cytokine release. In conclusion, siRNA-loaded nanoformulations effect their cellular uptake and immune stimulation in a manner that depends on the structure of the molecular assembly; therefore, nanoformulations should be optimized before extending studies into the in vivo environment. PMID:28790820

Uncovering Small RNA-Mediated Responses to Cold Stress in a Wheat Thermosensitive Genic Male-Sterile Line by Deep Sequencing1[W][OA

PubMed Central

Tang, Zhonghui; Zhang, Liping; Xu, Chenguang; Yuan, Shaohua; Zhang, Fengting; Zheng, Yonglian; Zhao, Changping

2012-01-01

The male sterility of thermosensitive genic male sterile (TGMS) lines of wheat (Triticum aestivum) is strictly controlled by temperature. The early phase of anther development is especially susceptible to cold stress. MicroRNAs (miRNAs) play an important role in plant development and in responses to environmental stress. In this study, deep sequencing of small RNA (smRNA) libraries obtained from spike tissues of the TGMS line under cold and control conditions identified a total of 78 unique miRNA sequences from 30 families and trans-acting small interfering RNAs (tasiRNAs) derived from two TAS3 genes. To identify smRNA targets in the wheat TGMS line, we applied the degradome sequencing method, which globally and directly identifies the remnants of smRNA-directed target cleavage. We identified 26 targets of 16 miRNA families and three targets of tasiRNAs. Comparing smRNA sequencing data sets and TaqMan quantitative polymerase chain reaction results, we identified six miRNAs and one tasiRNA (tasiRNA-ARF [for Auxin-Responsive Factor]) as cold stress-responsive smRNAs in spike tissues of the TGMS line. We also determined the expression profiles of target genes that encode transcription factors in response to cold stress. Interestingly, the expression of cold stress-responsive smRNAs integrated in the auxin-signaling pathway and their target genes was largely noncorrelated. We investigated the tissue-specific expression of smRNAs using a tissue microarray approach. Our data indicated that miR167 and tasiRNA-ARF play roles in regulating the auxin-signaling pathway and possibly in the developmental response to cold stress. These data provide evidence that smRNA regulatory pathways are linked with male sterility in the TGMS line during cold stress. PMID:22508932

AI-2 quorum-sensing inhibitors affect the starvation response and reduce virulence in several Vibrio species, most likely by interfering with LuxPQ.

PubMed

Brackman, Gilles; Celen, Shari; Baruah, Kartik; Bossier, Peter; Van Calenbergh, Serge; Nelis, Hans J; Coenye, Tom

2009-12-01

The increase of disease outbreaks caused by Vibrio species in aquatic organisms as well as in humans, together with the emergence of antibiotic resistance in Vibrio species, has led to a growing interest in alternative disease control measures. Quorum sensing (QS) is a mechanism for regulating microbial gene expression in a cell density-dependent way. While there is good evidence for the involvement of auto-inducer 2 (AI-2)-based interspecies QS in the control of virulence in multiple Vibrio species, only few inhibitors of this system are known. From the screening of a small panel of nucleoside analogues for their ability to disturb AI-2-based QS, an adenosine derivative with a p-methoxyphenylpropionamide moiety at C-3' emerged as a promising hit. Its mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of Vibrio harveyi AI-2 QS mutants. Our results indicate that this compound, as well as a truncated analogue lacking the adenine base, block AI-2-based QS without interfering with bacterial growth. The active compounds affected neither the bioluminescence system as such nor the production of AI-2, but most likely interfered with the signal transduction pathway at the level of LuxPQ in V. harveyi. The most active nucleoside analogue (designated LMC-21) was found to reduce the Vibrio species starvation response, to affect biofilm formation in Vibrio anguillarum, Vibrio vulnificus and Vibrio cholerae, to reduce pigment and protease production in V. anguillarum, and to protect gnotobiotic Artemia from V. harveyi-induced mortality.

STAT3 inhibition as a therapeutic strategy for leukemia.

PubMed

Kanna, Rubashruti; Choudhary, Gaurav; Ramachandra, Nandini; Steidl, Ulrich; Verma, Amit; Shastri, Aditi

2017-11-22

Leukemia is characterized by selective overgrowth of malignant hematopoietic stem cells (HSC's) that interfere with HSC differentiation. Cytoreductive chemotherapy can kill rapidly dividing cancerous cells but cannot eradicate the malignant HSC pool leading to relapses. Leukemic stem cells have several dysregulated pathways and the Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) pathway are prominent among them. STAT3 is an important transcription factor that regulates cell growth, proliferation, and inhibits apoptosis. High STAT3 expression in leukemia has been associated with an increased risk for relapse and decreased overall survival. Multiple strategies for interfering with STAT3 activity in leukemic cells include inhibition of STAT3 phosphorylation, interfering with STAT3 interactions, preventing nuclear transfer, inhibiting transcription and causing interference in STAT: DNA binding. A better understanding of key interactions and upstream mediators of STAT3 activity will help facilitate the development of effective cancer therapies and may result in durable remissions.

Sulforaphane Attenuated the Pro-Inflammatory State Induced by Hydrogen Peroxide in SH-SY5Y Cells Through the Nrf2/HO-1 Signaling Pathway.

PubMed

de Oliveira, Marcos Roberto; Brasil, Flávia Bittencourt; Fürstenau, Cristina Ribas

2018-02-23

Sulforaphane (SFN), an isothiocyanate obtained from cruciferous vegetables, exerts antioxidant, antiapoptotic, and antitumor activities in different cell types. Moreover, SFN has been viewed as an anti-inflammatory agent. Nonetheless, the mechanism underlying the ability of SFN in modulating the immune response in mammalian cells is not completely understood yet. Therefore, we investigated here whether and how SFN would be effective in preventing inflammation induced by a pro-oxidant agent (hydrogen peroxide, H 2 O 2 ) in the human neuroblastoma SH-SY5Y cells. The cells were treated with SFN at 5 μM for 30 min before a challenge with H 2 O 2 for an additional 24 h. Pretreatment with SFN reduced the secretion of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well as decreased the levels of cyclooxygenase-2 (COX-2) in H 2 O 2 -treated cells. SFN also decreased the activity of the transcription factor nuclear factor-κB (NF-κB) and the immunocontent of the p65 NF-κB subunit in the cell nucleus. The inhibition of heme oxygenase-1 (HO-1) by ZnPP-IX at 10 μM or the silencing of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor by small interfering RNA targeting Nrf2 attenuated the anti-inflammatory and cytoprotective effects induced by SFN. Therefore, SFN exerted an anti-inflammatory effect in H 2 O 2 -challenged SH-SY5Y cells by a mechanism dependent on the Nrf2/HO-1 signaling pathway.

32 CFR 234.6 - Interfering with agency functions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 2 2010-07-01 2010-07-01 false Interfering with agency functions. 234.6 Section 234.6 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS CONDUCT ON THE PENTAGON RESERVATION § 234.6 Interfering with agency functions. The following are...

47 CFR 73.185 - Computation of interfering signal.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 4 2010-10-01 2010-10-01 false Computation of interfering signal. 73.185... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.185 Computation of interfering signal. (a) Measured... paragraphs (a) (1) or (2) of this section. (b) For skywave signals from stations operating on all channels...

47 CFR 73.185 - Computation of interfering signal.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 4 2011-10-01 2011-10-01 false Computation of interfering signal. 73.185... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.185 Computation of interfering signal. (a) Measured... paragraphs (a) (1) or (2) of this section. (b) For skywave signals from stations operating on all channels...

47 CFR 73.185 - Computation of interfering signal.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 47 Telecommunication 4 2014-10-01 2014-10-01 false Computation of interfering signal. 73.185... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.185 Computation of interfering signal. (a) Measured... paragraphs (a) (1) or (2) of this section. (b) For skywave signals from stations operating on all channels...

47 CFR 73.185 - Computation of interfering signal.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 47 Telecommunication 4 2013-10-01 2013-10-01 false Computation of interfering signal. 73.185... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.185 Computation of interfering signal. (a) Measured... paragraphs (a) (1) or (2) of this section. (b) For skywave signals from stations operating on all channels...

47 CFR 73.185 - Computation of interfering signal.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 47 Telecommunication 4 2012-10-01 2012-10-01 false Computation of interfering signal. 73.185... RADIO BROADCAST SERVICES AM Broadcast Stations § 73.185 Computation of interfering signal. (a) Measured... paragraphs (a) (1) or (2) of this section. (b) For skywave signals from stations operating on all channels...

Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis

PubMed Central

Blevins, Todd; Pontes, Olga; Pikaard, Craig S.; Meins, Frederick

2009-01-01

5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays. PMID:19529764

Sumoylation of Smad3 stimulates its nuclear export during PIASy-mediated suppression of TGF-{beta} signaling

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

Imoto, Seiyu; Ohbayashi, Norihiko; Ikeda, Osamu

2008-05-30

Sma- and MAD-related protein 3 (Smad3) plays crucial roles in the transforming growth factor-{beta} (TGF-{beta})-mediated signaling pathway, which produce a variety of cellular responses, including cell proliferation and differentiation. In our previous study, we demonstrated that protein inhibitor of activated STATy (PIASy) suppresses TGF-{beta} signaling by interacting with and sumoylating Smad3. In the present study, we examined the molecular mechanisms of Smad3 sumoylation during PIASy-mediated suppression of TGF-{beta} signaling. We found that small-interfering RNA-mediated reduction of endogenous PIASy expression enhanced TGF-{beta}-induced gene expression. Importantly, coexpression of Smad3 with PIASy and SUMO1 affected the DNA-binding activity of Smad3. Furthermore, coexpression ofmore » Smad3 with PIASy and SUMO1 stimulated the nuclear export of Smad3. Finally, fluorescence resonance energy transfer analyses revealed that Smad3 interacted with SUMO1 in the cytoplasm. These results suggest that PIASy regulates TGF-{beta}/Smad3-mediated signaling by stimulating sumoylation and nuclear export of Smad3.« less

Downregulation of Id1 by small interfering RNA in gastric cancer inhibits cell growth via the Akt pathway

PubMed Central

YANG, GUANG; ZHANG, YAN; XIONG, JIANJUN; WU, JING; YANG, CHANGFU; HUANG, HONGBING; ZHU, ZHENYU

2012-01-01

Inhibitor of differentiation or DNA binding (Id1) is a member of the helix-loop-helix transcription factor family that is overexpressed in various types of cancer, including gastric carcinoma. Previous studies showed that Id1 is a prognostic marker in patients with gastric cancer. However, the role of Id1 in the proliferation of human gastric cancer cells has yet to be clarified. In the present study, we downregulated the Id1 gene in SGC-7901 gastric cancer cells by RNA interference, and we also constructed a recombinant plasmid-expressing Id1 to investigate its effects on the proliferation of SGC-7901 cells. Results showed that the downregulation of Id1 inhibited proliferation of SGC-7901 cells, while the upregulation of Id1 had no effect on SGC-7901 cell proliferation. The potential mechanism was also investigated. The changes of certain proteins associated with cell proliferation, apoptosis and the cell cycle were detected by western blotting. Furthermore, we demonstrated a positive correlation between Id1 and phospho-Akt expression in SGC-7901 cells. PMID:22245935

Context-based retrieval of functional modules in protein-protein interaction networks.

PubMed

Dobay, Maria Pamela; Stertz, Silke; Delorenzi, Mauro

2017-03-27

Various techniques have been developed for identifying the most probable interactants of a protein under a given biological context. In this article, we dissect the effects of the choice of the protein-protein interaction network (PPI) and the manipulation of PPI settings on the network neighborhood of the influenza A virus (IAV) network, as well as hits in genome-wide small interfering RNA screen results for IAV host factors. We investigate the potential of context filtering, which uses text mining evidence linked to PPI edges, as a complement to the edge confidence scores typically provided in PPIs for filtering, for obtaining more biologically relevant network neighborhoods. Here, we estimate the maximum performance of context filtering to isolate a Kyoto Encyclopedia of Genes and Genomes (KEGG) network Ki from a union of KEGG networks and its network neighborhood. The work gives insights on the use of human PPIs in network neighborhood approaches for functional inference. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Targeting NF-kB signaling with polymeric hybrid micelles that co-deliver siRNA and dexamethasone for arthritis therapy.

PubMed

Wang, Qin; Jiang, Hao; Li, Yan; Chen, Wenfei; Li, Hanmei; Peng, Ke; Zhang, Zhirong; Sun, Xun

2017-04-01

The transcription factor NF-kB plays a pivotal role in the pathogenesis of rheumatoid arthritis. Here we attempt to slow arthritis progression by co-delivering the glucocorticoid dexamethasone (Dex) and small-interfering RNA targeting NF-kB p65 using our previously developed polymeric hybrid micelle system. These micelles contain two similar amphiphilic copolymers: polycaprolactone-polyethylenimine (PCL-PEI) and polycaprolactone-polyethyleneglycol (PCL-PEG). The hybrid micelles loaded with Dex and siRNA effectively inhibited NF-kB signaling in murine macrophages more efficiently than micelles containing either Dex or siRNA on their own. In addition, the co-delivery system was able to switch macrophages from the M1 to M2 state. Injecting hybrid micelles containing Dex and siRNA into mice with collagen-induced arthritis led the therapeutic agents to accumulate in inflamed joints and reduce inflammation, without damaging renal or liver function. Thus, blocking NF-kB activation in inflammatory tissue using micelle-based co-delivery may provide a new approach for treating inflammatory disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana.

PubMed

Eun, Changho; Lorkovic, Zdravko J; Naumann, Ulf; Long, Quan; Havecker, Ericka R; Simon, Stacey A; Meyers, Blake C; Matzke, Antonius J M; Matzke, Marjori

2011-01-01

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

The histone code reader SPIN1 controls RET signaling in liposarcoma

PubMed Central

Franz, Henriette; Greschik, Holger; Willmann, Dominica; Ozretić, Luka; Jilg, Cordula Annette; Wardelmann, Eva; Jung, Manfred; Buettner, Reinhard; Schüle, Roland

2015-01-01

The histone code reader Spindlin1 (SPIN1) has been implicated in tumorigenesis and tumor growth, but the underlying molecular mechanisms remain poorly understood. Here, we show that reducing SPIN1 levels strongly impairs proliferation and increases apoptosis of liposarcoma cells in vitro and in xenograft mouse models. Combining signaling pathway, genome-wide chromatin binding, and transcriptome analyses, we found that SPIN1 directly enhances expression of GDNF, an activator of the RET signaling pathway, in cooperation with the transcription factor MAZ. Accordingly, knockdown of SPIN1 or MAZ results in reduced levels of GDNF and activated RET explaining diminished liposarcoma cell proliferation and survival. In line with these observations, levels of SPIN1, GDNF, activated RET, and MAZ are increased in human liposarcoma compared to normal adipose tissue or lipoma. Importantly, a mutation of SPIN1 within the reader domain interfering with chromatin binding reduces liposarcoma cell proliferation and survival. Together, our data describe a molecular mechanism for SPIN1 function in liposarcoma and suggest that targeting SPIN1 chromatin association with small molecule inhibitors may represent a novel therapeutic strategy. PMID:25749382

Knockdown of Zinc Transporter ZIP5 by RNA Interference Inhibits Esophageal Cancer Growth In Vivo.

PubMed

Li, Qian; Jin, Jing; Liu, Jianghui; Wang, Liqun; He, Yutong

2016-01-01

We recently found that SLC39A5 (ZIP5), a zinc transporter, is overexpressed in esophageal cancer. Downregulation of ZIP5 inhibited the proliferation, migration, and invasion of the esophageal cancer cell line KYSE170 in vitro. In this study, we found that downregulation of SLC39A5 (ZIP5) by interference resulted in a significant reduction in esophageal cancer tumor volume and weight in vivo. COX2 (cyclooxygenase 2) expression was decreased and E-cadherin expression was increased in the KYSE170K xenografts, which was caused by the downregulation of ZIP5. However, we did not find that the downregulation of ZIP5 caused a change in the relative expressions of cyclin D1, VEGF (vascular endothelial growth factor), MMP9 (matrix metalloprotein 9), and Bcl-2 (B-cell lymphoma/leukmia-2) mRNA or an alteration in the average level of zinc in the peripheral blood and xenografts in vivo. Collectively, these findings indicate that knocking down ZIP5 by small interfering RNA (siRNA) might be a novel treatment strategy for esophageal cancer with ZIP5 overexpression.

PACE4 regulates proliferation, migration and invasion in human breast cancer MDA-MB-231 cells.

PubMed

Wang, Feifei; Wang, Lin; Pan, Jihong

2015-01-01

PACE4 is one of the proprotein convertases (PC) that participate in the post-translational activation of inactive proteins, leading to mature, biologically active proteins. The processing reactions occur in pairs of basic amino acids. PACE4 is an extracellular PC that binds to growth factors and several components of the extracellular matrix contributing to tumor progression. In the present study, the PACE4 gene was silenced by small interfering RNA (siRNA), and the knockdown human breast cancer MDA-MB-231 cells showed significantly reduced proliferation, migration and invasion rates. Flow cytometry analysis indicated that downregulation of PACE4 increases the percentage of cells arrested at the G0/G1 phase. Moreover, the expression of genes involved in cell growth, invasion and adhesion, i.e., IGF-2, MMP9 and MPZL2 was significantly decreased following siRNA-mediated silencing of PACE4. Taken together, these results indicate that PACE4 plays an important role in human breast cancer, and that it might represent a novel target for breast cancer therapy.

On future's doorstep: RNA interference and the pharmacopeia of tomorrow.

PubMed

Gewirtz, Alan M

2007-12-01

Small molecules and antibodies have revolutionized the treatment of malignant diseases and appear promising for the treatment of many others. Nonetheless, there are many candidate therapeutic targets that are not amenable to attack by the current generation of targeted therapies, and in a small but growing number of patients, resistance to initially successful treatments evolves. This Review Series on the medicinal promise of posttranscriptional gene silencing with small interfering RNA and other molecules capable of inducing RNA interference (RNAi) is motivated by the hypothesis that effectors of RNAi can be developed into effective drugs for treating malignancies as well as many other types of disease. As this Review Series points out, there is still much to do, but many in the field now hope that the time has finally arrived when "antisense" therapies will finally come of age and fulfill their promise as the magic bullets of the 21st century.

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

PubMed

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

2015-01-01

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

Detection of protein-small molecule binding using a self-referencing external cavity laser biosensor.

PubMed

Meng Zhang; Peh, Jessie; Hergenrother, Paul J; Cunningham, Brian T

2014-01-01

High throughput screening of protein-small molecule binding interactions using label-free optical biosensors is challenging, as the detected signals are often similar in magnitude to experimental noise. Here, we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with sub-picometer wavelength accuracy. Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets with binding affinities or inhibition constants in the sub-nanomolar to low micromolar range. The demonstrated ability to perform detection in the presence of several interfering compounds opens the potential for increasing the throughput of the approach. As an example application, we performed a "needle-in-the-haystack" screen for inhibitors against carbonic anhydrase isozyme II (CA II), in which known inhibitors are clearly differentiated from inactive molecules within a compound library.

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

PubMed Central

Börner, Kathleen; Niopek, Dominik; Cotugno, Gabriella; Kaldenbach, Michaela; Pankert, Teresa; Willemsen, Joschka; Zhang, Xian; Schürmann, Nina; Mockenhaupt, Stefan; Serva, Andrius; Hiet, Marie-Sophie; Wiedtke, Ellen; Castoldi, Mirco; Starkuviene, Vytaute; Erfle, Holger; Gilbert, Daniel F.; Bartenschlager, Ralf; Boutros, Michael; Binder, Marco; Streetz, Konrad; Kräusslich, Hans-Georg; Grimm, Dirk

2013-01-01

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems. PMID:24049077

Work ability in patients with polymyositis and dermatomyositis: An explorative and descriptive study.

PubMed

Regardt, Malin; Welin Henriksson, Elisabet; Sandqvist, Jan; Lundberg, Ingrid E; Schult, Marie-Louise

2015-01-01

Polymyositis (PM) and dermatomyositis (DM) are rare, chronic inflammatory diseases leading to muscle weakness and low muscle endurance. The muscle weakness may lead to restrictions in daily activities and low health-related quality of life. This study aimed to investigate the work situation, work ability, work-related risk factors, and influence of the physical and psycho-social work environment in patients with PM and DM. Patients with PM/DM were assessed using the Work Ability Index (WAI), and the Work Environment Impact Scale (WEIS). Forty-eight patients (PM n = 25 and DM n = 23) participated (women/men: 29/19) with a mean age of 54 years (range 28-67 years, SD.10) and mean disease duration of nine years (SD.9). Forty-four percent worked full-time, 31% part-time and 25% were on full-time sick leave. More than 50% self-rated work ability as "poor" or "less good". Physically strenuous work components were present "quite to very often" in 23-79% and more in patients on sick leave ≥ 2 years. For those working, the interfering factors in the work environment concerned task and time demands. Supporting factors concerned meaning of work, interactions with co-workers and others. Self-rated work ability correlated moderately-highly positive with percentage of full-time employment, work-related risk factors and opportunities and constraints in the work environment. Poor self-rated work ability is common in patients with PM/DM indicating a need to identify interfering risk factors and support patients to enhance work performance.

RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis.

PubMed

Liu, Wanlu; Duttke, Sascha H; Hetzel, Jonathan; Groth, Martin; Feng, Suhua; Gallego-Bartolome, Javier; Zhong, Zhenhui; Kuo, Hsuan Yu; Wang, Zonghua; Zhai, Jixian; Chory, Joanne; Jacobsen, Steven E

2018-03-01

Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide small interfering RNAs (siRNAs) that bind to ARGONAUTE 4 (AGO4) and target genomic regions for silencing. RdDM also requires non-coding RNAs transcribed by RNA polymerase V (Pol V) that probably serve as scaffolds for binding of AGO4-siRNA complexes. Here, we used a modified global nuclear run-on protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5' adenine found in many AGO4-bound 24-nucleotide siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10 U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expression of wild-type AGO4 in ago4/6/9 mutants was able to restore slicing of Pol V transcripts, but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L), an elongation factor whose function is not well understood. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

District Superintendents and the School Improvement Problem of Addressing Barriers to Learning

ERIC Educational Resources Information Center

Center for Mental Health in Schools at UCLA, 2011

2011-01-01

This report begins with conclusions drawn from a wide range of research, reports, and other sources that convey what superintendents say is driving their work. The focus first is on what they identify as the challenges and frustrations of the job and what they say are factors interfering with student progress. Then, discussion turns to the…

NF-κB pathways are involved in M1 polarization of RAW 264.7 macrophage by polyporus polysaccharide in the tumor microenvironment

PubMed Central

Liu, Chun-Ping; Zhang, Xian; Tan, Qing-Long; Xu, Wen-Xing; Zhou, Chang-Yuan; Luo, Min; Li, Xiong; Zeng, Xing

2017-01-01

Bladder cancer is one of the most malignant tumors closely associated with macrophages. Polyporus polysaccharide (PPS) has shown excellent efficacy in treating bladder cancer with minimal side effects. However, the molecular mechanisms underlying the effects of PPS in inhibiting bladder cancer remain unclear. In this study, we used macrophages cultured alone or with T24 human bladder cancer cell culture supernatant as study models. We found that PPS enhanced the activities of IFN-γ-stimulated RAW 264.7 macrophages, as shown by the release of inducible nitric oxide synthase (INOS), secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-6, phagocytosis activity, as well as expression of M1 phenotype indicators, such as CD40, CD284 and CD86. PPS acted upstream in activation cascade of nuclear factor (NF)-κB signaling pathways by interfering with IκB phosphorylation. In addition, PPS regulated NF-κB (P65) signaling by interfering with Toll-like receptor (TLR)-4, INOS and cyclooxygenase (COX)-2. Our results indicate that PPS activates macrophages through TLR4/NF-κB signaling pathways. PMID:29155869

TRAF6 and the Three C-Terminal Lysine Sites on IRF7 Are Required for Its Ubiquitination-Mediated Activation by the Tumor Necrosis Factor Receptor Family Member Latent Membrane Protein 1▿

PubMed Central

Ning, Shunbin; Campos, Alex D.; Darnay, Bryant G.; Bentz, Gretchen L.; Pagano, Joseph S.

2008-01-01

We have recently shown that interferon regulatory factor 7 (IRF7) is activated by Epstein-Barr virus latent membrane protein 1 (LMP1), a member of the tumor necrosis factor receptor (TNFR) superfamily, through receptor-interacting protein-dependent K63-linked ubiquitination (L. E. Huye, S. Ning, M. Kelliher, and J. S. Pagano, Mol. Cell. Biol. 27:2910-2918, 2007). In this study, with the use of small interfering RNA and TNFR-associated factor 6 (TRAF6) knockout cells, we first show that TRAF6 and its E3 ligase activity are required for LMP1-stimulated IRF7 ubiquitination. In Raji cells which are latently infected and express high levels of LMP1 and IRF7 endogenously, expression of a TRAF6 small hairpin RNA construct reduces endogenous ubiquitination and endogenous activity of IRF7. In TRAF6−/− mouse embryonic fibroblasts, reconstitution with TRAF6 expression, but not with TRAF6(C70A), which lacks the E3 ligase activity, recovers LMP1's ability to stimulate K63-linked ubiquitination of IRF7. Further, we identify IRF7 as a substrate for TRAF6 E3 ligase and show that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo. Most important, we determine that the last three C-terminal lysine sites (positions 444, 446, and 452) of human IRF7 variant A are essential for activation of IRF7; these are the first such sites identified. A ubiquitination-deficient mutant of IRF7 with these sites mutated to arginines completely loses transactivational ability in response not only to LMP1 but also to the IRF7 kinase IκB kinase ɛ. In addition, we find that K63-linked ubiquitination of IRF7 occurs independently of its C-terminal functional phosphorylation sites. These data support our hypothesis that regulatory ubiquitination of IRF7 is a prerequisite for its phosphorylation. This is the first evidence to imply that ubiquitination is required for phosphorylation and activation of a transcription factor. PMID:18710948

36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

Code of Federal Regulations, 2014 CFR

2014-07-01

... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

Code of Federal Regulations, 2013 CFR

2013-07-01

... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

Code of Federal Regulations, 2012 CFR

2012-07-01

... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

Code of Federal Regulations, 2011 CFR

2011-07-01

... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

Code of Federal Regulations, 2010 CFR

2010-07-01

... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model.

PubMed

Drabsch, Yvette; He, Shuning; Zhang, Long; Snaar-Jagalska, B Ewa; ten Dijke, Peter

2013-11-07

The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells. We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy. Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells. The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.

11β-HSD1 Modulates LPS-Induced Innate Immune Responses in Adipocytes by Altering Expression of PTEN

PubMed Central

Lai, Wenfang; Tian, Xue; Xiang, Qing; Chu, Kedan; Wei, Yicong; Deng, Jingti; Zhang, Shaoping; Brown, John

2015-01-01

Inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) represents a therapeutic target for treating hyperglycemia in type 2 diabetes. Here, we investigate the effects of 11β-HSD1 on the innate immune response of adipocytes to produce proinflammatory cytokines. The 11β-HSD1 inhibitor emodin, or 11β-HSD1-targeted small interfering RNA, dose dependently suppressed IL-6, IL-1β, and TNF-α expression in lipopolysaccharide-treated 3T3-L1 adipocytes. Inhibiting 11β-HSD1 also reduced phosphatase and tensin homologue (PTEN) expression, a negative regulator of phosphatidylinositol 3-kinase effects, whereas 1pM cortisone or dexamethasone induced IL-6 and PTEN levels. PTEN-targeted small interfering RNA decreased IL-6, IL-1β, and TNF-α without affecting 11β-HSD1 levels. Correspondingly, emodin increased phosphorylated protein kinase B (p-PKB) (Ser473) to PKB ratio but not p-PKB (Thr308) to PKB ratio. Emodin did not increase the p-PKB (Ser473) to PKB ratio when the rapamycin-insensitive companion of mTOR was depleted, further supporting the involvement of mammalian target of rapamycin complex 2 in PKB phosphorylation. Moreover, emodin suppressed phosphorylated inhibitor of κB α (p-IκBα) to IκBα ratio and reduced nuclear factor κ B subunit p50 in the nuclear fraction. In contrast, 1pM cortisone or dexamethasone decreased p-PKB (Ser473) to PKB ratio, increased p-IκBα to IκBα ratio, and increased nuclear NF-κB subunit p50. Additionally, wortmannin had similar effects on IL-6, p-PKB (Ser473) to PKB ratio, and p-IκBα to IκBα ratio as 1pM cortisone or dexamethasone. Finally, emodin treatment of streptozotocin diabetic rats on a high-fat diet reduced levels of IL-6, PTEN, Cluster of Differentiation 68, and the ratio of p-IκBα to IκBα in visceral fat, indicating that our findings in vitro may also apply to visceral fat in vivo. Together, these results suggest that inhibiting 11β-HSD1 reduces lipopolysaccharide-induced proinflammatory innate immune responses in adipocytes by down-regulating PTEN expression, leading to activation of the PI3K/PKB pathway. PMID:25734515

Control of Collagen Production in Mouse Chondrocytes by Using a Combination of Bone Morphogenetic Protein-2 and Small Interfering RNA Targeting Col1a1 for Hydrogel-Based Tissue-Engineered Cartilage

PubMed Central

Perrier-Groult, Emeline; Pasdeloup, Marielle; Malbouyres, Marilyne; Galéra, Philippe

2013-01-01

Because articular cartilage does not self-repair, tissue-engineering strategies should be considered to regenerate this tissue. Autologous chondrocyte implantation is already used for treatment of focal damage of articular cartilage. Unfortunately, this technique includes a step of cell amplification, which results in dedifferentiation of chondrocytes, with expression of type I collagen, a protein characteristic of fibrotic tissues. Therefore, the risk of producing a fibrocartilage exists. The aim of this study was to propose a new strategy for authorizing the recovery of the differentiated status of the chondrocytes after their amplification on plastic. Because the bone morphogenetic protein (BMP)-2 and the transforming growth factor (TGF)-β1 are cytokines both proposed as stimulants for cartilage repair, we undertook a detailed comparative analysis of their biological effects on chondrocytes. As a cellular model, we used mouse chondrocytes after their expansion on plastic and we tested the capability of BMP-2 or TGF-β1 to drive their redifferentiation, with special attention given to the nature of the proteins synthesized by the cells. To prevent any fibrotic character of the newly synthesized extracellular matrix, we silenced type I collagen by transfecting small interfering RNA (siRNA) into the chondrocytes, before their exposure to BMP-2 or TGF-β1. Our results showed that addition of siRNA targeting the mRNA encoded by the Col1a1 gene (Col1a1 siRNA) and BMP-2 represents the most efficient combination to control the production of cartilage-characteristic collagen proteins. To go one step further toward scaffold-based cartilage engineering, Col1a1 siRNA-transfected chondrocytes were encapsulated in agarose hydrogel and cultured in vitro for 1 week. The analysis of the chondrocyte–agarose constructs by using real-time polymerase chain reaction, Western-blotting, immunohistochemistry, and electron microscopy techniques demonstrated that the BMP-2/Col1a1 siRNA combination is effective in reinitializing correct production and assembly of the cartilage-characteristic matrix in agarose hydrogel, without production of type I collagen. Because agarose is known to favor long-term expression of the chondrocyte phenotype and agarose-based hydrogels are approved for clinical trials, this strategy appears very promising to repair hyaline cartilage. PMID:23311625

Activation of β-catenin and Yap1 in human hepatoblastoma and induction of hepatocarcinogenesis in mice.

PubMed

Tao, Junyan; Calvisi, Diego F; Ranganathan, Sarangarajan; Cigliano, Antonio; Zhou, Lili; Singh, Sucha; Jiang, Lijie; Fan, Biao; Terracciano, Luigi; Armeanu-Ebinger, Sorin; Ribback, Silvia; Dombrowski, Frank; Evert, Matthias; Chen, Xin; Monga, Satdarshan P S

2014-09-01

Aberrant activation of β-catenin and Yes-associated protein 1 (Yap1) signaling pathways have been associated with the development of multiple tumor types. Yap functions as a transcriptional coactivator by interacting with TEA domain DNA binding proteins. We investigated the interactions among these pathways during hepatic tumorigenesis. We used immunohistochemical analysis to determine expression of β-catenin and Yap1 in liver cancer specimens collected from patients in Europe and the United States, consisting of 104 hepatocellular carcinoma, 62 intrahepatic cholangiocarcinoma, and 94 hepatoblastoma samples. We assessed β-catenin and Yap1 signaling and interactions in hepatoblastoma cell lines ((HuH6, HepG2, HepT1, HC-AFW1, HepG2, and HC-AFW1); proteins were knocked down with small interfering RNAs, and effects on proliferation and cell death were measured. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β-catenin (ΔN90/β-catenin) and Yap1 (YapS127A) in livers of mice; tissues were collected, and histological and immunohistochemical analyses were performed. We observed nuclear localization of β-catenin and Yap1 in 79% of hepatoblastoma samples but not in most hepatocellular carcinoma or intrahepatic cholangiocarcinoma samples. Yap1 and β-catenin coprecipitated in hepatoblastoma but not hepatocellular carcinoma cells. Small interfering RNA-mediated knockdown of Yap1 or β-catenin in hepatoblastoma cells reduced proliferation in an additive manner. Knockdown of Yap1 reduced its ability to coactivate transcription with β-catenin; β-catenin inhibitors inactivated Yap1. Overexpression of constitutively active forms of Yap1 and β-catenin in mouse liver led to rapid tumorigenesis, with 100% mortality by 11 weeks. Tumor cells expressed both proteins, and human hepatoblastoma cells expressed common targets of their 2 signaling pathways. Yap1 binding of TEA domain factors was required for tumorigenesis in mice. β-catenin and the transcriptional regulator Yap1 interact physically and are activated in most human hepatoblastoma tissues; overexpression of activated forms of these proteins in livers of mice leads to rapid tumor development. Further analysis of these mice will allow further studies of these pathways in hepatoblastoma pathogenesis and could lead to the identification of new therapeutic targets. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Characterization of Transcription Factor Gene OsDRAP1 Conferring Drought Tolerance in Rice

PubMed Central

Huang, Liyu; Wang, Yinxiao; Wang, Wensheng; Zhao, Xiuqin; Qin, Qiao; Sun, Fan; Hu, Fengyi; Zhao, Yan; Li, Zichao; Fu, Binying; Li, Zhikang

2018-01-01

HIGHLIGHTS Overexpressing and RNA interfering OsDRAP1 transgenic rice plants exhibited significantly improved and reduced drought tolerance, but accompanied with negative effects on development and yield. The dehydration responsive element binding (DREBs) genes are important transcription factors which play a crucial role in plant abiotic stress tolerances. In this study, we functionally characterized a DREB2-like gene, OsDRAP1 conferring drought tolerance (DT) in rice. OsDRAP1, containing many cis-elements in its promoter region, was expressed in all organs (mainly expressed in vascular tissues) of rice, and induced by a variety of environmental stresses and plant hormones. Overexpressing OsDRAP1 transgenic plants exhibited significantly improved DT; while OsDRAP1 RNA interfering plants exhibited significantly reduced DT which also accompanied with significant negative effects on development and yield. Overexpression of OsDRAP1 has a positive impact on maintaining water balance, redox homeostasis and vascular development in transgenic rice plants under drought stress. OsDRAP1 interacted with many genes/proteins and could activate many downstream DT related genes, including important transcription factors such as OsCBSX3 to response drought stress, indicating the OsDRAP1-mediated pathways for DT involve complex genes networks. All these results provide a basis for further complete understanding of the OsDRAP1 mediated gene networks and their related phenotypic effects. PMID:29449862

The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms.

PubMed

Athanasopoulos, Athanasios N; Economopoulou, Matina; Orlova, Valeria V; Sobke, Astrid; Schneider, Darius; Weber, Holger; Augustin, Hellmut G; Eming, Sabine A; Schubert, Uwe; Linn, Thomas; Nawroth, Peter P; Hussain, Muzaffar; Hammes, Hans-Peter; Herrmann, Mathias; Preissner, Klaus T; Chavakis, Triantafyllos

2006-04-01

Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked alphav-integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new anti-inflammatory and antiangiogenic therapies in several pathologies.

Pivotal Role of Brain-Derived Neurotrophic Factor Secreted by Mesenchymal Stem Cells in Severe Intraventricular Hemorrhage in Newborn Rats.

PubMed

Ahn, So Yoon; Chang, Yun Sil; Sung, Dong Kyung; Sung, Se In; Ahn, Jee-Yin; Park, Won Soon

2017-01-24

Mesenchymal stem cell (MSC) transplantation protects against neonatal severe intraventricular hemorrhage (IVH)-induced brain injury by a paracrine rather than regenerative mechanism; however, the paracrine factors involved and their roles have not yet been delineated. This study aimed to identify the paracrine mediator(s) and to determine their role in mediating the therapeutic effects of MSCs in severe IVH. We first identified significant upregulation of brain-derived neurotrophic factor (BDNF) in MSCs compared with fibroblasts, in both DNA and antibody microarrays, after thrombin exposure. We then knocked down BDNF in MSCs by transfection with small interfering (si)RNA specific for human BDNF. The therapeutic effects of MSCs with or without BDNF knockdown were evaluated in vitro in rat neuronal cells challenged with thrombin, and in vivo in newborn Sprague-Dawley rats by injecting 200 μl of blood on postnatal day 4 (P4), and transplanting MSCs (1 × 105 cells) intraventricularly on P6. siRNA-induced BDNF knockdown abolished the in vitro benefits of MSCs on thrombin-induced neuronal cell death. BDNF knockdown also abolished the in vivo protective effects against severe IVH-induced brain injuries such as the attenuation of posthemorrhagic hydrocephalus, impaired behavioral test performance, increased astrogliosis, increased number of TUNEL cells, ED-1+ cells, and inflammatory cytokines, and reduced myelin basic protein expression. Our data indicate that BDNF secreted by transplanted MSCs is one of the critical paracrine factors that play a seminal role in attenuating severe IVH-induced brain injuries in newborn rats.

Fascin Overexpression Promotes Cholangiocarcinoma RBE Cell Proliferation, Migration, and Invasion.

PubMed

Zhao, Haiying; Yang, Fuquan; Zhao, Wenyan; Zhang, Chunjv; Liu, Jingang

2016-04-01

Fascin is overexpressed in various tumor tissues and is closely related to tumor metastasis and invasion. However, the role of fascin in cholangiocarcinoma RBE cells has not been clearly reported. This study aimed to establish a cholangiocarcinoma cell line with stable and high expression of fascin to observe the effect of fascin on cell proliferation, migration, and invasion. A fascin overexpression vector, pcDNA3.1-Fascin, was constructed and transfected into the human cholangiocarcinoma RBE cell line. The results of real-time polymerase chain reaction, Western blot, and immunofluorescence indicated that fascin was steadily and highly expressed in RBE cells. The results of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and colony formation assay indicated that upregulated fascin expression could enhance cholangiocarcinoma cell proliferation. The results of wound healing assay and transwell assay indicated that fascin could promote cholangiocarcinoma cell migration and invasion, and a further study found that the nuclear factor-κB signaling pathway was activated after upregulation of fascin, whereas E-cadherin expression in these cells was significantly decreased. Additionally, E-cadherin expression was significantly increased after inhibiting nuclear factor-κB activity using inhibitor or small interfering RNA, and E-cadherin expression was decreased by fascin overexpression after nuclear factor-κB inhibition, suggesting that nuclear factor-κB signaling pathway was not involved in the regulation of E-cadherin by fascin. In summary, the results of this study demonstrated that fascin effectively promoted cholangiocarcinoma RBE cell proliferation, migration, and invasion. This study provides evidence for fascin as a potential target in the treatment of cholangiocarcinoma. © The Author(s) 2015.

Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

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

Matsuzaki, Shinichi; Ishizuka, Tamotsu, E-mail: tamotsui@showa.gunma-u.ac.jp; Yamada, Hidenori

Highlights: {yields} The involvement of extracellular acidification in airway remodeling was investigated. {yields} Extracellular acidification alone induced CTGF production in human ASMCs. {yields} Extracellular acidification enhanced TGF-{beta}-induced CTGF production in human ASMCs. {yields} Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. {yields} OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connectivemore » tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-{beta}-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G{sub q/11} protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP{sub 3}) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G{sub q/11} protein and inositol-1,4,5-trisphosphate-induced Ca{sup 2+} mobilization in human ASMCs.« less

Translation initiation on mRNAs bound by nuclear cap-binding protein complex CBP80/20 requires interaction between CBP80/20-dependent translation initiation factor and eukaryotic translation initiation factor 3g.

PubMed

Choe, Junho; Oh, Nara; Park, Sungjin; Lee, Ye Kyung; Song, Ok-Kyu; Locker, Nicolas; Chi, Sung-Gil; Kim, Yoon Ki

2012-05-25

In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.

The role of microtubule actin cross-linking factor 1 (MACF1) in the Wnt signaling pathway.

PubMed

Chen, Hui-Jye; Lin, Chung-Ming; Lin, Chyuan-Sheng; Perez-Olle, Raul; Leung, Conrad L; Liem, Ronald K H

2006-07-15

MACF1 (microtubule actin cross-linking factor 1) is a multidomain protein that can associate with microfilaments and microtubules. We found that MACF1 was highly expressed in neuronal tissues and the foregut of embryonic day 8.5 (E8.5) embryos and the head fold and primitive streak of E7.5 embryos. MACF1(-/-) mice died at the gastrulation stage and displayed developmental retardation at E7.5 with defects in the formation of the primitive streak, node, and mesoderm. This phenotype was similar to Wnt-3(-/-) and LRP5/6 double-knockout embryos. In the absence of Wnt, MACF1 associated with a complex that contained Axin, beta-catenin, GSK3beta, and APC. Upon Wnt stimulation, MACF1 appeared to be involved in the translocation and subsequent binding of the Axin complex to LRP6 at the cell membrane. Reduction of MACF1 with small interfering RNA decreased the amount of beta-catenin in the nucleus, and led to an inhibition of Wnt-induced TCF/beta-catenin-dependent transcriptional activation. Similar results were obtained with a dominant-negative MACF1 construct that contained the Axin-binding region. Reduction of MACF1 in Wnt-1-expressing P19 cells resulted in decreased T (Brachyury) gene expression, a DNA-binding transcription factor that is a direct target of Wnt/beta-catenin signaling and required for mesoderm formation. These results suggest a new role of MACF1 in the Wnt signaling pathway.

Arf6 Guanine Nucleotide Exchange Factor Cytohesin-2 Binds to CCDC120 and Is Transported Along Neurites to Mediate Neurite Growth*

PubMed Central

Torii, Tomohiro; Miyamoto, Yuki; Tago, Kenji; Sango, Kazunori; Nakamura, Kazuaki; Sanbe, Atsushi; Tanoue, Akito; Yamauchi, Junji

2014-01-01

The mechanism of neurite growth is complicated, involving continuous cytoskeletal rearrangement and vesicular trafficking. Cytohesin-2 is a guanine nucleotide exchange factor for Arf6, an Arf family molecular switch protein, controlling cell morphological changes such as neuritogenesis. Here, we show that cytohesin-2 binds to a protein with a previously unknown function, CCDC120, which contains three coiled-coil domains, and is transported along neurites in differentiating N1E-115 cells. Transfection of the small interfering RNA (siRNA) specific for CCDC120 into cells inhibits neurite growth and Arf6 activation. When neurites start to extend, vesicles containing CCDC120 and cytohesin-2 are transported in an anterograde manner rather than a retrograde one. As neurites continue extension, anterograde vesicle transport decreases. CCDC120 knockdown inhibits cytohesin-2 localization into vesicles containing CCDC120 and diffuses cytohesin-2 in cytoplasmic regions, illustrating that CCDC120 determines cytohesin-2 localization in growing neurites. Reintroduction of the wild type CCDC120 construct into cells transfected with CCDC120 siRNA reverses blunted neurite growth and Arf6 activity, whereas the cytohesin-2-binding CC1 region-deficient CCDC120 construct does not. Thus, cytohesin-2 is transported along neurites by vesicles containing CCDC120, and it mediates neurite growth. These results suggest a mechanism by which guanine nucleotide exchange factor for Arf6 is transported to mediate neurite growth. PMID:25326380

Insulin-like growth factor binding protein-3 induces angiogenesis through IGF-I- and SphK1-dependent mechanisms.

PubMed

Granata, R; Trovato, L; Lupia, E; Sala, G; Settanni, F; Camussi, G; Ghidoni, R; Ghigo, E

2007-04-01

Angiogenesis is critical for development and repair, and is a prominent feature of many pathological conditions. Based on evidence that insulin-like growth factor binding protein (IGFBP)-3 enhances cell motility and activates sphingosine kinase (SphK) in human endothelial cells, we have investigated whether IGFBP-3 plays a role in promoting angiogenesis. IGFBP-3 potently induced network formation by human endothelial cells on Matrigel. Moreover, it up-regulated proangiogenic genes, such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)-2 and -9. IGFBP-3 even induced membrane-type 1 MMP (MT1-MMP), which regulates MMP-2 activation. Decreasing SphK1 expression by small interfering RNA (siRNA), blocked IGFBP-3-induced network formation and inhibited VEGF, MT1-MMP but not IGF-I up-regulation. IGF-I activated SphK, leading to sphingosine-1-phosphate (S1P) formation. The IGF-I effect on SphK activity was blocked by specific inhibitors of IGF-IR, PI3K/Akt and ERK1/2 phosphorylation. The disruption of IGF-I signaling prevented the IGFBP-3 effect on tube formation, SphK activity and VEGF release. Blocking ERK1/2 signaling caused the loss of SphK activation and VEGF and IGF-I up-regulation. Finally, IGFBP-3 dose-dependently stimulated neovessel formation into subcutaneous implants of Matrigel in vivo. Thus, IGFBP-3 positively regulates angiogenesis through involvement of IGF-IR signaling and subsequent SphK/S1P activation.

The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor β-Stimulated Aortic Endothelial Cells ▿

PubMed Central

Daubon, Thomas; Buccione, Roberto; Génot, Elisabeth

2011-01-01

Podosomes are dynamic actin-rich adhesion plasma membrane microdomains endowed with extracellular matrix-degrading activities. In aortic endothelial cells, podosomes are induced by transforming growth factor β (TGF-β), but how this occurs is largely unknown. It is thought that, in endothelial cells, podosomes play a role in vessel remodeling and/or in breaching anatomical barriers. We demonstrate here that, in bovine aortic endothelial cells, that the Cdc42-specific guanine exchange factor (GEF) Fgd1 is expressed and regulated by TGF-β to induce Cdc42-dependent podosome assembly. Within 15 min of TGF-β stimulation, Fgd1, but none of the other tested Cdc42 GEFs, undergoes tyrosine phosphorylation, associates with Cdc42, and translocates to the subcortical cytoskeleton via a cortactin-dependent mechanism. Small interfering RNA-mediated Fgd1 knockdown inhibits TGF-β-induced Cdc42 activation. Fgd1 depletion also reduces podosome formation and associated matrix degradation and these defects are rescued by reexpression of Fgd1. Although overexpression of Fgd1 does not promote podosome formation per se, it enhances TGF-β-induced matrix degradation. Our results identify Fgd1 as a TGF-β-regulated GEF and, as such, the first GEF to be involved in the process of cytokine-induced podosome formation. Our findings reveal the involvement of Fgd1 in endothelial cell biology and open up new avenues to study its role in vascular pathophysiology. PMID:21911474

ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands.

PubMed

Tanaka, Motonari; Nanba, Daisuke; Mori, Seiji; Shiba, Fumio; Ishiguro, Hiroshi; Yoshino, Koichiro; Matsuura, Nariaki; Higashiyama, Shigeki

2004-10-01

A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.

Inhibition of insulin-like growth factor receptor-1 reduces necroptosis-related markers and attenuates LPS-induced lung injury in mice.

PubMed

Lee, Su Hwan; Shin, Ju Hye; Song, Joo Han; Leem, Ah Young; Park, Moo Suk; Kim, Young Sam; Chang, Joon; Chung, Kyung Soo

2018-04-15

Insulin-like growth factor-1 (IGF-1) levels are known to increase in the bronchoalveolar lavage fluid (BALF) of patients with acute respiratory distress syndrome. Herein, we investigated the role of IGF-1 in lipopolysaccharide (LPS)-induced lung injury. In LPS-treated cells, expressions of receptor-interacting protein 3 (RIP3) and phosphorylated mixed lineage kinase domain-like protein (MLKL) were decreased in IGF-1 receptor small interfering RNA (siRNA)-treated cells compared to control cells. The levels of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, tumour necrosis factor-α, and macrophage inflammatory protein 2/C-X-C motif chemokine ligand 2 in the supernatant were significantly reduced in IGF-1 receptor siRNA-treated cells compared to control cells. In LPS-induced murine lung injury model, total cell counts, polymorphonuclear leukocytes counts, and pro-inflammatory cytokine levels in the BALF were significantly lower and histologically detected lung injury was less common in the group treated with IGF-1 receptor monoclonal antibody compared to the non-treated group. On western blotting, RIP3 and phosphorylated MLKL expressions were relatively decreased in the IGF-1 receptor monoclonal antibody group compared to the non-treated group. IGF-1 may be associated with RIP3-mediated necroptosis in vitro, while blocking of the IGF-1 pathway may reduce LPS-induced lung injuries in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

Requirement for Chloride Channel Function during the Hepatitis C Virus Life Cycle

PubMed Central

Igloi, Zsofia; Mohl, Bjorn-Patrick; Lippiat, Jonathan D.; Harris, Mark

2015-01-01

Hepatocytes express an array of plasma membrane and intracellular ion channels, yet their role during the hepatitis C virus (HCV) life cycle remains largely undefined. Here, we show that HCV increases intracellular hepatic chloride (Cl−) influx that can be inhibited by selective Cl− channel blockers. Through pharmacological and small interfering RNA (siRNA)-mediated silencing, we demonstrate that Cl− channel inhibition is detrimental to HCV replication. This represents the first observation of the involvement of Cl− channels during the HCV life cycle. PMID:25609806

Sensors Locate Radio Interference

NASA Technical Reports Server (NTRS)

2009-01-01

After receiving a NASA Small Business Innovation Research (SBIR) contract from Kennedy Space Center, Soneticom Inc., based in West Melbourne, Florida, created algorithms for time difference of arrival and radio interferometry, which it used in its Lynx Location System (LLS) to locate electromagnetic interference that can disrupt radio communications. Soneticom is collaborating with the Federal Aviation Administration (FAA) to install and test the LLS at its field test center in New Jersey in preparation for deploying the LLS at commercial airports. The software collects data from each sensor in order to compute the location of the interfering emitter.

Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants

PubMed Central

Khraiwesh, Basel; Zhu, Jian-Kang; Zhu, Jianhua

2011-01-01

Small, non-coding RNAs are a distinct class of regulatory RNAs in plants and animals that control a variety of biological processes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved through a series of pathways. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs control the expression of cognate target genes by binding to reverse complementary sequences, resulting in cleavage or translational inhibition of the target RNAs. siRNAs have a similar structure, function, and biogenesis as miRNAs but are derived from long double-stranded RNAs and can often direct DNA methylation at target sequences. Besides their roles in growth and development and maintenance of genome integrity, small RNAs are also important components in plant stress responses. One way in which plants respond to environmental stress is by modifying their gene expression through the activity of small RNAs. Thus, understanding how small RNAs regulate gene expression will enable researchers to explore the role of small RNAs in biotic and abiotic stress responses. This review focuses on the regulatory roles of plant small RNAs in the adaptive response to stresses. PMID:21605713

COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis.

PubMed

Fang, Jingye; Liu, Ming; Zhang, Xuebao; Sakamoto, Takeshi; Taatjes, Douglas J; Jena, Bhanu P; Sun, Fei; Woods, James; Bryson, Tim; Kowluru, Anjaneyulu; Zhang, Kezhong; Chen, Xuequn

2015-08-01

Pancreatic β-cells possess a highly active protein synthetic and export machinery in the endoplasmic reticulum (ER) to accommodate the massive production of proinsulin. ER homeostasis is vital for β-cell functions and is maintained by the delicate balance between protein synthesis, folding, export, and degradation. Disruption of ER homeostasis by diabetes-causing factors leads to β-cell death. Among the 4 components to maintain ER homeostasis in β-cells, the role of ER export in insulin biogenesis is the least understood. To address this knowledge gap, the present study investigated the molecular mechanism of proinsulin ER export in MIN6 cells and primary islets. Two inhibitory mutants of the secretion-associated RAS-related protein (Sar)1 small GTPase, known to specifically block coat protein complex II (COPII)-dependent ER export, were overexpressed in β-cells using recombinant adenoviruses. Results from this approach, as well as small interfering RNA-mediated Sar1 knockdown, demonstrated that defective Sar1 function blocked proinsulin ER export and abolished its conversion to mature insulin in MIN6 cells, isolated mouse, and human islets. It is further revealed, using an in vitro vesicle formation assay, that proinsulin was packaged into COPII vesicles in a GTP- and Sar1-dependent manner. Blockage of COPII-dependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and β-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (p-eIF2α) and inositol-requiring protein 1 (IRE1)/x-box binding protein 1 (Xbp1) pathways but not via activating transcription factor 6 (ATF6). Collectively, results from the study demonstrate that COPII-dependent ER export plays a vital role in insulin biogenesis, ER homeostasis, and β-cell survival.

[Visual development and amblyopia prophylaxis in pediatric glaucoma].

PubMed

Steffen, H

2011-07-01

In children with congenital glaucoma the functional long-term result is often disappointing even if the intraocular pressure is well controlled. The reason for this discrepancy is attributed to amblyogenic factors responsible for interfering with normal visual development. These amblyogenic factors are corneal edema, irregular astigmatism and non-corrected ametropia as monocular causes. Binocular causes are anisometropia-induced suppression and strabismus. Full ametropic correction and a very early prophylaxis and treatment of amblyopia with a close follow-up are mandatory to reduce amblyogenic visual impairment in children with congenital glaucoma.

Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents.

PubMed

Ong, Christy C; Gierke, Sarah; Pitt, Cameron; Sagolla, Meredith; Cheng, Christine K; Zhou, Wei; Jubb, Adrian M; Strickland, Laura; Schmidt, Maike; Duron, Sergio G; Campbell, David A; Zheng, Wei; Dehdashti, Seameen; Shen, Min; Yang, Nora; Behnke, Mark L; Huang, Wenwei; McKew, John C; Chernoff, Jonathan; Forrest, William F; Haverty, Peter M; Chin, Suet-Feung; Rakha, Emad A; Green, Andrew R; Ellis, Ian O; Caldas, Carlos; O'Brien, Thomas; Friedman, Lori S; Koeppen, Hartmut; Rudolph, Joachim; Hoeflich, Klaus P

2015-04-23

Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis. PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n=980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting. We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P=1.29×10(-4) and P=0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis. Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.

Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes

PubMed Central

Watanabe, Toshiaki; Takeda, Atsushi; Tsukiyama, Tomoyuki; Mise, Kazuyuki; Okuno, Tetsuro; Sasaki, Hiroyuki; Minami, Naojiro; Imai, Hiroshi

2006-01-01

Small RNAs ranging in size between 18 and 30 nucleotides (nt) are found in many organisms including yeasts, plants, and animals. Small RNAs are involved in the regulation of gene expression through translational repression, mRNA degradation, and chromatin modification. In mammals, microRNAs (miRNAs) are the only small RNAs that have been well characterized. Here, we have identified two novel classes of small RNAs in the mouse germline. One class consists of ∼20- to 24-nt small interfering RNAs (siRNAs) from mouse oocytes, which are derived from retroelements including LINE, SINE, and LTR retrotransposons. Addition of retrotransposon-derived sequences to the 3′ untranslated region (UTR) of a reporter mRNA destabilizes the mRNA significantly when injected into full-grown oocytes. These results suggest that retrotransposons are suppressed through the RNAi pathway in mouse oocytes. The other novel class of small RNAs is 26- to 30-nt germline small RNAs (gsRNAs) from testes. gsRNAs are expressed during spermatogenesis in a developmentally regulated manner, are mapped to the genome in clusters, and have strong strand bias. These features are reminiscent of Tetrahymena ∼23- to 24-nt small RNAs and Caenorhabditis elegans X-cluster small RNAs. A conserved novel small RNA pathway may be present in diverse animals. PMID:16766679

Suppression of the epidermal growth factor receptor inhibits epithelial-mesenchymal transition in human pancreatic cancer PANC-1 cells.

PubMed

Chang, Zhi-Gang; Wei, Jun-Min; Qin, Chang-Fu; Hao, Kun; Tian, Xiao-Dong; Xie, Kun; Xie, Xue-Hai; Yang, Yin-Mo

2012-05-01

Aberrant expression of epidermal growth factor receptor (EGFR) has been detected in pancreatic cancer; however, the mechanisms of EGFR in inducing pancreatic cancer development have not been adequately elucidated. The objective of this study was to determine the role of EGFR in mediating epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. Pancreatic cancer cell line PANC-1 was transfected with small interfering RNA of EGFR by use of a lentiviral expression vector to establish an EGFR-knockdown cell line (si-PANC-1). PANC-1 cells transfected with lentiviral vector expressing negative control sequence were used as negative control (NC-PANC-1). Scratch assay and transwell study were used to analyze cell migration and invasion. Real-time PCR and Western blotting were used to detect the expression of EMT markers E-cadherin, N-cadherin, vimentin, and fibronectin and transcription factors snail, slug, twist1, and sip1 in PANC-1, NC-PANC-1, and si-PANC-1 cells. Immunofluorescent staining with these antibodies and confocal microscopy were used to observe their cellular location and morphologic changes. After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 cells decreased significantly. The expression of epithelial phenotype marker E-cadherin increased and the expression of mesenchymal phenotype markers N-cadherin, vimentin, and fibronectin decreased, indicating reversion of EMT. We also observed intracellular translocation of E-cadherin. Expression of transcription factors snail and slug in si-PANC-1 cells decreased significantly. Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 cells. The mechanism may be related with the down-regulation of the expression of transcription factors snail and slug.

Upregulation of the coagulation factor VII gene during glucose deprivation is mediated by activating transcription factor 4.

PubMed

Cronin, Katherine R; Mangan, Thomas P; Carew, Josephine A

2012-01-01

Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. Expression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/- SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/-15% to 188+/-27% and 100+/-8.8% to 176.3+/-17.3% respectively, p<0.001) at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress.

Calcitonin protects chondrocytes from lipopolysaccharide-induced apoptosis and inflammatory response through MAPK/Wnt/NF-κB pathways.

PubMed

Zhang, Lai-Bo; Man, Zhen-Tao; Li, Wei; Zhang, Wei; Wang, Xian-Quan; Sun, Shui

2017-07-01

Calcitonin (CT) is an anti-absorbent, which has long been used for treatment of osteoporosis. However, little information is available about the effects of CT on osteoarthritis (OA). This study was mainly aimed to explore the effects of CT on the treatment of OA, as well as the underlying mechanisms. Chondrocytes were isolated from immature mice and then were incubated with lipopolysaccharide (LPS), CT, small interfering (si) RNA against bone morphogenetic protein (BMP)-2, and/or the inhibitors of MAPK/Wnt/NF-κB pathway. Thereafter, cell viability, apoptosis, nitric oxide (NO) and inflammatory factors productions, and expression levels of cartilage synthesis protein key factors, cartilage-derived morphogenetic protein (CDMP) 1, SRY (sex-determining region Y)-box 9 protein (SOX9), and MAPK/Wnt/NF-κB pathways key factors were determined. CT significantly reversed LPS-induced cell viability decrease, apoptosis increase, the inflammatory factors and NO secretion, the abnormally expression of cartilage synthesis proteins and the activation of MAPK/Wnt/NF-κB pathways (P<0.05). In addition, we observed that administration of the inhibitors of MAPK/Wnt/NF-κB pathways statistically further increased the levels of CDMP1 and SOX9 (P<0.05). Suppression of BMP-2 decreased the levels of CDMP1 and SOX9 and activated MAPK/Wnt/NF-κB pathways, and could partially abolish CT-modulated the expression changes in CDMP1 and SOX9, and MAPK/Wnt/NF-κB pathways key factors (P<0.05). The results showed that CT protects chondrocytes from LPS-induced apoptosis and inflammatory response by regulating BMP-2 and thus blocking MAPK/Wnt/NF-κB pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tumor necrosis factor alpha induces gamma-glutamyltransferase expression via nuclear factor-kappaB in cooperation with Sp1.

PubMed

Reuter, Simone; Schnekenburger, Michael; Cristofanon, Silvia; Buck, Isabelle; Teiten, Marie-Hélène; Daubeuf, Sandrine; Eifes, Serge; Dicato, Mario; Aggarwal, Bharat B; Visvikis, Athanase; Diederich, Marc

2009-02-01

Gamma-glutamyltransferase (GGT) cleaves the gamma-glutamyl moiety of glutathione (GSH), an endogenous antioxidant, and is involved in mercapturic acid metabolism and in cancer drug resistance when overexpressed. Moreover, GGT converts leukotriene (LT) C4 into LTD4 implicated in various inflammatory pathologies. So far the effect of inflammatory stimuli on regulation of GGT expression and activity remained to be addressed. We found that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) induced GGT promoter transactivation, mRNA and protein synthesis, as well as enzymatic activity. Remicade, a clinically used anti-TNFalpha antibody, small interfering RNA (siRNA) against p50 and p65 nuclear factor-kappaB (NF-kappaB) isoforms, curcumin, a well characterized natural NF-kappaB inhibitor, as well as a dominant negative inhibitor of kappaB alpha (IkappaBalpha), prevented GGT activation at various levels, illustrating the involvement of this signaling pathway in TNFalpha-induced stimulation. Over-expression of receptor of TNFalpha-1 (TNFR1), TNFR-associated factor-2 (TRAF2), TNFR-1 associated death domain (TRADD), dominant negative (DN) IkappaBalpha or NF-kappaB p65 further confirmed GGT promoter activation via NF-kappaB. Linker insertion mutagenesis of 536 bp of the proximal GGT promoter revealed NF-kappaB and Sp1 binding sites at -110 and -78 relative to the transcription start site, responsible for basal GGT transcription. Mutation of the NF-kappaB site located at -110 additionally inhibited TNFalpha-induced promoter induction. Chromatin immunoprecipitation (ChIP) assays confirmed mutagenesis results and further demonstrated that TNFalpha treatment induced in vivo binding of both NF-kappaB and Sp1, explaining increased GGT expression, and led to RNA polymerase II recruitment under inflammatory conditions.

Upregulation of the Coagulation Factor VII Gene during Glucose Deprivation Is Mediated by Activating Transcription Factor 4

PubMed Central

Cronin, Katherine R.; Mangan, Thomas P.; Carew, Josephine A.

2012-01-01

Background Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. Methodology/Principal Findings Expression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/− SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/−15% to 188+/−27% and 100+/−8.8% to 176.3+/−17.3% respectively, p<0.001) at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. Conclusions/Significance Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress. PMID:22848420

Steps and Tools to Guide Planning and Implementation of a Comprehensive System to Address Barriers to Learning and Teaching

ERIC Educational Resources Information Center

Center for Mental Health in Schools at UCLA, 2007

2007-01-01

The data are clear: Too many students are not doing well in school. Too many are experiencing interfering barriers, most of which are not internal dysfunctions but are associated with neighborhood, family, school, and peer factors. If the situation is to change, schools must play a greater role in providing supports for students experiencing…

Comparison of Available Technologies for Fire Spots Detection via Linear Heat Detector

NASA Astrophysics Data System (ADS)

Miksa, František; Nemlaha, Eduard

2016-12-01

It is very demanding to detect fire spots under difficult conditions with high occurrence of interfering external factors such as large distances, airflow difficultly, high dustiness, high humidity, etc. Spot fire sensors do not meet the requirements due to the aforementioned conditions as well as large distances. Therefore, the detection of a fire spot via linear heat sensing cables is utilized.

Post-exposure treatments for Ebola and Marburg virus infections.

PubMed

Cross, Robert W; Mire, Chad E; Feldmann, Heinz; Geisbert, Thomas W

2018-06-01

The filoviruses - Ebola virus and Marburg virus - cause lethal haemorrhagic fever in humans and non-human primates (NHPs). Filoviruses present a global health threat both as naturally acquired diseases and as potential agents of bioterrorism. In the recent 2013-2016 outbreak of Ebola virus, the most promising therapies for post-exposure use with demonstrated efficacy in the gold-standard NHP models of filovirus disease were unable to show statistically significant protection in patients infected with Ebola virus. This Review briefly discusses these failures and what has been learned from these experiences, and summarizes the current status of post-exposure medical countermeasures in development, including antibodies, small interfering RNA and small molecules. We outline how our current knowledge could be applied to the identification of novel interventions and ways to use interventions more effectively.

On the effect of tilted roof reflectors in Martin-Puplett spectrometers

NASA Astrophysics Data System (ADS)

Schillaci, Alessandro; de Bernardis, Paolo

2012-01-01

In this paper we analyze theoretically and experimentally the effect of tilt of the roof mirrors in a double pendulum Martin-Puplett Polarizing Interferometer (MPI), focusing on the polarization of the interfering beams. In principle, the tilt affects the efficiency and polarimetric properties of the interferometer. The case of a moderate resolution spectrometer is analysed in detail. Using the Stokes formalism we recover the analytical expressions for the orientation angle and the ellipticity of the beam reflected from a metallic surface, and we compute these quantities for the roof-mirror of a MPI. We find that the polarization rotation and depolarization are small. Using the Jones formalism we propagate their effect on the measured interferogram and spectrum, and demonstrate that the performance degradation is small compared to other systematic effects.

Gas Chromatographic/Mass Spectrometric Differentiation of Atenolol, Metoprolol, Propranolol, and an Interfering Metabolite Product of Metoprolol

DTIC Science & Technology

2004-10-01

Gas Chromatographic/Mass Spectrometric Differentiation of Atenolol, Metoprolol , Propranolol, and an Interfering Metabolite Product of Metoprolol ...4. Title and Subtitle 5. Report Date October 2004 Gas Chromatographic/Mass Spectrometric Differentiation of Atenolol, Metoprolol , Propranolol...and an Interfering Metabolite Product of Metoprolol 6. Performing Organization Code 7. Author(s) 8. Performing Organization Report No. Angier MK

A small RNA activates CFA synthase by isoform-specific mRNA stabilization

PubMed Central

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-01-01

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability. PMID:24141880

A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

PubMed

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-11-13

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

[The influence of interfered circadian rhythm on pregnancy and neonatal rats].

PubMed

Chen, Wen-Jun; Sheng, Wen-Jie; Guo, Yin-Hua; Tan, Yong

2015-10-25

The aim of this study was to observe the influence of interfered circadian rhythm on pregnancy of rats and growth of neonatal rats, and to explore the relationship between the interfered circadian rhythm and the changes of melatonin and progesterone. Continuous light was used to inhibit melatonin secretion and therefore the interfered circadian rhythm animal model was obtained. The influence of interfered circadian rhythm on delivery of pregnant rats was observed. Serum was collected from rats during different stages of pregnancy to measure the concentrations of melatonin and progesterone. In order to observe the embryo resorption rate, half of pregnant rats were randomly selected to undergo a laparotomy, and the remainder was used to observe delivery and assess the growth of neonatal rats after delivery. The results showed that the interfered circadian rhythm induced adverse effects on pregnancy outcomes, including an increase of embryo resorption rate and a decrease in the number of live births; inhibited the secretion of melatonin along with decreased serum progesterone level; prolonged the stage of labor, but not the duration of pregnancy; and disturbed the fetal intrauterine growth and the growth of neonatal rats. The results suggest that interfered circadian rhythm condition made by continuous light could make adverse effects on both pregnant rats and neonatal rats. The results of our study may provide a way to modulate pregnant women's circadian rhythm and a possibility of application of melatonin on pregnant women.

Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies

PubMed Central

Kaur, Randeep; Chitanda, Jackson M; Michel, Deborah; Maley, Jason; Borondics, Ferenc; Yang, Peng; Verrall, Ronald E; Badea, Ildiko

2012-01-01

Purpose: Detonation nanodiamonds (NDs) are carbon-based nanomaterials that, because of their size (4–5 nm), stable inert core, alterable surface chemistry, fluorescence, and biocompatibility, are emerging as bioimaging agents and promising tools for the delivery of biochemical molecules into cellular systems. However, diamond particles possess a strong propensity to aggregate in liquid formulation media, restricting their applicability in biomedical sciences. Here, the authors describe the covalent functionalization of NDs with lysine in an attempt to develop nanoparticles able to act as suitable nonviral vectors for transferring genetic materials across cellular membranes. Methods: NDs were oxidized and functionalized by binding lysine moieties attached to a three-carbon-length linker (1,3-diaminopropane) to their surfaces through amide bonds. Raman and Fourier transform infrared spectroscopy, zeta potential measurement, dynamic light scattering, atomic force microscopic imaging, and thermogravimetric analysis were used to characterize the lysine-functionalized NDs. Finally, the ability of the functionalized diamonds to bind plasmid DNA and small interfering RNA was investigated by gel electrophoresis assay and through size and zeta potential measurements. Results: NDs were successfully functionalized with the lysine linker, producing surface loading of 1.7 mmol g−1 of ND. These modified NDs formed highly stable aqueous dispersions with a zeta potential of 49 mV and particle size of approximately 20 nm. The functionalized NDs were found to be able to bind plasmid DNA and small interfering RNA by forming nanosized “diamoplexes”. Conclusion: The lysine-substituted ND particles generated in this study exhibit stable aqueous formulations and show potential for use as carriers for genetic materials. PMID:22904623

The high mobility group AT-hook 1 protein stimulates bovine herpesvirus 1 productive infection.

PubMed

Zhu, Liqian; Jones, Clinton

2017-06-15

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle that causes clinical symptoms in the upper respiratory tract and conjunctivitis. Like most alpha-herpesvirinae subfamily members, BoHV-1 establishes latency in sensory neurons. Stress consistently induces reactivation from latency, which is essential for virus transmission. Recent studies demonstrated that a viral protein (ORF2) expressed in a subset of latently infected neurons is associated with β-catenin and the high mobility group AT-hook 1 protein (HMGA1), which correlates with increased expression of these proteins in latently infected neurons. Since HMGA1 is primarily expressed in actively growing cells, binds to the minor groove of A+T rich regions in double-stranded DNA, and mediates gene transcription, we hypothesized that HMGA1 regulates BoHV-1 productive infection. Studies in this report indicated that productive infection increased HMGA1 protein levels and re-localized the protein in the nucleus. Netropsin, a small molecule that binds to the minor groove of DNA and prevents HMGA1 from interacting with DNA inhibited viral replication and interfered with the ability of BoHV-1 to induce HMGA1 re-localization. Furthermore, netropsin reduced RNA and protein expression of two viral regulatory proteins (bICP0 and bICP22) during productive infection, but increased bICP4 levels. Small interfering RNAs (siRNAs) that specifically target HMGA1 reduced HMGA1 RNA levels and virus production confirming HMGA1 stimulates productive infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Local matrix metalloproteinase 2 gene knockdown in balloon-injured rabbit carotid arteries using nonviral-small interfering RNA transfection.

PubMed

Hlawaty, Hanna; San Juan, Aurélie; Jacob, Marie-Paule; Vranckx, Roger; Letourneur, Didier; Feldman, Laurent J

2009-01-01

Small interfering RNA (siRNA) delivery is a promising approach for the treatment of cardiovascular diseases. Matrix metalloproteinase (MMP) 2 over-expression in the arterial wall has been implicated in restenosis after percutaneous coronary intervention, as well as in spontaneous atherosclerotic plaque rupture. We hypothesized that in vivo local delivery of siRNA targeted at MMP2 (MMP2-siRNA) in the balloon-injured carotid artery of hypercholesterolemic rabbits may lead to inhibition of MMP2 expression. Two weeks after balloon injury, 5 micromol/l of Tamra-tagged MMP2-siRNA, scramble siRNA or saline was locally injected in the carotid artery and incubated for 1 h. Fluorescent microscopy studies showed the circumferential uptake of siRNA in the superficial layers of neointimal cells. MMP2 mRNA levels, measured by the real-time reverse transcriptase-polymerase chain reaction, was decreased by 79 +/- 25% in MMP2-siRNA- versus scramble siRNA-transfected arteries (p < 0.05). MMP2 activity, measured by gelatin zymography performed on the conditioned media of MMP2-siRNA versus scramble siRNA transfected arteries, decreased by 53 +/- 29%, 50 +/- 24% and 46 +/- 14% at 24, 48 and 72 h, respectively (p < 0.005 for all). No effect was observed on MMP9, pro-MMP9 and TIMP-2 levels. The results obtained in the present study suggest that significant inhibition of gene expression can be achieved with local delivery of siRNA in the arterial wall in vivo. (c) 2008 John Wiley & Sons, Ltd.

Small interfering RNA against the 2C genomic region of coxsackievirus B3 exerts potential antiviral effects in permissive HeLa cells.

PubMed

Luan, Ying; Dai, Hai-Li; Yang, Dan; Zhu, Lin; Gao, Tie-Lei; Shao, Hong-Jiang; Peng, Xue; Jin, Zhan-Feng

2012-01-01

Coxsackievirus B3 (CVB3) is the most important causal agent of viral heart muscle disease, but no specific antiviral drug is currently available. Small interfering RNA (siRNA) has been used as an antiviral therapeutic strategy via posttranscriptional gene silencing. In this study, eleven siRNAs were designed to target seven distinct regions of the CVB3 genome including VP1, VP2, VP3, 2A, 2C, 3C, and 3D. All of the siRNAs were individually transfected into HeLa cells, which were subsequently infected with CVB3. The impacts of RNA interference (RNAi) on viral replication were evaluated using five measures: cytopathic effect (CPE), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 50% tissue culture infectious dose (TCID(50)), real-time RT-PCR, and Western blot. Five of the eleven siRNAs were highly efficient at inhibiting viral replication. This was especially true for siRNA-5, which targeted the ATPase 2C. However, antiviral activity varied significantly among siRNA-9, -10, and -11 even though that they all targeted the 3D region. Our results revealed several effective targets for CVB3 silencing, and provided evidence that sequences except CRE within the 2C region may also be potential targets for CVB3-specific siRNAs design. These data supported a potential role of RNA interference in future antiviral intervention therapies. Copyright © 2011 Elsevier B.V. All rights reserved.

Antiviral Effects of Small Interfering RNA Simultaneously Inducing RNA Interference and Type 1 Interferon in Coxsackievirus Myocarditis

PubMed Central

Ahn, Jeonghyun; Ko, Ara; Jun, Eun Jung; Won, Minah; Kim, Yoo Kyum; Ju, Eun-Seon

2012-01-01

Antiviral therapeutics are currently unavailable for treatment of coxsackievirus B3, which can cause life-threatening myocarditis. A modified small interfering RNA (siRNA) containing 5′-triphosphate, 3p-siRNA, was shown to induce RNA interference and interferon activation. We aimed to develop a potent antiviral treatment using CVB3-specific 3p-siRNA and to understand its underlying mechanisms. Virus-specific 3p-siRNA was superior to both conventional virus-specific siRNA with an empty hydroxyl group at the 5′ end (OH-siRNA) and nonspecific 3p-siRNA in decreasing viral replication and subsequent cytotoxicity. A single administration of 3p-siRNA dramatically attenuated virus-associated pathological symptoms in mice with no signs of toxicity, and their body weights eventually reached the normal range. Myocardial inflammation and fibrosis were rare, and virus production was greatly reduced. A nonspecific 3p-siRNA showed relatively less protective effect under identical conditions, and a virus-specific OH-siRNA showed no protective effects. We confirmed that virus-specific 3p-siRNA simultaneously activated target-specific gene silencing and type I interferon signaling. We provide a clear proof of concept that coxsackievirus B3-specific 3p-siRNA has 2 distinct modes of action, which significantly enhance antiviral activities with minimal organ damage. This is the first direct demonstration of improved antiviral effects with an immunostimulatory virus-specific siRNA in coxsackievirus myocarditis, and this method could be applied to many virus-related diseases. PMID:22508300

Therapeutic effects of protein kinase N3 small interfering RNA and doxorubicin combination therapy on liver and lung metastases

PubMed Central

Hattori, Yoshiyuki; Kikuchi, Takuto; Nakamura, Mari; Ozaki, Kei-Ichi; Onishi, Hiraku

2017-01-01

It has been reported that suppression of protein kinase N3 (PKN3) expression in vascular and lymphatic endothelial cells results in the inhibition of tumor progression and lymph node metastasis formation. The present study investigated whether combination therapy of small interfering RNA (siRNA) against PKN3 and doxorubicin (DXR) could increase therapeutic efficacy against liver and lung metastases. In vitro transfection of PKN3 siRNA into PKN3-positive MDA-MB-231, LLC, and Colon 26 cells and PKN3-negative MCF-7 cells did not inhibit cell growth and did not increase sensitivity to DXR. However, following in vivo treatment, PKN3 siRNA suppressed the growth of liver MDA-MB-231 and lung LLC and MCF-7 metastases, although combination therapy with DXR did not increase the therapeutic efficacy. By contrast, in liver MCF-7 metastases, PKN3 siRNA or DXR alone did not exhibit significant inhibition of tumor growth, but their combination significantly improved therapeutic efficacy. Treatment of liver MDA-MB-231 metastases with PKN3 siRNA induced a change in vasculature structure via suppression of PKN3 mRNA expression. PKN3 siRNA may induce antitumor effects in lung and liver metastases by suppression of PKN3 expression in stroma cells, such as endothelial cells. From these findings, PKN3 siRNA alone or in combination with DXR may reduce the tumor growth of liver and lung metastases regardless of PKN3 expression in tumor cells. PMID:29098022

Inhibition of coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand.

PubMed

Yuan, Ji; Cheung, Paul K M; Zhang, Huifang M; Chau, David; Yang, Decheng

2005-02-01

Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic potential. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4 was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence match in the central region of the target, but mismatches were more tolerated near the 3' end than the 5' end of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new possibility for antiviral intervention.

Ultrasound-microbubble-mediated intercellular adhesion molecule-1 small interfering ribonucleic acid transfection attenuates neointimal formation after arterial injury in mice.

PubMed

Suzuki, Jun-ichi; Ogawa, Masahito; Takayama, Kiyoshi; Taniyama, Yoshiaki; Morishita, Ryuichi; Hirata, Yasunobu; Nagai, Ryozo; Isobe, Mitsuaki

2010-03-02

The purpose of this study was to investigate the efficiency of small interfering ribonucleic acid (siRNA) in murine arteries. We transfected it using a nonviral ultrasound-microbubble-mediated in vivo gene delivery system. siRNA is an effective methodology to suppress gene function. The siRNA can be synthesized easily; however, a major obstacle in the use of siRNA as therapeutics is the difficulty involved in effective in vivo delivery. To investigate the efficiency of nonviral ultrasound-microbubble-mediated in vivo siRNA delivery, we used a fluorescein-labeled siRNA, green fluorescent protein (GFP) siRNA, and intercellular adhesion molecule (ICAM)-1 siRNA in murine arteries. Murine femoral arteries were injured using flexible wires to establish arterial injury. The fluorescein-labeled siRNA and GFP siRNA showed that this nonviral approach could deliver siRNA into target arteries effectively without any tissue damage and systemic adverse effects. ICAM-1 siRNA transfection into murine injured arteries significantly suppressed the development of neointimal formation in comparison to those in the control group. Immunohistochemistry revealed that accumulation of T cells and adhesion molecule positive cells was observed in nontreated injured arteries, whereas siRNA suppressed accumulation. The nonviral ultrasound-microbubble delivery of siRNA ensures effective transfection into target arteries. ICAM-1 siRNA has the potential to suppress arterial neointimal formation. Transfection of siRNA can be beneficial for the clinical treatment of cardiovascular and other inflammatory diseases. Copyright 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Korean Red Ginseng inhibits apoptosis in neuroblastoma cells via estrogen receptor β-mediated phosphatidylinositol-3 kinase/Akt signaling

PubMed Central

Nguyen, Cuong Thach; Luong, Truc Thanh; Kim, Gyu-Lee; Pyo, Suhkneung; Rhee, Dong-Kwon

2014-01-01

Background Ginseng has been shown to exert antistress effects both in vitro and in vivo. However, the effects of ginseng on stress in brain cells are not well understood. This study investigated how Korean Red Ginseng (KRG) controls hydrogen peroxide-induced apoptosis via regulation of phosphatidylinositol-3 kinase (PI3K)/Akt and estrogen receptor (ER)-β signaling. Methods Human neuroblastoma SK-N-SH cells were pretreated with KRG and subsequently exposed to H2O2. The ability of KRG to inhibit oxidative stress-induced apoptosis was assessed in MTT cytotoxicity assays. Apoptotic protein expression was examined by Western blot analysis. The roles of ER-β, PI3K, and p-Akt signaling in KRG regulation of apoptosis were studied using small interfering RNAs and/or target antagonists. Results Pretreating SK-N-SH cells with KRG decreased expression of the proapoptotic proteins p-p53 and caspase-3, but increased expression of the antiapoptotic protein BCL2. KRG pretreatment was also associated with increased ER-β, PI3K, and p-Akt expression. Conversely, ER-β inhibition with small interfering RNA or inhibitor treatment increased p-p53 and caspase-3 levels, but decreased BCL2, PI3K, and p-Akt expression. Moreover, inhibition of PI3K/Akt signaling diminished p-p53 and caspase-3 levels, but increased BCL2 expression. Conclusion Collectively, the data indicate that KRG represses oxidative stress-induced apoptosis by enhancing PI3K/Akt signaling via upregulation of ER-β expression. PMID:25535479

Function and Evolution of a MicroRNA That Regulates a Ca2+-ATPase and Triggers the Formation of Phased Small Interfering RNAs in Tomato Reproductive Growth[W][OA

PubMed Central

Wang, Ying; Itaya, Asuka; Zhong, Xuehua; Wu, Yang; Zhang, Jianfeng; van der Knaap, Esther; Olmstead, Richard; Qi, Yijun; Ding, Biao

2011-01-01

MicroRNAs (miRNAs) regulate a wide variety of biological processes in most eukaryotes. We investigated the function and evolution of miR4376 in the family Solanaceae. We report that the 22-nucleotide miR4376 regulates the expression of an autoinhibited Ca2+-ATPase, tomato (Solanum lycopersicum) ACA10, which plays a critical role in tomato reproductive growth. Deep phylogenetic mapping suggested (1) an evolution course of MIR4376 loci and posttranscriptional processing of pre-miR4376 as a likely limiting step for the evolution of miR4376, (2) an independent phylogenetic origin of the miR4376 target site in ACA10 homologs, and (3) alternative splicing as a possible mechanism of eliminating such a target in some ACA10 homologs. Furthermore, miR4376 triggers the formation of phased small interfering RNAs (siRNAs) from Sl ACA10 and its Solanum tuberosum homolog. Together, our data provide experimental evidence of miRNA-regulated expression of universally important Ca2+-ATPases. The miR4376-regulated expression of ACA10 itself, and possibly also the associated formation of phased siRNAs, may function as a novel layer of molecular mechanisms underlying tomato reproductive growth. Finally, our data suggest that the stochastic emergence of a miRNA-target gene combination involves multiple molecular events at the genomic, transcriptional, and posttranscriptional levels that may vary drastically in even closely related species. PMID:21917547

Ubiquitin-proteasomal degradation of COX-2 in TGF-β stimulated human endometrial cells is mediated through endoplasmic reticulum mannosidase I.

PubMed

Singh, Mohan; Chaudhry, Parvesh; Parent, Sophie; Asselin, Eric

2012-01-01

Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-β. In addition, each TGF-β isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways.

Upregulated INHBA Expression May Promote Cell Proliferation and Is Associated with Poor Survival in Lung Adenocarcinoma1

PubMed Central

Seder, Christopher W; Hartojo, Wibisono; Lin, Lin; Silvers, Amy L; Wang, Zhuwen; Thomas, Dafydd G; Giordano, Thomas J; Chen, Guoan; Chang, Andrew C; Orringer, Mark B; Beer, David G

2009-01-01

Introduction The expression, mechanisms of regulation, and functional impact of INHBA (activin A) in lung adenocarcinoma (AD) have not been fully elucidated. Methods INHBA expression was examined in 96 lung samples (86 ADs, 10 normal lung) using oligonucleotide microarrays and 187 lung samples (164 ADs, 6 bronchioalveolar carcinomas, and 17 normal lung) using immunohistochemistry. The proliferation of AD cell lines H460 and SKLU1 was examined with WST-1 assays after treatment with recombinant activin A, follistatin, and INHBA-targeting small-interfering RNA. Cells were also treated with 5-aza-2′ deoxycytidine and trichostatin A to investigate the role of epigenetic regulation in INHBA expression. Results Primary ADs expressed 3.1 times more INHBA mRNA than normal lung. In stage I AD patients, high levels of primary tumor INHBA transcripts were associated with worse prognosis. Immunohistochemistry confirmed higher inhibin βA protein expression in ADs (78.7%) and bronchioalveolar carcinomas (66.7%) compared with normal lung (11.8%). H460 and SKLU1 demonstrated increased proliferation when treated with exogenous activin A and reduced proliferation when treated with follistatin or INHBA-targeting small-interfering RNA. INHBA mRNA expression in H460 cells was upregulated after treatment with trichostatin A and 5-aza-2′ deoxycytidine. Conclusions INHBA is overexpressed in AD relative to controls. Inhibin βA may promote cell proliferation, and its overexpression is associated with worse survival in stage I AD patients. In addition, overexpression of INHBA may be affected by promoter methylation and histone acetylation in a subset of lung ADs. PMID:19308293

Effective inhibition of HIV-1 production by short hairpin RNAs and small interfering RNAs targeting a highly conserved site in HIV-1 Gag RNA is optimized by evaluating alternative length formats.

PubMed

Scarborough, Robert J; Adams, Kelsey L; Daher, Aïcha; Gatignol, Anne

2015-09-01

We have previously identified a target site in HIV-1 RNA that was particularly accessible to a ribozyme and a short hairpin RNA (shRNA). To design small interfering RNAs (siRNAs) targeting this site, we evaluated the effects of siRNAs with different lengths on HIV-1 production. The potency and efficacy of these siRNAs were dependent on the length of their intended sense strand with trends for symmetrical and asymmetrical formats that were similar. Although a typical canonical format with a 21-nucleotide (nt) sense strand was effective at inhibiting HIV-1 production, Dicer substrate siRNAs (dsiRNAs) with the longest lengths (27 to 29 nucleotides) were the most effective. Induction of double-stranded RNA immune responses and effects on cell viability were not detected in cells transfected with different siRNAs, suggesting that the differences observed were not related to indirect effects on HIV-1 production. For the corresponding shRNA designs, a different trend in potency and efficacy against HIV-1 production was observed, with the most effective shRNAs having stem lengths from 20 to 27 bp. Our results highlight the importance of evaluating different designs to identify the best siRNA and shRNA formats for any particular target site and provide a set of highly effective molecules for further development as drug and gene therapies for HIV-1 infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Phosphorylation state of mu-opioid receptor determines the alternative recycling of receptor via Rab4 or Rab11 pathway.

PubMed

Wang, Feifei; Chen, Xiaoqing; Zhang, Xiaoqing; Ma, Lan

2008-08-01

Agonist-induced phosphorylation, internalization, and intracellular trafficking of G protein-coupled receptors are critical in regulating both cellular responsiveness and signal transduction. The current study investigated the role of receptor phosphorylation state in regulation of agonist-induced internalization and intracellular trafficking of mu-opioid receptor (MOR). Our results showed that after agonist stimulation, the recycle of a mutant MOR that lacks the C-terminal residues after Asn(362) (MOR362T) was greatly decreased, whereas a C-terminal phosphorylation sites-mutated MOR (MOR3A), which is deficient in agonist-induced phosphorylation recycled back to the membrane at a level comparable to that of the wild-type receptor, however, interestingly at a slower rate. Inhibition of functions of either Rab4 or Rab11 by dominant-negative mutants and small interfering RNA both significantly impaired the recycling of the wild-type MOR, whereas the recycling of the phosphorylation-deficient mutant was only inhibited by the dominant-negative mutant and small interfering RNA of Rab11, suggesting that the recycling of nonphosphorylated MOR is exclusively via Rab11-mediated pathway. Furthermore, phosphorylated MOR was observed accumulated in Rab5- and Rab4-, but not Rab11-positive vesicles. Our data indicate that both phosphorylated and nonphosphorylated MOR internalize via Rab5-dependent pathway after agonist stimulation, and the phosphorylated and nonphosphorylated MORs recycle through distinct vesicular trafficking pathways mediated by Rab4 and Rab11, respectively, which may ultimately lead to differential cellular responsiveness or downstream signaling.

The Application of Clinical Lithotripter Shock Waves to RNA Nucleotide Delivery to Cells.

PubMed

Nwokeoha, Sandra; Carlisle, Robert; Cleveland, Robin O

2016-10-01

The delivery of genes into cells through the transfer of ribonucleic acids (RNAs) has been found to cause a change in the level of target protein expression. RNA-based transfection is conceptually more efficient than commonly delivered plasmid DNA because it does not require division or damage of the nuclear envelope, thereby increasing the chances of the cell remaining viable. Shock waves (SWs) have been found to induce cellular uptake by transiently altering the permeability of the plasma membrane, thereby overcoming a critical step in gene therapy. However, accompanying SW bio-effects include dose-dependent irreversible cell injury and cytotoxicity. Here, the effect of SWs generated by a clinical lithotripter on the viability and permeabilisation of three different cell lines in vitro was investigated. Comparison of RNA stability before and after SW exposure revealed no statistically significant difference. Optimal SW exposure parameters were identified to minimise cell death and maximise permeabilisation, and applied to enhanced green fluorescent protein (eGFP) messenger RNA (mRNA) or anti-eGFP small interfering RNA delivery. As a result, eGFP mRNA expression levels increased up to 52-fold in CT26 cells, whereas a 2-fold decrease in GFP expression was achieved after anti-eGFP small interfering RNA delivery to MCF-7/GFP cells. These results indicate that SW parameters can be employed to achieve effective nucleotide delivery, laying the foundation for non-invasive and high-tolerability RNA-based gene therapy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Role of the Trypanosoma brucei HEN1 Family Methyltransferase in Small Interfering RNA Modification

PubMed Central

Shi, Huafang; Barnes, Rebecca L.; Carriero, Nicholas; Atayde, Vanessa D.

2014-01-01

Parasitic protozoa of the flagellate order Kinetoplastida represent one of the deepest branches of the eukaryotic tree. Among this group of organisms, the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser degree in Leishmania (Viannia) spp. The pathway is triggered by long double-stranded RNA (dsRNA) and in T. brucei requires a set of five core genes, including a single Argonaute (AGO) protein, T. brucei AGO1 (TbAGO1). The five genes are conserved in Leishmania (Viannia) spp. but are absent in other major kinetoplastid species, such as Trypanosoma cruzi and Leishmania major. In T. brucei small interfering RNAs (siRNAs) are methylated at the 3′ end, whereas Leishmania (Viannia) sp. siRNAs are not. Here we report that T. brucei HEN1, an ortholog of the metazoan HEN1 2′-O-methyltransferases, is required for methylation of siRNAs. Loss of TbHEN1 causes a reduction in the length of siRNAs. The shorter siRNAs in hen1−/− parasites are single stranded and associated with TbAGO1, and a subset carry a nontemplated uridine at the 3′ end. These findings support a model wherein TbHEN1 methylates siRNA 3′ ends after they are loaded into TbAGO1 and this methylation protects siRNAs from uridylation and 3′ trimming. Moreover, expression of TbHEN1 in Leishmania (Viannia) panamensis did not result in siRNA 3′ end methylation, further emphasizing mechanistic differences in the trypanosome and Leishmania RNAi mechanisms. PMID:24186950

Correlation of LNCR rasiRNAs Expression with Heterochromatin Formation during Development of the Holocentric Insect Spodoptera frugiperda

PubMed Central

Stanojcic, Slavica; Gimenez, Sylvie; Permal, Emmanuelle; Cousserans, François; Quesneville, Hadi; Fournier, Philippe; d'Alençon, Emmanuelle

2011-01-01

Repeat-associated small interfering RNAs (rasiRNAs) are derived from various genomic repetitive elements and ensure genomic stability by silencing endogenous transposable elements. Here we describe a novel subset of 46 rasiRNAs named LNCR rasiRNAs due to their homology with one long non-coding RNA (LNCR) of Spodoptera frugiperda. LNCR operates as the intermediate of an unclassified transposable element (TE-LNCR). TE-LNCR is a very invasive transposable element, present in high copy numbers in the S. frugiperda genome. LNCR rasiRNAs are single-stranded RNAs without a prominent nucleotide motif, which are organized in two distinct, strand-specific clusters. The expression of LNCR and LNCR rasiRNAs is developmentally regulated. Formation of heterochromatin in the genomic region where three copies of the TE-LNCR are embedded was followed by chromatin immunoprecipitation (ChIP) and we observed this chromatin undergo dynamic changes during development. In summary, increased LNCR expression in certain developmental stages is followed by the appearance of a variety of LNCR rasiRNAs which appears to correlate with subsequent accumulation of a heterochromatic histone mark and silencing of the genomic region with TE-LNCR. These results support the notion that a repeat-associated small interfering RNA pathway is linked to heterochromatin formation and/or maintenance during development to establish repression of the TE-LNCR transposable element. This study provides insights into the rasiRNA silencing pathway and its role in the formation of fluctuating heterochromatin during the development of one holocentric organism. PMID:21980354

Escherichia coli K1 promotes the ligation of CD47 with thrombospondin-1 to prevent the maturation of dendritic cells in the pathogenesis of neonatal meningitis.

PubMed

Mittal, Rahul; Gonzalez-Gomez, Ignacio; Prasadarao, Nemani V

2010-09-01

Dendritic cells (DCs) are professional APCs providing a critical link between adaptive and innate immune responses. Our previous studies have shown that Escherichia coli K1 internalization of myeloid DCs suppressed the maturation of the cells for which outer membrane protein A (OmpA) expression is essential. In this study, we demonstrate that infection of DCs with OmpA(+) E. coli significantly upregulates the expression of CD47, an integrin-associated protein, and its natural ligand thrombospondin 1 (TSP-1). Pretreatment of DCs with anti-CD47 blocking Ab or knocking down the expression of CD47 or TSP-1, but not signal regulatory protein alpha by small interfering RNA, abrogated the suppressive effect of E. coli K1. Ligation of CD47 with a mAb prevented the maturation and cytokine production by DCs upon stimulation with LPS similar to the inhibitory effect induced by OmpA(+) E. coli. In agreement with the in vitro studies, suppression of CD47 or TSP-1 expression in newborn mice by a novel in vivo small interfering RNA technique protected the animals against E. coli K1 meningitis. Reconstitution of CD47 knockdown mice with CD47(+) DCs renders the animals susceptible to meningitis by E. coli K1, substantiating the role of CD47 expression in DCs for the occurrence of meningitis. Our results demonstrate a role for CD47 for the first time in bacterial pathogenesis and may be a novel target for designing preventive approaches for E. coli K1 meningitis.

One-pot synthesis of pH-responsive hybrid nanogel particles for the intracellular delivery of small interfering RNA

PubMed Central

Parodi, Alessandro; Evangelopoulos, Michael; Corbo, Claudia; Scaria, Shilpa; Hu, Ye; Haddix, Seth G.; Corradetti, Bruna; Salvatore, Francesco; Tasciotti, Ennio

2016-01-01

This report describes a novel, one-pot synthesis of hybrid nanoparticles formed by a nanostructured inorganic silica core and an organic pH-responsive hydrogel shell. This easy-to-perform, oil-in-water emulsion process synthesizes fluorescently-doped silica nanoparticles wrapped within a tunable coating of cationic poly(2-diethylaminoethyl methacrylate) hydrogel in one step. Transmission electron microscopy and dynamic light scattering analysis demonstrated that the hydrogel-coated nanoparticles are uniformly dispersed in the aqueous phase. The formation of covalent chemical bonds between the silica and the polymer increases the stability of the organic phase around the inorganic core as demonstrated by thermogravimetric analysis. The cationic nature of the hydrogel is responsible for the pH buffering properties of the nanostructured system and was evaluated by titration experiments. Zeta-potential analysis demonstrated that the charge of the system was reversed when transitioned from acidic to basic pH and vice versa. Consequently, small interfering RNA (siRNA) can be loaded and released in an acidic pH environment thereby enabling the hybrid particles and their payload to avoid endosomal sequestration and enzymatic degradation. These nanoparticles, loaded with specific siRNA molecules directed towards the transcript of the membrane receptor CXCR4, significantly decreased the expression of this protein in a human breast cancer cell line (i.e., MDA-MB-231). Moreover, intravenous administration of siRNA-loaded nanoparticles demonstrated a preferential accumulation at the tumor site that resulted in a reduction of CXCR4 expression. PMID:26901429

NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

NASA Technical Reports Server (NTRS)

Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

2005-01-01

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

Layer-by-layer assembly of small interfering RNA and poly(ethyleneimine) for substrate-mediated electroporation with high efficiency.

PubMed

Fujimoto, Hiroyuki; Kato, Koichi; Iwata, Hiroo

2010-05-01

Electroporation microarrays have been developed for the high-throughput transfection of expression constructs and small interfering RNAs (siRNAs) into living mammalian cells. These techniques have potential to provide a platform for the cell-based analysis of gene functions. One of the key issues associated with microarray technology is the efficiency of transfection. The capability of attaining reasonably high transfection efficiency is the basis for obtaining functional data without false negatives. In this study, we aimed at improving the transfection efficiency in the system that siRNA loaded on an electrode is electroporated into cells cultured directly on the electrode. The strategy we adopted here is to increase the surface density of siRNA loaded onto electrodes. For this purpose, the layer-by-layer assembly of siRNA and cationic polymers, branched or linear form of poly(ethyleneimine), was performed. The multilayer thus obtained was characterized by infrared reflection-adsorption spectroscopy and surface plasmon resonance analysis. Transfection efficiency was evaluated in a system that siRNA specific for enhanced green fluorescent protein (EGFP) was electroporated on the electrode into human embryonic kidney cells stably transformed with the EGFP gene. The suppression of EGFP expression was assessed by fluorescence microscopy and flow cytometry. Our data showed that the layer-by-layer assembly of siRNA with branched poly(ethyleneimine) facilitated to increase the surface density of loaded siRNA. As a result, the expression of EGFP gene in the electroporated cells was suppressed much more on the electrodes with the multilayer of siRNA than that with the monolayer.

Working conditions, visual fatigue, and mental health among systems analysts in São Paulo, Brazil

PubMed Central

Rocha, L; Debert-Ribeiro, M

2004-01-01

Aims: To evaluate the association between working conditions and visual fatigue and mental health among systems analysts living in São Paulo, Brazil. Methods: A cross sectional study was carried out by a multidisciplinary team. It included: ergonomic analysis of work, individual and group interviews, and 553 self applied questionnaires in two enterprises. The comparison population numbered 136 workers in different occupations. Results: The study population mainly comprised young males. Among systems analysts, visual fatigue was associated with mental workload, inadequate equipment and workstation, low level of worker participation, being a woman, and subject's attitude of fascination by the computer. Nervousness and intellectual performance were associated with mental workload, inadequate equipment, work environment, and tools. Continuing education and leisure were protective factors. Work interfering in family life was associated with mental workload, difficulties with clients, strict deadlines, subject's attitude of fascination by the computer, and finding solutions of work problems outside work. Family support, satisfaction in life and work, and adequate work environment and tools were protective factors. Work interfering in personal life was associated with subject's attitude of fascination by the computer, strict deadlines, inadequate equipment, and high level of work participation. Satisfaction in life and work and continuing education were protective factors. The comparison population did not share common working factors with the systems analysts in the regression analysis. Conclusions: The main health effects of systems analysts' work were expressed by machine anthropomorphism, being very demanding, mental acceleration, mental absorption, and difficulty in dealing with emotions. PMID:14691269

MicroRNA-15b silencing inhibits IL-1β-induced extracellular matrix degradation by targeting SMAD3 in human nucleus pulposus cells.

PubMed

Kang, Liang; Yang, Cao; Yin, Huipeng; Zhao, Kangcheng; Liu, Wei; Hua, Wenbin; Wang, Kun; Song, Yu; Tu, Ji; Li, Shuai; Luo, Rongjin; Zhang, Yukun

2017-04-01

To determine the role of microRNA-15b (miR-15b) in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation in the nucleus pulposus (NP). MiR-15b was up-regulated in degenerative NP tissues and in IL-1β-stimulated NP cells, as compared to the levels in normal controls (normal tissue specimens from patients with idiopathic scoliosis). Bioinformatics and luciferase activity analyses showed that mothers against decapentaplegic homolog 3 (SMAD3), a key mediator of the transforming growth factor-β signaling pathway, was directly targeted by miR-15b. Functional analysis demonstrated that miR-15b overexpression aggravated IL-1β-induced ECM degradation in NP cells, while miR-15b inhibition had the opposite effects. Prevention of IL-1β-induced NP ECM degeneration by the miR-15b inhibitor was attenuated by small-interfering-RNA-mediated knockdown of SMAD3. In addition, activation of MAP kinase and nuclear factor-κB up-regulated miR-15b expression and down-regulated SMAD3 expression in IL-1β-stimulated NP cells. MiR-15b contributes to ECM degradation in intervertebral disc degeneration (IDD) via targeting of SMAD3, thus providing a novel therapeutic target for IDD treatment.

RORC2 is involved in T cell polarization through interaction with the FOXP3 promoter.

PubMed

Burgler, Simone; Mantel, Pierre-Yves; Bassin, Claudio; Ouaked, Nadia; Akdis, Cezmi A; Schmidt-Weber, Carsten B

2010-06-01

The process of Th cell differentiation toward polarized effector T cells tailors specific immunity against invading pathogens while allowing tolerance against commensal microorganisms, harmless allergens, or autologous Ags. Identification of the mechanisms underlying this polarization process is therefore central to understand how the immune system confers immunity and tolerance. The present study demonstrates that retinoic acid receptor-related orphan receptor C2 (RORC2), a key transcription factor in Th17 cell development, inhibits FOXP3 expression in human T cells. Although overexpression of RORC2 in naive T cells reduces levels of FOXP3, small interfering RNA-mediated knockdown of RORC2 enhances its expression. RORC2 mediates this inhibition at least partially by binding to two out of four ROR-responsive elements on the FOXP3 promoter. Knockdown of RORC2 promotes high FOXP3 levels and decreased expression of proinflammatory cytokines beta form of pro-IL-1, IL-6, IL-17A, IFN-gamma, and TNF-alpha in differentiating naive T cells, suggesting that the role of RORC2 in Th17 cell development involves not only induction of Th17-characteristic genes, but also suppression of regulatory T cell-specific programs. Together, this study identifies RORC2 as a polarizing factor in transcriptional cross-regulation and provides novel viewpoints on the control of immune tolerance versus effector immune responses.

Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3

PubMed Central

Yoneda-Kato, Noriko; Tomoda, Kiichiro; Umehara, Mari; Arata, Yukinobu; Kato, Jun-ya

2005-01-01

Myeloid leukemia factor 1 (MLF1) was first identified as the leukemic fusion protein NPM-MLF1 generated by the t(3;5)(q25.1;q34) chromosomal translocation. Although MLF1 expresses normally in a variety of tissues including hematopoietic stem cells and the overexpression of MLF1 correlates with malignant transformation in human cancer, little is known about how MLF1 is involved in the regulation of cell growth. Here we show that MLF1 is a negative regulator of cell cycle progression functioning upstream of the tumor suppressor p53. MLF1 induces p53-dependent cell cycle arrest in murine embryonic fibroblasts. This action requires a novel binding partner, subunit 3 of the COP9 signalosome (CSN3). A reduction in the level of CSN3 protein with small interfering RNA abrogated MLF1-induced G1 arrest and impaired the activation of p53 by genotoxic stress. Furthermore, ectopic MLF1 expression and CSN3 knockdown inversely affect the endogenous level of COP1, a ubiquitin ligase for p53. Exogenous expression of COP1 overcomes MLF1-induced growth arrest. These results indicate that MLF1 is a critical regulator of p53 and suggest its involvement in leukemogenesis through a novel CSN3–COP1 pathway. PMID:15861129

Cardiolipin-Specific Peroxidase Reactions of Cytochrome c in Mitochondria During Irradiation-Induced Apoptosis

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

Belikova, Natalia A.; Jiang Jianfei; Tyurina, Yulia Y.

2007-09-01

Purpose: To determine whether cytochrome c (cyt c) content and associated cardiolipin oxidation can be determinants of cell sensitivity to irradiation-induced apoptosis. Methods and Materials: The small interfering RNA (siRNA) approach was used to engineer HeLa cells with lowered contents of cyt c (14%, HeLa 1.2 cells). Cells were treated by {gamma}-irradiation (in doses of 5-40 Gy). Lipid oxidation was characterized by electrospray ionization mass spectrometry analysis and fluorescence high-performance liquid chromatography-based Amplex Red assay. Release of a proapoptotic factor (cyt c, Smac/DIABLO) was detected by Western blotting. Apoptosis was revealed by caspase-3/7 activation and phosphatidylserine externalization. Results: Irradiation causedmore » selective accumulation of hydroperoxides in cardiolipin (CL) but not in other phospholipids. HeLa 1.2 cells responded by a lower irradiation-induced accumulation of CL oxidation products than parental HeLa cells. Proportionally decreased release of a proapoptotic factor, Smac/DIABLO, was detected in cyt c-deficient cells after irradiation. Caspase-3/7 activation and phosphatidylserine externalization were proportional to the cyt c content in cells. Conclusions: Cytochrome c is an important catalyst of CL peroxidation, critical to the execution of the apoptotic program. This new role of cyt c in irradiation-induced apoptosis is essential for the development of new radioprotectors and radiosensitizers.« less

Long noncoding ribonucleic acid NKILA induces the endoplasmic reticulum stress/autophagy pathway and inhibits the nuclear factor-k-gene binding pathway in rats after intracerebral hemorrhage.

PubMed

Jia, Jiaoying; Zhang, Mingming; Li, Qi; Zhou, Qian; Jiang, Yugang

2018-06-12

Long noncoding RNAs (lncRNAs) have emerged as an important class of molecules that have been associated with brain function and neurological disease, but the expression profiles of lncRNAs after intracerebral hemorrhage (ICH) remain to be elucidated. In this study, we determined the expression pattern of nuclear factor-k-gene binding (NF-kB) interacting lncRNA (NKILA) after ICH and examined its respective effects on the endoplasmic reticulum stress (ERS)/autophagy pathway, hippocampal neuron loss, and the NF-kB pathway after type VII collagenase-induced ICH in rats. The regulatory mechanisms of NKILA were investigated by an intraperitoneal injection of small interfering (siRNA) against NKILA into rats after ICH. NKILA inhibition mediated by siRNA against NKILA was shown to significantly reduce ERS and autophagy, activate the NF-kB pathway, decrease neurological deficits, brain edema, and injury, and induce blood-brain barrier breakdown, further leading to hippocampal neuron loss and the production of inflammation cytokines. Taken together, the demonstration that NKILA induces the ERS/autophagy pathway and inhibits the NF-kB pathway after ICH supports the concept that NKILA functions as a novel target that is required for the attenuation of brain injuries after ICH. © 2018 Wiley Periodicals, Inc.

Modulation of Nrf2/Keap1 system by Wasabi 6-methylthiohexyl isothiocyanate in ARE-mediated NQO1 expression.

PubMed

Korenori, Yoshimi; Tanigawa, Shunsuke; Kumamoto, Takuma; Qin, Si; Daikoku, Yosuke; Miyamori, Koji; Nagai, Masashi; Hou, De-Xing

2013-05-01

6-Methylthiohexyl isothiocyanate (6-MTITC), one of the major bioactive ingredients in Japanese Wasabi, has revealed cytoprotective and cancer chemopreventive effects. This study aims to clarify the molecular mechanisms how 6-MTITC modulates nuclear factor E2-related factor 2 (Nrf2)/Kelchlike ECH-associating protein 1 (Keap1) system in antioxidant-responsive element (ARE)-mediated nicotinamide adenine dinucleotide phosphate (NADP): quinone oxidoreductase 1 (NQO1) expression. HepG2 cells were treated with 6-MTITC with varying time and dose. NQO1, Nrf2, and Keap1 proteins were detected by Western blotting. ARE transactivation was detected by electrophilic mobility shift assay and reporter gene assay. Nuclear localization of Nrf2 was determined by immunocytochemistry assay. Ubiquitination of Nrf2 and Keap1 was detected using immunoprecipitation after treatment with MG132. Small interfering RNA was used to knockdown Nrf2 or Keap1. The results revealed that 6-MTITC modulated Nrf2/ARE pathway by stimulating Keap1 modification, and inhibiting Nrf2 ubiquitination and protein turnover. These actions finally increased nuclear Nrf2 accumulation and ARE-binding activity. Moreover, silencing Nrf2 markedly reduced ARE-driven activity induced by 6-MTITC. 6-MTITC modulated ARE-driven NQO1 expression by stabilizing Nrf2 with enhanced Keap1 modification and decreased Nrf2 degradation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling

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

Matsumura, Kaori; Taketomi, Takaharu, E-mail: taketomi@dent.kyushu-u.ac.jp; Yoshizaki, Keigo

2011-01-28

Research highlights: {yields} Sprouty2-deficient mice exhibit cleft palate as a result of failure of palatal shelf elevation. {yields} We examined palate cell proliferation in Sprouty2-deficient mice. {yields} Palate mesenchymal cell proliferation was increased in Sprouty2 KO mice. {yields} Sprouty2 plays roles in murine palatogenesis by regulating cell proliferation. -- Abstract: Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, wemore » report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation.« less

Eukaryotic translational initiation factor 4AII reduces the replication of infectious bursal disease virus by inhibiting VP1 polymerase activity.

PubMed

Gao, Li; Li, Kai; Zhong, Li; Zhang, Lizhou; Qi, Xiaole; Wang, Yongqiang; Gao, Yulong; Wang, Xiaomei

2017-03-01

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Although an interaction between eukaryotic translational initiation factor 4AII (eIF4AII) of the host and viral protein 1 (VP1), the RNA-dependent RNA polymerase (RdRp) of IBDV, has been established, the underlying effects of this interaction on IBDV and the molecular mechanism remain unclear. We here report that interaction of the host eIF4AII with VP1 inhibits the RNA polymerase activity of IBDV to reduce its replication in host cells. We found that ectopically expressed eIF4AII markedly inhibited IBDV growth in DF1 cells, and knockdown of eIF4AII by small interfering RNA significantly enhanced viral replication in CEF cells. Furthermore, IBDV infection led to an increase in host eIF4AII expression, suggesting a feedback mechanism between the host and virus infection both in vitro and in vivo, which further confirmed the involvement of the host eIF4AII in the IBDV life cycle. Thus, via the interaction with VP1, eIF4AII plays a critical role in the IBDV life cycle, by inhibiting viral RNA polymerase activity, leading to a reduction of IBDV replication in cells. Copyright © 2016 Elsevier B.V. All rights reserved.

RhoA-ROCK and p38MAPK-MSK1 mediate vitamin D effects on gene expression, phenotype, and Wnt pathway in colon cancer cells.

PubMed

Ordóñez-Morán, Paloma; Larriba, María Jesús; Pálmer, Héctor G; Valero, Ruth A; Barbáchano, Antonio; Duñach, Mireia; de Herreros, Antonio García; Villalobos, Carlos; Berciano, María Teresa; Lafarga, Miguel; Muñoz, Alberto

2008-11-17

The active vitamin D metabolite 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits proliferation and promotes differentiation of colon cancer cells through the activation of vitamin D receptor (VDR), a transcription factor of the nuclear receptor superfamily. Additionally, 1,25(OH)(2)D(3) has several nongenomic effects of uncertain relevance. We show that 1,25(OH)(2)D(3) induces a transcription-independent Ca(2+) influx and activation of RhoA-Rho-associated coiled kinase (ROCK). This requires VDR and is followed by activation of the p38 mitogen-activated protein kinase (p38MAPK) and mitogen- and stress-activated kinase 1 (MSK1). As shown by the use of chemical inhibitors, dominant-negative mutants and small interfering RNA, RhoA-ROCK, and p38MAPK-MSK1 activation is necessary for the induction of CDH1/E-cadherin, CYP24, and other genes and of an adhesive phenotype by 1,25(OH)(2)D(3). RhoA-ROCK and MSK1 are also required for the inhibition of Wnt-beta-catenin pathway and cell proliferation. Thus, the action of 1,25(OH)(2)D(3) on colon carcinoma cells depends on the dual action of VDR as a transcription factor and a nongenomic activator of RhoA-ROCK and p38MAPK-MSK1.

Smad ubiquitination regulatory factor-2 controls gap junction intercellular communication by modulating endocytosis and degradation of connexin43.

PubMed

Fykerud, Tone Aase; Kjenseth, Ane; Schink, Kay Oliver; Sirnes, Solveig; Bruun, Jarle; Omori, Yasufumi; Brech, Andreas; Rivedal, Edgar; Leithe, Edward

2012-09-01

Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junction channels are made of a family of integral membrane proteins called connexins, of which the best-studied member is connexin43. Gap junctions are dynamic plasma membrane domains, and connexin43 has a high turnover rate in most tissue types. However, the mechanisms involved in the regulation of connexin43 endocytosis and transport to lysosomes are still poorly understood. Here, we demonstrate by live-cell imaging analysis that treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) induces endocytosis of subdomains of connexin43 gap junctions. The internalized, connexin43-enriched vesicles were found to fuse with early endosomes, which was followed by transport of connexin43 to the lumen of early endosomes. The HECT E3 ubiquitin ligase smad ubiquitination regulatory factor-2 (Smurf2) was found to be recruited to connexin43 gap junctions in response to TPA treatment. Depletion of Smurf2 by small interfering RNA resulted in enhanced levels of connexin43 gap junctions between adjacent cells and increased gap junction intercellular communication. Smurf2 depletion also counteracted the TPA-induced endocytosis and degradation of connexin43. Collectively, these data identify Smurf2 as a novel regulator of connexin43 gap junctions.

Synthetic Protection Short Interfering RNA Screen Reveals Glyburide as a Novel Radioprotector

PubMed Central

Jiang, Jianfei; McDonald, Peter R.; Dixon, Tracy M.; Franicola, Darcy; Zhang, Xichen; Nie, Suhua; Epperly, Laura D.; Huang, Zhentai; Kagan, Valerian E.; Lazo, John S.; Epperly, Michael W.; Greenberger, Joel S.

2009-01-01

To assist in screening existing drugs for use as potential radioprotectors, we used a human unbiased 16,560 short interfering RNA (siRNA) library targeting the druggable genome. We performed a synthetic protection screen that was designed to identify genes that, when silenced, protected human glioblastoma T98G cells from γ-radiation-induced cell death. We identified 116 candidate protective genes, then identified 10 small molecule inhibitors of 13 of these candidate gene products and tested their radioprotective effects. Glyburide, a clinically used second-generation hypoglycemic drug, effectively decreased radiation-induced cell death in several cell lines including T98G, glioblastoma U-87 MG, and normal lung epithelial BEAS-2B and in primary cultures of astrocytes. Glyburide significantly increased the survival of 32D cl3 murine hematopoietic progenitor cells when administrated before irradiation. Glyburide was radioprotective in vivo (90% of C57BL/6NHsd female mice pretreated with 10 mg/kg glyburide survived 9.5 Gy total-body irradiation compared to 42% of irradiated controls, P = 0.0249). These results demonstrate the power of unbiased siRNA synthetic protection screening with a druggable genome library to identify new radioprotectors. PMID:19772462

Application of small RNA technology for improved control of parasitic helminths.

PubMed

Britton, Collette; Winter, Alan D; Marks, Neil D; Gu, Henry; McNeilly, Tom N; Gillan, Victoria; Devaney, Eileen

2015-08-15

Over the last decade microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important regulators of post-transcriptional gene expression. miRNAs are short, non-coding RNAs that regulate a variety of processes including cancer, organ development and immune function. This class of small RNAs bind with partial complementarity to their target mRNA sequences, most often in the 3'UTR, to negatively regulate gene expression. In parasitic helminths, miRNAs are being increasingly studied for their potential roles in development and host-parasite interactions. The availability of genome data, combined with small RNA sequencing, has paved the way to profile miRNAs expressed at particular developmental stages for many parasitic helminths. While some miRNAs are conserved across species, others appear to be unique to specific parasites, suggesting important roles in adaptation and survival in the host environment. Some miRNAs are released from parasites, in exosomes or in protein complexes, and the potential effects of these on host immune function are being increasingly studied. In addition, release of miRNAs from schistosome and filarial parasites into host plasma can be exploited for the development of specific and sensitive diagnostic biomarkers of infection. Interfering with miRNA function, as well as silencing key components of the pathways they regulate, will progress our understanding of parasite development and provide a novel approach to therapeutic control. RNA interference (RNAi) by siRNAs has proven to be inconsistent in parasitic nematodes. However, the recent successes reported for schistosome and liver fluke RNAi, encourage further efforts to enhance delivery of RNA and improve in vitro culture systems and assays to monitor phenotypic effects in nematodes. These improvements are important for the establishment of reliable functional genomic platforms for novel drug and vaccine development. In this review we focus on the important roles of miRNAs and siRNAs in post-transcriptional gene regulation in veterinary parasitic helminths and the potential value of these in parasite diagnosis and control. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Study of physical conditions in protoplanetary disks by interferometry. Theory, instrumentation and first observations. -- étude des conditions physiques dans les disques protoplanétaires par interférométrie. Théorie, instrumentation et premières observations.

NASA Astrophysics Data System (ADS)

Malbet, Fabien

2007-10-01

Les étoiles se forment lors de l'effondrement de nuages de gaz et de poussière. Dans l'environnement proche de l'étoile naissante la matière se concentre dans un plan équatorial que l'on appelle disque protoplanétaire. Les astronomes pensent que les planètes se forment au sein de cette masse de gaz et de poussière orbitant autour de l'étoile. Pour sonder ces disques à des échelles correspondant aux orbites des futures planètes, il convient d'observer dans l'infrarouge à très haute résolution spatiale. L'interférométrie infrarouge est donc un outil idéal pour étudier les conditions physiques des disques protoplanétaires. Dans ce mémoire, je décris les premiers pas de l'interférométrie infrarouge, depuis la mise au point des petits interféromètres PTI et IOTA jusqu'à la construction de l'instrument AMBER au foyer de l'interféromètre du VLT. Je décris aussi les résultats d'une piste de recherche technologique particulièrement attrayante dans le cas de l'interférométrie infrarouge et issue des technologies des autoroutes de l'information: l'optique intégrée appliquée à la combinaison de plusieurs faisceaux en astronomie. Je montre ensuite comment à partir des observations obtenues à partir de ces instruments, il est possible de contraindre la physique des disques autour des étoiles jeunes. Gráce à la résolution spectrale nouvellement disponible sur ces instruments, pour la première fois nous pouvons séparer des phénomènes physiques aussi différents que l'accrétion de matière sur l'étoile et l'éjection de particules par des vents dont l'origine précise est encore mal connue. Les résultats présentés dans ce mémoire ont été obtenus principalement à partir d'observations sur les systèmes jeunes FU Ori et MWC 297 effectuées par AMBER sur le VLTI, mais aussi par les petits interféromètres infrarouges PTI et IOTA. Je développe aussi les travaux de modélisation de la structure verticale des disques associés afin de montrer la richesse des renseignements obtenus. Finalement je trace les contours d'un programme de recherche qui permettra tout d'abord de maximiser le retour astrophysique sur un instrument comme le VLTI, puis d'obtenir de premières images interférométriques de ces environnements circumstellaires. Je propose aussi la réalisation d'un instrument de seconde génération qui permettra de fournir des images interférométriques détaillées de ces sources compactes par synthèse d'ouverture. Stars are forming when clouds of gas and dust collapse. In the close environment of the new star, the matter is concentrated in an equatorial plane which is called protoplanetary disk. The astronomers think that planets are formed within this mass of gas and dust orbiting around the star. To probe these disks at scales corresponding to the orbits of the future planets, it is necessary to observe at very high spatial resolution in the infrared wavelength domain. Infrared interferometry is therefore an ideal tool to study the physical conditions in protoplanetary disks. In this document, I describe the first steps of infrared interferometry, from the beginning of the small interferometers PTI and IOTA until the construction of the AMBER instrument at the focus of the VLT Interferometer. I describe also the results of a technological research track, particularly attractive in the case of infrared interferometry, and coming from the information freeway: the integrated optics applies to the combination of several beams in astronomy. I show then how from observations obtained from these instruments, it is possible to constrain the physics of disks around young stars. Thanks to the spectral resolution recently available on these instruments, for the first time, we can separate the physical phenomena as different as accretion of matter onto the star and the ejection of particles by winds whose precise origin is still not well known. The results presented in this document were obtained mainly from observations on the young systems FU Ori and MWC 297 and performed by AMBER on the VLTI, but also by the small infrared interferometers PTI and IOTA. I tackle also the modeling of the vertical structure of those disks in order to show the wealth of obtained information. Finally I draw the contours of a research program that will allow first the VLTI astrophysical return to be maximized, and then the first interferometric images of these circumstellar environments to be obtained. I also propose to build a second generation instrument for the VLTI which will bring detailed interferometric images by aperture synthesis of these compact sources.

Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy

PubMed Central

Miele, Evelina; Spinelli, Gian Paolo; Miele, Ermanno; Di Fabrizio, Enzo; Ferretti, Elisabetta; Tomao, Silverio; Gulino, Alberto

2012-01-01

During recent decades there have been remarkable advances and profound changes in cancer therapy. Many therapeutic strategies learned at the bench, including monoclonal antibodies and small molecule inhibitors, have been used at the bedside, leading to important successes. One of the most important advances in biology has been the discovery that small interfering RNA (siRNA) is able to regulate the expression of genes, by a phenomenon known as RNA interference (RNAi). RNAi is one of the most rapidly growing fields of research in biology and therapeutics. Much research effort has gone into the application of this new discovery in the treatment of various diseases, including cancer. However, even though these molecules may have potential and strong utility, some limitations make their clinical application difficult, including delivery problems, side effects due to off-target actions, disturbance of physiological functions of the cellular machinery involved in gene silencing, and induction of the innate immune response. Many researchers have attempted to overcome these limitations and to improve the safety of potential RNAi-based therapeutics. Nanoparticles, which are nanostructured entities with tunable size, shape, and surface, as well as biological behavior, provide an ideal opportunity to modify current treatment regimens in a substantial way. These nanoparticles could be designed to surmount one or more of the barriers encountered by siRNA. Nanoparticle drug formulations afford the chance to improve drug bioavailability, exploiting superior tissue permeability, payload protection, and the “stealth” features of these entities. The main aims of this review are: to explain the siRNA mechanism with regard to potential applications in siRNA-based cancer therapy; to discuss the possible usefulness of nanoparticle-based delivery of certain molecules for overcoming present therapeutic limitations; to review the ongoing relevant clinical research with its pitfalls and promises; and to evaluate critically future perspectives and challenges in siRNA-based cancer therapy. PMID:22915840

Identification and molecular characterization of a trans-acting small interfering RNA producing locus regulating leaf rust responsive gene expression in wheat (Triticum aestivum L.).

PubMed

Dutta, Summi; Kumar, Dhananjay; Jha, Shailendra; Prabhu, Kumble Vinod; Kumar, Manish; Mukhopadhyay, Kunal

2017-11-01

A novel leaf rust responsive ta-siRNA-producing locus was identified in wheat showing similarity to 28S rRNA and generated four differentially expressing ta-siRNAs by phasing which targeted stress responsive genes. Trans-acting-small interfering RNAs (Ta-siRNAs) are plant specific molecules generally involved in development and are also stress responsive. Ta-siRNAs identified in wheat till date are all responsive to abiotic stress only. Wheat cultivation is severely affected by rusts and leaf rust particularly affects grain filling. This study reports a novel ta-siRNA producing locus (TAS) in wheat which is a segment of 28S ribosomal RNA but shows differential expression during leaf rust infestation. Four small RNA libraries prepared from wheat Near Isogenic Lines were treated with leaf rust pathogen and compared with untreated controls. A TAS with the ability to generate four ta-siRNAs by phasing events was identified along with the microRNA TamiR16 as the phase initiator. The targets of the ta-siRNAs included α-gliadin, leucine rich repeat, trans-membrane proteins, glutathione-S-transferase, and fatty acid desaturase among others, which are either stress responsive genes or are essential for normal growth and development of plants. Expression of the TAS, its generated ta-siRNAs, and their target genes were profiled at five different time points after pathogen inoculation of susceptible and resistant wheat isolines and compared with mock-inoculated controls. Comparative analysis of expression unveiled differential and reciprocal relationship as well as discrete patterns between susceptible and resistant isolines. The expression profiles of the target genes of the identified ta-siRNAs advocate more towards effector triggered susceptibility favouring pathogenesis. The study helps in discerning the functions of wheat genes regulated by ta-siRNAs in response to leaf rust.

We Can Still Be Friends: IFN-γ Breaks Up Macrophage Enhancers.

PubMed

Novakovic, Boris; Wang, Cheng; Logie, Colin

2017-08-15

Interferon (IFN)-γ can prime macrophages for inflammatory responses by several mechanisms, including enhancer establishment and gene activation. In this issue of Immunity, Kang et al. (2017) provide insight into the mechanisms of IFN-γ-mediated gene repression as they show that IFN-γ promotes the disassembly of select active enhancers by interfering with enhancer-binding transcription factor MAF. Copyright © 2017 Elsevier Inc. All rights reserved.

Determination of twenty-nine elements in eight argonne premium coal samples by instrumental neutron activation analysis

USGS Publications Warehouse

Palmer, C.A.

1990-01-01

Twenty-nine elements have been determined in triplicate splits of the eight Argonne National Laboratory Premium Coal Samples by instrumental neutron activtaion analysis. Data for control samples NBS 1633 (fly ash) and NBS 1632b are also reported. The factors that could lead to errors in analysis for these samples, such as spectral overlaps, low sensitivity, and interfering nuclear reactions, are discussed.

A novel class of small RNAs bind to MILI protein in mouse testes.

PubMed

Aravin, Alexei; Gaidatzis, Dimos; Pfeffer, Sébastien; Lagos-Quintana, Mariana; Landgraf, Pablo; Iovino, Nicola; Morris, Patricia; Brownstein, Michael J; Kuramochi-Miyagawa, Satomi; Nakano, Toru; Chien, Minchen; Russo, James J; Ju, Jingyue; Sheridan, Robert; Sander, Chris; Zavolan, Mihaela; Tuschl, Thomas

2006-07-13

Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes. A subgroup of the Argonaute proteins--known as the 'Piwi family'--is required for germ- and stem-cell development in invertebrates, and two Piwi members--MILI and MIWI--are essential for spermatogenesis in mouse. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5' uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26-31 nucleotides (nt) in length--clearly distinct from the 21-23 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)--and we refer to them as 'Piwi-interacting RNAs' or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis.

Incidence of sleep pattern disturbance (SPD) in a hemodialysis sample.

PubMed

Strangio, D; Locking-Cusolito, H

1999-01-01

Personal experience suggests that sleep pattern disturbance (SPD) is a serious problem for the patients we serve. The purpose of this study was to identify the scope of sleep problems among all willing patients in a medium-sized hemodialysis unit in a university teaching centre. This descriptive study examined SPD through the use of a sleep diary that subjects were asked to complete each morning for a week. Subjects were asked to describe sleep latency, sleep quantity, number of arousals, whether they awoke feeling rested, factors that interfered with sleep the night before, and sleep inducers employed the night before. They were also asked to record their dialysis schedule. Each subject's chart was reviewed with respect to medications and evidence of other medical problems that interfered with sleep. Findings were benchmarked with results from the literature. Information regarding facilitators and barriers to sleep has provided some basis for an interdisciplinary plan of care to address this distressing problem.

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

PubMed Central

Li, Changlin; Cai, Xiangyu; Sun, Haili; Bai, Ting; Zheng, Xilong; Zhou, Xing Wang; Chen, Xiongwen; Gill, Donald L.; Li, Jing; Tang, Xiang D.

2011-01-01

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a new promising target for prevention and treatment of cardiac hypertrophy and heart failure. There are 3 δ isoforms of CaMKII in the heart and previous studies focused primarily on δB and δC types. Here we report the δA isoform of CaMKII is also critically involved in cardiac hypertrophy. We found that δA was significantly upregulated in pathological cardiac hypertrophy in both neonatal and adult models. Upregulation of δA was accompanied by cell enlargement, sarcomere reorganization and reactivation of various hypertrophic cardiac genes including atrial natriuretic factor (ANF) and β-myocin heavy chain (β-MHC). Studies further indicated the pathological changes were largely blunted by silencing the δA gene. These results provide new evidence for selective interfering cardiac hypertrophy and heart failure when CaMKII is considered as a therapeutic target. PMID:21554860

Factors Affecting Gastrointestinal Absorption of Levothyroxine: A Review.

PubMed

Skelin, Marko; Lucijanić, Tomo; Amidžić Klarić, Daniela; Rešić, Arnes; Bakula, Miro; Liberati-Čizmek, Ana-Marija; Gharib, Hossein; Rahelić, Dario

2017-02-01

Levothyroxine (LT4) is a drug with a narrow therapeutic index, applied in small amounts (micrograms), which makes interactions in the absorption phase clinically significant. The main aim of this article was to review and present the latest information on factors that affect the gastrointestinal absorption of this drug. Relevant data were collected by using the MEDLINE, PubMed, EMBASE, Web of Science, Science Direct, and Scopus databases with the key words levothyroxine and absorption. Searches were not limited to specific publication types, study designs, dates, or languages. The reports were highly variable in the amount of information provided regarding study design and methods. Because of the heterogeneity of studies, no statistical analysis was performed. Many gastrointestinal disorders, such as celiac disease, atrophic gastritis, lactose intolerance, and Helicobacter pylori infection, may impede the absorption of levothyroxine. During treatment of these disorders, it is necessary to monitor serum thyroid-stimulating hormone and free T4 values to reduce the risk of developing iatrogenic hyperthyroidism. Soybeans and coffee have the greatest impact on the reduction of absorption, whereas vitamin C has the ability to increase it. Conversely, the effect of dietary fiber on the absorption of LT4 is not yet fully understood; further research is needed on this topic. A decrease in the absorption of LT4 is established and clinically significant when administered concomitantly with cholestyramine, colesevelam, lanthanum, calcium carbonate, calcium citrate, calcium acetate, iron sulfate, ciprofloxacin, aluminum hydroxide, sevelamer, or proton pump inhibitors. This effect should be taken into consideration when prescribing these drugs concomitantly with LT4. The effects of Giardia lamblia infection and the influence of orlistat, polystyrene sulfonate, raloxifene, and simethicone on absorption of LT4 have been poorly documented. For bariatric surgery, sucralfate and H 2 -antagonist interactions are not well founded or contradictory evidence is available regarding their existence; additional research should be conducted. The majority of the interactions are clinically significant. They are based on the LT4 adsorption on interfering substances in the digestive tract, as well as a consequently reduced amount of the drug available for absorption. These interactions can be avoided by separating the administration of LT4 and the interfering substance. Copyright © 2017 Elsevier HS Journals, Inc. All rights reserved.

RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding.

PubMed

Younis, Adnan; Siddique, Muhammad Irfan; Kim, Chang-Kil; Lim, Ki-Byung

2014-01-01

RNA interference (RNAi) is a promising gene regulatory approach in functional genomics that has significant impact on crop improvement which permits down-regulation in gene expression with greater precise manner without affecting the expression of other genes. RNAi mechanism is expedited by small molecules of interfering RNA to suppress a gene of interest effectively. RNAi has also been exploited in plants for resistance against pathogens, insect/pest, nematodes, and virus that cause significant economic losses. Keeping beside the significance in the genome integrity maintenance as well as growth and development, RNAi induced gene syntheses are vital in plant stress management. Modifying the genes by the interference of small RNAs is one of the ways through which plants react to the environmental stresses. Hence, investigating the role of small RNAs in regulating gene expression assists the researchers to explore the potentiality of small RNAs in abiotic and biotic stress management. This novel approach opens new avenues for crop improvement by developing disease resistant, abiotic or biotic stress tolerant, and high yielding elite varieties.

RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding

PubMed Central

Younis, Adnan; Siddique, Muhammad Irfan; Kim, Chang-Kil; Lim, Ki-Byung

2014-01-01

RNA interference (RNAi) is a promising gene regulatory approach in functional genomics that has significant impact on crop improvement which permits down-regulation in gene expression with greater precise manner without affecting the expression of other genes. RNAi mechanism is expedited by small molecules of interfering RNA to suppress a gene of interest effectively. RNAi has also been exploited in plants for resistance against pathogens, insect/pest, nematodes, and virus that cause significant economic losses. Keeping beside the significance in the genome integrity maintenance as well as growth and development, RNAi induced gene syntheses are vital in plant stress management. Modifying the genes by the interference of small RNAs is one of the ways through which plants react to the environmental stresses. Hence, investigating the role of small RNAs in regulating gene expression assists the researchers to explore the potentiality of small RNAs in abiotic and biotic stress management. This novel approach opens new avenues for crop improvement by developing disease resistant, abiotic or biotic stress tolerant, and high yielding elite varieties. PMID:25332689

EGFR-L858R mutant enhances lung adenocarcinoma cell invasive ability and promotes malignant pleural effusion formation through activation of the CXCL12-CXCR4 pathway

PubMed Central

Tsai, Meng-Feng; Chang, Tzu-Hua; Wu, Shang-Gin; Yang, Hsiao-Yin; Hsu, Yi-Chiung; Yang, Pan-Chyr; Shih, Jin-Yuan

2015-01-01

Malignant pleural effusion (MPE) is a common clinical problem in non-small cell lung carcinoma (NSCLC) patients; however, the underlying mechanisms are still largely unknown. Recent studies indicate that the frequency of the L858R mutant form of the epidermal growth factor receptor (EGFR-L858R) is higher in lung adenocarcinoma with MPE than in surgically resected specimens, suggesting that lung adenocarcinoma cells harboring this mutation tend to invade the adjacent pleural cavity. The purpose of this study was to clarify the relationship between the EGFR-L858R mutation and cancer cell invasion ability and to investigate the molecular mechanisms involved in the formation of MPE. We found that expression of EGFR-L858R in lung cancer cells resulted in up-regulation of the CXCR4 in association with increased cancer cell invasive ability and MPE formation. Ectopic expression of EGFR-L858R in lung cancer cells acted through activation of ERK signaling pathways to induce the expression of CXCR4. We also indicated that Inhibition of CXCR4 with small interfering RNA, neutralizing antibody, or receptor antagonist significantly suppressed the EGFR-L858R–dependent cell invasion. These results suggest that targeting the production of CXCR4 and blocking the CXCL12-CXCR4 pathway might be effective strategies for treating NSCLCs harboring a specific type of EGFR mutation. PMID:26338423

Progesterone receptor (PR) polyproline domain (PPD) mediates inhibition of epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer cells.

PubMed

Kawprasertsri, Sornsawan; Pietras, Richard J; Marquez-Garban, Diana C; Boonyaratanakornkit, Viroj

2016-05-01

Recent evidence has suggested a possible role for progesterone receptor (PR) in the progression of non-small cell lung cancer (NSCLC). However, little is known concerning roles of PR in NSCLC. PR contains a polyproline domain (PPD), which directly binds to the SH3 domain of signaling molecules. Because PPD-SH3 interactions are essential for EGFR signaling, we hypothesized that the presence of PR-PPD interfered with EGFR-mediated signaling and cell proliferation. We examined the role of PR-PPD in cell proliferation and signaling by stably expressing PR-B, or PR-B with disrupting mutations in the PPD (PR-BΔSH3), from a tetracycline-regulated promoter in A549 NSCLC cells. PR-B dose-dependently inhibited cell growth in the absence of ligand, and progestin (R5020) treatment further suppressed the growth. Treatment with RU486 abolished PR-B- and R5020-mediated inhibition of cell proliferation. Expression of PR-BΔSH3 and treatment with R5020 or RU486 had no effect on cell proliferation. Furthermore, PR-B expression but not PR-BΔSH3 expression reduced EGF-induced A549 proliferation and activation of ERK1/2, in the absence of ligand. Taken together, our data demonstrated the significance of PR extranuclear signaling through PPD interactions in EGFR-mediated proliferation and signaling in NSCLC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

p21-activated kinase 1: PAK'ed with potential.

PubMed

Ong, Christy C; Jubb, Adrian M; Zhou, Wei; Haverty, Peter M; Harris, Adrian L; Belvin, Marcia; Friedman, Lori S; Koeppen, Hartmut; Hoeflich, Klaus P

2011-06-01

The p21-activated kinases (PAKs) are central players in growth factor signaling networks and morphogenetic processes that control proliferation, cell polarity, invasion and actin cytoskeleton organization. This raises the possibility that interfering with PAK activity may produce significant anti-tumor activity. In this perspective, we summarize recent data concerning the contribution of the PAK family member, PAK1, in growth factor signaling and tumorigenesis. We further discuss mechanisms by which inhibition of PAK1 can arrest tumor growth and promote cell apoptosis, and the types of cancers in which PAK1 inhibition may hold promise.

Relevance of small GTPase Rac1 pathway in drug and radio-resistance mechanisms: Opportunities in cancer therapeutics.

PubMed

Cardama, G A; Alonso, D F; Gonzalez, N; Maggio, J; Gomez, D E; Rolfo, C; Menna, P L

2018-04-01

Rac1 GTPase signaling pathway has a critical role in the regulation of a plethora of cellular functions governing cancer cell behavior. Recently, it has been shown a critical role of Rac1 in the emergence of resistance mechanisms to cancer therapy. This review describes the current knowledge regarding Rac1 pathway deregulation and its association with chemoresistance, radioresistance, resistance to targeted therapies and immune evasion. This supports the idea that interfering Rac1 signaling pathway could be an interesting approach to tackle cancer resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

Native gel analysis for RISC assembly.

PubMed

Kawamata, Tomoko; Tomari, Yukihide

2011-01-01

Small-interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate expression of their target mRNAs via the RNA-induced silencing complex (RISC). A core component of RISC is the Argonaute (Ago) protein, which dictates the RISC function. In Drosophila, miRNAs and siRNAs are generally loaded into Ago1-containing RISC (Ago1-RISC) and Ago2-containing RISC (Ago2-RISC), respectively. We developed a native agarose gel system to directly detect Ago1-RISC, Ago2-RISC, and their precursor complexes. Methods presented here will provide powerful tools to biochemically dissect the RISC assembly pathways.

Delivery of large biopharmaceuticals from cardiovascular stents: a review

PubMed Central

Takahashi, Hironobu; Letourneur, Didier; Grainger, David W.

2008-01-01

This review focuses on the new and emerging large-molecule bioactive agents delivered from stent surfaces in drug-eluting stents (DES) to inhibit vascular restenosis in the context of interventional cardiology. New therapeutic agents representing proteins, nucleic acids (small interfering RNAs and large DNA plasmids), viral delivery vectors and even engineered cell therapies require specific delivery designs distinct from traditional smaller molecule approaches on DES. While small molecules are currently the clinical standard for coronary stenting, extension of the DES to other lesion types, peripheral vasculature and non-vasculature therapies will seek to deliver an increasingly sophisticated armada of drug types. This review describes many of the larger molecule and biopharmaceutical approaches reported recently for stent-based delivery with the challenges associated with formulating and delivering these drug classes compared to the current small molecule drugs. It also includes perspectives on possible future applications that may improve safety and efficacy and facilitate diversification of the DES to other clinical applications. PMID:17929968

A novel albumin nanocomplex containing both small interfering RNA and gold nanorods for synergetic anticancer therapy

NASA Astrophysics Data System (ADS)

Choi, Jin-Ha; Hwang, Hai-Jin; Shin, Seung Won; Choi, Jeong-Woo; Um, Soong Ho; Oh, Byung-Keun

2015-05-01

Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine.Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr00211g

A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis

PubMed Central

Fukunaga, Ryuya; Zamore, Phillip D

2014-01-01

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme. PMID:24787225

Transgenerational inheritance of an acquired small RNA-based antiviral response in C.elegans

PubMed Central

Rechavi, Oded; Minevich, Gregory; Hobert, Oliver

2011-01-01

Induced expression of the Flock House virus in the soma of C. elegans results in the RNAi-dependent production of virus-derived, small interfering RNAs (viRNAs), which in turn silence the viral genome. We show here that the viRNA-mediated viral silencing effect is transmitted in a non-Mendelian manner to many ensuing generations. We show that the viral silencing agents, viRNAs, are transgenerationally transmitted in a template-independent manner and work in trans to silence viral genomes present in animals that are deficient in producing their own viRNAs. These results provide evidence for the transgenerational inheritance of an acquired trait, induced by the exposure of animals to a specific, biologically relevant physiological challenge. The ability to inherit such extragenic information may provide adaptive benefits to an animal. PMID:22119442